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Cellular Respiration and Carcinogenesis

Shireesh P. Apte • Rangaprasad SarangarajanEditors

Cellular Respirationand Carcinogenesis

Foreword by Gregg L. Semenza

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EditorsShireesh P. ApteAlcon LaboratoriesFort Worth, [email protected]

Rangaprasad SarangarajanMassachusetts College ofPharmacy and Health SciencesWorcester, [email protected]

ISBN: 978-1-934115-07-7 e-ISBN: 978-1-59745-435-3DOI 10.1007/978-1-59745-435-3

Library of Congress Control Number: 2008937240

c© Humana Press, a part of Springer Science+Business Media, LLC 2009All rights reserved. This work may not be translated or copied in whole or in part without the written per-mission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street,New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis.Use in connection with any form of information storage and retrieval, electronic adaptation, computersoftware, or by similar or dissimilar methodology now known or hereafter developed is forbidden.The use in this publication of trade names, trademarks, service marks, and similar terms, even if they arenot identified as such, is not to be taken as an expression of opinion as to whether or not they are subjectto proprietary rights.

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Foreword

Cancer is remarkably heterogeneous when one considers pathogenesis of the diseasein affected individuals with the same diagnosis (such as “renal carcinoma”) or evenwhen cancer in a single individual is considered over time and space. This het-erogeneity is the single greatest obstacle to effective cancer therapy. As a result ofdisease heterogeneity, there are not too many universal truths in cancer biology. Oneof the most consistent findings, though, is the dramatic increase in glucose uptakeby metastatic cancer cells compared with that of their normal counterparts. Thisis such a reliable characteristic that it is used clinically to identify occult diseaseby positron emission tomography after injection of [18F]fluoro-2-deoxy-D-glucose.Increased glucose uptake reflects an increased rate of glycolysis, with conversion ofglucose to lactate and decreased conversion of pyruvate to acetyl coenzyme A, thesubstrate for mitochondrial oxidative phosphorylation. Because of the relative inef-ficiency of glycolysis compared with that of oxidative phosphorylation as a meansof generating ATP, flux through the glycolytic pathway must increase dramaticallyto maintain cellular energetics.

Because it is observed in more than 95% of advanced cancers, understandingthe mechanisms and consequences of this dramatic reprogramming of glucose andenergy metabolism in cancer cells is an important challenge for contemporary can-cer biology. The 12 chapters of this book provide the reader with state-of-the-artsummaries of recent exciting progress that has been made in the field of cancermetabolism. This story starts at the beginning of the 20th century with the workof Otto Warburg, whom Hans Krebs described as a “Cell Physiologist, Biochemist,and Eccentric.” Warburg invented a device, now known as the Warburg manometer,with which he demonstrated that tumor cells consume less O2 (and produce morelactate) than do normal cells under the same ambient O2 concentrations. A centurylater, the struggle to understand how and why metastatic cancer cells manifest theWarburg effect is still ongoing, and 12 rounds of this heavyweight fight await thereader beyond this brief introduction.

Why do cancer cells manifest altered energy metabolism? To answer this ques-tion, it is necessary to acknowledge that mitochondria, in addition to serving as themajor source of ATP production, provide two other critical cellular functions. Oneis that they also serve as the major source of reactive oxygen species (ROS), whichare generated when electrons donated to the respiratory chain are not successfully

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transferred from complex IV (cytochrome c oxidase) to O2 to form water but insteadreact with O2 prematurely to form superoxide anion, which is then converted tohydrogen peroxide through the action of mitochondrial superoxide dismutase. ROSare used by normal cells as signaling molecules (e.g., during cell proliferation).However, if generated in excess (amplitude or duration or both), ROS result in theoxidation of lipids, proteins, and nucleic acids leading to cell dysfunction or death,which introduces the other major role of mitochondria as arbiters of cell survival,as apoptosis is triggered through changes in mitochondrial structure and function.Thus, it appears that reduced mitochondrial respiration, which often is accompaniedby reduced mitochondrial mass, occurs in cancer cells as a result of selection againstexcess ROS production and/or apoptosis.

How do cancer cells reprogram energy metabolism? To answer this question(and to more completely answer “why?” beyond the pat generalization providedabove), it is necessary to return full circle to the issue of heterogeneity. Becausemitochondria play critical roles in energy production, redox balance, proliferation,and survival, many mutations that are selected for in advanced cancers impact mito-chondrial function in one or more ways, for although mitochondria control cell fate,the cell controls mitochondria by virtue of the fact that most of the components ofmitochondria are the products of nuclear, not mitochondrial, genes.

A danger of a reductionist approach to cancer biology that focuses on changesinvolving key oncogenes and tumor suppressor genes is the misconception that car-cinogenesis can be attributed to one or a few mutations in any given cell. As inevery other aspect of cancer biology, it is the net effect of all of the mutations innuclear and mitochondrial DNA of the cancer cell that will determine its metabolicphenotype. Another danger of a solely mutation-based approach to cancer is that itignores the effect of the tumor microenvironment. Like normal cells, cancer cellsreact to changes in the concentrations of O2, glucose, hydrogen ions, and free radi-cals. In fact, these changes represent the most important selective force acting on themutant gene pool of cancer cells. As a result, not all changes in cancer metabolismare hard-wired by genetic alteration but may instead represent adaptive responses tothe microenvironment. In other words, the reprogramming of energy metabolism isunique to each cancer cell.

The complex considerations that are covered so superficially in this brief intro-duction are discussed in detail in the 12 chapters that follow. There is tremendousinterest (and hope) that because altered glucose and energy metabolism appears tobe a virtually universal characteristic of metastatic cancer cells (i.e., the cells thatkill cancer patients), understanding the mechanisms and consequences of metabolicreprogramming may lead to novel therapeutic approaches that will more effectivelyfight this formidable foe. Ring the bell for Round 1 . . .

Baltimore, Maryland Gregg L. Semenza, M.D., Ph.D.

Contents

Oxidative Phosphorylation and Cancer: The OngoingWarburg Hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1Michael Ristow and Jose M. Cuezva

The Electron Transport Chain and Carcinogenesis . . . . . . . . . . . . . . . . . . . . . 19Jean-Jacques Briere, Paule Benit, and Pierre Rustin

Respiratory Control of Redox Signaling and Cancer . . . . . . . . . . . . . . . . . . . 33Pauline M. Carrico, Nadine Hempel, and J. Andres Melendez

Cellular Respiration and Dedifferentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45Roberto Scatena, Patrizia Bottoni, and Bruno Giardina

Cellular Adaptations to Oxidative Phosphorylation Defects in Cancer . . . . 55Sarika Srivastava and Carlos T. Moraes

Regulation of Glucose and Energy Metabolism in Cancer Cells byHypoxia Inducible Factor 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73Tulio Cesar Ferreira and Elida Geralda Campos

The Role of Glycolysis in Cellular Immortalization . . . . . . . . . . . . . . . . . . . . . 91Hiroshi Kondoh

Metabolic Modulation of Carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103Shireesh P. Apte and Rangaprasad Sarangarajan

Mitochondrial DNA Mutations in Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119Anna Czarnecka and Ewa Bartnik

Cellular Respiration and Tumor Suppressor Genes . . . . . . . . . . . . . . . . . . . . . 131Luis F. Gonzalez-Cuyar, Fabio Tavora, Iusta Caminha, George Perry,Mark A. Smith, and Rudy J. Castellani

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Uncoupling Cellular Respiration: A Link to Cancer Cell Metabolismand Immune Privilege . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145M. Karen Newell, Elizabeth M. Villalobos-Menuey, Marilyn Burnett,and Robert E. Camley

How Cancer Cells Escape Death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161Erica Werner

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179

Contributors

Shireesh P. ApteSenior Scientist, Process Research and Support, Mail stop: R4-11 AlconLaboratories, 6201 South Freeway, Fort Worth, Texas 76134, USA

Ewa BartnikDepartment of Genetics and Biotechnology, University of Warsaw; andInstitute of Biochemistry and Biophysics, Polish Academy of Sciences Warsaw,Pawinskiogo 5a. 02106, Poland

Paule BenitHopital Robert Debre, INSERM U676, Paris, France

Patrizia BottoniDipartimento di Medicina di Laboratorio, Universita Cattolica, Rome, Italy

Jean-Jacques BriereHopital Robert Debre, INSERM U676, Paris, France

Marilyn BurnettCU Institute of Bioenergetics and Immunology, University of Colorado at ColoradoSprings, Colorado Springs, Colorado

Iusta CaminhaDepartment of Pharmacology, University of Maryland, Baltimore, Maryland

Robert E. CamleyCU Institute of Bioenergetics and Immunology, University of Colorado at ColoradoSprings, Colorado Springs, Colorado

Elida Geralda CamposDepartamento de Biologia Celular, Universidade de Brasılia, Brasılia, DF, Brazil

Pauline M. CarricoOrdway Research Institute, Albany, New York

Rudy J. CastellaniDepartment of Pathology, University of Maryland, Baltimore, Maryland

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x Contributors

Jose M. CuezvaDepartamento de Biologıa Molecular, Centro de Biologıa Molecular “SeveroOchoa,” Centro de Investigacion Biomedica en Red de Enfermedades Raras,Universidad, Autonoma de Madrid, Madrid, Spain

Anna CzarneckaDepartment of Genetics and Biotechnology, University of Warsaw; Warsaw,Poland; and Postgraduate School of Molecular Medicine, Warsaw, Poland

Tulio Cesar FerreiraDepartamento de Biologia Celular, Universidade de Braselia, Braselia, DF, Brazil

Bruno GiardinaDipartimento di Medicina di Laboratorio, Universita Cattolica, Rome, Italy

Luis F. Gonzalez-CuyarDepartment of Pathology, University of Maryland, Baltimore, Maryland

Nadine HempelThe Center for Immunology & Microbial Disease, Albany Medical College,Albany, New York

Hiroshi KondohDepartment of Geriatric Medicine, Graduate School of Medicine, Kyoto University,Kyoto, Japan

J. Andres MelendezThe Center for Immunology & Microbial Disease, Albany Medical College,Albany, New York

Carlos T. MoraesDepartment of Neurology and Department of Cell Biology and Anatomy,University of Miami Miller School of Medicine, Miami, Florida

M. Karen NewellCU Institute of Bioenergetics and Immunology, University of Colorado at ColoradoSprings, Colorado Springs, Colorado

George PerryCollege of Sciences, University of Texas at San Antonio, San Antonio, Texas;and Department of Pathology, Case Western Reserve University, Cleveland, Ohio

Michael RistowDepartment of Human Nutrition, Institute of Nutrition, University of Jena, Jena,Germany

Pierre RustinHopital Robert Debre, Paris, France

Contributors xi

Rangaprasad SarangarajanAssociate Professor of Pharmacology & Toxicology, School of Pharmacy(Worcester) Massachusetts College of Pharmacy and Health Sciences,19 Foster Street, Worcester, MA 01608,

Gregg L. SemenzaDirector, Vascular Program, Institute for Cell Engineering; Professor, Departmentsof Pediatrics, Medicine, Oncology, Radiation Oncology, and the McKusick-NathansInstitute of Genetic Medicine, The Johns Hopkins University School of Medicine,Broadway Research Building, Suite 671, 733 North Broadway, Baltimore, MD21205 USA

Roberto ScatenaDipartimento di Medicina di Laboratorio, Universita Cattolica del Sacro Cuore,Rome, Italy

Mark A. SmithDepartment of Pathology, Case Western Reserve University, Cleveland, Ohio

Sarika SrivastavaDepartment of Neurology, University of Miami Miller School of Medicine, Miami,Florida

Fabio TavoraDepartment of Pathology, University of Maryland, Baltimore, Maryland

Elizabeth M. Villalobos-MenueyCU Institute of Bioenergetics and Immunology, University of Colorado at ColoradoSprings, Colorado Springs, Colorado

Erica WernerDepartment of Cell Biology, School of Medicine, Emory University, Atlanta,Georgia

Oxidative Phosphorylation and Cancer:The Ongoing Warburg Hypothesis

Michael Ristow and Jose M. Cuezva

Abstract More than eight decades ago, the German physiologist Otto Warburgobserved that cancer cells in the presence of oxygen produced large amounts of lac-tate and proposed that impaired oxidative metabolism may cause cancer. This for-mulation, later known as the Warburg hypothesis, was investigated and debated forseveral decades. The development of molecular biology and the discovery of onco-genes and tumor suppressor genes in subsequent years shifted the general interest inthe cancer field into directions other than metabolism and promoted the abandoningof the Warburg hypothesis or its consideration as an epiphenomenon of cell trans-formation. In recent years, a renaissance in the field of mitochondria has occurredin biological studies. This has happened mostly by the recognition of the key func-tional role that this organelle plays in the execution of cell death and, thus, for itsparticipation in the development of a vast array of human pathologies. The cancerfield was not indifferent to these changes, and the Warburg hypothesis was broughtback to the scene with renewed strength. Specifically, recent findings suggest thatcancer is associated with a decrease in the activity and expression of �-F1-ATPase,a key subunit of the mitochondrial ATP synthase. This alteration has been shown tolimit oxidative phosphorylation and to trigger the induction of glycolysis to provideenergy to the cell thus configuring the earlier Warburg observation in an additionalhallmark of the cancer cell. Moreover, increased and decreased cellular mitochon-drial activities are respectively associated with suppression and development of can-cer. We suggest that reactivating mitochondrial metabolism by pharmacologic ordietary measures and/or tackling the deviant glycolysis in cancer cells may effi-ciently suppress malignant growth.

M. Ristow (B)Chair, Department of Human Nutrition, Institute of Nutrition, University of Jena, D-07743 Jena,Germanye-mail: [email protected]

J.M. Cuezva (B)Departamento de Biologıa Molecular, Centro de Biologıa Molecular “Severo Ochoa”, CIBER deEntermedades Rasas, Universidad Autonoma de Madrid, 28049 Madrid, Spain.e-mail: [email protected]

S.P. Apte, R. Sarangarajan (eds.), Cellular Respiration and Carcinogenesis,DOI 10.1007/978-1-59745-435-3 1,C© Humana Press, a part of Springer Science+Business Media, LLC 2009

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2 M. Ristow, J.M. Cuezva

Keywords Mitochondria · Oxidative phosphorylation · H+-ATP synthase ·Glycolysis · Cancer

Nowadays, malignant disorders are a major cause of premature death. The incidenceof cancers increases with age, whereas metabolic activity and resting energy expen-diture resembling oxidative metabolism continuously decrease during aging ofhumans. Hence, an inverse relationship between mitochondrial activity and the pro-motion of tumor growth might possibly exist. To analyze whether this apparent linkis supported by scientific evidence rather than simply being a coincidence, tremen-dous efforts have been undertaken in the past to elucidate this possible connection.This chapter aims to summarize published evidence on mechanistic interdependen-cies between cancer biology and the activity of mitochondria. Other recent reviewsdealing with this subject are also available [1, 2].

In the past decades, glucose has become a major component of Western diets [3].The most frequently used nutritive sugar, the disaccharide sucrose, contains equalamounts of the monosaccharides fructose and glucose. Glucose is actively trans-ported into mammalian cells, and subsequently is (as well as fructose) biochem-ically converted into pyruvate (Fig. 1). Pyruvate either is converted into lactate,which prevents its further intracellular breakdown, or is completely oxidized to car-bon dioxide and water by mitochondrial activities linked to the supply of molecularoxygen (Fig. 1). The breakdown of glucose into pyruvate is termed glycolysis.Glycolysis takes place in the cytoplasm of eukaryotic cells, and it can proceed bothin the absence and presence of oxygen (Fig. 1). In order to carry on glycolysis inthe former case, the carbon skeleton of pyruvate needs to be reduced to lactate toregenerate the pool of oxidized NAD+ (Fig. 1). Many prokaryotes and lower eukary-otes including Saccharomyces cerevisiae (yeast) survive in the absence of oxygenby obtaining energy from anaerobic glycolysis only [4], whereas higher eukaryotes,especially mammals, require a continuous supply of oxygen for obtaining biologi-cal energy. Glycolysis in absence or presence of oxygen provides comparably littleenergy equivalents per mole of glucose oxidized when compared with its terminaloxidation in the mitochondria (Fig. 1). Nevertheless, anaerobic glycolysis suppliesthe precursors and building blocks for the biosynthesis of cellular macromolecules,and, in yeast, as well as in certain mammalian cell types, is sufficient to maintainviability for longer periods of time. Because highly developed organisms tend touse nutritive energy equivalents more efficiently than do less developed organisms[5], mammals have significantly more efficient ways to generate energy equivalents.This is illustrated by the further oxidation of a metabolite of glycolysis (pyruvate) inmitochondria, which yields an approximately 18-fold amount of energy equivalentsper mole of glucose oxidized when compared with anaerobic glycolysis (Fig. 1).Hence, the major advantage of glycolytic metabolism is to be independent fromoxygen, and the major disadvantage of this process is a comparably low yield ofenergy equivalents (Fig. 1).

For many decades, mitochondrial organelles were considered eminently impor-tant for the generation of biological energy and as the cellular site that allowed

Oxidative Phosphorylation and Cancer: The Ongoing Warburg Hypothesis 3

Glucose G-6-P F-6-P F-1,6-P G-3-P + DHAP G-3-P

1,3-BPG3-PG2-PGPEP

Pyruvate

Lactate

Net ATP produced: 2Net ATP produced: 36

CO2 + H2O

pyruvate

02

HK PGI PFK1 ALD

GAPDH

PGKPGMENO

PK

LDH

Glucose utilization: HIGHGlucose utilization: LOW

NADH

NAD+

AE

RO

BIO

SIS

/A

NA

ER

OB

IOS

IS

AE

RO

BIO

SIS

TPI

Fig. 1 Cellular ATP provision defines the rate of aerobic glycolysis. Abbreviations of glycolyticintermediates and enzymes of glycolysis are presented sequentially up to the pyruvate crossroad.The key glycolytic enzymes hexokinase (HK), phosphofructokinase 1 (PFK1), and pyruvate kinase(PK) are highlighted in bold. The reduction of pyruvate to lactate by lactate dehydrogenase (LDH)regenerates NAD+ and allows glycolysis to proceed with very low yield of energy and a highconsumption of glucose. Pyruvate could be oxidized to CO2 and water only in the presence ofoxygen by mitochondria. In this situation, the yield of energy obtained is high and consequentlyglucose consumption is low

the integration of the metabolism of macronutrients, specifically carbohydrates,fatty acids, and amino acids. Interconversion of carbohydrate derivatives (i.e., pyru-vate) into fatty acids, amino acids into carbohydrate precursors, and others occur inthe matrix of mitochondria, and these activities are integrated in the Krebs cycle.Concurrently, the reducing equivalents obtained from the terminal oxidation of themetabolic intermediates of the Krebs cycle in the form of NADH and FADH2 areshuttled into the complexes of the respiratory chain (Fig. 2). The respiratory chainis composed of proteins that are located in the inner mitochondrial membrane andhave, as overall function, the reoxidation of NADH and FADH2 to further allow theoxidation of more metabolic substrates (Fig. 2). The ordered transfer of electronsalong the carriers of the respiratory chain to molecular oxygen (Fig. 2), which isthe final electron acceptor, is the process known as respiration. Electron transferin complexes of the respiratory chain trigger the proton pumping activity of com-plexes CI, CIII, and CIV (Fig. 2), which extrudes protons from the matrix interior ofmitochondria and finally results in the generation of the proton electrochemical gra-dient (��H+) across the inner membrane of the organelle, which is the intermediateused for the synthesis of ATP [6] (Fig. 2). The reentry of protons to the matrix inte-rior through the proton channel of the H+-ATP synthase (CV in Fig. 2) drives the


O.M.

O2·

O2·

I.M.Q

NADHFADH2

NAD+

FAD O2 H2O

H+ H+ H+ ΔμH+

H+

Cyt c

CI

e–

e–

e–

e–

CIICIII

CIV

ATP

CV

CYTOPLASM

MATRIX

Fig. 2 Mitochondrial activities: respiration, oxidative phosphorylation, and the production of ROS.The oxidation of metabolic substrates promotes the reduction of redox coenzymes (NAD+ andFAD) that feed the electrons (e-) to the complexes of the respiratory chain (CI to CIV) placed inthe inner mitochondrial membrane (I.M.). Electron flow down the respiratory chain to molecularoxygen generates the water of respiration. Electron transfer is coupled with proton pumping fromthe matrix interior to the intermembrane space at CI, CIII, and CIV to generate the proton elec-trochemical gradient (��H+) across the inner membrane. ��H+ is the driving force used by theH+-ATP synthase (CV) for the synthesis of ATP. However, at complexes I and III, some of the elec-trons can combine with molecular oxygen to generate the toxic superoxide radical (dashed lines).Q, ubiquinone; Cyt c, cytochrome c; O.M., outer membrane

biosynthesis of adenosine triphosphate (ATP) from adenosine diphosphate (ADP)and inorganic phosphate (Pi) in the process known as oxidative phosphorylation(OXPHOS) (Fig. 2). Whereas the metabolism of nutrient intermediates andOXPHOS were thought to be the primary roles of mitochondria for manydecades, recent findings indicate that mitochondria play additional essential cellularfunctions. The protist Giardia lamblia carries remnant organelles resembling mito-chondria that, though they are unable to provide ATP, instead synthesize iron-sulfur (Fe-S) clusters [7]. Fe-S clusters are essential parts of specific mitochondrialenzymes, namely aconitase in the Krebs cycle as well as complexes of the respira-tory chain [8]. Briefly, Fe-S clusters are known to carry non-heme iron, which canboth donate and accept electrons [9]. Moreover, mitochondria are central regula-tors and executors of cell death [10], and the acquisition of a resistant phenotypeto apoptosis is a critical aspect in the biology of tumors and its therapy [11, 12].It is now firmly established that upon the activation of cell death by various typesof stimuli (genetic damage, ischemia, chemical and/or metabolic stresses), mito-chondria release proteins (cyt c, AIF, Endo G, Smac/DIABLO, Omi/HtrA2) and/orother molecules such as the superoxide anion (O2

.) that participate in signaling and


executing cell demise. It should be noted that in most cells, reactive oxygen species(ROS) are produced by single electron transfer reactions to molecular oxygen dur-ing mitochondrial respiration (Fig. 2). In summary, mitochondria are responsiblefor the production of Fe-S clusters, the conversion of primary energy substrates intoeach other, the generation of large amounts of ATP, and the signaling and executionof cell death. The major advantage of oxidative metabolism is the comparably highenergy yield, and the major disadvantage is its dependency on the availability ofoxygen and the subsequent production of ROS that could damage essential cellularconstituents.

Unaware of the above-mentioned facts, the physiologist Otto Warburg (Fig. 3and Fig. 4) proposed as early as 1924 that malignant growth might be causedby increased glycolysis accompanied by impaired oxygen consumption [13–16].This hypothesis was based on his original observations that cancer tissues showa low respiratory rate compared with that of adjacent healthy (i.e., nonmalignant)tissues. After becoming the Nobel laureate for medicine and physiology in 1931[17], his hypothesis on initiation of cancer obtained increasing attention and causedwidespread debate [18–22]. Warburg died in 1970 while his hypothesis was still

Fig. 3 Otto Warburg (dateunknown). (Photographcourtesy of the Archives ofthe Max Planck Society,Berlin, Germany.)


Fig. 4 Otto Warburg in hislaboratory (date unknown).(Photograph courtesy of theArchives of the Max PlanckSociety, Berlin, Germany.)

unconfirmed, denied, and/or neglected [23, 24] mostly because of the abandon-ment of metabolic studies in favor of the development of molecular biology andthe discovery of oncogenes, tumor suppressor genes, and other advances in cancerresearch. In any case, and after both OXPHOS and respiration had been attributed tomitochondria, electron microscopy–based techniques were developed to study themorphology of these organelles in tumor tissues. In summary, mitochondria fromrapidly growing cancers show less cristae and are smaller than those from better-differentiated cancers and normal tissues [25, 26]. Besides these structural differ-ences in the mitochondria of the cancer cell, it has been further emphasized thatsome cancer tissues also have a significant reduction in the number of mitochondriaper cell [25–27].

The abnormal high-aerobic glycolysis of tumors discovered by Warburg was con-firmed by most researchers studying cancer cells, and a significant number of poten-tial mechanisms have been put forward to explain the metabolic changes specificto cancer cells. These can be separated into mechanisms that impinge on cellu-lar glucose uptake and consumption by glycolysis (Fig. 1) and those affecting themitochondrial metabolism of pyruvate (Fig. 1 and Fig. 2). When comparing energymetabolism of tumors and healthy tissue, profound changes have been observed invirtually all cases of cancer or tissue types. Increased glycolysis has been found incancer cells at the genomic [28], proteomic [26, 29, 30–32], and metabolic levels:


increased glucose uptake [33, 34], as well as increased production of lactate as theend product of glycolysis [35, 36].

A modern noninvasive imaging technique called positron emission tomography(PET) is nowadays in clinical use for diagnosing and staging cancer patients [37]because it is remarkably sensitive to detect tumor tissue and its metastasis in situbased on the principles of the Warburg hypothesis [32]. By injecting the nonmetabo-lizable glucose analogue [18F]fluoro-2-deoxy-d-glucose (FDG) as a tracer substanceinto the human body, tissues with high FDG uptake (high glucose avidity) can easilybe identified from the surrounding normal tissue in images of PET scans [38]. Usingthis approach, we have recently showed that lung tumors with high glucose avid-ity have a profound alteration of the bioenergetic proteome [32]. The bioenergeticproteome (BEC index) provides a signature of the tumor that informs of the pre-ponderance of the metabolic pathways relevant for cellular energy provision at theprotein level [2, 26]. Remarkably, the analysis of the bioenergetic signature in tumorspecimens and in cells in culture [32] revealed that the downregulation of the bioen-ergetic function of mitochondria (Fig. 1 and Fig. 2), specifically of oxidative phos-phorylation, underlies the high rates of glucose uptake and consumption by aerobicglycolysis in the carcinomas (Fig. 1) [32]. These results have provided the first indi-cations that integrate molecular and functional data to confirm Warburg’s initialobservations [32].

In a recent review, Gatenby and Gillies put forward an evolutionary model toexplain the apparently high glycolytic rates of cancer cells [39]. Under conditions ofextensive hypoxia, which most likely occurs during hyperplastic growth, evolution-ary selection favors cells with elevated glycolytic capacity [40]. Moreover, increasedglycolysis causes increased lactate production and subsequent lactate-dependentacidification. Selection is in favor of those cells that exhibit increased resistancetoward an unfavorable acidic environment. Therefore, a premalignant cell will beless dependent on oxygen and is still able to expand under conditions that wouldkill a nonmalignant cell. In support of Warburg’s hypothesis, this simple model pro-vides an explanation on the benefit of cancer cells from an energetic trade-off (i.e.,preference for nonmitochondrial energy supply).

Numerous experimental findings further emphasize this assumption. Induction ofglucose-specific transporters (GLUTs), and, most importantly GLUT1, was severaltimes correlated with tumor aggressiveness [41–43]. Moreover, additional evidencelinks a number of established cancer genes to control of glycolytic flux. In thisregard, it has been shown that the c-myc oncogene induces expression of GLUT1and a number of additional genes involved in glycolytic flux, including glucose-6-phosphate isomerase, phosphofructokinase, glyceraldehyde-3-phosphate dehydro-genase, phosphoglycerate kinase, lactate dehydrogenase, and others (Fig. 1) [44].Of interest, c-Myc also acts to transactivate the lactate dehydrogenase A (LDHA)gene which is necessary for c-myc–mediated transformation in fibroblasts [35].Accordingly, expression of other oncogenes (ras, src, PDGF) causes increased glu-cose uptake in malignant cells [34, 35, 45]. Additionally, and acting as c-myc itself,such cells were capable of coactivating LDHA expression via proposed binding toa carbohydrate responsive element found in the LDHA promoter region [46].


Another relevant mechanism to regulate glycolysis and metabolic adaptation tooxygen depletion is that mediated by the hypoxia inducible factor 1 alpha (HIF-1�).Subsequent to its identification in 1992 [47], a significant body of evidence hasemerged proposing that HIF-1� might be relevant for many of the effects as sug-gested in the initial Warburg hypothesis [48]. HIF-1� forms dimers with its part-ner HIF-1� to become an active transcription factor binding to promoter elementsof several genes including those encoding for proteins of glucose transport [49]and activating the expression of several enzymes of the glycolytic pathway (Fig. 1)including LDHA [50, 51]. Furthermore, the active HIF-dimer promotes angiogen-esis and erythropoiesis [47, 52]. HIF-1� is constitutively expressed in most tissuesand degraded under conditions of normal oxygen pressure. In contrast, when oxygenpartial pressure is decreased, degradation of HIF-1� is blocked causing increasedtranscriptional activity to regulate the respective target genes following changesin oxygen availability [53]. Accordingly, deregulated HIF-1� or sustained stabil-ity under conditions of normal oxygen supply has been linked to numerous types ofcancer in past years [54]. Furthermore, the ubiquitin ligase responsible for HIF-1�inactivation, the von Hippel–Lindau tumor suppressor (VHL) protein, has been affil-iated with the von Hippel–Lindau syndrome, which promotes neuronal and kidneycancers [55]. Moreover, downregulation of pyruvate dehydrogenase might lead toaccumulation of pyruvate and increased lactate production, which contributes to sta-bilization of HIF-1� [56]. Hence, although hypoxia might play a key role in tumordevelopment, it seems not to be unambiguously required for sustained increasesin glycolysis. Putatively, this may promote a vicious cycle of enhanced glycolysisleading to increased accumulation of pyruvate, which subsequently may stimulateHIF-1� and lastly further increase the expression of glycolytic enzymes [57].

Another candidate pathway controlling the glycolysis-dependent phenotypeof malignancies, the Akt/PBK (protein kinase B) pathway, has been proposed.Akt/PKB has long been known for its capability of inducing glucose incorpora-tion and glycolytic flux independently of malignancy-promoting capacity [58]. Inmalignant cells, elevated glycolytic flux was linked to activation of Akt/PKB [59].Accordingly, blastoma cells lacking constitutive activation of Akt/PKB did not dis-play altered glycolytic flux, whereas expression of Akt/PBK promoted cell deathunder conditions of glucose deprivation. Moreover, it has recently been shown thatthe overexpression of Akt/PBK in noninvasive radial-growth melanoma cells trig-gers an increase in protein markers of glycolysis and in the glycolytic flux of themelanoma concurrently with its transformation to the aggressive vertical-growthinvasive behavior [60]. Altogether, these results suggest the possibility that deregu-lated Akt/PKB signaling might contribute to the Warburg effect in cancer cells.

Additionally, interactions of glycolytic enzymes with the well-characterized p53tumor suppressor protein have been described, further promoting the relevance ofcancer-related genes in causing a potential metabolic shift toward increased gly-colytic rate. In this regard, a putative role for hexokinase has been proposed [61]. Inhepatoma cells, hexokinase was closely associated with the mitochondria, whichwas not observed in normal (i.e., untransformed) liver cells [62]. A subsequentstudy revealed that mutations in the p53 tumor suppressor protein might activate


hexokinase II expression via binding to specific sites of the corresponding hexoki-nase promoter [63, 64]. In further support of these interesting observations, a proteinnamed TP53-induced glycolysis and apoptosis regulator (TIGAR ), an isoform ofphosphofructokinase that can be induced by p53, was identified in recent years[65]. TIGAR acts as an inhibitor of glycolysis by lowering levels of fructose-2,6-bisphosphate. Accordingly, in cases of p53 mutations as found in many cancers,activity of TIGAR is downregulated promoting an increase in glycolysis. In sum-mary, a number of cancer-related genes have been shown to promote alterations onthe glycolytic phenotype that could explain that particular aspect of the Warburgeffect.

Aspects other than deregulated glycolysis underscore that oxidative metabolismitself is also impaired in malignant cells, thereby representing another key aspectof Warburg’s hypothesis. The phenomenon of impaired mitochondrial activity [13,15, 25] appears to be a key feature of numerous cancer specimens [26, 29, 31, 32,66, 67]. Accordingly, mutations of mitochondrial DNA have repeatedly been asso-ciated with induction of tumor growth on a hypothetical basis [67, 68] and lateron a factual basis [69–73]. Complexes of the mitochondrial respiratory chain (Fig.2) including cytochrome c oxidase (COX) exhibit diminished activity in cancers,mainly due to decreased expression of the subunits encoded by the mitochondrialDNA [74]. Likewise, mutations in nuclear genes that affect the bioenergetic func-tion of mitochondria, and thus compromise the provision of metabolic energy, havealso been shown to predispose to different types of inherited neoplasia syndromes[75–77].

Moreover, over the past years it has been shown that most prevalent humantumors fit the original Warburg hypothesis because they show a decrease in theexpression of �-F1-ATPase, which is the catalytic subunit of the mitochondrial H+-ATP synthase and thus a bottleneck of oxidative phosphorylation (CV in Fig. 2),concurrently with an increased expression of several makers of the glycolytic path-way (26, 29, 31 and references therein). This proteomic feature, which was definedas “the bioenergetic signature” of cancer [26], illustrates both alterations in thebioenergetic proteome of the mitochondrion and on the overall mitochondrial activ-ity of the cancer cell [2] and provides markers of the two mechanisms that couldinterfere with mitochondrial activity in the various types of cancer cells [2]. Thisis especially relevant because depending upon the tissue type considered, a generalreduction in mitochondrial mass or a specific reduction on the expression of the ATPsynthase is associated with progression of the neoplasia [2, 26]. Interestingly, theserecent findings perfectly complement earlier findings on reduced ATP(synth)aseactivity in rodent models of liver carcinogenesis [78]. Additionally, Racker and co-workers also emphasized the putative relevance of ATP(synth)ase in the control oftumor proliferation [79, 80].

As indicated previously, we have recently documented both in vivo using FDG-PET imaging in lung cancer patients and in vitro that the rates of glucose cap-ture by lung tumors and of glucose utilization by aerobic glycolysis in cancercells depend on the expression level of the �-F1-ATPase protein and on the activ-ity of oxidative phosphorylation [32]. These results provide strong support to the


original Warburg hypothesis and to the principle of metabolic regulation known asthe Pasteur effect [81]. This principle of metabolism, which basically states that “themetabolic flux of glycolysis in eukaryotic cells depends on the energy provided inthe form of ATP by mitochondrial activity,” is what was used by Warburg as the“first stone” for the building of his seminal and outstanding hypothesis in the earlydates of biochemistry [2]. Remarkably, the bioenergetic signature has been shownto provide a relevant marker of disease progression in colon [26], lung [30, 32], andbreast [31] cancer patients. Furthermore, it has been documented that the bioener-getic signature also provides a marker of the cellular response to chemotherapy incolon [82] and liver [83] cancer cells that might allow its further use as a predictivemarker of therapeutic intervention and/or as a target of future cancer treatments.

Because it appears that alterations in mitochondrial energetic physiology con-tribute to the Warburg effect, that is, an impaired oxidative phosphorylation in mito-chondria promotes the upregulation of glycolysis (Fig. 1), at least two questionsarise: (i) what are the mechanism(s) that impair the OXPHOS activity of mitochon-dria, and (ii) what is the advantage and mechanistic contribution of the downreg-ulation of OXPHOS for the cancer cell? These will be partially addressed in thefollowing.

Supported by previous findings on p53 as mentioned before, it has been shownthat this tumor suppressor protein may directly be involved in regulating mitochon-drial oxygen consumption through regulation of COX activity [84]. Accordingly,cancer cell lines lacking p53 showed decreased rates of OXPHOS as reflected byimpaired ATP synthesis and decreased respiratory rate, and maintenance of cellu-lar ATP levels was dependent on a concurrent increase in glycolysis [85, 86]. Thisspecific regulation was promoted by Synthesis of Cytochrome c Oxidase 2 SCO2,which has previously been shown to be involved in the assembly of complex IV ofthe respiratory chain [86]. Nevertheless, it remains to be shown whether this par-ticular activation can be directly linked to mitochondrial activity. Therefore, thistumor suppressor, p53, not only regulates glycolysis as shown above but possiblyalso directly affects mitochondrial oxygen consumption.

Not only p53 has been shown to influence both glycolysis and mitochondrial activ-ity. Similar conjunct activities have been put forward for both HIF-1� and Akt/PKBsignaling pathways. In this regard, HIF-1� has previously been demonstrated to acti-vate pyruvate dehydrogenase kinase (PDK), which is known to inactivate pyruvatedehydrogenase, preventing the decarboxylation of pyruvate and hence its further oxi-dation in the mitochondria having a negative impact on mitochondrial oxygen con-sumption [87, 88]. Likewise, the above-mentioned tumor suppressor protein VHLnot only ubiquitinates HIF-1� but also promotes mitochondrial biogenesis and activ-ity. Accordingly, loss of VHL, as repeatedly described in various types of cancer,might possibly impair mitochondrial activity strongly promoting the Warburg effect[66]. Moreover, reduced OXPHOS capacity has previously been suggested to pro-mote phosphorylation and hence activation of Akt [89], a well-defined oncogene, ina Phosphatase and Tensin Homolog PTEN-dependent manner [90].

An additional alternative to explain the “Warburg phenotype” of the cancer cellis the one we have recently put forward [32] after the characterization of the biogen-esis and functional development of mitochondria during the cell cycle [91]. We have


suggested that the downregulation of oxidative phosphorylation and the concurrentupregulation of glycolysis is the phenotype that identifies the energetic metabolismof most proliferating cells. In other words, glycolysis is the metabolic pathway thatsupports proliferation and tumor growth. In this regard, it is well established thatlymphocytes switch to glycolysis as the energy-producing pathway during prolif-eration [92], and progression through the S/G2/M phases of the cell cycle, the so-called reductive phases of the “metabolic cycle” in which most of the mitochondrialcomponents are being synthesized [91], is supported by nonrespiratory modes ofenergy generation [93]. In agreement with these findings, it has recently been shownthat cyclin D1, which is involved in the phosphorylation and inactivation of theretinoblastoma protein marking in this way the entry of cells into the S phase,represses mitochondrial function in vivo [94, 95]. We presume that the effects thatoncogenes and tumor suppressors could have on mitochondrial activity might besuperimposed to the natural metabolic phenotype of proliferating cells. Mechanis-tically, and by analogy with findings on the biogenesis of mitochondria in rat hep-atomas [27], in fetal rat liver [96–98] and during the cell cycle [91], the repressionof �-F1-ATPase expression in human tumors [26, 29–31] could be exerted by trans-lation masking (silencing) of the �-F1-ATPase mRNA, a condition that might alsoaffect other OXPHOS mRNAs of the cancer cell [2].

According to Warburg’s hypothesis, impaired mitochondrial function should beconsidered a key property of cancer cells (i.e., an additional hallmark of cancer to beadded to the list) [99]. Strongly supporting this hypothesis, it was shown that induc-tion of mitochondrial energy conversion by forced expression of the mitochondrialprotein frataxin [100] leads to reduced growth of cancer cells [101]. Frataxin hasbeen shown to be involved in Fe-S cluster biogenesis [102] and induces OXPHOS[100]. In this regard, and as a novel pathway connecting induction of mitochondrialmetabolism and decreased growth rates, mitogen activated protein kinase p38 hasbeen proposed [101, 103]. This protein p38 has repeatedly been observed to act asan efficient tumor suppressor protein [104, 105]. In full accordance with these obser-vations, hepatocyte-specific inactivation of murine frataxin reduces the activity ofFe-S cluster–containing enzymes within the mitochondria, promoting hepatic ade-noma formation in such mice [103]. Consistently, defective Krebs cycle activity hasbeen observed in colorectal cancer samples including reduced activity of the Fe-Scluster–containing enzyme aconitase [106]. Taken together, these findings indicatethat activation of mitochondrial energy metabolism has a tumor suppressor effect thatopposes the Warburg phenotype and could be a therapeutic tool in cancer therapy.

Indeed, the execution of cell death requires an efficient oxidative phosphoryla-tion [107–110], the molecular components of the H+-ATP synthase being specif-ically required [111–113]. In this regard, we have recently shown that the activ-ity of the H+-ATP synthase is necessary for the efficient execution of apoptosisin cells that depend on oxidative phosphorylation for the provision of metabolicenergy, whereas it is dispensable in those cells that rely heavily on glycolysis [83].The role of the H+-ATP synthase in the execution of cell death is mediated bycontrolling the generation of ROS [83], which in turn promote a severe oxida-tive damage on mitochondrial proteins, favoring in this way the release of apop-togenic molecules from the organelle [83]. Consistently, highly glycolytic cells


with an arrested mitochondrial biogenesis and thus with no dependence on oxida-tive phosphorylation for energy provision do not produce ROS and are resistant tomitochondria-geared cell death stimuli [83]. Altogether, these results provide addi-tional evidence linking metabolism to cell death [114–117] and support stronglythat the acquisition of a Warburg phenotype is another strategy of the cancer cell inorder to ensure its perpetuation.

In recent years, AMP activated protein kinase (AMPK) has been established asa key regulator of cellular energy balance controlling a significant number of path-ways that control cellular ATP content [118]. AMPK is activated under conditions oflow energy supply, induced by stressors like reduced nutrient availability or hypoxia[118]. Activation of AMPK is initiated by the upstream tumor suppressor kinase,LKB1, which phosphorylates AMPK [119]. Of note, a mutation of LKB1 has beenshown to cause Peutz-Jeghers syndrome, a disease that has been linked to increasedincidence of cancers. Moreover, two other downstream targets of AMPK, the tumorsuppressors TSC1 and 2, have been shown to be mutated in tuberous sclerosis [120].One of the major outcomes of AMPK and/or activation of its downstream substratesis the induction of mitochondrial biogenesis and/or activity [121]. Not surprisingly,AMPK appears to be relevant in the control of protein synthesis and proliferation[122], and may activate p53 causing cell cycle arrest and/or cellular senescence instates of decreased glucose availability [123]. Given the connection of AMPK onthe one hand and several tumor suppressors, including TSCs and LKB1 (see above),on the other hand, as well as the fact that AMPK itself is of eminent relevance tomitochondrial activity and energy balance, disruption of this pathway might be con-sidered of importance in regard to the molecular causes of the Warburg effect instates of cancer and malignant growth.

In summary, though the Warburg hypothesis was formulated more than 80 yearsago, nowadays it still (or rather again) is a subject of extensive debate. The recentfindings on the metabolic signature of tumors and malignant cells on the one handand several molecular pathways linked to derangements of cellular energy flux intransformed state on the other hand strongly support the overdue importance of oneof the most long-standing hypotheses in oncology—Warburg’s hypothesis.

Acknowledgments We thank members of our laboratories for their contributions and encouragingdiscussions on the subject of this review. This review article was written while the research activityin the authors’ laboratory was supported by grants from the German government to M.R. andfrom the Ministerio de Educacion y Ciencia (BFU2007-65253) and Fundacion Mutua Madrilenato J.M.C.

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The Electron Transport Chainand Carcinogenesis

Jean-Jacques Briere, Paule Benit and Pierre Rustin

Abstract Major metabolic changes that affect the balance between respiration andglycolysis occur during carcinogenesis. It is therefore not surprising that it has longbeen suggested that abnormal activity of the mitochondrial electron transfer chaincould play a role in the underlying pathophysiologic process. However, it has onlyrecently been demonstrated that the specific impairment of one component of theelectron transfer chain, namely complex II, can indeed be the primary event trig-gering carcinogenesis. Unexpectedly, rather than superoxide overproduction, theorganic acid imbalance resulting from the complex II blockade appears to be instru-mental in the early step of tumorigenesis. Nevertheless, because an abnormal han-dling of oxygen is frequently suspected of being associated with electron transferdysfunction, a renewed interest is observed for the putative instability of the mito-chondrial genome often reported in tumor tissues. In this chapter, we review anddiscuss the evidence advocated for the implication of the electron transfer chain incarcinogenesis.

Keywords Carcinogenesis · Mitochondria · Organic acids · Superoxides · Electrontransfer

1 Introduction

Mitochondria play an irreplaceable role in all aerobic organisms (i.e., the handlingof oxygen by the electron transfer chain for the mutual benefit of the cell host andof these Proteobacteria-derived organelles) [1]. In addition, through a fascinatingand complex network of interactions, mitochondria have emerged as central exe-cutioners, or possibly even decision makers, for cell death under a large numberof conditions [2]. In keeping with this paradigm, a set of mitochondria-releasable

P. Rustin (B)INSERM U676, Hopital Robert Debre, 75019 Paris, Francee-mail: [email protected]


19

20 J.-J. Briere et al.

factors have been shown to be bifunctional proteins [3–5]. Involved in electrontransfer chain activity or maintenance, both cytochrome c and apoptosis inducingfactor (AIF) readily trigger apoptosis when released in the cytosol upon opening ofthe elusive mitochondrial permeability transition pore (MPTP) [6]. Furthermore, asevere shortage of mitochondrial ATP production, which presumably occurs whenthe electron transfer chain is deficient, is supposedly sufficient to cause cell necro-sis [7]. However, more recently, it was discovered that respiratory chain complexII deficiency can favor cell proliferation and tumor formation rather than cell death[8]. This has led to a renewed interest in the potential role of respiratory chain dys-function in tumor promotion.

2 Mitochondria and Cancer: A Long History

Otto Warburg performed the seminal observation that, even under aerobic condi-tions, cancer cells shift their metabolism from an oxidative one (through the mito-chondrial respiratory chain) to a highly glycolytic metabolism, thereby producingelevated levels of lactate [9]. This paradoxical effect, wherein oxygen is not utilizedeven though it is available, was thereafter referred to as the Warburg effect.Thisphenomenon is at variance with the Pasteur effect according to which glycolysisis naturally favored by conditions that turn off the oxygen-dependent mitochon-drial respiratory chain. As a result of the shift of metabolic flux from respirationto glycolysis, the former would become of secondary importance, and this logi-cally suggested the idea that the fate of the mitochondria in tumor tissues wouldbe somewhat irrelevant to tumor development. Accordingly, up to very recently, theword cancer was indeed largely absent from mitochondrial disease–devoted meet-ings and reviews. Even the reports of decreased respiratory chain activities or ofquantitative changes in mitochondrial DNA (mtDNA)-encoded mRNA in tumorswere insufficient to really draw the attention of the mitochondria-oriented scientificcommunity. However, several features of the mitochondrial electron transfer chainthat could play a role in triggering, or participating in, tumorigenesis deserve to bepresented.

3 The Respiratory Chain: Biochemical and Genetic Features

The respiratory chain (RC) is constituted by an association of supercomplexeswhose spatial organization favors a rapid exchange of electrons. Such a mecha-nism may serve to avoid—or at least to limit—direct reactions between reducedRC carriers and molecular oxygen that can produce superoxides [10]. Two majorentities have been described: the respirasome consisting of the association of RCcomplexes I/III/IV and presumably a subfraction of cytochrome c; and the ATPa-some, consisting of complex V plus the adenylate and the phosphate carriers. The

The Electron Transport Chain and Carcinogenesis 21

respirasome channels the electrons from respiratory substrates to molecular oxy-gen to form water with a concomitant proton extrusion from the mitochondrialmatrix space to the intermembrane space (Fig. 1). This creates an electrochem-ical gradient across the inner membrane that can be further used by the ATPa-some to generate ATP from ADP and inorganic phosphate. Additional processesmake use of the electrochemical gradient, ensuring the transport of various compo-nents through the membrane, including various cations (e.g., calcium), anions (e.g.,organic acids, fatty acids), or proteinaceous material (e.g., mitochondrial proteinprecursors) [1]. Dissipation of the electrochemical gradient can also be an aim perse, allowing the uncoupling of electron flow from the phosphorylation process [11].This process liberates heat and mechanically favors the oxidized state of the RC

Fig. 1 The dual genetic origin and the organization of the respiratory chain. (A) Respiratory chaincomponents are encoded by both the nuclear and the mitochondrial (mt) genomes. (B) A schematicview of the organization of the respirasome and the ATPasome. CI–CV, the various complexes ofthe respiratory chain; cyt c, cytochrome c; IM, inner membrane; Q: ubiquinone pools


components, thus reducing the chance to generate superoxides. UnCoupling Pro-teins (UCP), a large family of proteins, mediate this process, with a high tissuespecificity. The physiologic function of a number of these UCPs is still a subject ofdebate [12].

The respirasome interacts with several other dehydrogenases (e.g., complex II,the electron transfer flavoprotein [ETF], the glycerol-3-phosphate and the dihy-droorotate dehydrogenases) through the kinetically compartmentalized quinonepool in the inner mitochondrial membrane (Fig. 1) [12]. Depending on their spe-cific metabolic demand, tissues are variably endowed with these dehydrogenases[13]. Redox status of the quinone pool therefore depends on the respective activ-ities of the reducing enzymes (the various dehydrogenases) and of the oxidizingpart of the RC (i.e., the cytochromic segment). This redox status is also importantbecause it helps to reconstitute the reducing power of lipophilic antioxidants, suchas tocopherol (vitamin E) [14].

This complex machinery constituted of about 100 proteins is encoded by both themitochondrial genome (mtDNA) and the nuclear genome (Fig. 1) [15]. The mater-nally inherited mtDNA ranges from a few hundred (lymphocytes) to more than100,000 (oocytes) copies per cell [16]. Each circular molecule consists of 16,569base pairs with 37 genes encoding 13 proteins (polypeptides), 22 transfer RNA(tRNAs), and 2 ribosomal RNAs (rRNAs) [15]. In one cell, the mtDNA copies canbe identical (a situation referred as cellular homoplasmy) or not (a situation knownas heteroplasmy). In the particular case of deleterious mutations harbored by themtDNA, a threshold between wild type (wt) and mutant mtDNA species has to bereached before an RC phenotype can be observed [17]. Because of the random dis-tribution of mitochondria during cell division, an individual can have variable levelsof heteroplasmy from tissue to tissue. The involvement of a given tissue contain-ing mutant mtDNA will therefore depend on the actual level of heteroplasmy, thethreshold value required for RC impairment, and the susceptibility of the tissue tothe many consequences of an RC dysfunction (e.g., ATP shortage, superoxide over-production, metabolic blockade, etc.).

The remaining components of the RC are encoded by genes present in the nucleargenome. According to the theory of the symbiotic origin of the mitochondria, thesegenes have supposedly left the mitochondria to be inserted in the nuclear chro-mosomes during evolution [18]. Noticeably, nonfunctional copies of genes encod-ing mitochondrial proteins are frequently encountered in the nuclear genome [19].This also stands true for genes still harbored by the mtDNA, a feature that shouldbe carefully recorded when studying these genes [20]. Accordingly, the authentic-ity of the increased levels of mutant mtDNA frequently reported in tumor tissueshas been recently questioned [21]. Finally, if as mentioned earlier, the RC doesmuch more than to only transfer electrons and synthesize ATP, its functioning alsorequires much more than the hundreds of genes that encode its constituents. Indeedabout 1500 genes on the 30,000 genes of the human genome are believed to benecessary for the sustained building, function and maintenance of mitochondrialfunctions.


4 Mutations in Three Nuclear Genes Encoding RespiratoryChain Complex II Can Promote Tumor Formation

Mutations in three of the four genes encoding complex II have been shown tocause tumor formation and cancer. Mutations of succinate dehydrogenase (SDH)are frequently encountered in patients with an apparently sporadic form of paragan-gliomas (PGLs) or pheochromocytomas (PHEOs) [22]. PGLs are neuroendocrinetumors that may secrete catecholamines [23]. They occur most frequently in thehead (glomus tympanicum and jugulare), neck (carotid body and glomus vagale),adrenal medulla, and extra-adrenal sympathetic ganglia. PGLs are generally benign,highly vascularized tumors occurring close to the major blood vessels and cranialnerves [24]. About 10% of these tumors are malignant. PGLs are usually diagnosedon the basis of the presence of a mass in the neck, pulsatile tinnitus, or hearingloss. According to current nomenclature, the term pheochromocytomas should berestricted to secreting PGLs evolving from the adrenal medulla, whereas a func-tional PGL refers to an extra-adrenal secreting PGL, which was previously knownas an ectopic PHEO. The tumors now described as carotid body PGL were formerlyknown as chemodectoma due to the chemoreceptor function of the carotid body.PGLs seem to be inherited in about 30% of cases. The hereditary form of PGL isusually characterized by an early onset and a more severe presentation than that ofthe sporadic form. These tumors often display bilateral and multiple locations andmay be recurrent or malignant. The presence of one or several secreting tumors isnot rare and contributes to the severity of inherited PGL [23]. Such PGLs consti-tute a genetically heterogeneous group of diseases as four different loci have beenimplicated: PGL1 on 11q23, PGL2 on 11q13, PGL3 on 1q21, and PGL4 on 1p36. In2000, linkage analysis and positional cloning allowed Baysal et al. [8] to report thefirst deleterious mutations in the SDHD gene, corresponding with the PGL1 locus.The use of a candidate gene approach has led to the identification of mutations inSDHC (PGL3) and SDHB (PGL4) [24, 25]. Several mutations in these three geneswere subsequently reported in patients with hereditary PGL and in apparently spo-radic cases [26]. These observations fueled a vigorous debate on the mechanismlinking SDH deficiency to tumor formation.

Based on previous findings on the potential consequences of a RC blockade, ithas been suggested that superoxide overproduction may be at the origin of increasedcell proliferation [27]. Accordingly, the mev1 mutant of the worm Caenorhabditiselegans defective in the cytochrome b subunit of CII was found to have a reduced life-span that was attributed to overproduction of superoxides [28]. In the minds of manyinvestigators, the superoxide overproduction provided a simple explanation linkingRC defects to neoplastic transformation. However, no hyperplasia or indications ofabnormal cell proliferation were reported in the mev1 mutant at variance with someother C. elegans mutants for proteins that are prone to trigger tumorigenesis whenmutated in the homologous protein in mammals (e.g., cul1 mutant) [29].

Soon after the discovery that SDH mutations can result in tumor formation, itwas shown in a family harboring a SDHD mutation that these tumors were highly


vascularized and that this corresponded with hypoxia-inducible factor (HIF) stabi-lization and the activation of the hypoxia pathway [30]. HIFs are composed of twosubunits: an � subunit, which is regulated by oxygen, and a �-subunit (HIF-1�, alsocalled the aryl hydrocarbon receptor nuclear translocator, or ARNT), which is con-stitutively and ubiquitously expressed. Three �-subunits have been identified to date(HIF-1�, HIF-2�, and HIF-3�), each encoded by a different gene. Under normoxicconditions, HIF-� is continuously ubiquitinated and subsequently degraded by theproteasome [31]. The process of ubiquitination is started by their recognition by thevon Hippel–Lindau (VHL) protein, which in turn requires the hydroxylation of twoproline residues on HIF-� [32]. The very first step of HIF-� degradation under nor-moxic conditions is thus dependent on this hydroxylation, which is catalyzed by HIFprolyl hydroxylases (PHDs). PHDs belong to the superfamily of Fe(II)-dependentoxygenases and require reduced iron as a cofactor, alpha-ketoglutarate (�-KG) andoxygen as cosubstrates, with carbon dioxide and succinate being the products of thereaction [33]. Under hypoxic conditions, the absence of oxygen prohibits PHD activ-ity, and HIF-� is thus stabilized, allowing for its nuclear translocation and the sub-sequent activation of its target genes. The involvement of HIFs has been observedin numerous types of tumors, playing an active role in the progression of neoplasia[34]. Besides oxygen, others factors, which intervene in the reaction catalyzed bythe PHD enzyme, can also affect its activity. Consistent with this hypothesis, it wasshown that elevated levels of succinate, the product of the PHDs reaction, can resultin the blockade of its activity with the consequent stabilization of the HIF-1� protein(Fig. 2) [35, 36]. A significant accumulation of succinate was found in SDH-deficientcells and tumors. Moreover, the addition of �-ketoglutarate, the substrate of the PHD,

Fig. 2 The interactionbetween the mitochondrionand type II hexokinase. Thechanneling of mitochondrialATP by type II hexokinasefavors the production ofglucose 6-phosphate (G 6-P)and ultimately glycolysis.Ant, adenylate carrier; HKII,type II hexokinase; I–V, thevarious complexes of therespiratory chain; Q,ubiquinone; VDAC,voltage-dependent anionchannel


was shown to abolish the nuclear translocation of HIF-1� in the nucleus of SDH-defective cells [35]. This provided an alternative explanation linking SDH deficiencyto tumor formation and an explanation of the restriction of the induction of tumors toRC complex II blockade. Finally, the respiration of SDH-defective cells was shownto be not significantly decreased indicating that the electron flow to oxygen and thephosphorylation process are not significantly affected [37].

Additional support in favor of this latter hypothesis came from the observationthat fumarase defect can lead to leiomyomatosis and renal cell cancer (HLRCC) syn-drome [38]. In this latter case, fumarate, accumulated because of fumarase inactiva-tion, was found to act as a competitive inhibitor of the PHD, thus inducing the abnor-mal stabilization of HIF-1�. Noticeably, other structurally related organic acids canalso inhibit PHD [39]. Therefore, a tricarboxylic acid cycle (TCA) cycle blockademay result in the induction of angiogenesis and in the upregulation of glycolysisduring tumorigenesis, being at the origin of the Warburg effect. These observationssupport the view that rather than a blockade of the electron flow (potentially associ-ated with all subtypes of RC defects), the impairment of a particular segment of theKrebs cycle can be at the origin of tumor formation [40]. It would therefore be atleast rather imprudent to invoke SDH mutations as a general proof that a RC defectresults in tumor formation.

5 Does a Defective Respiratory Chain Trigger Cell Proliferation?

To date, there is no conclusive evidence that a perturbation of the electron flowthrough the RC is sufficient to increase cell proliferation and to constitute the pri-mary cause of tumor formation. On one hand, none of the nuclear genes encod-ing RC components or involved in the building or maintenance of the chain havebeen demonstrated to be tumor suppressors, with the exception of genes encod-ing RC complex II (or fumarase; see above) [41]. Noticeably, the accumulationof �-ketoglutarate is a feature frequently associated with RC dysfunction, whichwill not favor HIF-1� stabilization through PHD inhibition. On the other hand,RC-specific poisons are not known as carcinogenic (e.g., the complex I–specificinhibitor rotenone shown to possibly induce parkinsonism in mammals). Finally,patients harboring deleterious mutation in genes encoding RC components are notknown to be particularly at risk for tumor formation. This is even true for mutationsaffecting RC proteins such as ATPase (complex V) components that result in highlevels of superoxide production [42]. These observations lend less credibility to theidea that an impairment of the RC function could per se be at the origin of cellproliferation and tumor formation.

Even more puzzling is the fact that in a subset of tumor cells (liver and pancreatictumors), the enhanced glycolysis might well require an active production of ATPby the RC. The hexokinase specifically expressed in most tumor cells uses bothATP and glucose to produce ADP and glucose-6-phosphate. This tumor-specifichexokinase is characterized by its poor affinity (high Km, about 10 mM) for glu-cose, similar to the hexokinase IV (glucokinase) found in normal hepatocytes [43].


Fig. 3 Induction of the hypoxia path by succinate through HIF-1� stabilization. Upon succinatedehydrogenase (complex I) blockade, succinate accumulates and is exported to the cytosol. There,it inhibits the prolyl hydroxylase (PHD) triggering HIF-1� stabilization. The nuclear translocationof this latter factor induces an increased transcription of the hypoxia pathway components. VHL,von Hippel–Lindau; I–V, the various respiratory chain complexes


It possibly allows for an additional capacity for glucose uptake from plasma andincreased glucose phosphorylation by displacement of the cell glucose equilibrium.Interestingly, whereas the isoform with a low affinity is expressed in most tumorcells, in the tumoral liver, similarly to tumoral pancreatic cells, it is largely replacedby a high-affinity form (hexokinase II; HKII). This latter form can readily bindvoltage-dependent anion channel (VDAC) at the outer mitochondrial membrane andutilize the mitochondrial ATP to produce glucose-6-phosphate thus favoring an aer-obic glycolysis [44] so long as the electron transfer chain is functional (Fig. 3).Any significant impairment of RC activity would then tend to decrease the ATPproduced by the RC and oppose the upregulation of glycolysis. In addition, it hasbeen suggested that, upon binding to VDAC, HKII would be protected from productinhibition because of a conformational change and in turn may reduce the chance ofVDAC to open and to facilitate release of proapoptotic factors [43, 45]. In this case,not only would carcinogenesis not be triggered by mitochondrial dysfunction, butalso it would require the preservation of the respiratory chain function.

Finally, there is ample evidence that an impairment of RC function under mostconditions leads to cell death, tissue necrosis, and the related wide range of clinicalsymptoms observed in patients harboring mutations in genes necessary for the syn-thesis of the electron transfer chain [46]. Potential overproduction of superoxideshas been shown to be responsible for triggering apoptotic cell death, even if lowamount of superoxides might be required for normal cell division, and can targetvarious cell cycle checkpoints.

It therefore appears that a defective RC is probably not per se a primary cause oftumor formation. On the contrary, it could in some cases even constitute an obstacleto this process!

6 Mitochondria, Superoxides, Hypoxia

The possibility nevertheless exists that low oxygenation in tumors may secondar-ily affect mitochondrial functioning favoring the formation of superoxides andperoxides by the RC (Fig. 4). In principle, activated oxygen species may in turnupregulate both oncogene growth factors and their tyrosine kinase receptors thusdriving cell transformation [47], simultaneously promoting HIF-1� stabilizationby inhibiting prolyl hydroxylase. In any event, superoxide production that maypromote tumorigenesis would have to be restricted to a very narrow window ofconcentration, large overproduction readily favoring cell death rather than prolif-eration. The suggested role of superoxides in triggering tumorigenesis has longbeen advocated in support for a therapeutic use of antioxidants [48]. Actually, awide range of antioxidant molecules is available that can potentially interfere withcell free radicals, possibly released by mitochondria. However, contrasting resultsfrom in vitro and in vivo (intervention trials) experiments have raised some doubtabout the ability of antioxidants to actually fight cancer by such a mechanism.Indeed, most antioxidant molecules also act as prooxidants; this possibly accounts


Fig. 4 Linking electron transfer chain to cell death and proliferation. (A) According to a firsthypothesis, the level of mitochondrial superoxide production largely determines the fate of thecells. A low superoxide production favors cell division, whereas increasing this production trig-gers dysplastic lesion and tumor formation. An excessive production rather results in cell death.(B) According to this second scheme, the blockade of the prolyl hydroxylase by organic acids(succinate, fumarase) results in HIF-1� stabilization and the activation of the hypoxia path allow-ing for cell proliferation. Release of mitochondrial proapoptotic factors (AIF, cytochrome c) andoverproduction of superoxide (blockade of the ATPase; complex V) result in cell death

for their potential antitumoral activity. For instance, an antioxidant molecule likemelatonin would exercise its antiproliferative effect on the growth of rat pituitaryprolactin-secreting tumor cells in vitro by damaging mitochondria rather than byquenching superoxides [49]. Thus even if an increased superoxide production isobserved in a subset of cancer cells, more evidence is needed to establish that,as a general rule, these superoxides are instrumental in triggering tumorigene-sis. Recent studies however have established that overexpression of thioredoxin,manganese-dependent superoxide dismutase (MnSOD), glutathione peroxidase, orcatalase all tend to reduce cell proliferation in different animal or cell models


suggesting that handling of superoxides or their derivatives might be critical at somepoint in a subset of cases [50, 51] (see, however, Refs. 52, 53].

7 Targeting the Respiratory Chain to Kill Tumors

Although it is not clear that a defective respiratory chain actually favors tumor for-mation, mitochondria might represent the Achilles’ heel of cancer cells (Fig. 5).Indeed, mitochondria house several proapoptotic factors that are simultaneously

Fig. 5 Mitochondria and apoptosis. Release of intermembrane space components (such as AIFor cytochrome c) and/or the loss of membrane potential (��) under the effect of numerous stim-uli trigger cell commitment to die through an apoptotic process. Loss of membrane potential andof various matrix cofactors can result from the opening of the mitochondrial permeability transi-tion pore (PTP). The balance between the proapoptotic and antiapoptotic members of the Bcl-2family controls the opening of the pore. Alternatively, the members of this family can form chan-nels that may also allow for the release of proapoptotic components present in the intermembranespace. PTP would be a complex formed between the voltage-dependent anion channel (VDAC;outer membrane) and the adenylate carrier (ANT; inner membrane), associated with several addi-tional proteins. AIF, apoptosis inducing factor; Br, benzodiazepine receptor; CI–CV, the variouscomplexes of the respiratory chain; c, cytochrome c; IM, inner membrane; OM, outer membrane


components of (or closely associated with) the electron transfer chain, and target-ing tumors with reagents that induce release of these components has became afashionable idea [54]. Accordingly, cisplatin, one of the most important chemother-apeutic agents ever developed, has been shown to readily interact with mitochondriato trigger apoptosis [55]. Resveratrol, a natural polyphenolic antioxidant, has beenreported to possess a cancer chemopreventive potential that has been attributed toits ability to trigger mitochondrial dysfunction and cell apoptosis [56]. Actually,an exploding list of promising therapeutic and preventative agents is being shown(or has been suggested) to actually act through a quite similar mechanism, with orwithout the involvement of superoxide production.

8 Conclusion

Mutations in both nuclear and mitochondrial genes encoding respiratory chain com-ponents have been suggested to be causative for triggering cancer. However, despitethe numerous mtDNA mutations reported in many tumors, only germ-line muta-tions in the complex II–encoding genes (SDHB,C,D) have been conclusively shownas the primary cause of tumor formation. Interestingly enough, no inherited mtDNAmutation has been observed to cause tumor to date, contrasting the high frequencyof mutant mtDNA in various type of tumors (see, however, Ref. 21). Although thesemay have a role in the oncogenic process at some point, they may be a secondaryevent totally irrelevant for this process. As a general rule, it seems that targetingrespiratory chain function, except for complex II activity, provides a mechanism toinhibit rather than to promote tumor formation.

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Respiratory Control of Redox Signalingand Cancer

Pauline M. Carrico, Nadine Hempel and J. Andres Melendez

Abstract In the past several decades, elegant work has been performed in a varietyof organisms suggesting that decreases in oxidant burden can prolong life span anddecrease the severity of age-associated degenerative disorders (Finkel T, HolbrookNJ. Nature 2000; 408:239–247). Cancer is primarily an age-related disease andis associated with the altered production of oxidants (Cerutti PA. Science 1985;227:375–381). Compounds that generate reactive oxygen species (ROS) promoteskin tumors in mice. Treatment with antioxidants that terminate the chain reactionsinitiated by ROS antagonizes this process. Many tumor cell types have also beenshown to have an increased oxidant radical load relative to normal tissue. Thus,tumor cells have adapted to cope with an increased oxidant burden compared withthat of normal nonmalignant tissue and may even harness the increased oxidantload to modulate signaling pathways that are critical for their survival. With thetight connection between oxidant production and tumor promotion and growth, itis quite surprising that antioxidant-based therapeutics have not become pervasive intreatments for many cancers (for a concise review on this topic, see Seifried HE,et al. Cancer Research 2003; 63:4295–4298).

Hydrogen peroxide (H2O2) is the electron-neutral, 2e- reduction product of oxy-gen that is generated both chemically and enzymatically and can modulate theactivity of a variety of signaling molecules. This chapter will summarize how themetabolic production of H2O2 can impact distinct signaling pathways that are essen-tial for successful tumorigenesis and metastatic progression and will identify redox-sensitive pathways that may be targets for antioxidant-based cancer therapy.

Keywords Reactive oxygen species · Cancer · Antioxidants · Redox signaling ·Hydrogen peroxide

J.A. Melendez (B)Center for Immunology and Microbial Disease MC151, Albany Medical College, Albany, NY12208e-mail: [email protected]


33

34 P.M. Carrico et al.

1 ROS and Redox Signaling

All aerobic organisms generate ROS as by-products of normal cellular metabolism.Although the concentrations of ROS are normally low but measurable, accumulationof these species can be toxic to the cells. To protect themselves from the deleteriouseffects of ROS, cells have evolved an impressive repertoire of enzymatic and nonen-zymatic antioxidant defenses. In normal cells, a balance between the rates of ROSproduction and the scavenging capacity by antioxidants is required to maintain thecellular redox homeostasis. An imbalance resulting from either an increase in ROSconcentration or a decrease in the antioxidant capacity leads to the modulation ofredox-dependent signaling events.

ROS production can arise from a variety of sources in nonphagocytic cells. MostROS are produced as by-products of the electron transport chain of the mitochon-dria at the level of complex I (NADH/ubiquinone oxidoreductase) and complex III(ubiquinol/cytochrome c oxidoreductase). Outside the mitochondria, ROS can orig-inate from enzymes such as NADPH oxidase, dual oxidases, and lipoxygenases.At the cell surface, ROS production can be initiated after a variety of stimuli, suchas integrin signaling, growth factors, and cytokines, such as TNF-�, which acti-vate NADPH oxidase. ROS can also arise from arachidonic acid metabolism via5-lipoxygenase. Both cytoplasmic and mitochondrial pathways create the highlyunstable superoxide anion (O2

-.), which is either chemically or enzymatically con-verted to H2O2. The enzyme responsible for this dismutation is superoxide dismu-tase, isoforms of which can be found in the cytoplasm (copper zinc/sod1) or themitochondria (manganese /sod2).

Conditions where mitochondrial-derived ROS are produced at a rate that exceedstheir scavenging capacity are commonly associated with metabolic perturbationsthat impact signaling. Recent evidence also suggests that ROS produced as a resultof normal metabolic flux affect signaling. For example, Nemoto et al. [1] demon-strated that the addition of extracellular pyruvate to HeLa cells resulted in a dramaticincrease in the phosphorylation/activation of JNK1, a modest increase in ERK activ-ity, and no effect on JNK2 or p38. This activation was accompanied by an increasein mitochondrial oxidants and was reversible by the addition of N-acetylcysteine(NAC), overexpression of glutathione S-transferase, or extracellular catalase. Thesedata suggest that the addition of pyruvate results in an increase in H2O2 levels andthe subsequent phosphorylation of JNK1. Furthermore, the activation of JNK1 coin-cided with the inhibition of glycogen synthase kinase 3-� and glycogen synthase.This work provided convincing evidence that ROS produced as a result of normalcellular metabolism could act as a regulator of signal transduction.

The principle mediator of ROS-dependent signaling is H2O2. H2O2 generated inresponse to receptor stimulation has been shown to be an efficient signal transducingmolecule by its ability to reversibly oxidize active site cysteines [2]. Many proteintyrosine phosphatases (PTPs) are particularly susceptible to H2O2-dependent inac-tivation because of the lowered pKa of the active site cysteine. The essential cys-teine residue in the PTP signature active-site motif Cys-(X)5-Arg exists as a thiolateanion (Cys-S-), which, at neutral pH, is susceptible to nucleophilic attack by H2O2.

Respiratory Control of Redox Signaling and Cancer 35

Oxidation of the active-site cysteine generates a sulfenic derivative (Cys-SOH)leading to enzyme inactivation that can be reversed by cellular thiols. This mode ofinactivation is similar to that described for both bacterial and eukaryotic peroxire-doxin family members that are responsible for detoxification of organic hydroper-oxides, H2O2 and ONOO- [3]. A unique feature of the eukaryotic peroxiredoxins(PRXs) is their sensitivity to inactivation by H2O2 levels that exceed steady state.This sensitivity to inactivation has led to the “floodgate” hypothesis, whereby PRXinactivation can lead to a rapid rising burst of signaling H2O2, which may leadto PTP inactivation and subsequent enhancement of kinase signaling [4]. SeveralPTP family members have been shown to be oxidatively inactivated by physio-logic stimuli in vivo including the dual lipid protein phosphatase and tensin homologdeleted on chromosome 10 (PTEN), the low-molecular-weight PTPs (LMW-PTPs),and the mitogen activated protein (MAP) kinase phosphatase family members [5].Such redox-based signaling, arising from a chronic shift in ROS homeostasis thatresults from metabolic changes or altered antioxidant scavenging, can impact criticalcomponents of the malignant phenotype. Tumor cell proliferation, survival, vascu-lar recruitment, and migration are components that drive the malignant phenotype.Below we discuss how alterations in metabolic ROS production can regulate thesemechanisms.

2 Mitochondrial ROS and Cell Cycle

ROS have been shown to be potent mitogens. Effective detoxification of H2O2 bycatalase inhibits proliferation in Her-2/neu–transformed Rat-1 fibroblasts. Similarly,addition of exogenous H2O2 at low concentrations and its intracellular productionby the noninflammatory NADPH oxidase family members (Nox1-5) is also mito-genic [6].

H2O2 may directly induce cell proliferation, however, it also acts as a down-stream effector of platelet-derived growth factor (PDGF)-induced mitogenic signal-ing. In Rat-1 cells treated with PDGF, the SH2 domain–containing PTP, SHP-2,can be oxidatively inactivated, resulting in the phosphorylation and activation ofMAPK. Both the inactivation of SHP-2 and resulting activation of MAPK is abro-gated by treatment of cells with the H2O2 scavenger NAC, demonstrating a regu-latory role of H2O2 in cell proliferation through the reversible oxidation of PTPs[7]. In this context, antioxidant strategies can attenuate the proliferative capacity ofnumerous tumor cell lines [8]. H2O2 detoxification by the use of adenoviral cata-lase can effectively inhibit DNA synthesis and decrease the proliferation of humanaortic endothelial cells. Goswami and co-workers have demonstrated that intracel-lular H2O2 is also required for G1-S transit in mouse embryonic fibroblasts. Thus,in contrast with the growth inhibition commonly associated with the bolus additionof H2O2, the intracellular production of submicromolar concentrations of H2O2 ismitogenic and stimulates proliferation, and its effective removal can prevent cellcycle progression. It is likely that the intracellular redox state fluctuates as a resultof alterations in production or removal of mitochondrial-derived ROS. Menon and


Goswami have proposed that periodic fluctuations in metabolic ROS productionlikely control cell proliferation via redox-sensitive cell cycle regulatory proteins [9].The rapid turnover of antioxidants may give rise to acute shifts in cellular oxidantproduction that can control normal cell cycle progression. The elucidation of oxi-dant scavenging or oxidant generating enzymes whose levels fluctuate with respectto cell cycle would give credence to this hypothesis. During the process of carcino-genesis, a similar response may occur as a result of the loss of tumor suppressorfunction or oncogene activation. Thus, the enhanced proliferative capacity of tumorcells may be attributed to chronic shifts in respiratory ROS production due to per-turbations in metabolic or antioxidant function.

3 Mitochondrial ROS and Cell Survival

H2O2 has been implicated as a strong inducer of apoptosis in various cell types.Apoptosis or programmed cell death refers to a series of tightly coordinated eventsdesigned to eliminate potentially dangerous cells and cells that have reached theend of their life cycle. Apoptosis allows an organism to tightly control cell numberand tissue size, as well as to protect itself from cells that interfere with homeosta-sis. Apoptosis is a tightly regulated process whereby a set of cysteine proteases(caspases) are made active through a complex signaling cascade resulting in degra-dation of cellular nuclear DNA [10]. In contrast with necrosis, apoptosis destroysonly the cell in question with no effect on surrounding cells. Apoptosis can be trig-gered by both extracellular stimuli as well as intracellular events that mark cellulardysfunction. The hallmarks of apoptosis include membrane blebbing, cytoskeletaldisorganization, and loss of mitochondrial integrity and DNA fragmentation. Theultimate step in apoptosis is the activation of caspases, which act on a number ofdownstream cellular proteins inactivating them by catalytic cleavage at an Asp-Xxxsequence. The pro/inactive forms of these enzymes are activated via cleavage byother upstream caspases or via autoactivation. Caspases are involved in the degrada-tion of important cellular proteins as well as activation of caspase-activated DNAsesthat cleave mitochondrial DNA, ultimately leading to cell death. Neoplastic cells areable to evade apoptosis and circumvent cell death, an essential survival step duringtumorigenesis.

TNF-� is produced by cells under stress and induces signaling via binding to itscell surface receptor. TNF-� along with cycloheximide (CHX) has been establishedas a potent inducer of apoptosis in various tumor cell lines. One of the primarymechanisms of TNF-�/CHX–induced apoptosis involves activation of the receptor-associated death domains that recruit and activate caspase-8, leading to activation ofdownstream caspases and finally of the executioner caspase, caspase-3. In addition,CHX enhances the induction of apoptosis via TNF-� by inhibition of new proteinsynthesis. One of the proteins that was first discovered to be essential for pro-tection from TNF-mediated cell death is the mitochondrial manganese containingsuperoxide dismutase (Sod2) [11]. Since this initial observation by Wong and


Goeddel, numerous reports have demonstrated a role for Sod2 in protection frommany apoptotic stimuli. The antiapoptotic effects of Sod2 suggest that either theremoval of O2

-. or the generation of H2O2 via Sod2 may have an inhibitory effecton programmed cell death.

The mitochondrial generation of H2O2 has been shown to confer resistanceto apoptotic stimuli. Procaspases contain an active site cysteine that is suscep-tible to oxidation thereby preventing their activation via proteolytic cleavage.This may explain the ability of oxidants in some studies to deter apoptotic celldeath. Cederbaum and Bai have demonstrated that catalase overexpression inHepG2 hepatocarcinoma cells increases their susceptibility to TNF-�–inducedapoptosis. Fas-induced apoptotic cell death can be enhanced by overexpressionof mitochondrial-targeted catalase. The antioxidant flavonoid and terpenoid com-pounds derived from the Ginkgo biloba plant have been shown to enhance theexpression of proapoptotic molecules as a result of its ability to scavenge H2O2

in distinct cancer cell types. Recent studies from this lab indicate that efficient andmitochondrial-targeted H2O2 detoxification can reverse the antiapoptotic propertiesof Sod2 overexpression [12].

Another mechanism whereby H2O2 may augment cell survival is via oxidativeinactivation of the LMW-PTPs. LMW-PTP activity is dependent upon the tyrosinephosphorylation on Tyr131 and Tyr132; phosphorylation on Tyr131 results in a 25-fold increase in enzymatic activity, and phosphorylation of Tyr132 serves as a scaf-fold for the SH2 domain of the Grb2 adaptor protein. In PDGF-treated NIH-3T3or human prostatic carcinoma PC3 cells, inactivation of LMW-PTP by glucose oxi-dase (G/O)-induced oxidative stress inhibits LMW-PTP auto-dephosphorylation onTyr131 and Tyr132, thus enhancing the recruitment of Grb2 to LMW-PTP and, sub-sequently, ERK activation and cell survival [13].

Thus, even though it has been observed that bolus additions of H2O2 induceapoptosis, these studies have established that submicromolar concentrations ofmitochondrial-derived H2O2 may provide a selective inhibition of programmed celldeath and that effective H2O2 detoxification may also sensitize cancer cells to apop-totic stimuli.

4 Mitochondrial ROS and Angiogenesis

H2O2 has been shown to regulate proangiogenic responses in a variety of systemsincluding human retinal pigment epithelial cells, cultured keratinocytes, and bovinepulmonary artery endothelial cells. Monte et al. have demonstrated that H2O2 con-tributes to promoting the angiogenic activity of tumor-bearing lymphocytes and thatthis activity can be inhibited by coadministration of H2O2-detoxifying enzyme cata-lase but not superoxide dismutase. In vitro angiogenesis of bovine thoracic aorta canbe induced with relatively low concentrations (1 �M) of H2O2 and blocked by cata-lase. The discovery of a noninflammatory NADPH oxidase (Nox1-5) has providedan additional source for the production of O2

-. and H2O2 in a variety of tumor cell


types [14]. The Nox1-dependent generation of H2O2 is a potent trigger of angiogen-esis, increasing the vascularity of tumors and inducing molecular markers of angio-genesis including vascular endothelial growth factor (VEGF), VEGF receptors, andmatrix metalloproteinase (MMP) activity in cultured cells and in tumors. Coexpres-sion of catalase blocks the increased activity of the angiogenic markers, indicat-ing that H2O2 signals part of the switch to the angiogenic phenotype. Furthermore,MMPs are important contributors to the angiogenic switch, and their expression isredox-responsive [15].

The transcription factor Ets-1 is also an important regulator of angiogenesis andis sensitive to H2O2 production [15]. The role of Ets-1 family members in tumori-genesis is largely due to their ability to activate the transcription of a number ofgenes involved in matrix degradation, such as MMP-1, 2, 3, and 9 as well as uroki-nase plasminogen activator. These proteases contribute to tumor invasion and theearly phases of blood vessel formation [16]. In cisplatin-resistant ovarian carcinomavariants, elevations in H2O2 are attributable to alterations of mitochondrial functionrelative to parental cell lines leading to an increase in transcription of Ets-1. Theincrease in transcription enhances binding of Nrf2, a key transcription factor in thecellular response to oxidative stress, to an antioxidant response element (ARE) inthe promoter of Ets-1. Ets-1 expression has been correlated with poor prognosis inbreast and ovarian cancer. Furthermore, the development of cisplatin resistance is amajor limitation of this chemotherapy regime, which is widely used in the treatmentof testicular and ovarian cancer. These observations suggest that shifts in the steady-state production of H2O2 in response to receptor engagement or altered mitochon-drial function resulting from the development of cisplatin resistance can enhancethe expression of the angiogenic transcription factor Ets-1.

VEGF is a key mediator in regulating the vascular recruitment of tumor endothe-lial cells. The transcriptional activity of VEGF is directly responsive to H2O2 andinvolves a GC-rich region of the promoter between −95 and −51. Detailed char-acterization of the promoter has identified two SP1 and SP3 sites at −73/−66 and−58/−52 that represent the core mechanism of oxidative stress–triggered VEGFtransactivation. Connor et al. have also established that mitochondrial-derived H2O2

resulting from the dismuting activity of Sod2 was responsible for enhanced VEGF-dependent angiogenic activity. The mechanism involved the H2O2-dependent oxida-tive inactivation of the dual lipid-protein phosphatase PTEN, leading to increase inactivity of the serine/threonine-specific protein kinase AKT and subsequent VEGFexpression [17].

Hypoxia inducible factor 1� (HIF-1�), the master regulator of cellular responsesto hypoxia, responds to shifts in the metabolic production of ROS [18]. HIF-1�regulates the transcriptional activity of genes that participate in angiogenesis, ironmetabolism, glucose metabolism, and cell proliferation/survival. The regulation ofHIF-1� in response to hypoxia is controlled by its hydroxylation state. Hydroxyla-tion is controlled by a family of prolyl hydroxylases (PHDs) that require molecularoxygen for full activity. In the presence of oxygen, PHD activity is maximal, leadingto HIF-1� hydroxylation. Hydroxylated HIF-1� is then targeted for polyubiquitina-tion leading to its degradation by the cellular proteasome complex. In the absence of


O2, hydroxylation is inhibited, leading to HIF-1� stabilization and activation of itstarget genes. Studies have determined that efficient mitochondrial ROS scavengingcan prevent the hypoxic induction of HIF-1�. This ROS-dependent stabilization ofHIF-1� involves mitochondrial complex III–dependent production of superoxideand subsequent inhibition of PHD activity. The mechanisms for how mitochondrialROS regulate HIF-1� are not clear but may involve a shift in the oxidation stateof PHD-bound iron. It also possible that the hypoxic production of ROS may leadto the oxidative inactivation of phosphatases, such as PTEN, that contribute to themaximal activation of VEGF.

Thus, it appears that angiogenic signaling is particularly sensitive to alterationsin the metabolic production of oxidants and that mitochondrial free radicals mayplay a role in preserving levels of their initial substrate, oxygen, via HIF-1�.

5 Mitochondrial ROS and Invasion/Migration

A hallmark of malignancy and a primary cause of morbidity and mortality in cancerpatients is metastasis. Tumor metastasis is a multistep process initiated by migrationand invasion of cells into the surrounding vasculature. This is followed by extrava-sation from the circulation and proliferation at a secondary site where the formationof a metastatic lesion occurs. A key process in the initial movement and subsequentinvasion of neoplastic cells into the circulatory system is the remodeling of the extra-cellular matrix (ECM). Emerging evidence has implicated ROS and the activation ofredox-sensitive signaling pathways in invasion and migration. Intrinsic antioxidantenzymes are vital to the regulation of oxidative stress within cells. In vitro stud-ies have shown that a number of cancer cell lines contain elevated levels of Sod2and decreased levels of catalase and that this change in steady-state levels of H2O2

correlates with increased metastasis, proliferation, and resistance to apoptosis.An integral part of migration and adhesion is the dynamic rearrangement of

focal adhesion complexes in the cell. The role of Rho GTP-binding proteins (Rho,Rac, and Cdc42) in regulating cytoskeletal dynamics by activation of Src and focaladhesion kinase (FAK) and initiating remodeling of integrins has been firmly estab-lished. In adherent cells, mature focal adhesions have been shown to be regulatedby RhoA, whose activation results in actin stress fiber formation. Rac and Cdc42are considered to be promigratory resulting in the formation of focal complexes oflamellipodia and filopodia, respectively. It has been shown that ROS modulate Racand Rho signaling. Rac can specifically downregulate RhoA in a ROS-dependentmanner through oxidative inactivation of LMW-PTP, which normally dephosphory-lates and inactivates the Rho GTPase-activating protein, p190Rho-GAP. Activationof p190Rho-GAP results in the conversion of the GTP-bound to the GDP-boundform of Rho, and its inactivation contributes to a loss of adhesion and a transitionto a migratory phenotype. While ROS appear to disrupt the formation of maturefocal adhesions of adherent cells, there is evidence that suggests a role of ROS inthe turnover of leading edge focal complexes observed in lamellipodia of migratingcells. Rac has been shown to activate NADPH oxidase, which is spatially localized


to membrane ruffles of the leading edge of migrating cells, and was shown to beassociated with cell migration. In this context, oxidants can reversibly inactivate theprotein tyrosine phosphatase PTP-PEST that normally dephosphorylates and inac-tivates FAK, leading to a more rapid turnover of active FAK at focal complexes. Itis also possible that mitochondrial-derived ROS are involved in disrupting maturefocal adhesions yet contribute to active remodeling of focal complexes of the lamel-lipodia required for migration.

Lipid signaling molecules also play an important role in regulating the migra-tory phenotype. Studies in phagocytic cells and the amoeba Dictyostelium dis-coideum demonstrate that phosphoinositide (3,4,5) 3-phosphate (PtdIns[3,4,5]P3)accumulates at the front of chemotaxing cells. Concomitant with the distributionof PtdIns(3,4,5)P3 to the leading edge is a redistribution of PTEN to the remainingoutside perimeter of the cell where it degrades PtdIns(3,4,5)P3 by removing the 3-phosphate. Alterations in the steady-state levels of H2O2 can inactivate the tumorsuppressor PTEN leading to an augmentation of AKT signaling and redistributionof phosphoinositides at the plasma-lamellar surface [17]. It is intriguing to hypoth-esize that receptor engagement in response to chemotactic triggers may lead to thelocal oxidant-dependent inactivation of PTEN and rapid reproportioning of lipidsignaling molecules that promote the migratory phenotype.

An essential and rate-limiting step in metastasis is the remodeling and degrada-tion of the ECM and basement membrane by proteolytic enzymes. In this model,cells must interact with surrounding stromal cells, leading to the loss of matrixfunction and resulting in a compromised matrix boundary. MMPs are major con-tributors of stromal degradation and are vital to the process of cellular invasion.MMP-1/interstitial collagenase has a specificity for type I collagen, the primary col-lagen of the interstitial matrix; though it has been shown to degrade collagen typesII, III, VII, and X. Studies using antisense mRNA against MMP-1 in melanomacells have shown a significant attenuation in the capacity of these cells to invadetype I collagen and Matrigel in vitro. Moreover, the expression of MMP-1 has beenshown to be a definitive prognostic marker for breast lesions that will develop intocancer suggesting that the expression and regulation of MMP-1 may be importantin malignancy.

Studies from this and other laboratories have demonstrated that the Sod2-dependent production of H2O2 leads to increased expression of MMP family mem-bers and that there is a strong correlation between this increase in MMP levels andenhanced metastasis. Interestingly, the Sod2-dependent increases in MMP expres-sion can be reversed by the H2O2 detoxifying enzyme, catalase, or glutathione per-oxidase [15]. Thus, H2O2-dependent upregulation of MMPs may, in part, contributeto increased invasion and metastatic capacity of tumors displaying elevated Sod2levels.

Maximal mitochondrial H2O2-dependent transcription of MMP-1 transcriptionis achieved by a proximal activator protein 1 (AP-1) site at –1602 and a singlenucleotide polymorphism (SNP) guanine insertion at –1607, which creates an Ets-1binding site [15]. This redox-sensitive MMP-1 protein expression requires activa-tion of both ERK1/2 and JNK pathways. JNK signaling is largely responsible for


the H2O2 sensitivity of the MMP-1 promoter, whereas ERK1/2 contributes to bothits basal and H2O2 dependence [19].

The ability of a tumor cell to migrate and invade through the extracellular matrixis attributed to local shifts in signaling that regulate the distribution of phospho-lipid signaling molecules, rearrangement of focal adhesion complexes, and matrixdegradation. All of these processes are redox-sensitive and are likely responsiveto metabolic shifts in the steady-state production of H2O2. As many of the pro-cesses are highly compartmentalized, it is possible that vicinal mitochondria mayparticipate in these migratory signaling events by augmenting their local productionof H2O2. With the development of fluorescent reporter molecules that detect focalshifts in the production of many of the migratory signaling intermediates and newredox-sensing mitochondrial targeted GFP constructs, we will be able to determineif these events are juxtaposed.

6 Mitochondrial ROS and Cancer Stem Cells

Recently, the hypothesis has emerged that conventional therapies may fail to com-pletely eradicate cancer because cancer stem cells, which are thought to act ascancer-initiating cells, are insensitive to these treatments. Work in leukemia, breast,brain, and lung cancers have provided further understanding of the role cancer stemcells play in malignancies. Although cancer stem cells appear to have some of thephenotypic and functional properties of embryonic stem cells, such as the potentialto give rise to a larger population of cells, survival of these two different cell typesrequires different pathways that are frequently at odds with one another. Recentresearch indicates that signaling by ROS may regulate growth and differentiation inboth hematopoietic stem cells and cancer stem cells.

PTEN, like p53 is one of the most common targets for mutation in sporadichuman cancers. The oxidative inactivation of PTEN and subsequent activation of theAKT signaling pathway may lead to the transformation of adult hematopoietic stemcells (HSCs) into leukemias. Transgenic mice with conditional deletions of PTEN inadult HSCs develop leukemias within 4 to 6 weeks along with a depletion of normalHSCs. These defects are dependent upon the downstream effector mammalian targetof rapamycin (mTOR), a serine/threonine kinase that regulates metabolism and thathas also been shown to generate ROS [20].

The oxidative inactivation of PTEN and subsequent activation of the AKT sig-naling pathway leads to the phosphorylation of the class O of forkhead transcrip-tion factors (FoxO), resulting in the retention of FoxO in the cytoplasm throughinteractions with 14-3-3 proteins [21]. FoxOs, which have also been postulated toact as tumor suppressors, regulate the transcription of genes involved in cell cyclearrest, apoptosis, glucose metabolism, and stress response. In C. elegans, the FoxOortholog DAF-16 has been shown to affect life span through resistance to oxida-tive stress and adaptation to food shortage. DAF-16 promotes resistance to oxida-tive stress by activating transcription of Sod2 and catalase. In mammals, the FoxO


subgroup consists of four members (FoxO1, FoxO3, FoxO4, and FoxO6), of whichthree (FoxO1, FoxO3, FoxO4) overlap in function and expression patterns. Indi-vidual deletion of FoxO proteins in mice does not result in a tumor-prone pheno-type, however, mice containing conditional deletions of FoxO1, FoxO3, and FoxO4display a cancer-prone condition characterized by thymic lymphomas and heman-giomas [22]. In addition, mice with conditional deletions of FoxO1, FoxO3, andFoxO4 exhibited premature aging of HSCs, which was shown to be at least partlydue to an increase in ROS levels in the HSC compartment and attributable to adecrease in expression of antioxidant genes [23]. Treatment of these mice with NACrestored the HSC compartment size, reestablished stem cell cycling, and normalizedHSC apoptosis.

FOXO3a is also regulated via p66Shc, which belongs to a family of adaptors thatfunction in signal transduction of mitogenic and apoptotic responses, and whoseabsence has been shown to prolong life span both in cell culture and mouse mod-els. Although p66Shc is activated through tyrosine phosphorylation in response toextracellular signals such as EGF and insulin, phosphorylation of p66Shc at ser-ine 36 in response to oxidative stress leads to the subsequent phosphorylation andsequestration of FOXO3a in the cytosol [24]. This may further increase levels ofROS as FOXO3a has been shown to activate a reporter under the control of thehuman catalase promoter [24].

In a hypoxic environment, p66shc functions to promote survival of stem cellsand mammary cancer stem cells through the upregulation of Notch-3 and carbonicanhydrase IX (CA-IX) [25]. In contrast, as discussed above, inhibition of FOXOproteins in response to H2O2 augments apoptosis of stem cells.

Recently, p66shc was also shown to regulate mitochondrial metabolism [26]. Inimmortalized mouse embryonic fibroblasts (MEFs) lacking p66shc, oxygen con-sumption is reduced 30% to 50% with a coincident increase in glycolysis anddecrease in NADH metabolism. In addition, Giorgio et al. [27] demonstrated thatp66shc utilizes reducing equivalents of the mitochondrial electron transfer chainthrough the oxidation of cytochrome c to generate mitochondrial H2O2, which func-tions as a signaling molecule for apoptosis.

7 Conclusion

The role of ROS in the process of carcinogenesis has evolved from damaging lipid,proteins, and nucleic acids that in turn promote tumor development to regulators ofsignal transduction events that control key facets of the malignant phenotype. Manyof the redox-sensitive signaling proteins drive the expression or activation of genesand proteins that regulate tumor cell proliferation, survival, vascular recruitment,and migration. The source of the ROS that regulate these signaling networks has inmany cases been assigned to the ubiquitous family of noninflammatory respiratoryburst oxidases. However, emerging data indicates that mitochondrial-derived ROSalso play a prominent role in regulating the tumorigenic phenotype. A number of


signaling proteins like the dual lipid protein phosphatase PTEN and the transcriptionfactor Ets-1 are sensitive to perturbations in mitochondrial oxidant production andare involved in regulating numerous aspects of the tumorigenic phenotype. H2O2-dependent activation of Ets-1 leads to enhanced tumor angiogenesis, migration, andinvasion. Oxidative inactivation of PTEN leads to an increase in active AKT therebypromoting tumor cell survival, proliferation, and angiogenesis. This implies thatalterations in mitochondrial function that enhance respiratory ROS production cancontrol signaling molecules that synergize and potentially drive the malignant phe-notype. PTEN contains an active site cysteine that renders it redox-sensitive. Ets-1contains several cysteines that are essential for its DNA binding, and it is likely thatoxidation would lead to its inactivation. The fact that oxidants promote Ets-1 activitysuggest some as yet undefined oxidant-sensitive signaling molecule may control theredox-responsiveness of this transcription factor independent of its DNA-bindingactivity.

It is clear that many facets of the tumorigenic phenotype are ROS responsive.The angiogenic factor VEGF is controlled by oxidants through promoter activation,stabilization of transcription factors (HIF-1�), and activation of upstream signals.Ets-1 activation also contributes to the early phases of blood vessel formation. Thus,angiogenic signals appear to be highly sensitive to shifts in oxidant production andmay be highly responsive to shifts in respiratory oxidant production. Further char-acterization and identification of oxidant-responsive molecular targets that controltumor progression and eradication of cancer stem cells will undoubtedly lead to thedevelopment of novel antioxidant-based therapies for the treatment of malignancy.

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8. Floyd RA. Antioxidants, oxidative stress, and degenerative neurological disorders. Proc SocExp Biol Med 1999; 222(3):236–245.

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12. Dasgupta J, Subbaram S, Connor KM, et al. Manganese superoxide dismutase protects fromTNF-alpha-induced apoptosis by increasing the steady-state production of H2O2. AntioxidRedox Signal 2006; 8(7–8):1295–1305.

13. Chiarugi P, Buricchi F. Protein tyrosine phosphorylation and reversible oxidation: two cross-talking posttranslation modifications. Antioxid Redox Signal 2007; 9(1):1–24.

14. Lambeth JD, Cheng G, Arnold RS, Edens WA. Novel homologs of gp91phox. TrendsBiochem Sci 2000; 25(10):459–461.

15. Nelson KK, Melendez JA. Mitochondrial redox control of matrix metalloproteinases. FreeRadic Biol Med 2004; 37(6):768–784.

16. Lincoln DW, Bove K. The transcription factor Ets-1 in breast cancer. Front Biosci 2005;10:506–511.

17. Connor KM, Subbaram S, Regan KJ, et al. Mitochondrial H2O2 regulates the angiogenicphenotype via PTEN oxidation. J Biol Chem 2005; 280(17):16916–16924.

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20. Kim JH, Chu SC, Gramlich JL, et al. Activation of the PI3K/mTOR pathway by BCR-ABL contributes to increased production of reactive oxygen species. Blood 2005; 105(4):1717–1723.

21. Brunet A, Bonni A, Zigmond MJ, et al. Akt promotes cell survival by phosphorylating andinhibiting a Forkhead transcription factor. Cell 1999; 96(6):857–868.

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26. Nemoto S, Combs CA, French S, et al. The mammalian longevity-associated gene productp66shc regulates mitochondrial metabolism. J Biol Chem 2006; 281(15):10555–10560.

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Cellular Respiration and Dedifferentiation

Roberto Scatena, Patrizia Bottoni, and Bruno Giardina

Abstract Mitochondria are far more than the “powerhouse” of the cell as theyhave classically been described. They are also the location where various catabolicand anabolic processes, calcium fluxes, reactive oxygen and nitrogen species, andnumerous signal transduction pathways interact to maintain cell homeostasis andto modulate cellular responses to diverse stimuli. Because of the mitochondrion’svital roles in cell physiology, this organelle squarely falls within the ambit of can-cer pathophysiology. Indeed cancer-associated alterations in cell energy metabolismrelated to mitochondrial dysfunction have been recognized for many years in the so-called Warburg effect. Furthermore, these organelles appear to play a fundamentalrole in determining whether a cell will undergo cell death by apoptosis or necrosis;and key metabolic enzymes of the tricarboxylic acid (TCA) cycle, the fundamentaloxidative pathway of the cell, may act as oncosuppressors. Recent findings indicatethat modulation of mitochondrial activities in general, and of its electron respiratorychain in particular, could induce differentiation and/or death of cancer cells. Thusit may be possible to induce anticancer activities by deliberate modulation of cellu-lar respiration. Elucidation of the role of mitochondria in cancer cell dedifferentia-tion processes may provide a new definition of the dedifferentiation/differentiationof cancer cells as well as important information about the pathophysiologyof so-called stem cells, with important diagnostic, prognostic, and therapeuticconsequences.

Keywords Cancer cell differentiation · Oxidative phosphorylation · NADHdehydrogenase · Heat shock proteins · Reactive oxygen species · Warburg effect ·Nitric oxide · Oncogenes · Oncosuppressors · Biomarkers

R. Scatena (B)Dipartimento di Medicina di Laboratorio, Universita Cattolica del Sacro Cuore, 0168 Rome, Italye-mail: [email protected]


45

46 R. Scatena et al.

1 Introduction

Cancer can be defined as a genetic disease characterized by dysregulation of var-ious cellular pathways that orchestrate cell proliferation, differentiation, and death[1]. A cell becomes cancerous when a series of oncogenes and/or oncosuppres-sors become dysfunctional and hence induce a neoplastic phenotype in that cell. Inthis dramatic clonal disorder, the cell acquires high proliferation kinetics and losescell-cell contact inhibition. These properties are progressively achieved by a seriesof genetic “hits,” including mutations and or other epigenetic modifications [1].The final result is often defined as dedifferentiation of a neoplastic cell that mor-phologically becomes anaplastic. The grade of dedifferentiation (anaplasia) differsamong cancer cell specimens and is often correlated with the malignancy of theneoplasia.

In the dramatic perturbation of cell homeostasis that accompanies carcinogene-sis, mitochondria are involved in various relevant, interconnected, but also indirectpathophysiologic roles. In this context, the significance of mitochondrial respirationhas received inadequate attention. In spite of an abundance of data that link mito-chondria to cancer, there have been few studies that have investigated the interrela-tionships between cellular respiration and cellular dedifferentiation. Here we brieflysummarize some molecular mechanisms, which will be extensively considered inother chapters, that link cancer cell dedifferentiation to mitochondrial oxidativemetabolism to provide an introduction to concepts that facilitate the understandingof these aspects of oncology:

1. Peculiar modification of cell metabolism (Warburg effect). Cancer cells gener-ally produce ATP mainly through so-called aerobic glycolysis, a metabolic shiftcharacterized by high glucose uptake and increased production of lactate. Orig-inally, this altered metabolic state was considered to be the result of adaptation(via HIF and/or AKT) to the new potentially anoxic environment of a neoplasticlesion [2]. However it has become evident that the aerobic glycolysis of cancercells is an epiphenomenon that results from a more complex metabolic rear-rangement in which not only glycolysis but also Krebs cycle, beta-oxidation,and anabolic metabolism in general are altered to support the new primary func-tion of the cell (e.g., uncontrolled proliferation) by providing not only energybut also the building blocks for the synthesis of nucleic acids, peptides, andlipids [3–5].

2. Distinct properties of cancer cell mitochondria. Emerging data suggest that mito-chondria have an active pathogenic role in the induction and maintenance of theWarburg effect. This role is emphasized by some typical metabolic peculiari-ties of mitochondria that enable cancer cells to be functionally distinguishedfrom normal cells. Cancer cells exhibit reduced mitochondrial respiration (bothin terms of ATP turnover and proton leakage), increased NADH/NAD+ ratios,and a pronounced increase in membrane potential [6]. Other morphologic(number, size, shape) and structural differences (phospholipids, cholesterol, and

Cellular Respiration and Dedifferentiation 47

membrane proteins) have been described, but their pathogenic roles are notunderstood [7].

3. Mitochondrial DNA (mtDNA) mutations. Mutations of mtDNA have beenlinked to some tumors. In particular, intragenic deletions, missense and chain-terminating point mutations, and alterations of homopolymeric sequences havebeen described in various sporadic tumors. Intriguingly, mutations have beendescribed in the hypervariable regions of the mitochondrial D-loop, the sectionof DNA that controls mtDNA transcription and replication. Mutations have alsobeen described in all tRNAs, in rRNAs, and in all 13 of the mtDNA-encodedsubunits of the electron respiratory chain [8]. However, Salas et al. [9] recentlysuggested that most of the reported mtDNA mutations in tumors may be dueto laboratory-induced artifacts. Others contend that mtDNA mutations play areal pathogenetic role in cancer [10]. Hence there is ongoing debate about therole of mtDNA mutations and the consequent respiratory dysfunction in cancercells.

4. Nuclear DNA mutations and mitochondrial proteins. Succinate dehydrogenase(SDH) and fumarate hydratase (FH), mitochondrial enzymes of the tricarboxylicacid (TCA) cycle, can act as classic tumor suppressors, and mutations of thegenes that encode them have been associated with various benign and malignanttumors. These observations have drawn attention to the potential pathogeneticrole of the mitochondrion, especially of mitochondrial oxidative metabolism,in cancer [11, 12]. Given the role of SDH (complex II) in the electron respi-ratory chain, such findings suggest that there may be critical interrelationshipsbetween mitochondrial energy metabolism and timely surveillance of the differ-entiation/dedifferentiation state of cells.

5. Apoptosis. Mitochondria have a well-defined role in caspase-mediated induc-tion of the intrinsic pathway of apoptosis initiated by the release of cytochromec and second mitochondrial activator of caspases (SMAC) from the mitochon-drial intermembrane space in response to a variety of noxious stimuli, includingDNA damage, loss of adherence to the extracellular matrix (ECM), oncogene-induced proliferation, and growth factor deprivation. Perturbation in the releaseof apoptosis-inducing proteins regulated by proapoptotic and antiapoptotic mem-bers of the Bcl-2 family (e.g., Bcl-2, Bcl-XL, and Mcl-1) seems to influencenot only etiopathogenesis but also cancer prognosis and therapeutic efficacy.Accordingly, derangements of the permeability transition pore (PTP) and elec-trochemical gradient, which strictly depend on mitochondrial respiration, havebeen implicated in these phenomena [13].

6. Free radicals (reactive oxygen species [ROS] and reactive nitrogen species[RNS]). Mitochondria are the main source of free radicals in cells. Thus mito-chondrial dysfunction, especially at the level of the electron respiratory chain,can result in increased production of ROS and RNS. ROS are considered classiccarcinogens because they can directly induce mutagenesis and thereby promotetumorigenesis and cancer progression [14]. However, the role of ROS and RNSin cancer is complex. In fact, the growth-promoting effects of ROS have beenalso related to activation of redox-responsive signaling cascades (MAPK, PKC,


AP-1, NF-�B). Paradoxically, ROS can also act as tumor suppressors, activat-ing via a p16ink4a-Rb pathway an irreversible cellular process of senescence thatestablishes stable G1 phase cell-cycle arrest [15].

7. Heat shock proteins (HSPs). HSPs represent a ubiquitous and evolutionarilyconserved family of proteins (classified according to their molecular weight)that were originally discovered because they can be induced by heat stress.Their production confers a peculiar cellular resistance to further stresses (heat orchemical). Initially, these proteins were considered to be chaperones—molecularguides acting to ensure proper protein folding. Other functions have since beendiscovered, including roles in gene expression regulation, DNA replication, sig-nal transduction, cell differentiation, metastasis, apoptosis, senescence, immor-talization, and, intriguingly, multidrug resistance development. However, theprecise pathophysiologic roles of HSPs in general, and of mitochondrial HeatShock Proteins (mitHSPs) in particular, are not well understood. Studies employ-ing several methodological approaches (immunochemical, immunohistochemi-cal, proteomic) have shown that HSP expression gradually increases from normalto dysplastic and then to neoplastic tissues, such that HSP overexpression corre-lates with cancer evolution. Hence some of these proteins (i.e., HSP60, HSP10,HSP70, PHB1, and PHB2) have been implicated in cancer diagnosis, prognosis,and therapy [16]. Overexpression of HSP70 (also known as 75-kDa glucose reg-ulated protein, HSP70-9, HSP70-9B, mortalin 2) may be particularly importantin cancer cell proliferation and dedifferentiation; its role may go beyond genericcytoprotection with respect to apoptosis and involve a more specific role in thepathogenesis of the peculiar cellular metabolic shift that characterizes the cancercell phenotype [17].

8. Hexokinase II. The cancer cell glycolytic phenotype is supported by overexpres-sion of the hexokinase II isoenzyme, which is linked to the outer mitochondrialmembrane via the porin-like protein voltage-dependent anion channel (PPVAC).Hexokinase II-PPVAC binding may have significant functional implications incancer because it facilitates glucose phosphorylation and thus glucose utilizationfor energetic and biosynthetic purposes [18].

9. Calcium. Because of the pivotal roles mediated by calcium ions in signaltransduction pathways, calcium has been considered to be a potential phys-iopathogenetic marker in several chronic and acute cellular diseases. How-ever, research findings have not confirmed the originally grand expectationsfor calcium in disease processes, at least in oncology. When Scorrano et al.[19] examined the interrelationships between calcium, mitochondrial respira-tion, and dedifferentiation, they found only an indirect role of Bax/Bak viaregulation of calcium release from the endoplasmic reticulum. Interestingly,however, chemotherapeutic-resistant tumor cells sequester cytosolic calcium(Cacyt) more efficiently than do normal cells; they show reduced release of cal-cium ions from intracellular storage sites upon apoptosis induction [20]. It hasbeen proposed that transition to more invasive cellular behavior may be relatedto impaired release of calcium from the endoplasmic reticulum of various kindsof cancer cells. On the contrary, Amuthan et al. [21] showed in tumorigenic


human pulmonary carcinoma A549 cells that mitochondrial stress induced bydamaging mtDNA (with ethidium bromide treatment) activates a mitochondriato nucleus signaling pathway mediated by increased Cacyt concentration and thatactivation of this pathway induces cell dedifferentiation associated with pheno-typic changes and cell invasion.

2 Cellular Respiration and Dedifferentiation

Although the connection between mitochondrial respiration and cancer cell ded-ifferentiation (i.e., resistance to apoptosis, cell proliferation, metabolic shift, cellinvasion, genetic instability) is fundamental to cancer pathogenesis, the precisemolecular interrelationships and regulatory processes are far from clarified. Thislack of understanding is related, at least in part, to the traditional view that the so-called Warburg effect was essentially a reflection of mitochondria being damagedand dysfunctional organelles in cancer cells. Recent studies are reexamining mito-chondrial function, especially cellular respiration, in cancer. However, a consensusamong the findings has yet to be achieved [6, 21–25].

Herein some important findings linking cellular respiration to cell dedifferen-tiation in cancer are provided. It is worth noting that this putative link may havecrucial pharmacologic and clinical implications. The work of Amuthan et al. [21] isparticularly relevant in this regard because the findings provide mechanistic insightinto how cancer progression and tumor invasion may be directly related to chemi-cal damage of mtDNA that results in mitochondrial respiratory chain dysfunction,loss of mitochondrial membrane potential, and reduction of ATP synthesis. Thismitochondrial derangement also causes calcium release into the cytoplasm, whichactivates a cell dedifferentiation program. Amuthan et al. [21] showed in C2C12myoblasts and in human pulmonary carcinoma A549 cells that thus-induced dedif-ferentiation programs can transform cells into a more aggressive phenotype with thefollowing properties:

• induction of hexokinase, the enzyme that primes glucose by consuming ATP;• induction of phosphoenolpyruvate carboxykinase (PEPCK), which regulates glu-

coneogenesis, glyceroneogenesis, and many other anaplerotic reactions;• induction of tumor invasion markers (i.e., cathepsin L, TGF-�1, ERK1, ERK2,

calcineurin) and antiapoptotic proteins (Bcl2 and Bcl-XL);• reduction of levels of the proapoptotic proteins Bid and Bax;• reduction of cell growth and occurrence of morphologic changes.

In summary, the findings of Amuthan et al. [21] demonstrate that, at least in thisset of experimental conditions, a generic mitochondrial stress caused by mtDNAdepletion deranges cellular respiration and alters patterns of nuclear gene expressionand thereby induces tumorigenic properties.


In general, it is well-known that mtDNA depletion can also render cells moresensitive to apoptotic stimuli. Amuthan et al. [21] explain that the outcome of thestress (cell dedifferentiation vs. apoptosis) could be related to the relative resistanceto apoptosis and/or to the capacity of the adopted cellular models to buffer efficientlythe secondary metabolic stresses. Furthermore, evidence obtained from this combi-nation of methodological approaches demonstrated the existence of mitochondria-to-nucleus stress signaling, through which alterations in cellular respiration havethe ability to change nuclear gene expression and selectively promote the growth ofmore aggressive clones.

These results compel us to consider other research demonstrating that mitochon-dria can mediate the proliferative or inhibitory actions of different physiologic orpharmacologic molecules that influence cellular respiration. Interestingly, it hasbeen reported recently that some “nongenomic” effects of estrogens, that is; effectsmediated by mitochondria rather than estrogen receptors, seem to modulate theexpression of nuclear cell cycle genes and human breast tumor growth. Estrogenscause increases in the transcription of various complex subunits of the electron res-piratory chain in mitochondria. For example, estradiol treatment (0.5 nM for 6 days)is associated with a 16-fold increase in cytochrome oxidase II (COII) mRNA in ratpituitary tumor cells. Similarly, ethinyl estradiol treatment (0.5 to 10 �M for 40hours) caused a two- to threefold increase in cytochrome oxidase I (COI), COII, andNADH dehydrogenase subunit I. Estrogens seem to negatively affect mitochondriaat the protein level as well. Several studies have shown that estrogens can inhibitmitochondrial respiratory complexes I, II, III, IV, and V. This perturbation of theelectron respiratory chain can induce mitochondrial ROS production, which couldpromote the generation and progression of some cancers via oxidative stress and/orROS-related signaling pathways [23]. Given the suggested role of estrogens in somecancers (i.e., breast cancer) and the discussed pathogenetic mechanisms at the basisof oncopromotion and oncoprogression activity, further evidence should be gatheredto determine the relevance of these findings.

The ability of lipophilic molecules to derange cellular respiration and alter celldifferentiation/dedifferentiation processes is a neglected pathophysiologic facet ofthe mitochondrial respiratory chain. On the basis of the cytopathology of liver dis-ease associated with use of fibrates (lipid-reducing drugs) and some experimentaldata indicating that fibrate binding to human hemoglobin can functionally mod-ify the hemoprotein, we postulated that fibrates, and their thiazolidinedione deriva-tives, could interact with many hydrophobic protein domains and especially withcomplex I subunits of the mitochondrial electron respiratory chain. We found thatthis molecular interaction is rapid (<1 hour from administration) and occurs innumerous cell types in varying degrees. The following human cell lines showedderangement of varying degrees of complex I and cell differentiation: K-562human erythroleukemia, HL-60 human myeloid leukemia, U-937 human monocyticleukemia, TE-671 human rhabdomyosarcoma, HepG2 human hepatocarcinoma, andSK-N-BE[2] human neuroblastoma [22, 24, 25].

In fact, these molecules, by their lipophilic properties, diffuse freely into lipidbilayers in general and in mitochondria in particular also because of interplay


between their logD and mitochondrial electrochemical gradient, altering in sucha way the function of proteins embedded in membranes. One of the larger proteincomponents of mitochondria membranes is complex I (NADH-ubiquinone oxidore-ductase, subunits >30, MW 850 kDa), which because of these features may act asthe main lipophilic molecular sink in mitochondria [26].

The rapid iatrogenic respiratory derangement caused by introduction of theselipophilic molecules can induce metabolic and ROS-mediated stress that provokesa differentiation process, also mitochondrial HSP-mediated, in human tumor celllines. In particular, the derangement of mitochondrial complex I may induce ROSproduction and may impair NADH oxidation resulting in an energetic shortage incancer cells. Importantly, considering that complex I is only partially deranged, theoxidative and energetic stress are in general not lethal. In this condition, cell growthis hampered and phenotypic differentiated features may reappear. This implies thatthere is modification of the actual differentiation/dedifferentiation definition andthus suggests that a redefinition of the role of some oncogenes and oncosuppres-sors is appropriate. Data confirming that fibrates and thiazolidinediones inhibit, vianongenomic activity, complex I–impairing NADH oxidation at the mitochondriallevel can explain some of the pharmacologic activities of this class of drugs (i.e.,antihyperlipidemia, insulin sensitization). The findings also may account for someadverse side effects of fibrates and thiazolidinediones, including myocardiotoxicity,hepatotoxicity, and, most relevant to this discussion, peroxisome proliferation, car-cinogenicity, and cancer differentiating properties, considered typical peroxisomeproliferator activated receptor (PPAR)-�–mediated effects in rodents [27].

The differentiating activity of fibrates and thiazolidinediones on human tumorcell lines has been shown to be tightly correlated with the level of mitochondrialcomplex I derangement. This differentiating activity is independent of the expres-sion level of PPARs, as well as the embryonic origin of the tissue. Stress-related dif-ferentiation in human tumor cell lines, including some human leukemia cell lines,is a well-known occurrence [28].

Fibrate/thiazolidinedione-induced differentiation is unusual; it is linked tothe metabolic status of differentiated cells that shift toward a more glycolyticmetabolism (paradoxical Warburg effect) and thus showing that original neoplasticphenotype yet used mitochondria. Moreover, metabolic parameters recorded dur-ing drug-induced cell differentiation (residual activity of the mitochondrial electronrespiratory chain components glycerol 3-phosphate dehydrogenase and succinatedehydrogenase; levels of glycolytic and nonglycolytic acetate, pyruvate, and ala-nine) suggest that the so-called Warburg effect is really an epiphenomenon of a morecomplex metabolic rearrangement of the neoplastic cell. This cancer-associatedmetabolic rearrangement is induced by the cancer’s rigid genetic selection program,characterized by a high level of cell proliferation and diffusion, which is normallypresent only during embryonic and fetal development.

Our understanding of the functional connections between alterations in cellmetabolism and mitochondrial physiology in cancer is under profound revision. Andall mitochondrial physiology is strongly affected by electron respiratory chain activ-ities. The recent results of Fantin et al. [6] are consistent with the view that tumor


progression is strongly associated with the so-called Warburg effect and mitochon-drial metabolism. These authors showed that attenuation of lactate dehydrogenase A(LDH-A) expression by LDH-A short hairpin RNAs in tumor cell lines reactivatesmitochondrial respiration, in terms of proton leakage and ATP turnover, and restoresthe normally depolarized state of mitochondrial membranes. In this situation, cancercells can no longer oxidize NADH via LDH but must rely on the mitochondrial res-piratory chain. Interestingly, this abruptly induced change in metabolism inhibitedcell growth. The authors concluded that these results demonstrate a fundamentalrole of aerobic glycolysis in general and LDH in particular in tumor maintenanceand progression and further show that cancer-associated slowdown of mitochondrialrespiration is not due to structural damage of the organelles but rather results fromaltered metabolic signals. NADH/NAD+ ratio and modulation of related mitochon-drial shuttle systems are likely involved in these signals.

The fundamental role of cellular respiration in cancer cell dedifferentiation is fur-ther emphasized by a recent study that examined energy metabolism in breast cancerbrain metastasis, which undoubtedly represents a clear in vivo model of bioener-getic adaptation of dedifferentiated cells [29]. Interestingly, these metastatic cellsshowed increased expression of enzymes involved in glycolysis, in accordance withthe Warburg effect, but also showed increased expression of enzymes involved inthe TCA cycle, pentose phosphate pathways, beta-oxidation, and oxidative phos-phorylation. Intriguingly, the authors also reported induction of enzymatic pathwaysrelated to glutathione synthesis, which together with activation of the pentose phos-phate pathway could indicate the presence of antioxidant defense mechanisms tominimize production of ROS derived from enhanced oxidative metabolism [29].

3 Conclusion

Emerging evidence, despite conflicts likely due to experimental differences, is shed-ding light on the interrelationships between cellular respiration and cancer cell ded-ifferentiation, in particular:

• Confirms a more active role of mitochondrial respiration to maintain the cellulardifferentiated phenotype and at the same time underline the pathogenetic signif-icance of perturbations of mitochondrial electron respiratory chain in promotionand progression of cancer.

• Stresses the importance of the shift of all cellular metabolism to ensure the newenergetic and structural needs of neoplastic cell.

• Justifies some debated metabolic and functional evidence peculiar to cancer cell(i.e., induction of hexokinase II and its binding to ANT).

• Enhances the role of NADH and NADH/NAD+ ratio in modulation of a signaltransduction pathway that controls differentiation/dedifferentiation status.

• Emphasizes the interplay of complex I and complex II to adapt cell metabolismto different cellular physiopathologic conditions.


• Last but not least, addresses the new research, which explores the interrelation-ships between cellular respiration and cell dedifferentiation, toward the regu-latory function of complex I, complex II, and above all pushes to investigatedeeper, in terms of cancer cell metabolism adaptions, the other pathways of elec-tron transport (i.e., alpha glycerophosphate and fatty acyl CoA).

In conclusion, it is clear that there exists a significant link between cellular res-piration and cell differentiation/dedifferentiation. Elucidating the molecular mech-anisms at the basis of this link may have critical clinical implications in terms ofcancer diagnosis, prognosis, and, most importantly, therapy. Disentangling the rela-tionship between cancer cell oxidative metabolism and cellular differentiation stateis fundamental not only for oncology but also for properly understanding and usingall potential clinical applications related to the physiopathology of stem cells.

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Cellular Adaptations to OxidativePhosphorylation Defects in Cancer

Sarika Srivastava and Carlos T. Moraes

Abstract Mitochondrial DNA (mtDNA) somatic mutations or mutations in nucleargenes encoding mitochondrial proteins important for the assembly, activity, or main-tenance of the individual oxidative phosphorylation (OXPHOS) complexes havebeen observed in tumors. Although the functional consequence of such mutationsis unclear at the moment, retrograde signaling in response to OXPHOS defectscan activate various nuclear genes and signaling pathways that alter mitochon-drial function, tumor invasion, metastasis, redox-sensitive pathways, programmedcell death pathways, calcium signaling pathways, and cellular pathways leadingto global changes in cellular morphology and architecture. In addition, we havefound that some cancer cell lines harboring deleterious mtDNA mutations upregu-late the expression of members of the peroxisome-proliferator activated � coactiva-tor 1 family of coactivators, probably to sustain the necessary ATP production forcell proliferation. In this chapter, we describe such cellular adaptations and changesin response to OXPHOS defects that are associated with a variety of cancer celltypes.

Keywords mtDNA · Retrograde signaling · OXPHOS · Cancer · Mitochondria

1 Mammalian Oxidative Phosphorylation

Mammalian mitochondrial oxidative phosphorylation (OXPHOS) occurs in fivemultimeric enzyme complexes (complexes I, II, III, IV, and V) using two electrontransport carriers (ubiquinone or coenzyme Q10 and cytochrome c). Electrons fromreduced equivalents (NADH and FADH2) are transported along these complexes viaubiquinone and cytochrome c to molecular oxygen, producing water. At the sametime, protons are pumped across the mitochondrial inner membrane (from the matrixto intermembrane space) by enzyme complexes I, III, and IV thereby generating a

C.T. Moraes (B)University of Miami Miller School of Medicine, Miami, FL 33136e-mail: [email protected]


55

56 S. Srivastava, C.T. Moraes

CoQ10 Cyt c

O2 H2O

e- e-

e-

e-e- e-

H+ H+ H+

H+

Complex VComplex IVComplex IIIComplex IIComplex I

ADP ATP

Intermembrane space

Matrix

Fig. 1 Schematic representation of mammalian mitochondrial OXPHOS. Mammalian electrontransport chain (ETC) consists of four multimeric complexes (complexes I, II, III, and IV). Elec-trons from the reduced equivalents, NADH and FADH2, enter the ETC and reduce complex I andcomplex II, respectively. Coenzyme Q10 or ubiquinone is an electron carrier in the inner mito-chondrial membrane that accepts an electron from either complex I or complex II and donates itto complex III. Cytochrome c, another electron carrier in the mitochondrial intermembrane space,accepts an electron from complex III and donates it to complex IV. Complex IV donates electronsto molecular O2, which results in the formation of H2O. During the electron flow, complexes I, III,and IV pump the protons from the matrix toward the intermembrane space thereby generating anelectrochemical gradient across the mitochondrial inner membrane. The energy in the electrochem-ical gradient is harnessed by complex V to generate ATP from ADP (oxidative phosphorylation)

proton gradient or membrane potential (��m). The energy in this gradient is har-nessed by complex V (ATP synthase) to synthesize ATP from ADP, a phenomenontermed OXPHOS, during which protons flow back from the mitochondrial inter-membrane space to the mitochondrial matrix (Fig. 1).

Mitochondria are also important for cytosolic Ca+2 buffering and are the predom-inant source of ROS production in most cell types. Superoxide anions are generatedat the level of complex I and complex III in the inner mitochondrial membrane.

2 OXPHOS Defects in Cancer

OXPHOS defects in cancer cells were first reported more than 50 years ago byWarburg who stated that “cancer cells are impaired in respiratory chain functionand are very glycolytic” [1]. Studies on various types of cancer cells have found

Cellular Adaptations to OXPHOS Defects in Cancer 57

this hypothesis to be correct in several but not all tumor cells. Several independentgroups have also reported that many cancer cells possess a full complement of res-piratory chain enzymes and can couple electron transport chain to ATP production[2]. Isolated tumor mitochondria have also been found to respire on a variety of sub-strates. Specific changes that occur in the energy metabolism of tumor cells have notyet been established.

In the past 10 years, studies on mitochondrial DNA (mtDNA) mutations thatcause OXPHOS defects and their potential role in cancer have regained momentum.Mitochondrial DNA mutations have been increasingly identified in various types ofcancer cells [3, 4]. Polyak et al. were first to report a detailed analysis of somaticmtDNA mutations in human colorectal cancer cells by sequencing their completemitochondrial genome [3] (somatic mtDNA mutations are those present in the tumortissue but absent from the adjacent normal tissue). After this initial finding, mtDNAfrom various tumor cell types was sequenced by different groups, and mtDNA muta-tions have now been reported in esophageal, ovarian, thyroid, head, neck, lung, blad-der, and renal cancer cells [4–6]. The majority of the mtDNA mutations reported indifferent tumor cell types are transitions (G-to-A or T-to-C), a feature that is char-acteristic of reactive oxygen species (ROS)-derived mutations. Further, most of thesomatic mtDNA mutations have been reported to be homoplasmic suggesting theirdominance at intra- and intercellular levels.

2.1 Functional Significance of OXPHOS Defects in Cancer

Although in the past decade, the list of mtDNA mutations affecting OXPHOS incancer cells has increased steadily, the functional relevance of these mutations intumor promotion or formation process are yet obscure. Because the majority of thesomatic mtDNA mutations found in tumor cells have been reported to be homoplas-mic, one of the existing hypotheses is that the homoplasmic levels of these mutationswere probably achieved through some kind of replication or growth advantage thatthe mutant mtDNA harbored over the wild type in the tumor progenitor cells. It hasalso been suggested that mtDNA mutations may cause a moderate increase in ROSproduction that in turn stimulates cell growth [3, 4]. Low levels of ROS have beenshown to stimulate mitosis in various cell types [7, 8]. In contrast with this hypoth-esis, Coller and co-workershave demonstrated that there is a sufficient opportunityfor a tumor progenitor cell to achieve homoplasmy through unbiased mtDNA repli-cation and segregation during cell division [9]. Coller et al.constructed a theoreticalmodel based on computer simulation studies and showed that there is a good cor-relation between the reported frequency of homoplasmic mutations in tumor cellsand the predicted frequency of homoplasmy in tumor progenitor cells that can beattained in the absence of any selection [9]. The authors suggested that mtDNAmutations such as silent substitutions of amino acid coding regions or alterations inthe length of polynucleotide tracts that are polymorphic in healthy individuals arethe ones that are unlikely to confer any selective advantage to the mtDNA or the


host cell. However, the authors did not exclude the possibility that certain mtDNApoint mutations that cause functional alterations (e.g., mutations affecting the con-served amino acid regions in the coding region of a protein) might be segregatedin a nonrandom manner and may provide a selective growth advantage to the hostcell [9].

mtDNA mutations leading to OXPHOS dysfunction have also been shown toincrease tumorigenicity in certain tumor cell types. Mutations in the mtDNA COXIgene were shown to increase tumorigenicity in prostate cancer patients [10]. Petroset al. reported that ∼11% to 12% of prostate cancer patients harbor pathogenicCOXI mtDNA mutations that alter the conserved amino acid regions in the pro-tein and suggested that these mutations may play a role in the etiology of prostatecancer [10]. mtDNA ATP6 gene mutation (T8993G) has also been shown to increasetumorigenicity by two independent groups. The first study was from Petros and co-workers who showed that PC3 prostate cancer cybrid cells harboring T8993G ATP6mutant mtDNA when introduced in nude mice formed tumors that were ∼7 timeslarger than cells harboring the wild-type mtDNA suggesting a role of mtDNA muta-tion in tumor promotion process [10]. They also showed that tumor cells harboringATP6 mutant mtDNA generated significantly higher levels of ROS than did thoseharboring wild-type mtDNA, further suggesting that high levels of ROS promotedtumor cell growth. The second study was from Shidara and co-workers. They con-structed HeLa transmitochondrial cybrids harboring homoplasmic T8993G ATP6mtDNA mutation and showed that these cybrids grew much faster in culture aswell as in nude mice forming tumors that were larger in size compared with thecybrids harboring the wild-type mtDNA suggesting that the mtDNA ATP6 mutationin these cells conferred a selective growth advantage [11]. Further, transfection ofa wild-type nuclear version of the mitochondrial ATP6 gene in these HeLa cybridsharboring homoplasmic ATP6 mutant mtDNA followed by their transplantation innude mice was shown to slow down the tumor cell growth. Conversely, the expres-sion of a nuclear version of the mutant ATP6 gene in the wild-type cybrids wasfound to accelerate tumor cell growth thereby suggesting that the growth advantagein these tumor cells depended on the mitochondrial ATP6 function [11].

OXPHOS dysfunction caused by partial mtDNA depletion (genetic stress) ortreatment with mitochondrial-specific inhibitors (metabolic stress) has been shownto induce cell invasion and tumor progression in otherwise noninvasive cells [12,13]. A correlation between OXPHOS impairment and tumor aggression has alsobeen reported in renal carcinomas [14]. Simonnet et al. studied the mitochondrialrespiratory chain enzyme content in three different renal tumors and found that theOXPHOS impairment increased from the less aggressive to the most aggressivetypes of renal carcinomas [14]. They also found that renal oncocytomas (benigntumors) are exclusively deficient in mitochondrial complex I activity, whereas theactivity of all other OXPHOS complexes are increased in these tumors [15]. Thesetumors further showed dense mitochondrial proliferation suggesting an increasein mitochondrial biogenesis as an attempt to compensate for the potential loss ofOXPHOS function. Complex I deficiency was also found in normal cells adjacent tothe tumor tissue leading the authors to suggest that the complex I deficiency could be


an early event in the formation of renal oncocytomas [15]. Similarly, OXPHOS dys-function and mitochondrial proliferation have also been reported in thyroid oncocy-tomas [16].

The current studies on potential effects of mtDNA mutations and OXPHOSdysfunction in cancer cells therefore suggest their role in tumor promotion and/orprogression. It is believed that nuclear DNA mutations and not mtDNA muta-tions control the tumor initiation process. Akimoto and co-workers have tested thishypothesis [17]. They showed that cells carrying nuclear DNA from tumor cellsand mtDNA from normal cells form tumors in nude mice whereas those carryingnuclear DNA from normal cells and mtDNA from tumor cells do not form tumors[17]. However, mutations in the mitochondrial enzymes that are encoded by thenuclear genome have been associated with certain types of cancer. Germ-line muta-tions in the tricarboxylic acid (TCA) cycle enzyme fumarate hydratase have beenshown to be associated with uterine fibroids, skin leiomyomata, and papillary renalcell cancer [18], and mutations in the succinate dehydrogenase (SDH) subunits B,C, and D have been shown to be associated with hereditary paragangliomas andpheochromocytomas [19–21]. The potential role of these mutations in the tumorformation process is yet unclear.

3 Cellular Adaptations to OXPHOS Defects in Cancer

The cross-talk between mitochondria and nucleus plays an important role in main-taining mitochondrial function and integrity. Mitochondrial dysfunction affects themitochondrial-nuclear cross-talk leading to altered signaling cascades (Fig. 2). Thenuclear-mitochondrial stress signaling, also called retrograde signaling, is a phe-nomenon that involves changes in the nuclear gene expression in response to

OXPHOS Dysfunction

Nucleus

Altered GeneExpression

Retrograde Signaling

OXPHOS regulatory or structural genes

Tumor invasion or metastatic genes

Calcium signaling pathway

Redox signaling / antioxidant defense pathway

Cellular morphology and architectureFig. 2 Mitochondrial-nuclearintergenomic signaling inresponse to OXPHOSdefects. MitochondrialOXPHOS dysfunction incancer cells can activate aretrograde signaling cascade.Altered retrograde signalingcan affect the expression ofvarious nuclear genes andcellular pathways that allowtumors cells to adapt to theenvironment and/or promotetumor cell growth orprogression


OXPHOS dysfunction. These nuclear gene expression changes allow the tumor cellsto adapt to the environment that may promote tumor cell growth and/or progres-sion. Studies have shown that a variety of nuclear genes are altered in responseto OXPHOS defects in different tumor cell types and have implicated their role intumorigenesis.

3.1 Retrograde Signaling in Cancer Cells

Retrograde signaling has been implicated as an important mechanism in carcinogen-esis although the precise mechanism(s) of this pathway is yet elusive in mammaliansystems. Studies have shown that retrograde signaling in response to mitochon-drial dysfunction can alter the expression of nuclear genes involved in energymetabolism, tumor invasion and metastasis, calcium signaling, redox signaling, anti-apoptotic genes, as well as genes involved in regulating cellular morphology andarchitecture.

3.1.1 Altered Expression of OXPHOS Regulatory Genes and Subunits

The mitochondrial OXPHOS complexes (I, III, IV, and V) comprise subunitsencoded by both mtDNA and nuclear DNA. The expression of nuclear encodedOXPHOS subunits is under a tight regulation by regulatory genes, namely tran-scription factors and transcriptional coactivators. Nuclear respiratory factors 1 and2 (NRF-1 and NRF-2) are the nuclear transcription factors, and the members ofthe peroxisome-proliferator activated � coactivator 1 (PGC-1) gene family arethe nuclear transcriptional coactivators that directly or indirectly modulate theexpression of nuclear encoded OXPHOS genes, respectively. Besides regulating theexpression of nuclear encoded OXPHOS genes, the NRFs also exert a regulatorycontrol over the expression of mitochondrial transcription and replication machin-ery components, the protein import machinery components, and heme biosynthe-sis. A change in the expression levels or activity of NRFs and/or PGC-1 family oftranscriptional coactivators would therefore influence the expression of OXPHOSsubunits and mitochondrial biogenesis.

Altered expression of OXPHOS subunits and transcriptional regulatory genes hasbeen reported in different tumor cell types [22, 23]. We studied a human colorectaltumor cell line (termed V425) harboring a homoplasmic nonsense mutation in themitochondrial COXI gene. Despite the mtDNA mutation load, these cells were foundto maintain a very high rate of mitochondrial respiration (∼75% of control cells) and alow level of COX activity (∼10% of control cells). Interestingly, osteosarcoma cybridsharboring the V425 mutant mtDNA showed a significant decline in respiration andCOX activity [22]. Previous studies have shown that ∼10% COX activity cannot sus-tain a functional respiratory chain [24]. We found that the steady-state transcript levelsof a transcriptional coactivator PGC-1� and its homolog PGC-1� were highly upregu-lated in V425 cells. In parallel, we also observed an increase in the steady-state levelsof several mitochondrial proteins (e.g., SDH, COXIV, and cytochrome c) in these


cells. Further, the overexpression of PGC-1� in osteosarcoma cybrids harboring theV425 mutant mtDNA showed a significant improvement in mitochondrial respirationand an increase in the steady-state levels of several mitochondrial proteins suggest-ing that PGC-1� upregulation can improve mitochondrial respiration in a colorectalcancer cell line harboring COX deficiency [22]. It has also been shown that PGC-1�stimulates mitochondrial biogenesis and respiration by activating the expression ofNRFs, augmenting their transcriptional activity or activating both the expression andtranscriptional activity [25].

Altered expression of PGC-1 related coactivator (PRC), NRF-1, and mitochon-drial transcription factor A (TFAM) have also been observed in thyroid oncocytomasharboring an OXPHOS defect [23]. Savagner et al. found that dense mitochondrialproliferation in 90% of the tested oncocytic tumors were associated with overexpres-sion of the PRC compared with the controls suggesting that increased mitochondrialbiogenesis might be a feedback mechanism to compensate for the presence ofOXPHOS deficit in these tumors. They also found that the increase in PRC lev-els was associated with a 5-fold increase in NRF-1 transcripts, 10-fold increase inTFAM transcripts, and a 3-fold increase in COX activity further suggesting that theoverexpression of PRC pathway is likely responsible for the increased mitochon-drial proliferation in thyroid oncocytomas [23].

In contrast with the above studies, low levels of expression of peroxisome-proliferator activated receptor gamma (PPAR-�) and PGC-1 have also been reportedin breast cancer tissues [26]. Jiang et al. showed that the human metastatic breastcancer tissues have low levels of PPAR-� and PGC-1 transcripts compared withthose of the normal tissues. Further, lower levels of these molecules were found tobe associated with poor clinical outcomes in breast cancer patients [26]. Interest-ingly, agonists of PPAR-� have also been shown to inhibit growth and proliferationof cancer cells by inducing apoptosis [27].

3.1.2 Activation of Genes Involved in Tumor Invasion and Progression

OXPHOS dysfunction has been shown to modulate tumor invasive properties bytranscriptional upregulation of genes coding for specific members of matrix remod-eling pathway and activation of tumor-specific marker genes. van Waveren et al.showed that OXPHOS dysfunction can alter the expression of nuclear genes cod-ing for factors involved in extracellular matrix remodeling in human osteosarcomacells [28]. These genes included members of the matrix metalloproteinases (MMPs)and tissue inhibitors of metalloproteinases (TIMP) family, urokinase plasminogenactivator and its inhibitor, plasminogen-activator inhibitor 1 (PAI-1), and CTGFand CYR61 (members of the cysteine-rich 61, connective tissue growth factor andnephroblastoma-overexpressed [CCN] gene family of growth regulators). Changesat the protein level for some of these factors have also been observed, though at alesser magnitude. The study also showed that osteosarcoma cells harboring mtDNAmutations were associated with an increased matrigel invasion [28].

Activation of tumor-specific marker genes in response to an OXPHOS dysfunc-tion was observed by Amuthan et al. [12, 13]. They found that partial mtDNA


depletion (genetic stress) or treatment of cells with mitochondrial-specific inhibitors(metabolic stress) activated Ca2+-dependent kinases (PKC, ERK1, ERK2, and cal-cineurin) and signaling pathways that in turn activated the expression of nucleargenes involved in tumor invasion and progression. Among these genes, the extracel-lular matrix protease cathepsin L, transforming growth factor � (TGF-�), and mousemelanoma antigen (MMA) were found to be upregulated in both genetically andmetabolically stressed rhabdomyosarcoma and pulmonary carcinoma cells. Using invitro Matrigel invasion assays and in vivo rat tracheal xenotransplants in Scid mice,they further showed that these genetically and metabolically stressed cells were ∼4-to 6-fold more invasive than were their respective controls thereby implicating arole of OXPHOS dysfunction in inducing genes involved in tumor invasion and pro-gression. The invasive behavior of mtDNA-depleted rhabdomyosarcoma and pul-monary carcinoma cells was reverted by the restoration of the mtDNA content [12,13]. These findings clearly established a role of mitochondrial retrograde signal-ing in inducing phenotypic changes and tumor progression in osteosarcoma, rhab-domyosarcoma, and human pulmonary carcinoma cells. OXPHOS dysfunction wasalso shown to be associated with tumor aggressiveness in renal carcinoma cells [14].Retrograde signaling in breast cancer has been reported by Delsite et al. They per-formed a comparative microarray analysis of the nuclear gene expression changes ina human breast cancer cell line (MDA-435) and its o derivative devoid of mtDNA.They found that the expression of several nuclear genes involved in cell signaling,cell growth, energy metabolism, cell architecture, cell differentiation, and apoptosiswere altered in the o derivative of the breast cancer cell line [29]. Functional char-acterization of these genes is further required to gain a clear insight to the potentialrole of retrograde signaling in tumorigenesis.

3.1.3 Activation of Calcium Signaling Pathway

Changes in intracellularcalcium(fromtheapproximate100nMatrest)mediateanum-ber of processes that affect tumorigenesis, including angiogenesis, motility, cell cycle,differentiation, transcription, telomerase activity, and apoptosis [30]. Mitochondriatake up Ca2+ in a process dependent on a ��m [31]. Ca2+ uptake by mitochondria con-trols organelle metabolic activity, as pyruvate, �-ketoglutarate, and isocitrate dehy-drogenases are activated by Ca2+. In addition, some metabolite transporters have beenshown to be regulated by Ca2+ as well and to enhance aerobic metabolism. Mitochon-dria, by buffering local [Ca2+] (generated by Ca2+ channels on the plasma membraneor the ER/SR), can also modulate intracellular Ca2+ [31]. Therefore, Ca2+ can regu-late cell growth and survival by modulating OXPHOS function. Likewise, a defec-tive OXPHOS impairs Ca2+ buffering. As described above, impaired Ca2+bufferingcapacity by defective mitochondria can activate proteins involved in tumor invasionand progression, including PKC, ERK1, ERK2, and calcineurin.

OXPHOS dysfunction has been shown to activate calcium-dependent signal-ing events in several tumor cell types. Avadhani and co-workers investigated thenuclear gene expression changes in C2C12 rhabdomyosarcoma and human A549pulmonary carcinoma cells in response to OXPHOS dysfunction and found that


calcium-dependent signaling events were activated in these cells [12, 13, 32].OXPHOS defects associated with decreased mitochondrial membrane potential(��m) and ATP synthesis caused a sustained increase in intracellular calcium[Ca2+]i levels in these cells. They found that the expression of ryanodine receptor-1(RyR-1) and ryanodine recptor-2 (RyR-2) genes was upregulated in C2C12 rhab-domyosarcoma and A549 pulmonary carcinoma cells, respectively. Increased RyRgene expression correlated with high levels of Ca2+release from ER to the cytosolin response to caffeine stimulation (RyR agonist). Avadhani and co-workers sug-gested that the defect in ��m was associated with the observed increase in [Ca2+]i

levels as mitochondria are key players in sequestering cytosolic Ca2+ and a defectin ��m would affect mitochondrial Ca2+uptake ability. Restoration of the ��m inboth C2C12 rhabdomyosarcoma and A549 pulmonary carcinoma cells was foundto revert the ryanodine receptor gene expression and [Ca2+]i levels to near normallevels suggesting a direct link between OXPHOS dysfunction and elevated [Ca2+]i

in these cancer cells. Ca2+ responsive factors such as calcineurin, calcineurin-dependent NFATc (cytosolic counterpart of activated T-cell–specific nuclear fac-tor), and JNK (c-Jun N-terminal kinase)-dependent ATF2 (activated transcriptionfactor 2) were also found to be elevated in both C2C12 rhabdomyosarcoma andpulmonary carcinoma cells. Further, Ca2+-dependent PKC (protein kinase C) andMAPK (mitogen activated protein kinase) genes were activated in C2C12 rhab-domyosarcoma and lung carcinoma cells, respectively. Another study by Avadhaniand co-workers has shown that calcineurin that is activated in response to elevatedcytosolic Ca2+ inactivates I-�B� thereby leading to an activation of the NF-�B/Relfamily of transcription factors in the OXPHOS-deficient C2C12 rhabdomyosarcomacells [33]. They suggested that the NF-�B/Rel family of transcription factors mightbe involved in the activation of nuclear genes in response to OXPHOS dysfunctionin these cells [33]. Rise in cytosolic Ca2+ in response to OXPHOS dysfunction hasalso been observed in rat pheochromocytoma PC12 cells. Luo et al. showed thatthe treatment of PC12 cells with mitochondrial uncoupler FCCP releases Ca2+frominternal stores leading to a rise in cytosolic Ca2+, which in turn activates MAPKalso suggesting a potential link between OXPHOS dysfunction and Ca2+signalingin these cells [34].

3.1.4 Activation of Redox Signaling Pathway

Changes in nuclear gene expression in response to oxidative stress have beenobserved in a variety of tumor cells. mtDNA mutations or OXPHOS dysfunctionpredispose mitochondria to the generation of ROS leading to oxidative stress. Cel-lular oxidative stress has been implicated in initiation, promotion, and progressionof carcinogenesis [35]. Low levels of ROS generation have been shown to be mito-genic in a variety of human and mouse cell types [36, 37]. For example, under cellculture conditions, 10 nM to 1 �M concentrations of both O2

•-and H2O2 have beenshown to stimulate the growth of hamster and rat fibroblasts [37]. Low levels ofROS were also shown to stimulate the growth of mouse epidermal cells, humanfibroblasts, and human astrocytoma cells [38, 39]. The increased tumorigenesis of


NARP cells with a pathogenic mtDNA mutation described above [10] could also berelated to increased ROS, as the mutation in ATPase 6 was reported to significantlyincrease ROS production [40]. Further, many chemical carcinogens initiate carcino-genesis by stimulating ROS production, which in turn activates the expression ofearly growth related genes such as c-fos, c-myc, and c-jun [41].

Although the molecular mechanism(s) of ROS-mediated cell growth are yetunclear, cellular ROS have been shown to activate the expression of redox-sensitivenuclear genes and signaling pathways. It has been shown that ROS activate theexpression of nuclear factor kappa B (NF-�B) and activator protein (AP-1) tran-scription factors [42, 43]. Both NF-�B and AP-1 are redox-sensitive transcriptionfactors. NF-�B in its active form consists of two subunits, p50 and p65. In theabsence of a stimulus, it is present in the cytoplasm in association with its inhibitorysubunit I-�B. In response to a stimulus that leads to the I-�B subunit phosphory-lation, NF-�B dissociates from the I-�B inhibitory complex and translocates to thenucleus where it activates the expression of specific target genes [44]. AP-1 is a het-erodimer of transcription factors composed of Jun, Fos, or activating transcriptionfactor (ATF) subunits and binds the AP-1 sites in the promoter region to activate thetarget gene expression [45].

Constitutive activation of NF-�B and Ap-1 genes in response to oxidative stresshas been observed in a variety of cancer cell types. Shi et al. demonstrated thatOH radicals activated the expression of NF-�B in Jurkat cells, macrophages, andmouse epidermal cells, and antioxidants that scavenge OH radicals inhibited its acti-vation [46]. High levels of ROS were also shown to cause a constitutive elevation ofNF-�B and AP-1 transcription factors in transformed mouse keratinocyte cells [7].Constitutive elevation of AP-1 activity has also been shown to be associated withthe conversion of benign papillomas to malignant carcinomas in mouse epidermalcells suggesting that the target genes induced by activated AP-1 were involved incell growth and metastasis [47]. Inhibition of Ap-1 activity by a dominant negativec-Jun mutant was shown to suppress the tumorigenic phenotype of the malignantmouse epidermal cells thereby blocking the tumor formation in nude mice [48].Constitutive activation of NF-�B has also been shown to induce genes involvedin tumor progression and metastasis [49]. Further, treatment with an antioxidant,N-acetylcysteine (NAC), has been shown to inhibit the elevated expression of NF-�B and AP-1 levels in tumor cells implicating the role of ROS in the activation ofredox-sensitive signaling pathways [7].

The activities of NF-�B and AP-1 transcription factors are modulated by phos-phorylation. Although the precise signaling events that activate the phosphoryla-tion of these transcription factors in response to increase in cellular ROS levels areyet not fully understood, protein kinases are central in their activation. It has beenobserved that MAPK pathways are involved in signaling the activation of both NF-�B and Ap-1 transcription factors. There are three subtypes of MAPKs; the extra-cellular signal regulated kinases (ERKs), the c-jun N-terminal kinases (JNKs), andthe p38 MAPKS. Gupta et al. showed that the NF-�B activation in malignantly pro-gressed mouse keratinocytes was associated with an increase in ERK-1/2 and p38MAPK activities and that NAC treatment rapidly abolished the increase in MAPKactivities [7]. Direct activation of I-�B by MEKK1 (MAPK/ERK kinase kinase-1)


has also been demonstrated in vitro [50]. Activation of AP-1 by MAPK in responseto various stimuli has been demonstrated [51]. There is also evidence that antioxi-dants can attenuate MAPK activation suggesting that MAPK signaling cascades areactivated in response to cellular oxidative stress conditions [7].

3.1.5 Altered Antioxidant Defense Pathway

The antioxidant defense enzymes have been found to be altered in several humantumors. Superoxide dismutases (SODs) are the first line of cellular defense againstincrease in intracellular ROS. There are two forms of intracellular SODs: amanganese-containing SOD (MnSOD) that localizes to the mitochondria and acopper-zinc–containing SOD that localizes to the cytosol. Most human tumor celltypes have been found to be deficient in their antioxidant defense function. Antiox-idant enzymes including catalase and glutathione peroxidase have also been shownto be lowered in cancer cells [52, 53] suggesting that tumor cells have a deficientantioxidant function.

Studies have shown that antioxidant treatment prevents the malignant transfor-mation of cancer cells. Increased expression of MnSOD has been shown to sup-press cancer phenotypes in several murine and human cancer cells. St Clair et al.showed that overexpression of human MnSOD in mouse cells significantly reducedthe frequency of radiation-induced neoplastic transformation implicating a directlink between mitochondrial antioxidants and neoplastic transformation [54]. Zhaoet al. demonstrated that in response to tumor promoters (phorbol esters), transgenicmice expressing human MnSOD in the skin showed a significant reduction in papil-loma formation compared with that of the non-transgenic controls [55]. They fur-ther found that the decreased papilloma formation was associated with a delay andreduction in AP-1 transcription factor binding activity suggesting that the MnSODoverexpression suppressed the tumor formation and Ap-1 activation in these trans-genic animals [55]. Overexpression of human MnSOD has also been shown tosuppress the malignant phenotype of human pancreatic and breast cancer cells. Liet al. demonstrated that MnSOD-overexpressing MCF-7 breast cancer cells showeda marked inhibition in growth rate in vitro under culture conditions and in vivo uponinoculation in nude mice when compared with the wild-type MCF-7 cells [56]. Inanother study, they demonstrated that the transcriptional and DNA binding ability ofNF-�B and AP-1 were reduced by ∼50% in MnSOD-overexpressing MCF-7 cells[57]. They further showed that the expression of NF-�B and AP-1 responsive geneswas downregulated in these cells when compared with that of the wild-type MCF-7cells suggesting that the tumor-suppressive effects of MnSOD were associated withthe inhibition of NF-�B and Ap-1 activities that in turn downregulated the genesinvolved in malignant progression [57].

3.1.6 Altered Mitochondrial Morphology, Cell Surface, and Architecture

Mitochondrial fission and fusion are the central events that regulate mitochondrialmorphology. Changes in mitochondrial morphology appear to be a key event duringretrograde signaling. Studies have implicated a role of mitochondrial morphology in


modulating respiratory activity. For example, downregulation of mitofusin 2 (Mfn 2)decreases mitochondrial respiration and increases the cellular glucose uptake levelsfor glycolysis. Further, ectopic upregulation of Mfn 2 increases the expression ofOXPHOS subunits, glucose oxidation, and ��m [58]. Optic Atrophy 1 (Opa 1) isan important player in the formation of mitochondrial cristae. Downregulation ofOpa 1 has been shown to induce the loss of mitochondrial respiration and increasedmitochondrial fragmentation [59].

Changes in mitochondrial morphology and ultrastructure were reported in dif-ferent tumor cell types over several decades. Tumor cell mitochondria were foundto contain abnormal size, shape (dumbbell or cup), cristae organization, or inclu-sions. For example, rapidly growing hepatomas were found to contain small-sizemitochondria with fewer cristae, whereas slowly growing hepatomas were found tocontain large-size mitochondria with densely packed cristae [60]. Changes in themitochondrial morphology in tumor cells could therefore affect their respiratoryactivity. Conversely, it is also possible that in response to altered respiratory activityor OXPHOS dysfunction, the retrograde signaling events in tumor cells affect theexpression of nuclear genes involved in the regulation of mitochondrial morphologyand ultrastructure.

Changes in cell morphology and architecture are a characteristic feature of manycancer cells. These changes occur in cell surface charge, glycoprotein composition,membrane organization, and/or the ability of cells to agglutinate on lectin. The ini-tial evidence of a potential role of OXPHOS dysfunction in inducing cell surfacechanges was obtained in yeast. In 1980, Evans and colleagues showed that mito-chondrial petite mutations in yeast that cause respiratory deficiency lead to drasticchanges in cell surface charge, cell wall organization, and lectin agglutinability incontrast with the wild-type yeast cells suggesting that the OXPHOS deficiency inyeast altered the retrograde signaling pathway leading to gene expression changesand hence altered cell surface properties [61]. Preliminary evidence in literature hasalso suggested a link between OXPHOS dysfunction and cell surface properties inmammalian cells. For example, Soslau et al. showed that baby hamster kidney cellswhen treated with ethidium bromide (Etbr) show an altered glycoprotein composi-tion in the plasma membrane [62]. Further, they also showed that the glycopeptideelution profile of these Etbr-treated cells was similar to that of the cells transformedwith Rous sarcoma virus. Low concentrations of Etbr are known to intercalate tomtDNA and abrogate mtDNA replication. Exponentially growing cells when treatedwith Etbr (25 ng/mL to 2 �g/mL) undergo mtDNA loss leading to either partial orcomplete loss of mtDNA molecules, generating an OXPHOS defect. These find-ings therefore suggest that altered retrograde signaling events during OXPHOS dys-function could activate nuclear genes or pathways that cause global changes in cellsurface properties.

3.1.7 Activation of Antiapoptotic Genes

Activation of antiapoptotic genes is a common occurrence in most human can-cers. However, retrograde signaling pathway has also been shown to activate


antiapoptotic genes (e.g., Bcl-xL and Bcl-2) in certain cancer cell types [12, 63].Increased expression of the antiapoptotic genes may also contribute to metabolichomeostasis. It has been shown that members of the Bcl-2 antiapoptotic family ofproteins regulate ATP/ADP exchange across the mitochondrial membranes and canprevent the loss of mitochondrial respiration during apoptosis [64]. Manfredi andcolleagues showed that Bcl-2/Bcl-xL can improve OXPHOS function in cells har-boring pathogenic mtDNA mutations. The effect of Bcl-2 overexpression in mutatedcells was found to be independent from apoptosis and was suggested to modulate theadenine nucleotide exchange between mitochondria and cytosol. This study there-fore provided evidence that when OXPHOS function is reduced, Bcl-2 expressioncan improve it by regulating the levels of adenine nucleotides inside the mitochon-dria [64].

3.2 Mechanism of Retrograde Signaling

The mechanism of retrograde signaling was first identified in yeast and it is stillpoorly understood in other systems. Three retrograde regulatory genes (RTG1,RTG2, and RTG3) play a central role in retrograde signaling in yeast [65] (Fig. 3).Rtg1p and Rtg3p are transcription factors, whereas Rtg2p is mitochondrial functionsensor. During normal mitochondrial function, Rtg1p and Rtg3p form heterodimers

MitochondrialDysfunctionSensor

Rtg2p

Rtg3p Rtg1p

P

Rtg3p Rtg1p

P P

P

P

Cytoplasm

GTCAC

R box

Rtg3p Rtg1p

P

Nucleus Target gene

Fig. 3 Schematic model of retrograde signaling in yeast. RTG genes play a central role in ret-rograde signaling. Rtg1p and Rtg3p are sequestered in cytoplasm during normal mitochondrialfunction. Rtg3p is highly phosphorylated during this state. In response to mitochondrial dysfunc-tion (sensed by Rtg2p), the Rtg3p undergoes partial dephosphorylation. Both Rtg3p and Rtg1ptranslocate to the nucleus and activate target gene expression leading to changes in nuclear genesand/or signaling pathways that in turn can alter cellular function and homeostasis


and are sequestered in the cytoplasm. Rtg3p is phosphorylated at multiple sitesduring this state. In response to mitochondrial dysfunction (sensed by Rtg2p), aretrograde signaling pathway gets activated. The Rtg3p undergoes partial dephos-phorylation and translocates to the nucleus. Rtg1p also follows and translocates tothe nucleus. Both Rtg1p and Rtg3p activate transcription at the target genes. In theabsence of Rtg2p, the Rtg1p/Rtg3p complex remains in the cytoplasm during activeretrograde signaling pathway implicating its role as mitochondrial function sensorand in signal relay to the Rtg1p/Rtg3p complex [65].

The precise mechanism of retrograde signaling in mammalian systems is yetnot known. The mammalian homologs of RTG genes have yet not been identified.However, human MYC protein has been found to share a significant homologywith yeast Rtg3p, and it is involved in the regulation of many cellular functionssuch as regulation of glycolysis, cellular stress response cell cycle progression, andapoptosis [66, 67].

4 Conclusion

Mitochondrial dysfunction has been reported in a variety of human cancers.Mitochondrial-nuclear intergenomic signaling is an important phenomenon in reg-ulating mitochondrial and cellular function and homeostasis. Although the role ofmtDNA mutations or OXPHOS defects in cancer progression is poorly understood,a considerable body of evidence has now shown that mitochondrial function has animportant role in different aspects of tumorigenesis. Processes such as cell survivaland cell invasion can be directly influenced by OXPHOS function. Less clear isthe association between cell cycle progression and an OXPHOS defect. However,specific examples, such as SDH mutations in paragangliomas and pheochromocy-tomas, provide proof of principle that OXPHOS defects can somehow stimulate celldivision.

It appears likely that some properties related to OXPHOS dysfunction can havean advantageous consequence for tumor development. However, this feature couldimpair cell performance at a different level. Therefore, tumor cells with an OXPHOSdefect, in some cases, have compensatory mechanisms, such as an increase in mito-chondrial biogenesis via overexpression of PGC-1 family members. Studies on theinterplay between these metabolic and signaling pathways will help us better under-stand the basic mechanisms of tumor progression.

Note

A recent study by Ishikawa et al. (Ishikawa K, Takenaga K, Akimoto M, et al.ROS-generating mitochondrial DNA mutations can regulate tumor cell metastasis.


Science. 2008;320(5876):661–664) showed that specific mtDNA changes in com-plex I subunit can increase the metastatic properties of tumor cells.

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Regulation of Glucose and Energy Metabolismin Cancer Cells by Hypoxia Inducible Factor 1

Tulio Cesar Ferreira and Elida Geralda Campos

Abstract A crucial aspect of carcinogenesis is the adaptation of cells to the chang-ing environmental conditions of the tumor mass. To grow and proliferate, the cellsneed nutrients that have to be delivered by surrounding vessels. In addition, theyhave to cope with a gradient of oxygen tension as the tumor mass grows. Hypoxiainducible factor 1 (HIF-1) mediates important responses to physiologic and patho-logic processes that allow angiogenesis, erythropoiesis, cell proliferation and sur-vival, and metabolic changes to take place. It is a heterodimeric transcription factorregulated by a complex network. Among the major metabolic changes evident incancer cells is the activation of the glycolytic pathway, even in the presence of oxy-gen. HIF-1 regulates glucose and energy metabolism by directly activating the geneexpression of glycolytic enzymes, regulatory enzymes, and glucose transporters.HIF-1� (the regulated subunit of the HIF-1 complex) is regulated mainly at theposttranscriptional level through mechanisms that block its proteasomal degradationdepending on the oxygen concentration. HIF-1 is overexpressed in many tumors,and evidence is mounting that HIF-1 regulation of glucose and energy metabolismis important in carcinogenesis and tumor progression.

Keywords HIF-1 · HIF-1� · Glycolysis · von Hippel–Lindau tumor suppressorprotein (pVHL) · Lactate · Glucose transporter · Regulation of gene expression ·Hypoxia response element (HRE) · Prolyl hydroxylases

1 Oxygen Homeostasis

Cells respond to extracellular and intracellular stimuli to maintain homeostasis, andhypoxia is one of the most fundamental environmental stimuli. Oxygen homeosta-sis is crucial to maintain the approximately 1014 cells in the adult human body with

E.G. Campos (B)Departamento de Biologia Celular, Universidade de Brasılia, CEP 70910-900, Brasılia, DF, Brazile-mail: [email protected]


73

74 T.C. Ferreira, E.G. Campos

an adequate supply of O2. The maintenance of the O2 levels within strict limits isessential throughout life but is particularly important during periods of rapid cellularproliferation, such as in normal growth or during the development of neoplasias. Innormal tissues, the average oxygen tension is 40 to 50 mm Hg, whereas in tumorsthese levels are 5 to 10 mm Hg. Control of O2 levels in the tissues of animalsoccurs through the combination of gene regulation and biochemical and physio-logic mechanisms. Low levels of oxygen in the environment lead cells and tissuesto adapt to this adverse condition. Significant changes in gene expression and activa-tion of signaling pathways occurs in response to hypoxia, a characteristic conditionin solid tumors, resulting in an elevated transcription of angiogenic, hematopoietic,and some metabolic genes. Hypoxia inducible factor 1 (HIF-1) was discovered in1992 by Gregg L. Semenza and Guang L. Wang at the Johns Hopkins UniversitySchool of Medicine. It is currently known as the central regulator of the responseto low levels of oxygen. HIF-1 is a heterodimeric transcription factor and regulatesgenes whose products participate in glycolysis, glucose transport, angiogenesis, cellproliferation and survival, pH regulation, and cell migration.

2 HIF-1 Structure

HIF-1� protein is one of the subunits that form HIF-1 and belongs to the basichelix-loop-helix (bHLH)-PAS family of transcription factors. PAS is an acronymthat refers to the Drosophila proteins Period (or PER), ARNT, and Single-mindedor (SIM), the first proteins in which this motif was identified. HIF-1� has the bHLH-PAS domain, which is in the N-terminal region of the protein and has two repeats(A and B) in its structure. This subunit binds to the second subunit that formsHIF-1, the aryl hydrocarbon receptor nuclear translocator (ARNT), also referredas HIF-1�. ARNT is a nuclear protein constitutively expressed that also partici-pates in the transcriptional response to xenobiotics when interacting with the arylhydrocarbon receptor (AHR). As with HIF-1�, the � subunit also belongs to thebHLH-PAS family of transcription factors. HIF-1� and HIF-1� share some aminoacid sequence similarities. The bHLH-PAS domains in both subunits are crucial forthe heterodimerization and binding of these proteins to DNA.

HIF-1� is an 826-amino-acid protein that has two transactivation domains(TADs), one in the N-terminal region (TAD-N) that overlaps with the oxygen degra-dation dependent (ODD) domain and another in the C-terminal region (TAD-C), thelatter also found in the HIF-1� (Fig. 1). The TAD domains are localized at aminoacids 531 to 575 (TAD-N) and 786 to 826 (TAD-C) of the HIF-1�. The TAD-Cdomain of HIF-1� interacts with the transcriptional activators CREB binding pro-tein (CBP) and p300. Maintenance of cysteine 800 in the TAD-C in a reduced stateis essential for p300/CBP binding. Therefore, HIF-1� redox state is involved in itsactivation. HIF-1� and p300 interaction requires a leucine-rich hydrophobic inter-face that is regulated by the reversible change between hydrophobic and hydrophilicCys800 of HIF-1�. The reduced state of this cysteine is maintained by redox factor-1

Regulation of Glucose and Energy Metabolism in Cancer Cells by HIF-1 75

Fig. 1 HIF-1 proteins � and � have similar structural domains. HIF-1� (upper drawing) is a826-amino-acid protein with a basic helix-loop-helix-PAS (bHLH-PAS) domain in the N-terminaland repeats A and B. There are two transactivation domains (TADs), one located in the N-terminalregion (TAD-C), which overlaps with the oxygen degradation dependent (ODD) domain, and otherin the C-terminal region (TAD-C). Both TADs are connected by an inhibitory domain (ID). HIF-1� (lower drawing) is a 789-amino-acid protein, which also belongs to the bHLH-PAS family oftranscription factors. As with HIF-1�, HIF-1� also has the bHLH-PAS domain in the N-terminaland repeats A and B. However, it has only one TAD, which is located in the C-terminal of theprotein (see Color Insert)

[1] and mediated by thioredoxin (TRX). Therefore, compounds that inhibit thiore-doxin cause inhibition of HIF-1�–mediated transactivation of HIF-1 target genes.Both TAD-C and TAD-N domains are connected by a inhibitory domain (ID). TheODD domain is a proline-serine-threonine–rich domain involved in the regulationof protein stability as a function of O2 concentration. In HIF-1�, the main targetamino acids for hydroxylation are localized in this domain (described below). HIF-1� protein contains two nuclear localization signals (NLS), one in the N-terminal(NLS-N) and the other in the C-terminal, which encompass the amino acids 17 to 23and 718 to 721, respectively [1].

3 HIF-1� Regulation

HIF-1� regulation is complex and is governed by posttranslational mechanisms(Fig. 2). Protein stability and activity are regulated by covalent modifications suchas hydroxylation, acetylation, S-nitrosation, and phosphorylation. Among them,hydroxylation and phosphorylation are the most studied. In normoxia conditions,hydroxylated HIF-1� is recognized by the von Hippel–Lindau tumor suppressorprotein (pVHL), which is part of a polyubiquitination complex. HIF-1� is hydroxy-lated by a HIF prolyl hydroxylase (PHD). pVHL recognizes the hydroxylated pro-line residues (Pro402 and Pro564) located in the human HIF-1� ODD domain.PHD is a dioxygenase that uses O2 and 2-oxoglutarate (2-OG) as substrates, thelatter generated by the tricarboxylic acid (TCA) cycle or by the transamination ofamino acids. This enzyme transfers the first oxygen atom to the proline residue, andthe second oxygen atom reacts with the 2-OD producing succinate. In addition tooxygen and 2-OG, the PHD proteins require Fe2+ and ascorbate as cofactors [2].

The first 2-OG dioxygenase to be identified was procollagen prolyl hydroxy-lase, which participates in collagen synthesis and catalyzes trans-4-hydroxylationreactions. There are three PHD isoenzymes: 1, 2, and 3. PHDs hydroxylate specificproline residues inside a highly conserved domain whose sequence is LXXLAP


Fig. 2 Mechanisms of HIF-1� regulation. Under normoxia conditions, HIF-1� is hydroxylatedby prolyl hydroxylase (PHD), an enzyme that belongs to the family of dioxygenases and requiresO2 and 2-oxoglutarate (2-OG) as substrates. This enzyme also uses iron as cosubstrate, which ismaintained in the reduced form (Fe+2) by ascorbic acid. Iron can be scavenged by treatment ofcells in normoxia with desferrioxamine (DFO), mimicking a hypoxia condition. Nickel and cobaltalso promote HIF-1� stabilization in normoxia condition by competing with iron. The pVHL rec-ognizes the hydroxylated (OH) proline residues 402 and 564. pVHL is a member of E3-ubiquitin-ligase complex formed by elongin C (EloC), elongin B (EloB), and cullin 2 (CUL2) and RBX1.


(where X indicates any amino acid; P indicates the hydroxylated proline; L, leucineand A, alanine). Concerning the cellular localization, PHD1 is found exclusivelyin the nucleus, PHD2 in the cytoplasm, and PHD3 is found in both cytoplasmand nucleus, with predominance in the nucleus. Selective blockage of HIF-1�expression using RNA interference technology leads to a complete loss of PHD2and PHD3 gene expression under hypoxia, whereas PHD1 mRNA levels remainunchanged [2]. It seems that hypoxic induction of PHD2 and PHD3 is criticallydependent on HIF-1�. This negative feedback mechanism probably limits HIF-1�accumulation in hypoxia and leads to accelerated degradation on reoxygenationafter long period of hypoxia. RNA interference showed the participation of PHD2 onHIF-1� hydroxylation. Indeed, the specific silencing of PHD2 stabilized the HIF-1� in normoxia conditions in several tested human cells. However, the silencingof PHD1 and PHD3 has no effect in HIF-1� stabilization in both normoxia andreoxgenation of cells briefly exposed to hypoxia. Therefore, it seems that PHD2is the critical oxygen sensor that maintains HIF-1� in a low level in normoxicconditions [3].

Under normoxia, HIF-1� activity is also regulated by hydroxylation ofasparagine 803 in the TAD-C domain, catalyzed by an enzyme known as factorinhibiting HIF-1 (FIH), asparaginyl hydroxylase. FIH also belongs to the super-family of 2-OD–dependent dioxygenases whose activity requires O2 as substrate.The hydroxylation of Asn803 does not lead to HIF-1� degradation. Therefore, thisresidue is not directly involved in the O2-sensing mechanism. Hydroxylation ofAsn803 abolishes the interaction between HIF-1� and its transcriptional coactiva-tors CBP and p300. CBP and p300 are coactivators that participate in the activitiesof many different transcription factors. The CBP/p300 proteins have a key role incoordinating and integrating multiple signal-dependent events. They make possiblethe adequate expression levels of genes in response to diverse physiologic stimuliand influences; for example, proliferation, differentiation, and apoptosis.

�

Fig. 2 (Continued) This complex is responsible for HIF-1� polyubiquitination prior to degrada-tion by the proteasome. Factor inhibiting HIF-1 (FIH-1) hydroxylates the asparagine 803 (N803)residue of HIF-1� preventing interaction with the CBP/p300. ARD1 acetylates the lysine 532residue of HIF-1� leading to its degradation. Hypoxia, oncogenes, and growth factors can activatethe phosphatidylinositol 3-kinase (PI3K) pathway, which has AKT as a downstream target. AKTcan also be activated by phosphatidylinositol-dependent protein kinase 1 (PDK-1). PI3K/AKTactivation causes HIF-1� overexpression mediated by mTOR (mammalian target of rapamycin).MEK (MAPK kinase) participates in the phosphorylation pathway activating the extracellularsignal–regulated kinases (ERK1/2), which phosphorylate HIF-1�. Phosphorylated HIF-1� is sta-bilized and activated, enters into the nucleus, forms the heterodimer with HIF-1�, is coactivated byCBP/p300 complex, and induces the transcription of more than 100 target genes. A cysteine residueis kept in the reduced state by redox factor-1 (REF-1) mediated by thioredoxin (TRX), which par-ticipate in HIF-1� stabilization. Under hypoxia, nitric oxide (NO) disfavors HIF-1� accumulation(activates PHDs), and under normoxia condition, NO “mimics” hypoxia conditions (inhibits PHDs)and leads to HIF-1� accumulation . Some evidence points to the role of reactive oxygen species(ROS) as messengers that regulate HIF-1 activity (see Color Insert)


pVHL is a component of the E3 ubiquitin-protein ligase complex that drives theproteolysis in the proteasome, preventing the transcriptional activation of its targetgenes. E3 ubiquitin ligases are a family of proteins that participate in the degra-dation and activity of many cellular proteins and function together with ubiquitin-activating enzyme E1 and ubiquitin-conjugating enzyme E2. In the hydroxylatedHIF-1� degradation pathway, E3 ubiquitin-protein ligase complex is formed bypVHL, elongin C, elongin B, cullin 2, and RBX1 (Fig. 2). The E3 complex iscapable of functioning with E1 ubiquitin-activation and E2 ubiquitin-conjugatingenzymes to mediate the ubiquitination of HIF-1� leading to its rapid proteosomaldegradation. HIF-1� has a half-life of approximately 5 minutes. However, whenHIF-1� escapes the degradation pathway, its detection by Western blot experi-ments occurs within minutes. Indeed, HIF-1� is detectable in the nucleus of cellsafter less than 2 minutes of exposure to anoxia or hypoxia. In this case, HIF-1�escapes ubiquitination and proteosomal degradation, is stabilized by posttransla-tional modifications, and enters into the nucleus. A comprehensive study about HIF-1� turnover (analyzed by electrophoretic mobility shift assay; EMSA) showed thatits maximal protein expression occurs in cells exposed for 1 hour in anoxia/hypoxia.These expression levels are maintained after 4 hours at low levels of oxygen expo-sure. However, after reoxygenation of cells, a decrease in the HIF-1 DNA bindingactivity is observed after 2 minutes. In addition, the nuclear HIF-1� protein wasreduced within 4 to 8 minutes, down to a level below the detection limit within32 minutes [4].

Under hypoxia conditions, HIF-1� is no longer degraded; it is phosphorylatedin the cytoplasm and activates its target genes inside the nucleus. Once the tar-get genes are expressed, a physiologic response to low oxygen concentration isachieved. Little is known about the fine-tuning of the posttranslational modificationsand their role in HIF-1� activity. The MAPK (mitogen-activated protein kinase)and phosphatidylinositol-3-kinase (PI3K) pathways are certainly involved in theregulation of HIF-1�. ERK1/2 (also called p44/42) activation, which is a resultof activation of the upstream proteins Ras/Raf-1/MEK-1/ERK1/2, leads to HIF-1� phosphorylation. Although MAPK phosphorylates HIF-1� in vitro, studies onmutations in the phosphorylation sites in HIF-1� protein show that its transactiva-tion capacity is not affected or directly mediated by phosphorylation. Regulation ofHIF-1� by MAPK is still an open issue [5]. Loss of tumor suppressor activity mayalso contribute to HIF-1� overexpression. For example, interaction of the p53 tumorsuppressor gene with HIF-1� negatively regulates HIF-1� protein levels. There-fore, HIF-1� overexpression correlates with expression of mutant p53 in humancancer.

AKT (protein kinase B), a serine/threonine kinase, is commonly activatedin cancer cells. This protein functions downstream to PIP3 (phosphatidylinosi-tol 3,4,5-triphosphate) in the PI3K phosphorylation pathway (Fig. 2). PI3K/AKTsignaling pathway regulates vascular endothelial growth factor (VEGF) expres-sion through HIF-1� in several types of transformed cells, including ovariancancer and human gastric adenocarcinoma. Cancer cells have constitutive AKT


activity through amplification of PI3K signaling pathway or deletion of phos-phatase and tensin homolog deleted on chromosome 10 (PTEN), a PI3K antago-nist. AKT induction is an important event in tumorigenesis as this pathway leadsto a higher level of glycolysis in a dose-dependent manner that correlates witha more aggressive malignancy in vivo. Apparently, AKT has an effect more pro-nounced on glucose metabolism than it effects on proliferation and survival ofthese transformed cells. This phenomenon was observed in established humanglioblastoma cells, which showed a switch to aerobic glycolysis and glucosedependence [6].

HIF-1� is also modified in the lysine residue 532 localized in the ODD domainof HIF-1� by an acetyl transferase called arrest defective-1 (ARD1). ARD1 wasoriginally identified in the yeast Saccharomyces cerevisiae and received this namedue to the phenotype of mutant yeast defective in the mitotic cell cycle. A humanvariant of ARD1 is able to partially decrease HIF-1� accumulation in hypoxia[7]. Another HIF-1� posttranslational modification is S-nitrosation. Nitric oxide(NO) promotes HIF-1� stabilization, DNA binding, and transactivation of targetgenes under normoxic conditions. This NO effect on HIF-1� activity was verifiedin several cell lines, including swine tubular cells (LLC-PK1), human glioblastomaand hepatoma, as well as endothelial cells from bovine pulmonary artery. Thesecells, when treated with different NO donors, such as S-nitroglutathione, (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (NOC-18),1-hydroxy-2-oxo-3-(3-aminopropyl)-3-isopropyl-1-triazene (NOC-5), and S-nitroso-N-acetyl-D,L-penicillamine (SNAP), accumulate HIF-1� protein [8]. Therefore,under hypoxia, NO disfavors HIF-1� accumulation, whereas under normoxicconditions, NO “mimics” hypoxia conditions and leads to HIF-1� accumulation.

4 HIF-1 Isoforms

There are two other members of the bHLH-PAS superfamily that share struc-tural similarity to HIF-1�: HIF-2�, also named endothelial PAS-domain protein1 (EPAS1), and HIF-3�. The roles of the 2� and 3� subunits are poorly under-stood. Similarly to HIF-1�, HIF-2� is also regulated by hydroxylation of the con-served proline residue, which causes its degradation under normoxic conditions viaubiquitin-E3-ligase. HIF-2� has 48% amino acid sequence identity with HIF-1�.HIF-2� and HIF-3� also have the common ability to heterodimerize to HIF-1�and bind to the hypoxia response element (HRE) motif in the regulatory regions oftarget genes. Additionally, an alternative splice variant of HIF-3� exists, a proteinthat lacks the transactivation domain. This splice variant was termed inhibitory PAS(IPAS) protein, and it was identified as the dominant-negative regulator of HIF-1.IPAS interacts with the amino-terminal region of HIF-1� and prevents its DNAbinding. The effect of the different members of the HIF-1 family on gene expres-sion can vary depending on the cell type. For example, HIF-1� and HIF-2� arestrongly expressed in the kidney, vascular cells, bone marrow, macrophages, lungs,


endothelium, and carotid body, whereas HIF-3� is mainly expressed in Purkinjecells of the cerebellum and mouse cornea epithelial cells. HIF-2� has more restric-tive distribution inside tissues than does HIF-1 [9].

5 HIF-1� Induction by Nonhypoxic Stimuli

In addition to hypoxia, HIF-1 is also responsive to several nonhypoxic stim-uli, including insulin, platelet-derived growth factor (PDGF), transforming growthfactor � (TGF-�), insulin-like growth factor (IGF-1), thrombin angiotensinII, cytokines, and carbachol (activates the muscarinic acetylcholine receptors).Whereas growth factor stimulation of HIF-1� is cell type specific, hypoxia increasesHIF-1� expression in all cell types. In addition, HIF-1 is activated by some metalssuch as cobalt, chromium, nickel, arsenic, and iron. Desferrioxamine also activatesHIF-1 by mimicking hypoxic conditions. Some evidences point to the role of reac-tive oxygen species (ROS) as messengers that regulate HIF-1 activity [10].

The oxidative status of the iron ion bound to the PHDs might also regulateHIF-1� activity, as these enzymes are Fe2+-dependent. ROS generated by mitochon-dria and released into the cytoplasm of cells exposed to hypoxia promote oxidationof Fe2+ to Fe3+. A decrease in the Fe2+ availability would cause reduction in thePHD activity. This mechanism allows HIF-1 accumulation and stabilization [11].

6 HIF-1 in Cancer

Once stabilized and activated, inside the nucleus, HIF-1 recognizes and binds tohypoxia response elements (HREs) present in the regulatory regions of its tar-get genes. The consensus HRE has the sequence 5′-BRCGTGVBBB-3′, where theRCGTG core is the HIF-1 binding site (HBS). Microarray experiments suggest thatmore than 200 HIF-1 target genes might exist. However, not all of them are likely tobe directly regulated by an HRE in their regulatory regions [10]. More than 100 HIF-1 target genes are known, including the genes that encode erythropoietin, VEGF,glucose transporters, and glycolytic enzymes.

Erythropoietin (EPO) and VEGF play crucial roles in adaptive responses to sys-temic and local hypoxia. Both are implicated in tumor progression. EPO stimulateserythropoiesis, which increases O2 delivery, whereas VEGF stimulates angiogene-sis, which increases delivery of both O2 and energy substrates such as glucose. Rig-orous analysis has confirmed the role of HIF-1 in EPO and VEGF transcriptionalactivation, and consequently in carcinogenesis.

HIF-1� is overexpressed in many human cancers [12]. Immunohistochemicalstaining analysis of surgical biopsy specimens show overexpression of HIF-1� pro-tein in colon, ovary, lung, prostate, skin, stomach, brain, and breast cancers. Signif-icant associations between HIF-1� overexpression and patient mortality have beenshown in cancers of the breast, brain, cervix, oropharynx, esophagus, ovary, and


uterus. Premalignancy and neomalignant lesions as well as cancer metastases ofsome cancer types also show increased HIF-1� protein. For example, HIF-1� isoverexpressed in the majority of breast and colon cancer metastases. By contrast,association between HIF-1� overexpression and decreased mortality were reportedfor patients with head and neck cancer and non–small cell lung cancer. Thus, theeffect of HIF-1� overexpression seems to be dependent on the cancer type. HIF-1�can have antiapoptotic or proapoptotic effects on tumor cells [9].

Mutation in the VHL gene results in the so called von Hippel–Lindau syndrome,characterized by the formation of tumors and fluid-filled sacs (cysts) in many dif-ferent parts of the body. Clear cell renal carcinoma (CCRC) cells lacking pVHLtumor suppressor constitutively express HIF-1� and HIF-1 target genes under non-hypoxic conditions [13]. CCRCs present all the main characteristics supposed to bemaintained by proteins encoded by genes regulated by HIF-1: increased glycolysis,decreased respiration, increased glucose uptake and lactate production. pVHL loss-of-function also occurs in hemangioblastoma. Therefore, at least in these two typesof tumors, deregulation of HIF-1� regulatory pathway plays a causal role.

7 HIF-1, Cancer, and the Glycolytic Pathway

The way cells change their metabolism under hypoxia shares many similarities tothe way tumors cells change their metabolism to survive and proliferate. Indeed,many of the known oncogenic signaling pathways overlap with hypoxia-inducedpathways. Cancer cells have special metabolic needs for their proliferation and sur-vival. They need to cope with different levels of oxygen and adjust their metabolismto guarantee cell proliferation and minimize apoptosis. The vast majority of humanand animal tumors display a high rate of glycolysis, increased glucose uptake,increased lactate production, and decreased respiration under aerobic conditions(a phenomenon known as the Warburg effect) [14]. Aerobic glycolysis providescancer cells (both nonmetastatic and metastastic) a high competitive capacity overnormal cells. This adaptive response can be driven by hypoxia, as well as by muta-tions that inactivate tumor suppressor genes or activate oncogenes. Hypoxia is apotent inducer of gene expression, especially of genes involved in glycolysis formaintaining cellular energy. The higher glycolytic rate in tumor cells comparedwith that of nontumorigenic cells can be attained by the increased transcription,followed by translation, of glycolytic genes and glucose transporters associated ornot with inhibition of oxidative phosphorylation. Hypoxia has HIF-1 as the keyregulator. Evidence is surmounting that HIF-1 regulation of glycolysis is impor-tant in carcinogenesis. However, there is much controversy as to whether HIF-1–induced glycolysis is a reactive process or a “cause” of cancer. A definitive solutionto this key question is complicated by the fact that hypoxia and HIF-1 are not exclu-sive regulators of the glycolytic enzymes, and HIF-1 activation is not associatedonly with hypoxia (oncogenes and growth factors can also activate this pathway).In addition, high pyruvate concentrations result in HIF-1� stabilization indepen-


dently of hypoxia [15].Therefore, although hypoxic HIF-1 activation alone doesnot explain aerobic glycolysis, other factors may provide feedback to enhance nor-moxic HIF-1 induction. The structure, regulation, and action of HIF-1 was discussedin detail above and shows a very complex network (Fig. 2). Regardless of whichpathway leads to HIF-1 activation, its effect on glucose metabolism is due to theupregulation of specific target genes.

Regulation of metabolism by HIF-1� was evidenced through investigation of itsfunction in mouse embryonic stem (ES) cells in which the Hif1� gene was inac-tivated by homologous recombination. Analysis of Hif1a+/+, Hif1a+/-, and Hif1a-/-

ES cells revealed that HIF-1� is required for the induction of several genes whoseproducts are involved in glucose metabolism; more specifically, aldolase A and C,enolase 1, hexokinase I and II, phosphofructokinase L, phosphoglycerate kinase I,pyruvate kinase M, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydroge-nase A, GLUT1 and GLUT3 [16]. HRE has been characterized in the aldolase A,enolase 1, and lactate dehydrogenase A human promoters and also in the aldolase Cmouse promoter. Complete identification of the key gene products specifying HIF-dependent tumor metabolism is crucial for therapeutic targeting of HIF-1 signalingin cancer.

In the presence of oxygen, glucose is converted to pyruvate by the glycolyticenzymes. Pyruvate is transported to the mitochondrial matrix, where it is convertedto acetyl-CoA and metabolized in the TCA cycle. Under hypoxia, pyruvate is con-verted to lactate through lactate dehydrogenase (LDH) A in the cytoplasm, whichis a target for HIF-1 regulation. In cancer cells, aerobic glycolysis and lactate pro-duction is favored to the detriment of oxidative phosphorylation. To understand therole of HIF-1 in the regulation of glucose and energy metabolism in cancer cells,we reviewed the literature in search of experimental data demonstrating the directinvolvement of HIF-1 in the induction of such genes in these cells. In some cases,the link between HIF-1 and upregulation of a gene in cancer cells was based on thepresence of HRE motifs in their regulatory regions. In the next sections, we describethe link found between HIF-1 and the proteins involved in glucose metabolism incancer cells.

8 Hexokinase

Hexokinases (HKs) catalyze the first step in the glycolytic pathway, conversionof glucose to glucose-6-phosphate (G6P). Human cells express four isozymes ofhexokinases: types I, II, III, and IV (glucokinase). The key hexokinase isoenzymeinvolved in cancer promotion is the type II (HKII), the major isozyme overexpressedin tumors exhibiting the high glycolytic phenotype. HKII has been extensively stud-ied by Dr. Peter Pedersen from Johns Hopkins University School of Medicine andhis co-workers [17]. They found that HKII binds to transmembrane channels formedby the porin-like protein voltage-dependent anion channel (VDAC) located withinthe outer mitochondrial membrane. This interaction reduces the enzyme’s sensitivity


to product inhibition by G6P and allows the enzyme to have direct and rapid accessto mitochondrially generated ATP. It also protects HKII against proteolytic degra-dation. These combined properties, together with the high content of the enzymein highly malignant tumors (>100-fold increase), result in the rapid production ofG6P. This key metabolic intermediate-precursor serves as a major carbon sourcefor most biosynthetic pathways that are essential for the growth and rapid pro-liferation of tumors. It also serves as the initial substrate for glycolysis and ATPgeneration.

Enhanced transcription of HKII occurs in malignant tumors, implicating theHKII promoter activation and upregulation during tumorigenesis. Functional HREs(HIF-1 responsive) are located in the distal region of the HKII promoter. HKII canalso be transcriptionally activated by mutant p53 or through demethylation of itspromoter. Regulation of the gene encoding hepatoma HKII occurs through activa-tion of its promoter that contains in its distal region two overlapping E-box/HIF-1sequences separated by approximately 50 base pairs. These sequences are knownto be associated in other gene promoters with responsiveness to glucose (E-box) orhypoxic conditions (HIF-1). The response of the HKII gene promoter to both glu-cose and hypoxic conditions is not confined to its distal region but involves signif-icantly the proximal region as well. Elements in this region remain to be identifiedand studied in detail.

9 Glucose Phosphate Isomerase

Glucose phosphate isomerase (GPI) is a housekeeping cytosolic enzyme of glucosemetabolism that plays a key role in both glycolysis and gluconeogenesis path-ways, catalyzing the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate. Upon secretion, GPI also acts as a cytokine also referred to as autocrinemobility factor (AMF) or NLK [18]. AMF was originally isolated as a cytokineautocrine factor that stimulates cell mobility and NLK as a lymphokine and neu-rotrophic factor for spinal and sensory neurons. GPI/AMF/NLK is associated withthe development of tumors, and an increased amount of this protein is present in theurine and serum of patients with various malignancies. Therefore, in addition to itsrole in the glycolytic pathway, the induction of this gene under hypoxia may play arole in stimulating cell mobility, invasion, and metastasis in tumor cells.

In human pancreatic cancer PC-3 cells, GPI mRNA is weakly expressed in cellscultured in normoxic conditions, but its synthesis is markedly increased in hypoxicconditions (1% oxygen plus CoCl2). YC-1, 3-[5′-hydroxymethyl-2′furyl]-1-benzylindazole, a guanylate cyclase activator, reduces the mRNA levels of GPI in PC-3cells in a dose-dependent manner under hypoxic conditions. Guanylate cyclase acti-vation can increase production of NO, which inhibits the accumulation and activityof HIF-1 in hypoxia. Therefore, GPI transcriptional activation is intimately linkedto HIF-1 activity.


10 Phosphofructokinase

Phosphofructokinase 1 (PFK-1) catalyses the rate-limiting phosphorylation offructose-6-phosphate to fructose-1,6-biphosphate, which is an energy-consumingstep in glycolysis. The fructose-2,6-biphosphate is the most potent allostericactivator of glycolysis and exerts control over the rate of glucose utilization. Itactivates PFK-1 and inhibits the gluconeogenic enzyme fructose-1,6-biphosphatase.Because of the antagonistic effects in these enzymes, the product of the reactioncatalyzed by 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKBF) playsan essential role in the opposing glycolytic and gluconeogenic pathways.

In human cells, there are at least four different genes encoding PFKFBs(PFKBP-1, PFKBP-2, PFKBP-3, and PFKBP-4). They all respond to hypoxia invivo, and this response is diverse in different organs. Tissue-specific isoforms ofPFKBP are not completely exclusive, and several tissues express more than oneisoform. Among the PFKFB isoforms of mammalian origin, PFKFB3 has the high-est kinase:phosphatase activity (K/B) and is the most highly expressed isozyme intransformed cells. The PFKFB-4 isozyme lacks a serine phosphorylation residuethat is critical for the downregulation of its kinase activity. Therefore, one can con-clude that it may have a high K/B ratio, and it should greatly promote glycolysisunder conditions of limited oxygen supply. The use of dimethyloxalylglycine (aspecific competitive inhibitor of PHDs) demonstrated the role of HIF-1� in hypoxicinduction of the genes coding for PFKFB3 and PFKFB-4 isoforms (in human hep-atoma Hep-3B and HepG2 cell lines, human prostate cancer cell, PC-3 and HeLacells). A splice variant of PFKFB-4 (PFKFB-4 s) is also induced by hypoxia in DB-1melanoma, besides the full-length PFKFB-4. Hypoxic induction of the transcriptionof these genes is mediated by HREs located in their promoter regions. In addition,PFKFB3 and PFKFB4 isoforms are overexpressed in solid malignant tumors fromthe breast and colon, overexpression of the PFKFB-4 protein being higher comparedwith that of PFKBF-3 [19].

11 Aldolase

Aldolase (ALD) catalyzes the reversible aldol cleavage of fructose-6-phosphate intotwo trioses, glyceraldehyde-3-phosphate and dihydroxyacetone. It has a role in theglycolytic, gluconeogenic, and fructose metabolic pathways. Three tissue-specificforms exist in higher vertebrates: aldolase A (predominately in muscle and red bloodcells), aldolase B (predominately in liver, kidney, and intestine), and aldolase C (pre-dominately in brain). The direct association of aldolase gene expression and HIF-1in tumors was observed in a study with a mouse hepatoma cell line, Hepa1c1c7,and its mutant derivative, ARNT-deficient c4T. The absence of the ARNT proteincompletely abolished the oxygen regulation of the aldolase A and phosphoglycer-ate kinase 1 (PGK1) gene expression, indicating that these genes share a commonpathway of response to hypoxia involving activation of HIF-1. Among the three


isoenzymes, aldolases A and C seem to have evolved to perform the glycolyticreaction, fructose-1,6-phosphate cleavage, more efficiently than does aldolase B.Aldolase B seems to be more involved with the gluconeogenic pathway [20].

12 Glyceraldehyde Phosphate Dehydrogenase

Glyceraldehyde phosphate dehydrogenase (GAPDH) is a key enzyme in glycolysisand also a multifunctional protein. It converts glyceraldehyde-3-phosphate into 1,3-bisphosphoglycerate. Its gene is constitutively expressed in many tissues. Variationsin GAPDH expression levels occurs in tissues at different developmental stages andhave been observed in cells treated with a variety of agents such as mitogens, dex-amethasone, calcium, insulin, and in cells under hypoxia conditions. Overexpressionof GAPDH in tumor cells as a consequence of the development of a hypoxic cellu-lar microenvironment is cell type specific. It has been observed in human prostateadenocarcinoma cells (cell line LNCap), human spontaneous cervical cancer cells(SiHA), but not in human malignant glioma cells (cell lines U373-MG, GaMG, andU251) ([21], [22]). It is not clear if the reason for the upregulation of GAPDH intumor cells is only for increasing glycolysis. The role of GAPDH as a NADH gen-erator and as a mediator of cell death has been highlighted in several studies, whichadds to the complexity of its upregulation in tumor cells.

13 Phosphoglycerate Kinase

Phosphoglycerate kinase (PGK) catalyzes the transfer of the phosphoryl groupfrom the acyl phosphate of 1,3-bisphosphoglycerate to ADP. ATP and 3-phosphoglycerate are the products. Human PGK1 gene harbors HREs in the 5′

flanking and 5′ UT regions, and mouse PGK1 harbors three HREs in the 5′ flank-ing sequence [10]. The HREs of the PGK promoter mediate the transcriptionalresponse to hypoxia. Indeed, the induction of PGK in different solid tumor cell linesin hypoxic conditions is under the control of HIF-1�. Expression of PGK1 increasesin mouse hepatoma Hepa1c1c7 cell lines exposed to hypoxia or to treatment withdesferrioxamine [20].

14 Enolase

Enolase (ENO) is also upregulated in many cancers, including breast, liver, colon,and lung. Direct evidence of HIF-1 upregulation of the enolase gene was obtainedusing YC-1, a drug that targets HIF-1. Levels of aldolase and enolase mRNAsdecrease in YC-1–treated Hep3B hepatoma cells. The inhibitory action of YC-1on these glycolytic genes probably inhibits cell survival under hypoxia and maypromote cell death in hypoxic areas [23].


15 Pyruvate Kinase

Pyruvate kinase (PK) is responsible for net ATP production within the glycolyticpathway. It is consistently altered during tumorigenesis. Four isoenzymes of pyru-vate kinase have been identified: type L (present in tissues with gluconeogenesissuch as liver and kidney); type R (expressed in erythrocytes); type M1 (muscleand brain); and type M2 (expressed in lung tissues and all cells with high ratesof nucleic acid synthesis, such as embryonic cells, adult stem cells, and especiallytumor cells). Investigation of the mRNA levels of HIF-1�, PGK1 and muscle-typepyruvate kinase 2 (PKM2) were performed in various hepatoma cell lines as wellas in mouse and human tumor specimens and the corresponding normal tissues. Infive of eight human colorectal cancers investigated, PGK1 and PKM2 mRNA lev-els were increased in comparison with the corresponding normal tissues, whereasHIF1-� was not significantly changed [24]. Though probable, these studies do notconclusively demonstrate that the increase in PKM2 gene expression is, in fact,mediated by HIF-1–induced transcriptional activation. However, HIF-1� plays acritical role in PK expression during hypoxic challenge in glial cells.

16 Pyruvate Dehydrogenase

The pyruvate dehydrogenase (PDH) complex catalyzes the conversion of pyruvateto acetyl-coenzyme A, which enters the TCA cycle, producing NADH and carbondioxide. The PDH complex is composed of three subunits, E1 (pyruvate decarboxy-lase), E2 (dihydrolipoamide acetyltransferase), and E3 (dihydrolipoamide dehy-drogenase), and is under the control of pyruvate dehydrogenase kinases (PDKs)1 to 4. PDKs phosphorylate the E1 subunit of PDH causing its inhibition. Theactivity of PDK is regulated by the concentration of the metabolic products ofpyruvate (NADH and acetyl-coA). HIF-1 upregulates the expression of PDK1[25]. Upregulation of the expression of PDK1 by HIF-1 illustrates how it favorsa metabolic switch toward glycolysis to the detriment of oxidative phosphoryla-tion. Hypoxic cells compensate the inefficient glycolytic energy output by increas-ing glucose uptake, overexpressing and increasing the activities of the glycolyticenzymes.

17 Lactate Dehydrogenase and Lactate Carrier MCT4

Lactate dehydrogenase (LDH) converts pyruvate into lactate. LDH-A converts pyru-vate to lactate during glycolysis under anaerobic conditions in normal cells. Theother isoenzyme, LDH-B, kinetically favors the conversion of lactate to pyruvateand is found at high levels in aerobic tissues such as heart. Elevated LDH-A expres-sion contributes to the ability of tumor cells to undergo aerobic glycolysis. Onceproduced, lactic acid exits cancer cells through the MCT4 lactate carrier that is also


induced in these cells. Under hypoxia, both pyruvate transformation into lactatewith concomitant regeneration of NAD+ and lactate exit from the cell are stimulatedbecause the genes encoding the LDH-A isoform and the lactate carrier MCT4 areHIF-1 targets.

The c-myc gene is frequently activated in human cancers. This transcrip-tion factor forms a heterodimer with another protein (MAX) and binds to a5′-CACGTG-3′ consensus sequence in its target genes inducing gene expression. Inaddition to HIF-1, the LDH-A gene is also a c-MYC target. Therefore, the molecularbasis for altered tumor glycolysis and lactate production cannot be solely explainedby HIF-1 activation of specific genes [26]. In addition to its role in lactate pro-duction, LDH-A may also be involved in the regulation of gene expression and/orDNA replication. Therefore, its modulation by HIF-1 and c-MYC could be linkedto effects other than to increased lactate production.

18 Glucose Transporters and HIF-1

Glucose enters animal cells through facilitated diffusion via glucose transporters(GLUT1 to GLUT5) and is subsequently phosphorylated by hexokinase. GLUT1and GLUT3, present in nearly all mammalian cells, are responsible for basal glu-cose uptake and continually transport glucose into cells at an essentially con-stant rate. They have a high affinity for glucose, allowing transport of glucose ata high rate under normal physiologic conditions. Cancer cells overexpress bothGLUT1 and GLUT3 to increase glucose uptake. Upregulation of glucose trans-porters has been observed during carcinogenesis in esophageal, gastric, breast,and colon cancers. A major technique used to measure glucose intake in tumorcells is positron emission tomography (PET) with [18F]fluoro-2-deoxy-D-glucose(FDG). PET is a noninvasive imaging technique used in the clinic to detect malig-nant tumors, which measures the glucose metabolism in vivo. FDG is a fluori-nated glucose analogue and accumulates in tumor cells in proportion to the rateof glucose metabolism. A high uptake of FDG is associated with a higher GLUT1expression.

The enhanced uptake of glucose often reflects tumor aggressiveness in patientswith different types of tumors. The glucose transfer from the bloodstream to theinside of cells mediated by GLUT1 plays a central role in the development andmalignant behavior of cancer cells. HIF-1 binding to the promoter of glucose trans-porters was seen in human HepG2 cells (GLUT1 and GLUT3) and mouse hepatomacells exposed to hypoxia (GLUT1). Studies of GLUT mRNA expression in hypoxiccells have shown that the level of GLUT1 mRNA in human placenta choriocarci-noma cell line BeWo (JCRB911) increases 1.4-fold in the presence of 5% O2 andby 2.2-fold in the presence of 250 �M CoCl2 compared with that under 20% O2.In the rat choriocarcinoma Rcho-1 cell line, the level of GLUT1 mRNA increases1.8-fold in the presence of 5% O2 and by 3.2-fold in the presence of 250 μM CoCl2compared with that under 20% O2. The GLUT1 protein levels also increase under


hypoxia and CoCl2 treatment, and these results suggest that GLUT mRNA and pro-tein levels were directly mediated through HIF-1 induction [27].

19 HIF-1� Upregulation by TCA Intermediates Is Linkedto Carcinogenesis

Succinate dehydrogenase (SDH) is a TCA enzyme, which is also part of themitochondrial respiratory chain. Succinate dehydrogenase deficiency causes eithersevere encephalomyopathy or tumor formation [28]. Germ-line heterozygote muta-tions in the SDH subunits cause inherited pheochromocytomas (adrenal glandtumors) and paragangliomas. The effects of SDH deficiency are postulated to beassociated with the disturbance of two signaling pathways: one involving the super-oxide radical and another involving HIF-1. SDH defect can increase superoxide rad-ical production by the mitochondrial respiratory chain, which is able to trigger bothcell death and proliferation. On the other hand, when succinate accumulates insidethe mitochondria, it is transported into the cytosol where it can inhibit prolyl hydrox-ylases (PHD1 to PHD3), the enzymes responsible for HIF-1 destabilization. UponPHD inhibition, HIF-1� is stabilized under normoxic conditions. Heterodimeriza-tion of stabilized HIF-1� and HIF-1� forms active HIF-1, which induces transcrip-tion of nuclear genes involved in tumor progression.

Mutations in another TCA enzyme, fumarate hydratase (FH), cause cutaneousand uterine leiomyomas, as well as renal cell carcinomas. Mitochondrial proteinsmay also play a role in the development of sporadic kidney tumor oncocytoma. Highconcentrations of cytosolic fumarate may inhibit prolyl hydroxylases and have thesame consequences as excess succinate on HIF-1 destabilization. Defective SDHand FH activity lead, respectively, to the accumulation of succinate and fumarate.These TCA intermediates, in addition to oxaloacetate, are capable of inhibiting thePHD activity and subsequently inhibiting HIF-1� degradation [29].

20 HIF-1 Activity as a Therapeutic Target

The prognostic significance of HIF-1� expression in cancer cells, its central role inthe adaptive response to hypoxia, and its participation in the Warburg effect makeit a potential target for anticancer drug development. It is a reasonable specula-tion that inhibition of HIF-1 activity may be a useful cancer therapy. A number ofanticancer agents that may target HIF-1 activity have been described (some citedin this text) and are treated more fully by Patiar and Harris [30]. The challengeremains to prove that the antitumorigenic effect of these drugs is due to HIF-1�inhibition. Selectivity may be resolved by the fact that tumor cells are more hypoxicthan are normal cells and have specific intrinsic markers of HIF-1� activation. Fur-ther increase in this difference is possible through the use of angiogenesis inhibitors(e.g., thrombospondin-1) in association with HIF-1� inhibition.


21 Conclusion

HIF-1� can be activated by hypoxic and nonhypoxic stimuli, including growth fac-tors, oncogenes, metals, and ROS. Metabolism of glucose and oxidative phospho-rylation are directly affected by the HIF-1 signaling pathway. A change in ATPproduction pathway from oxidative phosphorylation to aerobic glycolysis is a hall-mark of cancer cells. There is an evident overlap between HIF-1 function and cancercell metabolism. Cancer cells have special needs for rapid proliferation and survival.Some of these needs are fulfilled by the HIF-1 signaling cascade. In addition, HIF-1is located at the center of the major pathways that define cell destiny toward adap-tation to fast growth or apoptosis. The available data show that cancer cell survivalis a much more pronounced HIF-1 effect than is cell death. Therefore, HIF-1 willcontinue to be explored as a potential target for cancer therapy.

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3. Berra E, Benizri E, Ginouves A, Volmat V, Roux D, Pouyssegur J. HIF prolyl-hydroxylase2 is the key oxygen sensor setting low steady-state levels of HIF-1� in normoxia. EMBO J2003; 22:4082–4090.

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9. Lee J-W, Bae S-H, Jeong J-W, Kim S-H, Kim K-W. Hypoxia-inducible factor (HIF-1) �: itsprotein stabilization and biological functions. Exp Mol Med 2004; 36:1–12.

10. Wenger RH, Stiehl DP, Camenisch G. Integration of oxygen signalling at the consensus HRE.Sci Stke 2005;re12.

11. Pouyssegur J, Mechta-Grigoriou F. Redox regulation of the hypoxia-inducible factor. BiolChem 2006; 387:1337–1346.

12. Zhong H, De Marzo AM, Laughner E, et al. Overexpression of hypoxia-inducible factor1alpha in common human cancers and their metastases. Cancer Res 1999; 59:5830–5835.

13. Semenza GL. HIF-1 mediates the Warburg effect in clear cell renal carcinoma. J BioenergBiomembr 2007; 39:231–234.

14. Warburg O. On the origin of cancer cells. Science 1956; 123:309–314.15. Lu H, Forbes RA, Verma A. Hypoxia-inducible factor 1 activation by aerobic glycolysis impli-

cates the Warburg effect in carcinogenesis. J Biol Chem 2002; 277:23111–23115.16. Iyer NV, Kotch LE, Agani F, et al. Cellular and developmental control of O2 homeostasis by

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17. Mathupala SP, Ko YH, Pedersen PL. Hexokinase II: cancer’s double-edged sword acting asboth facilitator and gatekeeper of malignancy when bound to mitochondria. Oncogene 2006;25:4777–4786.

18. Funasaka T, Yanagawa T, Hogan V, Raz A. Regulation of phosphoglucose isomerase/autocrinemotility factor expression by hypoxia. FASEB J 2005; 19:1422–1430.

19. Minchenko OH, Ochiai A, Opentanova IL, et al. Overexpression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-4 in the human breast and colon malignant tumors.Biochimie 2005; 87:1005–1010.

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30. Patiar S, Harris AL. Role of hypoxia-inducible factor-1alpha as a cancer therapy target.Endocr Relat Cancer 2006; 13(Suppl 1):S61–S75.

The Role of Glycolysis in CellularImmortalization

Hiroshi Kondoh

Abstract The clinical significance of the Warburg effect is well established,although it is not clear why and when cancer cells start to display a high glycolyticrate in vivo. The discovery of hypoxia-inducible transcriptional factor as a criticalregulatory factor for glycolytic genes along with the recent advances in senescencebiology imply that the metabolic shift to enhanced glycolysis would be observed inmultiple stages during tumorigenesis in vivo. Increased glycolysis would be requiredin the early step of immortalization to avoid oxidative stress–mediated senescence,followed by the adaptation to hypoxic conditions through increased enhancementof glycolysis during transformation. Discovery of regulatory mechanisms for sucha metabolic shift could provide important information for the future development ofcancer diagnosis and anticancer therapy.

Keywords Phosphoglycerate mutase · Glycolysis · Immortalization · p53 ·Warburg effect

1 In Which Stage of Carcinogenesis Is the Warburg EffectRequired?

1.1 Immortalization and Transformation During MultistepCarcinogenesis

It is well-known that tumorigenesis is a multistep process that involves a seriesof genetic and epigenetic alterations [1]. Chemically induced skin tumor is themost established animal model of multistep tumorigenesis. The sequential appli-cation of carcinogen can effectively induce tumors in mice. The application of 7,

H. Kondoh (B)Department of Geriatric Medicine, Graduate School of Medicine, Kyoto University,54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japane-mail: [email protected]


91

92 H. Kondoh

12-dimethyl-benzanthracene (DMBA), followed by treatment with phorbol esters(e.g., TPA), can result in skin tumors. In these experimental models, each step iscalled initiation, promotion, and progression, respectively, supporting the notion thatcancer progression includes multistep events.

From the pathologic and clinical viewpoint, the process of tumorigenesis invivo can be divided into three or more stages: immortalization, transformation,and metastasis [2]. Among other factors, the acquisition of unrestricted prolifera-tive potential, called cellular immortalization, would be involved in the very earlystage in vivo. During immortalization, several biological events are supposed to berequired; bypassing senescence, evasion of apoptosis and antigrowth signals, growthfactor independence, and so on. Then, to grow more aggressively in vivo, theseimmortalized cells should be transformed to be anchorage-independent, resistant tocontact inhibition, proangiogenic, and so forth. In the end stage, they easily detachfrom each other, more easily attach to and degrade matrix components, and invadeand migrate to other tissues (metastasis).

All these properties are also distinct hallmarks of cancerous cells compared withtheir normal counterpart [3]. Another candidate for inclusion in this list would beenhanced glycolysis, noted by Otto Warburg more than seven decades ago. He firstreported that cancerous tissues or cells display increased glycolysis by an unknownmechanism. A high glycolytic rate, even under high oxygen conditions, is referred toas the Warburg effect. This property is well used in clinical practice for the detectionof metastatic tumor mass by positron-emission scanning of [18F]fluoro-2-deoxy-D-glucose. Thus there is no dispute about its clinical significance, whereas there hasbeen a controversy as to exactly when cancer cells become highly glycolytic invivo. In other words, one big emerging question is the following: In which stage ofmultistep oncogenesis would the enhancement of glycolysis be involved?

1.2 Enhanced Glycolysis Is Required During Transformationto Adapt to Hypoxic Conditions

It was widely assumed that cancer cells maintain upregulated glycolytic metabolismto adapt to the hypoxic conditions in vivo, as solid aggressive tumors outgrow theblood supply of the feeding vasculatures [4]. Alternatively, it has been proposedthat the increased glucose flux might improve efficiency of glucose utilization ina microenvironment in which glucose is limited. In such a context, the glycolyticresponse represents a successful metabolic adaptation of cancer cells in vivo.

The concomitant induction of angiogenesis and enhancement of glycolysis withcell proliferation is mediated partly by activating the hypoxia-inducible transcrip-tional factor (HIF-1) [5]. Hypoxia increases HIF-1� levels in most cell types, andHIF-1 mediates adaptive responses to changes in tissue oxygenation. Thus, HIF-1can directly upregulate expression of a set of genes involved in both local and globalresponses to hypoxia, including angiogenesis, erythropoiesis, respiration, and mostof the glycolytic enzymes: Hexokinase 1 (HK1), Hexokinase 2 (HK2), Autocrine

The Warburg Effect and Immortalization 93

Motility Factor/Glucose-6-Phosphate Isomerase (AMF/GPI), Enolase 1 (ENO1),glucose transporter 1 (GLUT1), glyceraldehyde-3-P dehydrogenase (GAPDH), lac-tate dehydrogenase (LDH), 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase3 (PFKBF3), phosphofructokinase liver form (PFKL), phosphoglycerate kinase 1(PGK1), pyruvate kinase muscle form (PKM), triose phosphate isomerase (TPI).Interestingly, both HIF-1 and glycolytic enzymes are overexpressed in many tumorsand cancer cells. Moreover, ectopic expression of glycolytic enzyme LDH can trans-form culture cells, whereas the inactivation of LDH ablates the transformation abil-ity in cancer cells [6]. Altogether, these data support a functional link betweenenhanced glycolysis and cellular adaptation during tumor formation and expansion.In conclusion, the transformation during multistep oncogenesis would require theenhancement of glycolysis (Fig. 1).

1.3 Oncogene and Glycolysis: ras and c-Myc

However, the Warburg effect cannot be explained solely by cellular adaptation tohypoxic conditions for the following reasons.

1. The cellular adaptation model does not explain the constitutive metabolic changethat maintains high glycolytic rates in cultured cancer cells even under 20% oxy-gen in vitro [7].

A

Hypoxia

HIF-1

Glycolysis

Immortalization

Transformation

Metastasis

Hypoxia

HIF-1

Glycolysis

Immortalization

Transformation

Metastasis

c-Mycp53 KO

Glycolysis

B

Fig. 1 Two models of glycolysis involvement during multistep tumorigenesis.(A) Enhanced gly-colysis during transformation. In the growing solid tumors, the core of tumors would suffer hypoxiaas they outgrow the feeding capacity of the neovasculature. To adapt to hypoxic conditions, HIF-1is activated; which in turn enhances glycolysis. (B) Enhanced glycolysis during immortalizationand transformation. Other than the model in (A), glycolysis would be involved also in the earlierstep of immortalization. Immortalizing genes, induction of c-Myc, or knock-down of p53 (p53KO)would enhance glycolysis in cancerous cells

94 H. Kondoh

2. Ectopic expression of HIF-1 in culture cells induces cell cycle arrest, whichargues against the proposition that glycolysis in cancer cells is regulated onlyby HIF-1 or hypoxia.

3. Indeed, emerging evidence indicates that glycolysis is regulated in a cellu-lar context–dependent manner and by multiple genetic factors: oncogenes (ras,c-Myc), tumor suppressor genes (p53), signaling kinases (AMP kinase, Aktkinase, Pak1 kinase), and so forth.

These facts suggest that there would be possible other mechanisms to increaseglycolysis during multistep oncogenesis other than adaptation to hypoxia. Interest-ingly, several groups reported that c-Myc plays a critical role in cellular immortal-ization of human primary epithelial cells and fibroblasts via telomerase activation[8], and c-Myc could also enhance glycolysis through transcriptional upregulationof several glycolytic enzymes: HK, PFK, TPI, GAPDH, ENO, and LDH [9]. Takentogether, it is possible that glycolysis could be enhanced in the earlier step of tumori-genesis before cellular transformation occurs.

2 Enhanced Glycolysis Is Also Required in Immortalization

2.1 Senescence Is a Barrier for Tumorigenesis

Historically, senescence and aging research came into their own around the 1960 s.Hayflick and Moorhead reported replicative senescence in tissue cultured primarycells in 1967 [10], and Harman proposed a radical theory of aging in 1956 [11].Recently, there has been a resurgence in senescence biology, as cancer cells areknown to be immortal both in vivo and in vitro.

Most somatic cells have a limited replicative capacity under standard tissue cul-ture conditions and suffer a permanent cell cycle arrest, called replicative senescence[12]. Replicative senescence is induced by telomere erosion upon reaching replica-tive exhaustion, which can be bypassed by the ectopic expression of telomerasein human fibroblasts. It is well established that senescence can also be induced ina telomere-independent manner, called stress-induced senescence (SIS) [13]. Cellssuffering either replicative or stress-induced senescence (SIS) are phenotypicallysimilar: they adopt an enlarged and flattened appearance; deposit increased amountsof extracellular matrix; express elevated levels of inhibitor of plasminogen activatortype 1 (PAI-1); develop lipofuscin granules, single prominent nuclei, senescence-associated heterochromatin, senescence-associated �-galactosidase activity; andshow negligible DNA synthesis (Fig. 2).

Interestingly, although mouse telomeres are much longer than their human coun-terparts (60 kb vs. 12 kb), mouse embryonic fibroblasts (MEFs) stop growingwithin a shorter time when compared with human primary fibroblasts [14]. Apartfrom telomere structure, there are several other factors influencing cellular lifespan, as some cell types reach a proliferative barrier long before telomere erosionbecomes critical. It is now recognized that cells can arrest virtually at any age, in


Immortal

Replicativesenescence

Primary cells

glycolysisdeclined

reducedROSRadical

Scavanger

Enhancedglycolysis

Oxidative, orCulture stress

Telomeric erosion

Prematuresenescence

Fig. 2 Glycolysis and cellular senescence. Senescence can be induced in telomere-dependent or-independent manner. In both cases, glycolysis declines. Enhanced glycolysis can immortalizeprimary cells with significant reduction of oxidative damage

a telomere-independent manner. For example, oncogenic stimuli can induce pre-mature senescence, called oncogene-induced senescence. Thus, cellular senescencealso constitutes a potent anticancer mechanism [15].

2.2 Oxidative Stress and Senescence

Recent studies suggest that oxidative damage and the accumulation of reactive oxy-gen species (ROS) are closely involved in senescence. ROS, such as superoxideanion and hydroxyl radical, are produced during cellular metabolism mainly in themitochondria. ROS are also produced in response to different environmental stim-uli such as UV, IR, chemicals, hyperoxia, or hydrogen peroxide treatment. Abnor-mal ROS accumulation and its effects on intracellular macromolecules (oxidation oflipid, protein, and DNA) induces cumulative damage at the cell, tissue, and organ-ism level. Mild oxidative stress (e.g., treatment with low concentrations of hydrogenperoxide) is enough to induce senescence in primary cells. Interestingly, prematuresenescence induced by culture stress or oncogene-induced stress is associated withoxidative damage in cells [16].

Increased ROS accumulation is also observed during replicative senescence. Thereplicative potential of both murine and human fibroblasts is significantly enhancedunder low oxygen, associated with less oxidative damage inflicted than under

96 H. Kondoh

normoxia (O2 20%) [17]. Immortalized cells suffer less oxidative damage than doprimary fibroblasts when cultured under 20% O2. Moreover, immortalized cells aremore resistant to the deleterious effects of hydrogen peroxide than are primary cells.Thus, ability to resist oxidative stress could be a clue for explaining the immortalityof cancer cells.

Several radical scavengerscanprotectcellsagainstoxidativestress.Thesuperoxidedismutase enzyme (SOD) converts superoxide anions into hydrogen peroxide, andhydrogen peroxide can be detoxified by catalase. Consequently, these antioxidantenzymes can impact the proliferation of both primary and immortal cells as theycan counteract ROS effects. The ability of SOD to bypass senescence has been wellstudied and established in various cells or organisms. Increased expression of SODcan extend the life span of primary fibroblasts [18]. Conversely, knockdown of SODusing small interfering RNA (siRNA) induces premature senescence accompanied byp53 activation. Transgenic fly overexpressing SOD [19] or the detoxifying enzymecatalase [20]presentsanextendedorganismlifespan.Althoughit isclearlyestablishedthat these antioxidant scavengers are essential for proliferation of immortal cells, littleis known to date on the specific regulation operating in cancer cells.

2.3 Senescence-Bypassing Effect of Enhanced Glycolysis

We recently found that glycolytic enzymes can modulate the cellular life span ofMEFs [21]. In a senescence-bypassing screening in MEFs using a retroviral cDNAlibrary, we isolated the glycolytic enzyme phosphoglycerate mutase (PGM). PGMconverts 3-phosphoglycerate to 2-phosphoglycerate during glycolysis. Analysis ofthe impact of other glycolytic enzymes on senescence in MEFs showed that glu-cophosphate isomerase (GPI) could also drive immortalization of MEFs. Ectopicexpression of PGM or GPI increases glycolytic flux, decreases the oxidative damagethat MEFs are exposed to, and extends the life span of primary MEFs. Conversely,knockdown of PGM or GPI via specific siRNA induces premature senescence.Moreover, we and others found that the glycolytic flux declines during senescenceboth in murine and human fibroblasts [22].

How can an increase in glycolysis immortalize primary cells? From the datapresented above, it appears that enhanced glycolysis can protect cells from oxidativestress and as a consequence avoid senescence [21]. MEFs immortalized by PGMor GPI suffer less oxidative damage than do control cells as estimated by cytosolicROS staining, or quantification of 8-hydroxydeoxyguanosine (8-OHdG), a hallmarkof oxidative DNA damage lesions.

Moreover, mouse embryonic stem (ES) cells also present a surprisingly high gly-colytic rate [23]. Thus similarly to cancer cells, ES cells display the Warburg effect.ES cells are an exception that can bypass culture stress–induced senescence amongother primary cells. We hypothesized that reversible modifications such as a specificfactor playing a role in ES cell immortality enhances the glycolytic rate resultingin an increased protection from oxidative stress. This metabolic protection mightcontribute to preserve the genome integrity of ES cells thereby avoiding genetic


alterations and allowing them to maintain their self-renewal capacity. Also, thesemetabolic rates can be reversed, which would explain why differentiated cells donot present this enhanced metabolic level.

2.4 Glycolysis as Radical Scavenger

How can enhanced glycolysis protect cells from oxidative damage? One clue is thecommon metabolic feature shared between several immortalized cells: cancer cells,immortalized primary cells, and ES cells [22]. All these cells display enhanced gly-colysis with decreased mitochondrial respiration. Thus, increased glycolysis dur-ing immortalization could be accompanied by the downregulation of mitochondrialfunction by unknown mechanism(s) and would result in less intrinsic ROS produc-tion, which will be discussed later.

A second possible mechanism is that increased glycolysis could imitate or acti-vate radical scavengers. Interestingly, the antioxidant function of some scavengers(such as reduced gluthation (GSH) or Thioredoxin (TRX)) is closely coupled tothe NADPH/NADP balance. Most of the NADPH/NADP is produced through thepentose phosphate pathway (PPP), a branching metabolic pathway derived from theglycolytic pathway. Enhanced glycolysis might activate PPP and increase the levelof NADPH as by-products (Fig. 2).

Although this remains a hypothesis, several reports support it. The impact ofglucose-6-phosphate dehydrogenase (G6PD) activity on cell proliferation is wellestablished [24]. G6PD catalyzes the rate-limiting step in the pentose phosphate PPP,which is responsible for the recycling of NADPH and maintenance of the redox bal-ance as described above. G6PD-deficient human fibroblasts have a reduced life spanthat is attributable to oxidative stress and can be corrected by the ectopic expressionof this enzyme [25]. Both G6PD activity and the NADPH pool decline during contin-ued culture passage, presumably as a consequence of the accumulation of oxidativedamage. Importantly, ES cells ablated from G6PD expression are extremely sensitiveto oxidative damage, showing massive apoptosis at low concentration of oxidants thatare nonlethal for wild-type ES cells. It would, therefore, be worth exploring in thefuture whether enhanced glycolysis can then promote increased NADPH productionvia the PPP thereby exerting its antisenescence function.

3 Opposing Effect of Glycolysis on Life Span Observedin Different Organisms

3.1 Discovery of Sir2 as Longevity Gene in Yeast

Any discussion about longevity and glycolysis merits evaluation of the resultsobserved in yeast. The hypothesis that enhanced glycolysis can promote prolifer-ation would not be applied in every species. It seems quite opposite in the case

98 H. Kondoh

of budding yeast. In yeast, Sir2 gene was isolated as a longevity gene, which canbe activated in a calorie-restricted condition [26]. Calorie restriction is also well-known to extend the life span of mouse and yeast. In a calorie-restricted condition,glycolysis declines in yeast, accompanied by an elevation of the respiratory rate.Mutations in the glycolytic pathway have also been reported to extend life span inyeast, suggesting that decreased glycolysis has a causal effect on extension of yeastlife span.

Sir2 works as an NAD-dependent histone deacetylase. In yeast, restricting foodavailability affects two pathways that activate Sir2 enzymatic activity [27]. First,calorie restriction turns on a gene called PNC1, which produces an enzyme that ridscells of nicotinamide, a small molecule similar to vitamin B3 that normally repressesSir2. Second, activated respiration during calorie restriction is a mode of energyproduction that creates NAD as a by-product and lowers NADH. In consequence,NAD-dependent Sir2 activity is induced by calorie restriction. Thus, Sir2 is a keymolecule linking calorie restriction and longevity in yeast.

3.2 Calorie Restriction and Sir2

It seems that glycolysis has an opposing effect on yeast life span, in sharp contrastwith the Warburg effect observed in cancerous or immortalized mammalian cells. Todate, it is quite difficult to understand these differences in glycolytic profile betweenlong-lived yeast in calorie restriction and cancer cells in tissue culture, but there aresome possible explanations.

First, the mammalian homolog of Sir2 gene, called Sirt1, has less impact onaging of mammalian primary cells in tissue culture. Sirt1 can directly bind and mod-ify several DNA binding proteins: histone, p53, Peroxisome Proliferator-ActivatedReceptor-� Coactivator-1 (PGC-1), and possibly other unknown transcriptionalfactors. At least p53 is not conserved in yeast. Mammalian Sirt1 might targetmore unknown different factors, which would mask its senescence-bypassing effectobserved in yeast.

Second, aging of budding yeast is quite different from senescence observed inprimary culture cells. The mother yeast cells suffer the accumulation of buddingscars on their surface during the division process. In parallel, aging yeast cellsshow ballooning nucleolus, which is caused by accumulation of circular riboso-mal DNAs [28]. These aging phenotypes are quite specific to yeast, which mightexplain the difference of aging mechanisms between yeast and mammalian culturecells.

Third, Sir2 could affect mammalian organismal aging rather than senescence ofmammalian culture cells. Resveratrol is a chemical reagent that activates Sirt1 andis enriched in red wine. Resveratrol improves health and survival of mice on a high-calorie diet. In this sense, calorie restriction–induced longevity observed in yeastshould be more relevant to mammalian organismal longevity in calorie-restrictedcondition rather than the Warburg effect observed in cancer cells.


ACalorie

restrictionCalorie

restriction

Glycolysis declined

Glycolysis?

Others?

Sir2 activated

Sir2 activated

B

Respiration increased

Longevity Longevity

Fig. 3 Impact of calorie restriction and Sir2 on yeast longevity and mammalian organismal aging.(A) In budding yeast, life span is extended in calorie-restricted condition. In such a case, glycolysisdeclined associated with enhanced respiration. Enhanced respiration will promote NAD produc-tion, leading to activation of Sir2. (B) In mouse model, calorie restriction can delay organismalaging. In contrast, high-calorie diet can shorten the life span, which would be restored by treat-ment with resveratrol, activating chemicals for Sir2. The significance of glycolysis in this situationis still not clear

Collectively, Sir2 is a highly conserved protein and serves as a key to modulatemetabolic switching. Sir2 would work as a longevity gene in calorie-limited con-ditions, but its effect on replicative and stress-induced senescence remains to beclarified (Fig. 3). Thus, yeast might not be a good model to explore and explainthe Warburg effect that occurs primarily in cancerous and immortalized mammaliancells.

4 Novel Regulatory Mechanism of Glycolysis

4.1 p53 and Glycolysis

Recent advances in understanding the regulatory mechanism of glycolysis also sup-ports our hypothesis that the Warburg effect is a very early event in tumorigenesis.Inactivation of tumor suppressor gene p53 increases glycolytic flux in vitro andinvivo. Ablation of p53 significantly downregulates mitochondrial respiration in vitroand in vivo.

p53 is the most frequently mutated gene in various types of cancer and functionsas a transcriptional factor to induce cell cycle arrest, apoptosis, and so forth. Inac-tivation of p53 immortalizes primary cells in vitro, implicating that p53 can alsoaffect the senescence process. p53 knockout mice are cancer prone, possibly due tothis senescence effect. Partial activation of p53 induces premature organismal aging

100 H. Kondoh

p53 KO

Short lifespan due to cancer prone

Activated allele of p53

Premature ageing

Old wild-type

Cancer prone

Extra single copy of p53

Longevity with less tumor events

Fig. 4 p53 and organismal life span. p53 null mice are cancer prone, suffering short life span. p53activating allele knock-in mice display premature aging phenotype. In contrast, mice knocked-infor an extra copy of p53 acquire organismal longevity due to less oxidative damage and fewertumor events KO-knock out

in vivo[29] (Fig. 4). One possible interpretation would be that one of the major tar-gets of p53 involved in senescence induction impacts metabolic regulation, whichrenders cells sensitive to oxidative stress. TP53-induced glycolysis and apoptosisregulator (TIGAR) may be such a target through which p53 can modulate oxidativestress in vivo. Recent works suggest that mice knocked-in for an extra copy of p53acquire longevity associated with a resistance to oxidative damage [30]. It is possi-ble that the metabolic shift caused by p53 might modulate cellular senescence andorganismal aging through reduction of oxidative damage in vivo and in vitro.

p53 regulates both glycolysis and mitochondrial respiration through different tar-gets. p53 can target glycolytic enzymes (HK and PGM) and modulate the overallflux of glycolysis in primary cells. On the other hand, p53 knockdown can decreasemitochondrial respiration [23]. This is mainly due to inhibition of cytochrome coxidase 2 (SCO2) [31], a direct target of p53. SCO2 is critical in regulating thecytochrome c oxidase complex, the major site of oxygen utilization. In this way, p53could modulate both glycolysis and respiration in a concerted manner and wouldaffect cellular life span. This fact suggests that stable alterations at the genetic orepigenetic levels, including inactivation of p53, provide a possible explanation forthe enhanced glycolysis that cancer cells present in vitro.

4.2 PGM and Its Regulation

Since Dr. Warburg’s discovery, research on glycolysis has been mainly performed byusing established cancer and muscle cell lines. But these cells are already immortalwith several genetic alterations and presumably not suitable to dissect the impact ofimmortalizing genes, such as c-Myc or p53 knockdown, on the glycolytic pathway.

For example, phosphofructokinase (PFK) is described as the rate-limitingenzyme for glycolysis, while its overexpression has no effect on glycolytic flux


in immortalized and nonimmortalized cells. Ectopic expression of other enzymes,PGM or GPI, can increase glycolytic flux drastically in primary cells, indicating thatregulation of glycolysis pathway is largely cellular context–dependent. For example,PGM protein levels are tightly regulated by the ubiquitination pathway in primarycells, while they are not so regulated in immortalized cells. Chemical screening ofa breast cancer drug identified the PGM inhibitor as the most potent one, suggest-ing again that PGM can be a key step in the glycolytic pathway in a tissue-specificor cellular context–dependent manner. Interestingly, HIF-1 is a key transcriptionalfactor that upregulates many glycolytic enzyme genes under hypoxic conditions,but PGM is the only exception. The regulatory mechanism for PGM remains to beclarified.

5 Conclusion

Glycolysis serves not only as an energy source for cells but also is essential in thesteps of immortalization and transformation during multistep tumorigenesis, as itsenhancement can render cells resistant both to oxidative stress and hypoxic con-ditions, respectively. Cancerous cells show a concerted metabolic shift, includingenhanced glycolysis with reduced mitochondrial respiration by an as yet poorlycharacterized mechanism. The regulation of glycolysis relevant to the senescenceprocess is probably more complicated than we have expected. Future work to clar-ify its molecular mechanism should be employed in various cell types, includingprimary cells and ES cells—not just in cancer cells. p53 might be a key molecule asit plays a significant role both in organismal and cellular aging. These findings willalso contribute to improve and identify new anticancer therapies in the future.

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Metabolic Modulation of Carcinogenesis

Shireesh P. Apte and Rangaprasad Sarangarajan

Abstract Emerging evidence inextricably links cellular pathways that involveglucose metabolism (via oxidative phosphorylation and glycolysis), oxygen sens-ing, apoptosis, cell cycle regulation, and immune recognition and response. Thisaccounts for the epidemiologic and epigenetic effects of calorie intake and qual-ity, obesity, diabetes, immunosuppression, chronic infection or inflammation, aging,and viral diseases on carcinogenic rates and trends. The mitochondrion is a criticalorganelle that coordinates and integrates the above-mentioned signaling pathwaysso as to ultimately affect cell proliferation or cell death. The differential regula-tion of these pathways not only depends on the varying levels of stress inducedby the above-mentioned epigenetic factors but also on acquired genetic mutations,thereby attempting the prediction of cell survival or death, an exceedingly diffi-cult proposition. Concepts that have not received much attention in the literature—such as the role of the exogenous cytochrome C/NADH pathway in apoptosis andoxidative phosphorylation; the switch to glycolysis at lower pyruvate thresholds incancer cells and the paradox that this glycolytic switch may be necessary to pro-vide ATP and matrix alkalinization necessary for apoptosis to occur; reconciling theobservations that caloric restriction causes a generalized decrease in apoptosis yetincidences of cancer are significantly decreased in calorie-restricted animals; theunsaturation patterns of the fatty acids comprising the mitochondria-specific phos-pholipids; and the variation of the intermediary metabolism of tumors—with timewill be addressed from the viewpoint of altered cellular respiration. The implicationsof attenuation of cellular respiration on the mitochondrial adhesion and localizationof key immunoregulatory and glycolytic-apoptotic enzymatic or protein complexeswill be discussed.

Keywords Glycolysis · Oxidative phosphorylation · Apoptosis · Carcinogenesis ·Cell cycle · Hypoxia · Warburg · Calorie restriction · Cytochrome c

S.P. Apte (B)Alcon Laboratories, 6201 South Freeway, Forth Worth, Texas 76134, USAe-mail: [email protected]


103

104 S.P. Apte, R. Sarangarajan

Oxidative phosphorylation (OXPHOS), the last of the major steps in aerobic respi-ration and energy generation, occurs in the mitochondrion, an intracellular organellethat also controls and modulates apoptosis. A dysfunctional OXPHOS or an inade-quate oxygen supply can therefore modulate apoptotic function. It can also increasethe relative or absolute contribution of an oxygen-independent pathway of respi-ration and energy generation—glycolysis—to meet cellular energy requirements.This adaptive response temporally progresses from being proapoptotic to becom-ing antiapoptotic in nature. The proapoptotic effect may predominate in the earliertime period/stage of adaptation if tumor suppressors and apoptotic machinery arefunctional. The antiapoptotic effect may predominate in the latter times/stages ofadaptation if either the tumor suppressors and apoptotic machinery is dysfunctionalor is made so by this adaptive response itself.

1 A Decreased OXPHOS Phenotype Attenuates Apoptosis

A decreased content of OXPHOS complexes II, III, and IV, reduced level of ATP-synthase, and suppression of NDUFA1, an accessory component of OXPHOS, areall associated with the development of tumors, increased tumor aggressiveness,and antiapoptotic effects. Deficiencies in mtETC, ATP synthesis machinery (F0F1-ATPase), or ANT abrogate the apoptotic effects of Bax. OXPHOS dysfunction isassociated with a proinvasive and pro-proliferative modeling of the ECM by alter-ing transcriptional regulation of genes coding for members of MMP/TIMP, UPA,and CCN in human osteosarcoma cells.

An impaired mtETC may increase cellular NADH levels because electrons canno longer be transferred as efficiently to oxygen and NADH cannot be oxidizedback to NAD+. Increased levels of NADH induce protection from apoptosis anddecreased cell adhesion by promoting the binding of the tumor suppressor E1A tothe (NADH activated) corepressor CtBP.

Progression toward a more tumorigenic state is associated with increased anddecreased sensitivity to 2-deoxyglucose and oligomycin, respectively, consistentwith an increase and decrease in glycolysis and OXPHOS, respectively [1].

Dysfunction or mutations in tumor suppressors may cause attenuation inOXPHOS. A decrease in COX activity due to p53 attenuation can lead to a dimin-ished OXPHOS phenotype [2]. In hepatomas caused by mutated p53, the mitochon-drial porin bound hexokinase II is increased [3]. This enzyme isoform binds to themitochondrial membrane through interaction with VDAC and inhibits apoptosis bysuppressing the release of intramembrane space proteins in part by preventing theopening of the PTP [4]. In CCRC, VHL deficiency is one of the factors responsiblefor downregulation of the biogenesis of OXPHOS complexes, as well as for upreg-ulation of HIF-1�. AIF deficiency has been shown to produce a reduced OXPHOSphenotype.

Metabolic Modulation of Carcinogenesis 105

2 Reactive Oxygen Species and Carcinogenesis

A general picture that emerges from the literature is that cellular ROS can modulatecell-signaling pathways, carcinogenesis, or cell death depending upon their cellularlevels. Whether a particular cellular level of ROS triggers proliferation or apoptosisalso seems to depend on the magnitude of the cellular stress—which in turn dependson substrate and oxygen supply, the existence of mutations in key tumor suppressorgenes, OXPHOS function, and the local cellular environment including the levels ofantioxidant enzymes such as SOD and catalase.

Mitochondria can generate ROS not only accidentally but also via a regulatedenzymatic mechanism, thereby attesting to the importance of ROS in apoptosis. Thismechanism involves the electron transfer between the proapoptotic signal p66Shc

and cyt-C that generates ROS, which in turn triggers apoptosis by opening the PTPand subsequent caspase activation.

Interestingly, the redox reaction between p66Shc and cyt-C is favored by an excessof reduced cyt-C; as may occur under hypoxia when COX activity is decreased.Proapoptotic molecules such as Bax and tBid cause an increase of free intermem-brane cyt-C, which is then fully reduced by the rotenone-insensitive NADH-cyt b5

reductase (see Section 8). Therefore, apoptotic induction in attenuated OXPHOSor hypoxic cells routes through the exogenous NADH/cyt-C electron transportpathway.

Mitogenic signals during low-stress conditions activate E2F—which in turndecreases ROS—so as to induce proliferation. In conditions of high cellular stress,however, tumor suppressor genes such as p53 and p16INK4a are activated, leading toinhibition of E2F. Consequently, ROS levels accumulate to senescence-promotinglevels. High levels of ROS also induce a PKC-mediated block of cytokinesis.

Under conditions where either OXPHOS or ROS scavenging is dysfunctional,increased ROS levels may inactivate certain tumor suppressors such as PTEN lead-ing to increased activation of Akt and its downstream targets, HIF-1� and VEGF.The activation of the Akt oncogene has been demonstrated to be sufficient tostimulate the switch from OXPHOS to aerobic glycolysis, in part by increasingmitochondria-associated hexokinase activity. Akt is a negative regulator of AMPKactivity and hence can overcome the tumor-suppressor activity of LKB1. ROS medi-ated impaired OXPHOS induced PTEN oxidation may also suppress p53 activity byallowing nuclear translocation of Mdm2.

3 The Hexosamine Biosynthetic Pathway

Adaptive glycolysis triggered by impaired OXPHOS, increased ROS generation,hypoxia, or deregulation of nutrient uptake pathways may siphon more glucose intothe hexosamine biosynthetic pathway (HBP). Apart from causing insulin resistance,an increased flux through the HBP may cause increased O-GlcNAc glycosylation


and inactivation of the proapoptotic protein Bnip3 [5], thereby conferring a survivaladvantage on glycolytic cells [6].

Furthermore, increased posttranslational modification of transcriptional factorssuch as Sp1—mediated by O-GlcNAc glycosylation—may result in downregulationof OXPHOS gene expression and decreased mitochondrial function by impairingthe activity or expression of known regulators of OXPHOS transcription PGC-1�and NRF [7]. This phenomenon may further exacerbate the shift from OXPHOS toglycolysis.

4 Diversion of Pyruvate from Mitochondria

The prevalence of the Crabtree effect (a preference to ferment glucose into ethanol)in the bakers’ yeast, Saccharomyces cerevisiae, has been attributed in part to differ-ences in enzymes at the branch point between respiration (PDH) and fermentation(PDC). The concentration of pyruvate that gives half maximal activity (Km) of PDHis lower than that for PDC. The latter enzyme exhibits cooperativity with sub-strate concentration and also has a higher maximal activity. The net result is tomake metabolic flux through PDH favored at low pyruvate concentrations and fluxthrough PDC favored at higher ones. It is instructive to compare aerobic metabolismin this organism to that exhibited by tumor cells.

A decreased NAD:NADH ratio, which occurs in part as a consequence ofimpaired mtETC and which is characteristic of highly glycolytic malignant cells,downregulates the PDH complex [8] so that entry of pyruvate into the TCA cycle isimpaired.

The unusual metabolite, acetoin, unique to tumors and formed by the nonox-idative decarboxylation of pyruvate with acetic acid, is a competitive inhibitor ofintramitochondrial tumor PDH. Km values for pyruvate for the PDH of tumor celllines were significantly lower (up to threefold) than were those for the PDH ofnon-transformed cells [9]. Broadly interpreted, the concentration of pyruvate belowwhich metabolic flux is favored through OXPHOS may be lower in tumor cells,thereby contributing toward an oxygen supply–independent “pyruvate siphoning”into glycolysis. This interpretation also implies that changes in metabolic enzymeactivity/polymorphism may be sufficient to initiate the “switch to glycolysis” inde-pendent of the cellular oxygen concentration.

Nonhypoxic activation of HIF-1 through loss of VHL or activation of Akt orother signal transduction pathways could increase PDK1 and trigger the Warburgeffect. HIF-1–mediated increased PDK1 activity has been shown to inhibit PDH,causing the shunting of pyruvate from the mitochondria and subsequently attenuat-ing mitochondrial respiration and ROS production in MEFs.

The influx of respiratory substrate into mitochondria has been shown to be animportant determinant of cell sensitivity to oxidant-induced apoptosis. ReducedOXPHOS may hence induce low sensitivity to oxidative stress.


5 Organelle and Cell Membrane Perturbations

Complexation of the mitochondria-specific phospholipid cardiolipin (CL) with cyt-C enables the latter to act as a CL-oxygenase, which is activated during apoptosisand selectively oxidizes CL. Oxidized CL is required for the release of proapop-totic factors from the mitochondria into the cytosol. Progression of the apoptoticprogram is hence dependent on the susceptibility of CL to oxidation; which in turnis dependent on its polyunsaturation pattern [10]. Consistent with this observation,enrichment of CL with the highly unsaturated fatty acid docosahexaenoic acid con-ferred HL-60 cells with increased sensitivity to apoptosis induced by staurosporine[11].

Hypoxia may be partly the effect of the incorporation of adulterated, nonoxy-genating, or oxidized polyunsaturated fatty acids into the phospholipids of cellularand mitochondrial membranes [12]. Such incorporation is hypothesized to causechanges in membrane permeability that impair oxygen transmission into the cell.Trans fats, partially oxidized PUFA entities, and inappropriate omega 6:omega 3ratios are all potential sources of unsaturated fatty acids that can disrupt the nor-mal membrane structure and oxygen diffusivity. Significant differences have beenobserved between the phospholipid fatty acid profiles of the human colorectalmucosa among the control, adenoma, and carcinoma groups [13].

Acetoin has been shown to have a potentiating effect on citrate formation, whichin turn is responsible for cytoplasmic cholesterol synthesis from the truncatedtumoral Krebs cycle [14]. Deregulation of cholesterol synthesis has been observedin various cancer cell lines leading to its accumulation in every type of cell mem-brane. Cholesterol enrichment in membranes induces changes in biophysical proper-ties, such as increased rigidification and decreased passive proton permeability. Theaccumulation of cholesterol, in combination with altered ratios of omega 6:omega3 fatty acids, partially oxidized PUFA entities, and trans-esters of fatty acids havebeen hypothesized to impair oxygen transmission into the cell.

6 How Calorie Restriction Inhibits Carcinogenesis

Nowhere is the link between metabolism and carcinogenesis more profoundlydemonstrated than in studies of caloric restriction. CR has unequivocally been asso-ciated with increased life span and decreased carcinogenesis. In contrast, metabolicdisorders such as obesity [15] and diabetes [16] are known risk factors for the devel-opment of cancer. CR has been shown to promote mitochondrial biogenesis andincrease bioenergetic efficiency by modulating eNOS, PGC-1�, and SIRT1 expres-sion [17]. IGF1 inhibition by ER downregulates the longevity inhibitory gene prod-uct p66Shc and upregulates the induction of NRF1 and NRF2�� mRNA.

The expression of mammalian SIRT1 is induced in CR rats as well as inhuman cells that are treated with serum from such animals. SIRT1 deacetylates theDNA repair factor ku70, causing it to sequester the proapoptotic factor Bax away


from mitochondria, thereby inhibiting stress-induced apoptotic cell death. CR ratsshowed less apoptosis in skeletal muscle compared with their age-matched cohorts.

CR produces a reduction in body temperature of rodents and nonhuman pri-mates. This has been suggested to occur by an increase in the coupling of oxidativephosphorylation to ATP synthesis. Energy-deprived HepG2 hepatoma cells showedincreased mitochondrial biogenesis and OXPHOS. OXPHOS efficiency, as indi-cated by a higher ADP:O ratio, lower H2O2 production, and lower mtDNA contentwas found to correlate with Drosophila simulans survival [18]. Nutrient-induceddownregulation of OXPHOS genes in skeletal muscle has been suggested to repre-sent a thrifty response to nutrient excess, which attempts to limit the compensatoryincrease in energy expenditure normally designed to counterbalance the increase inenergy intake.

If caloric restriction causes a generalized decrease in apoptosis, then it shouldbe conducive to carcinogenesis. However, incidences of cancer are significantlydecreased in calorie-restricted animals. This apparent paradox can be explained byinvoking the hypothesis that growth advantage for mutated cells (along with theprobability of monoclonality and malignancy) is considerably increased (in partdue to decoupling and consequent deregulation of nutrient intake pathways inde-pendent of the extracellular matrix) if the normal multiclonal environment is dam-aged by a pathologic proapoptotic process that spares the apoptosis-resistant clones.If the multiclonal environment is more stress resistant and robust—as would beexpected to be the case with CR—the selective growth advantage of mutated cellsis diminished.

As an example, senescence creates a local tissue environment that promotes thegrowth of initiated or preneoplastic epithelial cells both in culture and in vivo [19].

To illustrate this effect further, CR results in a negative regulation of p53-mediated transcriptional activation due to its deacetylation by SIRT1. A slight reduc-tion in p53 activity permits the expression of several genes involved in cell-cyclearrest (p21WAF1/CIP1), DNA repair (p53R2), and those involved in protection againstoxidative stress (TIGAR, sestrins, GPX1), thereby allowing cell survival. In con-trast; a proapoptotic process mediated by elevated levels of p53 leads to the induc-tion of pro-oxidant genes (PIG3, praline oxidase), the repression of transcription ofantioxidant genes (PGM, NQO1), and results in elevated intracellular ROS levelsand cell death or senescence in nonmutated cells or tumorigenesis in mutated orapoptosis-resistant clones. This differential regulation of target genes in response tovarying levels of stress seems to be generally applicable also to ROS and Ca2+ inthe context of cell survival or death.

Apoptosis in the earlier stage of carcinogenesis may be necessary to increasethe selective pressure on the initiated cells to favor the emergence of proliferative,apoptosis-resistant cells. For example, it has been hypothesized that Bax toxicitymay be a result of a Bax-induced impairment in the ability to perform OXPHOS.Consistent with this hypothesis, it has been reported that Bax-induced lethality isdiminished under conditions that favor fermentation and that the cells that recoverfrom growth arrest after Bax expression frequently exhibit a permanent loss of res-piration competence [20]. Deregulated activity of bcl-2 family members enhances


tumor formation when overexpressed in the upper layers of the epidermis but retardstumor formation when overexpression is restricted to the basal layer.

Furthermore, apoptosis-induced selection pressure in the early stage of carcino-genesis is supported by the finding that coexpression of two proapoptotic genes(transforming growth factor �1 and the hepatitis B virus X) potentiates c-Myc–induced hepatocarcinogenesis.

Clonal expansion of potentially malignant p53-deficient cell variants was sup-pressed in Bcl-2 overexpressing tumors, presumably due to the lack of selectiveadvantages for p53-deficient cells inside a population of cells resistant to apoptosis.

There is an age-related increase in the proportion of liver mitochondria withfragile outer membranes as evidenced by an increased release of adenylatekinase in hypotonic medium. Peroxidized cardiolipid in the mitochondrial mem-brane lowers the threshold of Ca2+ for PTP induction and cyt-C release. Agingmice exhibit enhanced PTP activation in lymphocytes, brain, and liver. Theseage-related, chronic, mitochondrial degenerative, apoptosis-favoring phenomenafurther increase the probability of growth advantage of a subset of apoptosis-resistant mutant clones.

That may be why pathologic proapoptotic processes such as paroxysmal noc-turnal hemoglobinuria, myelodysplastic syndromes, chronic myeloid leukemia,secondary acute leukemias, and immunosuppression-related non–Hodgkin’s lym-phomas may be interpreted as “opportunistic” clonal and malignant diseases [21].

CR may cause a generalized increase in efficiency of OXPHOS because it causesa decreased respiratory substrate flux through the HBP and decreased levels of UDP-GlcNAc, the main substrate for protein glycosylation (see Section 3).

7 Chronic Infection and Carcinogenesis

Hypoxia is a characteristic feature of the tissue microenvironment during bacterialinfection. HIF-1� has been shown to be induced by bacterial infection, even undernormoxia, and regulates the production of key immune effector molecules, includ-ing granule proteases, antimicrobial peptides, nitric oxide, and TNF-�. Mice lack-ing HIF-1� in their myeloid cell lineage showed decreased bactericidal activity andfailed to restrict systemic spread of infection from an initial tissue focus. Conversely,activation of the HIF-1� pathway through deletion of VHL tumor-suppressor pro-tein or pharmacologic inducers supported myeloid cell production of defense fac-tors and improved bactericidal capacity. Therefore, any chronic infectious condition,whether or not triggered by pathogens, may have the ability to upregulate HIF in thevicinity of the affected cells.

Among agents that have been reported to cause uncoupling of OXPHOS toATP synthesis or a decrease in OXPHOS are Helicobacter pylori and hepatitis Band the Epstein-Barr viruses; these are also selectogens for non–Hodgkin′s lym-phoma, liver cancer, and Burkitt′s lymphoma, respectively. Epstein-Barr virus hasalso been shown to induce synthesis of HIF-1�, and the protein is also upregulated in


macrophages at normoxia after exposure to bacteria. Uncoupling protein 2 (UCP2)expression is increased in most human colon cancers, and the level of expressioncorrelates with the degree of neoplastic changes. Bacterial lipopolysaccharide hasbeen shown to increase the expression of mitochondrial UCP2 in several tissues.

Differential compartmentalization of signaling molecules in cells is emerging asan important mechanism for regulating the specificity of signal transduction path-ways. Altered stress tolerance (and therefore altered heat shock protein levels) incells with dysfunctional OXPHOS can affect protein folding and thereby disrupttheir membrane localization or rate of ubiquitination [22]. Candidates subject tosuch a pathologic response include the antiviral protein MAVS, whose disruptionof mitochondrial localization renders it unable to activate NF-�B or IRF3 to inducetype-I interferons. The resultant attenuation of innate immunity can sensitize thecell to carcinogenesis [23].

Increased MHC class I expression serves to alert the immune system to cells withmitochondrial mutations [24]. Mice deficient in MHC I accumulate mitochondrialDNA deletions in various tissues. Therefore, immunocompromised individuals maynot be effectively able to eliminate cells containing mitochondrial DNA mutations.Indeed, NHL has been reported to occur more frequently in AIDS and in posttrans-plant immunocompromised patients.

8 Electron Transport Modulation

It has been shown that the desorbed intermembrane fraction of endogenous cyt-Ccan function as an electron shuttle between the outer and inner membranes of intactmitochondria. The oxidation of extramitochondrial NADH can proceed throughthe rotenone-insensitive respiratory chain of the outer membrane, leading to cyt-b5 reduction. From cyt-b5, electrons are channeled via the intermembrane cyt-C toCOX. In physiologic conditions, cyt-C is constitutively released outside the mito-chondria to activate the exogenous NADH/cyt-C electron transport pathway. Thispathway, at least part of which routes through the VDAC, may attempt to maintainthe membrane electrochemical potential that is necessary for ATP synthesis in theevent of dysfunction of the respiratory chain upstream of complex IV [25].

It has been demonstrated that electrons from the external Nde1p have the rightof way over those coming from either Gut2p or Ndip in Saccharomyces cerevisiae.Might a differential regulation for electron transfer in mammalian cells exist, suchthat under normal conditions, electrons originating from the citric acid cycle arepreferentially used over those originating from the exogenous NADH/cyt-C path-way? Consistent with this proposition is the observation from control analysis thatNADH oxidation has negligible control over the carbon flux through OXPHOS.Under these circumstances, blockage of the citric acid cycle or a general reductionof mitochondrial OXPHOS could shift the bulk of the burden of electron transfer tothe exogenous NADH/cyt-C pathway. This may necessitate a greater concentrationof extramitochondrial cyt-C to maintain mitochondrial ATP levels, which, coinci-dently, may be enough to activate procaspase 9 and cause apoptosis. This may also


explain why cyt-C has been chosen by biological evolution to be a mediator of bothrespiration and apoptosis and why gene promoters regulating the cell cycle containHRE elements (see Section 11).

9 The “Switch to Glycolysis” Is Necessary to Induce Apoptosis

Lymphocytes, enterocytes, and fetal tissues that exhibit rapid growth are poorlyoxidative, whereas highly oxidative tissues such as kidney cortex or brain are nor-mally quiescent.

It has long been noted that tumor cells display elevated rates of glycolysis,despite oxygen availability. Crabtree observed that even though most tumor cellsare capable of oxidative phosphorylation, extracellular glucose represses oxida-tive phosphorylation in tumor cells via an unknown mechanism. Self-sufficiency ingrowth signals and the ability to take up nutrients independently of the extracellularenvironment has been postulated to be a primary acquired capability of cancer.

It has been demonstrated that eosinophils die by apoptosis even in the absenceof a functioning OXPHOS. They do so by importing and hydrolyzing glycolyticATP into mitochondria to maintain a transmembrane potential. This may be accom-plished by operating the bidirectional FoF1-ATPase proton pump in reverse. Stimulitriggering apoptosis have been shown to be partly attributable to the reverse oper-ation of this H+ pump, which causes an increase in mitochondrial matrix pH—anearly event associated with mitochondrial dependent apoptosis.

Several reports in the literature suggest that the increase in ATP levels in cellsen route to apoptosis derives from both OXPHOS and glycolysis. It has also beenshown that apoptosis can occur in cells that lack functional mitochondria and thatATP synthesized via glycolysis plays a critical role in preventing the collapse ofthe mitochondrial membrane potential thereby allowing the cell to die by apoptosis.Further support for this hypothesis comes from the observation that staurosporine-induced apoptotic ATP elevation in caspase-competent HeLa cells occurs via Ca2+-dependent activation of glycolytic ATP synthesis, with little or no contributionfrom mitochondrial OXPHOS. TNF-induced hepatic apoptosis could be selectivelyblocked by ketohexose-mediated ATP depletion.

Because apoptosis is generally recognized as an ATP-dependent process, glycoly-sis may provide adequate ATP to activate caspases. Procaspase 9 may be activated byan increase in cytosolic cyt-C levels, which in turn are increased due to age-related orpathogenic attenuation of mitochondrial OXPHOS (discussed in Section 8).

Cytosolic acidification caused by the glycolytic product, lactic acid, has beenshown to be required for induction of caspase activation and apoptosis; it alsodecreases the ability of hexokinase to bind to the outer mitochondrial membrane.Increased susceptibility to stress-induced apoptosis in c-myc transformed cells hasbeen linked to the overexpression of the LDH gene.

This leads to the intriguing—as yet experimentally unproven—proposition thattransient aerobic glycolysis may precede apoptosis in OXPHOS-deficient non-transformed cells and may even be necessary for such cells to undergo programmedcell death.


On the other hand, in transformed or preneoplastic cells, where caspase-dependent apoptotic pathways are blocked or rendered nonfunctional by other non–OXPHOS-related mutations (such as PTEN, P53, TSC2, VHL, etc.), the shift toglycolysis to supply adequate ATP to fuel caspase-dependent apoptotic pathwaysmay, ironically, give these cells a selective growth advantage over non-transformedneighboring cells. Because glucose phosphorylation, the first committed step of gly-colysis, is sufficient to promote cell survival by Akt, it seems possible that even atransient shift to glycolysis in the presence of non–OXPHOS-related mutations maypromote cell immortalization.

10 Regulation of the Cell Cycle and the Apoptotic PathwayIs Sensitive to Glucose Concentration

Increased rates of glycolysis in cultured proliferating human lymphocytes and ratthymocytes have been reported to peak in concert with DNA synthesis. The gly-colytic enzymes GAPD and LDH have been found in transcriptional coactivatorcomplexes with the OCT-S transcription factor, which stimulates the expression ofhistone H2A as cells enter the S-phase. Similar to these glycolytic enzymes that dis-play nonglycolytic functions among their regulatory DNA sequences, inhibition ofPFK-2, coded by the pfkfb3 gene, produces proapoptotic effects in HeLa cells. Thepfkfb3 gene promoter contains HIF-1 binding sites.

The cyclin D1 promotor contains two consensus HREs. Cyclin D1 has beenshown to be overexpressed in VHL-deficient RCC cell lines. A cyclin D1–dependentkinase was found to phosphorylate and inactivate NRF1 with a consequent decreasein OXPHOS. The proapoptotic protein, BAD, is required to assemble a mitochon-drially located complex that also contains hexokinase IV. Furthermore, the phos-phorylation status of BAD regulates hexokinase IV activity.

The GlcRE within the PK promoter was found to be sufficient to confer the mod-ulation by HIF-1 of the glucose-dependent induction of the PK gene expression.HIF-1 also activates telomerase via transcriptional activation of the human telom-erase reverse transcriptase gene.

These results suggest that regulatory points exist in the cell cycle, oxygen sensingand apoptotic pathways that are sensitive to glucose metabolism thereby providingdirect biochemical and genetic links between oxygen availability, metabolism, andcarcinogenesis.

11 Temporal and Spatial Effects in Tumorigenesis

In VHL-deficient RCC cell lines, the resulting stabilization of HIF-1� negativelyregulates mitochondrial biogenesis by inhibiting the transcription of the gene encod-ing the coactivator factor PGC-1�. This is achieved by the downregulation of c-myc.The concomitant stabilization of HIF-1�, on the other hand, increases c-myc activ-


ity and promotes cell-cycle progression in part through increased c-myc promoterbinding. Even though such opposing transcription factors (HIF-1� and HIF-1�) maycoexist during hypoxia, the differential activation both temporally and spatially oftheir downstream gene targets may still drive tumor growth instead of a net “cancel-lation” of their effects [26].

The variation of the intermediary metabolism of tumors with time has been pro-posed with respect to the cellular energy sensor, AMPK. This kinase facilitates glu-cose and fatty acid metabolism. However, it also switches off anabolic processes,including proliferation through mTOR via TSC2. Active AMPK, therefore, hasproperties that facilitate advanced tumorigenesis yet reduce the selective advantageof cancer cells. The solution to this paradox is that some advanced cancers oftenretain a sufficient complement of defective downstream tumor suppressors, whichallow a degree of AMPK activation while mitigating its growth-limiting effect. Forexample, some cancers harbor TSC2 mutations, allowing AMPK activation withoutmTOR suppression. On the other hand, for premalignant cells that are not limitedby substrate supply and that retain intact cellular senescence machinery such as p53,TSC2, and mTOR, active AMPK is a disadvantage because it slows cellular prolif-eration. The inhibition of AMPK at this stage in tumorigenesis may be achieved,in part, by increasing mitochondrial ATP production. Indeed, mitochondrial biogen-esis has been shown to increase to its greatest extent at an intermediate degree oftransformation rather than at the fully transformed state.

12 The Warburg Model

The sequence of events arising from the defective functioning of the electron trans-fer chain in mitochondria may thus be projected to occur thus: Because the elec-tron transfer chain uses reducing equivalents in the form of NADH as a source ofelectrons, a defect in this chain (due to multiple reasons) leads to an intramitochon-drial elevation of (unused) NADH levels. This leads to retrograde product inhibitionof the citric acid cycle, which in turns leads to a similar retrograde inhibition ofthe NADH shuttle that transfers reducing equivalents from the cytosol to the mito-chondrion [8, 27]. The increase in mitochondrial NADH levels is thus spread to thecytoplasm. The increased NADH level in the cytosol facilitates the conversion ofpyruvate into lactate via aerobic glycolysis, thereby diverting the pool of aerobicpyruvate away from the mitochondria to compensate for the lack of electron trans-port and oxidative phosphorylation. At the same time, this increased NADH/NAD+

ratio also acts to limit respiratory flux through glycolysis and promotes senescence;both effects are due to inhibition of key glycolytic enzymes, especially GAPD.

The forced conversion of pyruvate to lactate via aerobic glycolysis [1] usesNADH thereby serving as a mechanism to decrease NADH-mediated Akt activa-tion [28] and [2] generates NAD+, which can in turn activate PARP to force the cellto undergo necrosis [29] partly by a mechanism that induces poly(ADP)ribosylationand inactivation of GAPD [30]. An increased concentration of lactate acidifies the


cytosol, which is conducive to activation of caspases and the accumulation of theproapoptotic protein BNIP3 [31]. Cytosolic acidification also decreases the abilityof hexokinase to bind to the outer mitochondrial membrane. 2-Methyl glyoxal, thefinal product of aerobic glycolysis, acts to inhibit further increase in glycolysis bya retrograde product inhibition. Lastly, the aerobic route of glycolysis makes thecell more susceptible to death by glucose deprivation and death-receptor–mediatedapoptosis [32] (Table 1).

The increased cytosolic NADH levels are also postulated to increase the burdenof electron transfer to the exogenous NADH/cyt-C electron transfer pathway. Thisnecessitates an increase in the concentration of extra-mitochondrial cyt-C, whichcoincidently may be enough to trigger activation of APAF and the activation ofprocaspase 9—ultimately activating apoptosis.

Because the mitochondria cannot produce ATP from the oxidation of glucose viathe electron transfer chain, it attempts to bring in glycolytic ATP into the mitochon-dria via the ANT/VDAC channels. Because the import of phosphate into mitochon-

Table 1 Apoptotic Pathways Activated by Glycolysis

Attribute Effect

GAPD NADH / NAD+

ratio Exogenous NADH/cyt-C

electron pathway

ROS PARP, GAPD

NADH Akt

NAD+ PARP

Lactate pH, BNIP3, Caspases

Hexokinase binding to mitochondrial membrane

PTP flux

Reverse operation of ATPase

Mitochondrial pHATP

Disproportionate decrease in flux through glycolysis than through OXPHOS

Increasingfluxthroughglycolysis

Glycolysis, LDH Susceptibility to stress,glucose deprivation anddeath receptor

Increasedapoptosis,necrosis

Increasing metabolic flux through glycolysis activates proapoptotic pathways and mechanisms.These may induce death in cells whose OXPHOS is impaired but which possess functional tumorsuppressors and apoptotic machinery. Metabolic deviation may therefore act as a “self-regulatingmechanism” in such cells to prevent carcinogenesis. On the other hand, OXPHOS dysfunctionin association with other mutations may tip the balance toward proliferation, thereby causingcarcinogenesis.


dria is blocked, the mitochondrial transmembrane potential can only be maintainedby operating the FoF1-ATPase pump in reverse, by hydrolyzing the mitochondrialATP pool to pump protons out into the intermembrane space. This results in thealkalinization of the mitochondrial matrix, which, in tandem with the acidificationof the cytosol, is generally recognized as the first step to initiate apoptosis. Controlanalysis studies show that ATP consumption exercises significantly more controlover the flux through glycolysis than it does through OXPHOS [33]; consequently,an overall decreased cellular ATP level should disproportionately decrease carbonflux through glycolysis than it does through OXPHOS.

13 Conclusion

Although aberrant functioning of the mitochondrial OXPHOS respiratory apparatusis well documented in the literature beginning with the observations by Warburg,emerging evidence suggests that the respiratory contribution from OXPHOS andfrom the cell surface may not be negligible, when compared with that from gly-colysis, in proliferating transformed cells. These observations, when taken togetherwith evidence suggesting that glycolysis may be a cell-death mediator of last resort[34]—capable of activating cell death pathways either on its own or in conjunctionwith OXPHOS—suggest that deficient OXPHOS may be sufficient but not neces-sary to initiate malignancy, and the “switch to glycolysis” may be coincidental tobut not causative of this transformation. In fact, it may be speculated that cells withadequately functioning tumor suppressors and apoptotic machinery may be able toderive the majority of their cellular energy requirements by “switching” back andforth between OXPHOS and glycolysis numerous times over their life span depend-ing on substrate or oxygen availability or as a response to moderate oxidative stresswithout undergoing transformation.

In pancreatic beta cells, the ATP derived from mitochondrial pyruvatemetabolism does not significantly contribute to the glucose-stimulated insulin secre-tion; instead, the glycolytic metabolism of glucose is critically involved in this sig-naling process [35]. If the cellular energy sensor AMPK in tumor cells were alteredsimilarly, its “setpoint” would be decreased significantly, allowing the cancer cell tomaintain respiratory flux through anabolic metabolic processes even in the presenceof significantly reduced cellular ATP (due to impaired OXPHOS).

More studies such as the one cited in reference [36] are needed that specificallyinduce a selective block of aerobic glycolysis to ascertain its effect on the initiationand maintenance of the malignant phenotype. The contribution of pathways otherthan OXPHOS and glycolysis, such as the electron transport pathway activated byexogenous cyt-C and the trans plasma membrane electron transport pathway, shouldbe investigated in much more detail with regard to substrate utilization, oxygen con-sumption, and energy generation. Experiments should be designed to supply cellcultures not only with just glucose or glutamine but also with the whole comple-ment of other substrate fuels at physiologic concentrations. Metabolic alterations


should be examined in the absence of growth factor stimulation because of its facil-itating effects on the metabolic shift toward aerobic glycolysis [36]. Such metabolicalterations can be quantitatively measured through control analysis, and this tech-nique could be expanded to include hypoxic and substrate-enhancing or -limitingconditions as well. Levels of the whole complement of glycolytic enzymes shouldbe measured in order to be able to make an accurate assessment of deviations ofmetabolic flux in non-transformed as well as in transformed cells. Effects shouldbe compared between primary, immortalized, and neoplastic cells to dissect cell-specific effects.

14 Abbreviations

AIF, apoptosis inducible factor; Akt, v-akt murine thymoma viral oncogene;ALDH2, aldehyde dehydrogenase allele 2; AMPK, AMP activated protein kinase;ANT, adenine nucleotide translocator; BCC, basal cell carcinoma; Bnip3, Bcl-2/adenovirus E1B 19 kDa interacting protein 3; CCN, connective tissue growthfactor and nephroblastoma–overexpressed gene; CCRC, clear cell renal carci-noma; cGDPH, cytosolic glycerol-3-phosphate dehydrogenase; CHO, Chinese ham-ster ovary; COX, cytochrome c oxidase; CR, calorie restriction; CREB, cAMPresponsive element binding; CtBP, C-terminal binding protein; cyt-C, cytochromeC; DM2, type 2 diabetes mellitus; E1A, adenovirus 5 early region 1A onco-protein; ECM, extracellular matrix; eNOS, endothelial nitric oxide synthase; ES,embryonic stem; FCCP, carbonylcyanide-p-trifluoromethoxyphenylhydrazone; FH,fumarate hydratase; GAPD, glyceraldehyde-3-phosphate dehydrogenase; GlcRE,glucose responsive element; GPI, glucose phosphatase isomerase; GPX1, glu-tathione peroxidase 1; Gut2p, glycerol 3-phosphate dehydrogenase; HBP, hex-osamine biosynthetic pathway; HIF, hypoxia inducible factor; HRE, hypoxia reg-ulating elements; IAP-2, inhibitor of apoptosis protein 2; IGF-1, insulin growthfactor; IRF3, interferon regulating factor 3; LDH, lactate dehydrogenase; LKB1, ser-ine threonine protein kinase 11; MAVS, mitochondrial antiviral signaling protein;Mdm2, mouse double minute 2; MDR, multidrug resistance; MEF, mouse embryofibroblasts; MELAS, mitochondrial encephalomyopathy, lactic acidosis and stroke-likeepisodes;MMP,matrixmetalloproteinase;mtDNA,mitochondrialDNA;mtETC,mitochondrial electron transport chain; mTOR, mammalian target of rapamycin;NAD+/NADH, nicotinamide adenine dinucleotide (oxidized and reduced forms,respectively); Nde1p and Nde2p, external NADH dehydrogenases; NDUFA1, NADHdehydrogenase (ubiquinone) alpha subcomplex 1; NF-�B, nuclear factor kappa B;NHL, non–Hodgkin’s lymphoma; NQO1, NADPH dehydrogenase quinone 1; NRF,nuclear respiratory factor; NSCLC, non–small cell lung cancer; O-GlcNAc, O-linkedacetylglucosamine; OXPHOS, oxidative phosphorylation; p21Waf1/Cip1, cyclin depen-dent kinase inhibitor 1A; PDC, pyruvate decarboxylase; PDH, prolyl hydroxylase;PDH, pyruvate dehydrogenase; PDK1, pyruvate dehydrogenase kinase 1; PFK-2,6-phosphofructo-2 kinase/fructose-2,6-bisphosphatase; PGC, peroxisome prolifer-


ator activator protein–�–coactivator-1�; PGM, phosphoglycerate mutase; PIG3, p53inducible gene 3; PK, pyruvate kinase; PNP, purine nucleoside phosphorylase; PPAR,peroxisome proliferator activated receptor; PTEN, phosphatase and tensin homolog;PTP, permeability transition pore; PUFA, polyunsaturated fatty acids; RCC, renalcell carcinoma; ROS, reactive oxygen species; SCC, squamous cell carcinoma; SDH,succinyl dehydrogenase; SIRT1, silent information regulator 2-homolog 1; SOD,superoxide dismutase; TCA, tricarboxylic acid; TIGAR, TP53 induced glycolysis andapoptosis regulator; TIMP, tissue inhibitor of matrix metalloproteinase; TNF, tumornecrosis factor; TSC2, tuberous sclerosis complex; UCP2, uncoupling protein 2;UDP-GlcNAc, uridine diphosphate-N-acetylglucosamine; UPA, urokinase plasmino-gen activator; VDAC, voltage-dependent anion channel; VHL, von Hippel–Lindau;VLB, vinblastine.

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Mitochondrial DNA Mutations in Tumors

Anna Czarnecka and Ewa Bartnik

Abstract Mitochondria are subcellular organelles with the most well-known andbest-characterized function of adenosine triphosphate (ATP) production throughoxidative phosphorylation (OXPHOS). Mitochondria play an important role inapoptosis, a fundamental biological process by which cells die in a programmedmanner and are the strategic point in the cell death cascade. Alterations in respira-tory activity and mitochondrial DNA (mtDNA) abnormalities appear to be a generalfeature of malignant cells. The presence of mtDNA mutations has been reported inmost types of cancer. However, the functional consequences and clinical relevanceof these mutations are not clear.

Keywords Mitochondria · Mutations · Cancer · Tumor · DNA

1 Introduction

Mitochondria are eukaryotic organelles involved in many metabolic pathways andhave a principal function of generating most of the cellular ATP through oxidativephosphorylation (OXPHOS). Mitochondria are semiautonomous organelles that notonly perform essential functions in cellular metabolism but also play an importantrole in the regulation of cell death, signaling, and free radical generation [1]. Mito-chondria possess a double-membrane structure and their own genome along withtheir own transcription, translation, and protein assembly machinery [2]. Neverthe-less, mitochondrial functions involve an interplay between the mitochondrial andnuclear genomes [3]. Mitochondria are called “cellular powerhouses”; the primaryfunction of these organelles is to produce ATP in the process of oxidative phospho-rylation. At the same time, many housekeeping metabolic reactions take place in this

E. Bartnik (B)Department of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, and Instituteof Biochemistry and Biophysics, Polish Academy of sciences, Pawinskiego 5a, 02106 Warsaw,Polande-mail: [email protected]


119

120 A. Czarnecka, E. Bartnik

cell compartment including production of intermediates of carbohydrate, nucleotide,fatty acid, and amino acid metabolism. Mitochondria are indispensable for the cellbecause of their key role in homeostasis, which is probably the reason for theirmaintenance within cells lacking mtDNA (called o) [4]. Processes believed to beaffected by mitochondrial function include climatic adaptation, aging, longevity,degenerative disease, and cancer [3].

Numerous diseases are known to be caused by mutations in the mtDNA. A break-through in mitochondrial pathophysiology occurred in 1988 when Wallace et al. [5]found the first point mutation in the mtDNA of patients with Leber’s hereditary opticneuropathy. To date, the etiology and pathophysiology of mitochondrial diseaseshas remained perplexing. The essential role of mitochondrial oxidative phosphory-lation in cellular energy production, the generation of reactive oxygen species, andthe initiation of apoptosis has suggested a number of novel mechanisms for mito-chondrial pathology. Mutations in mtDNA appear to be the primary factor respon-sible for mitochondria-related diseases and syndromes. The number of known pointmutations, deletions, and duplications of mtDNA is more than several hundred andnew mutations are annotated every month, and the number of mtDNA polymor-phisms has exceeded 1000 [6]. The recent resurgence of interest in the study ofmitochondria has been fueled in large part by the recognition that genetic and/ormetabolic alterations in this organelle are causative or contributing factors in a vari-ety of human diseases including cancer. The role for mitochondria in tumorigenesiswas hypothesized when tumor cells were found to have an impaired respiratorysystem and high glycolytic activity [7]. Recent findings elucidating the role of mito-chondria in apoptosis and the high incidence of mtDNA mutations in cancer samplesfurther support this hypothesis [3].

Defects in mitochondrial function have long been suspected of contributing tothe development and progression of cancer. In the early 1920s, Warburg pioneeredthe research on mitochondrial respiration alterations in the context of cancer andproposed a mechanism to explain how they evolve during cell transformation. Hehypothesized that a key event in carcinogenesis involved the development of aninjury to the respiratory machinery, resulting in compensatory increases in gly-colytic ATP production. Eventually, malignant cells would satisfy their energy needsby producing a large portion of their ATP through glycolysis rather than throughoxidative phosphorylation [7]. Because of the inherent inefficiency of glycolyticATP generation, cancer cells represent a unique metabolic state and require highconsumption of glucose to fulfill cellular energy requirements [4]. Hypoxia itselfhas been studied as a carcinogenic factor. It has been proved that aerobic glycolysisis highly active in malignant cancers. In benign tumors, the rate of aerobic gly-colysis is only one third of the malignant cancer aerobic glycolytic activity. Highaerobic glycolysis activity must correlate with glucose absorption by the cell. Invivo measurements have been performed, and high glucose uptake by cancerous tis-sue in comparison with normal tissues was reported for gliomas, meningiomas, andsarcomas [7].

To date, mitochondrial failure has been reported at all levels of their structureand function, including abnormal ultrastructure and metabolism deregulation. First

Mitochondrial DNA Mutations in Tumors 121

of all, aerobic energy metabolism alterations have been assigned to disruption of thenuclear encoded enzymes fumarate hydratase and succinate dehydrogenase expres-sion (complex II of the respiratory chain). It is worth noting that mutations in thesetwo genes can also behave as classic tumor suppressors linking mitochondrial phys-iology with cancer [8]. In addition, subunits of complexes of the respiratory chainare also encoded by the mitochondrial genome (mtDNA) [3]. Accordingly, mtDNAalterations are frequently observed in tumor cells [4, 9]. Mutations in the mtDNAmay result in altered structure of mitochondrial proteins and thus disrupt OXPHOS,which might shift metabolism toward anaerobic respiration. However, evidence fordirect linkage of respiratory deficiency in a specific tumor type with a specificmtDNA mutation is still missing [8].

2 The Mitochondrial Genome

In 1981, the complete sequence of human mtDNA was determined by Sanger andco-workers [10]. All genes have been identified and characterized [3]. HumanmtDNA is a double-stranded, closed-circular molecule of approximately 16.6 kb,which corresponds with a molecular weight of about 10 million Da, and in mostcells it represents only about 0.5% to 1% of total DNA content [11]. The normalmtDNA state is thought to be a supercoiled structure, and it is poorly associatedwith proteins in comparison with nuclear DNA. The two strands of mtDNA can bedistinguished due to their different G content and can be separated by density indenaturing gradients into a heavy or H-strand and a light or L-strand [2]. Each mito-chondrion contains 2 to 10 copies of its genome, and a mammalian cell typicallycontains 200 to 2000 mitochondria. As a consequence of mtDNA multiplication inthe cell, mitochondrial genomes can tolerate very high levels (up to 90%) of dam-aged DNA through complementation by the remaining wild type [2]. MitochondrialDNA encodes a small, but essential number of polypeptides of the OXPHOS sys-tem. The coding sequences for two ribosomal RNAs (rRNAs), 22 transfer RNAs(tRNAs) and 13 polypeptides, which are subunits of four OXPHOS complexes,are contiguous and lack introns (see Fig. 1 and Table 1). The tRNAs are regu-larly interspersed between the rRNA and protein-coding genes, playing a crucialrole in RNA maturation from the polycistronic transcripts. A single major 1.1-kb-long noncoding region, called the displacement loop (D-loop), contains the mainregulatory sequences for transcription and replication initiation [2]. The D-loop is atriple-stranded structure in which a nascent H-strand DNA segment of 500 to 700nucleotides remains annealed to the parental L-strand. This is the region that ismost variable in sequence. However, the whole mtDNA is much more variable insequence than nuclear genome (nDNA), as it is more vulnerable to mutations dueto the lack of histone protection, limited repair capacity, and close proximity to theelectron transport chain, which constantly generates superoxide radicals. A higherrate of mutation is accompanied by higher ratio of nonsynonymous to synonymoussubstitution in mtDNA than in nuclear genes [12]. As mtDNA lacks introns, mostmutations occur in the coding sequences and are thus likely to be of biological


Fig. 1 Human mitochondrial DNA molecule. ATP6/8, ATP synthase F0 subunit 6/8; COX I,cytochrome c oxidase subunit I; COX III, cytochrome c oxidase subunit III; CYT B, cytochromeb; HSP, heavy-strand promoter; LSP, light-strand promoter; ND1, NADH dehydrogenase subunit1; ND2, NADH dehydrogenase subunit 2; ND3, NADH dehydrogenase subunit 3; ND4L, NADHdehydrogenase subunit 4L; ND4, NADH dehydrogenase subunit 4; ND5, NADH dehydrogenasesubunit 5; ND6, NADH dehydrogenase subunit 6; OH, H-strand origin; OL, L-strand origin; A-W,tRNAs.

consequence. The mitochondrial genome has other characteristics that distinguishmtDNA from nDNA. These include not only a higher rate of mutation but also theuse of a divergent genetic code and transmission by maternal inheritance [3].

It has been proposed that dysregulation of mtDNA genes may contribute tobioenergetic imbalances as it is known that some rapidly growing tumors displayalterations of oxidative metabolism and high rates of aerobic glycolysis. Furtherexperiments have shown that mutations in D-loop, OXPHOS genes, and mitochon-drial tRNA are characteristic for cancer cells. Moreover, mutations and deletions inmtDNA that have been observed in various solid tumors and hematologic malig-nancies are associated with abnormal expression of mtDNA-encoded proteins [11].Mutations in a single mtDNA genome are silent, but as soon as the proportion ofmutant mtDNA exceeds a critical threshold concentration, a defect of OXPHOS,integrity of inner mitochondrial membrane, electron leakage, and increased reactiveoxygen species (ROS) production result [3]. Clonal expansion of a single somaticmtDNA mutation has substantial implications for a cell, as mtDNA mutations are atthe basis of a number of human pathologies, not only cancer. This state of art is aderivative of the fact that a combination of different respiratory chain complexes (I,


Table 1 Mitochondrial Genes Encoded in the Mitochondrial Genome (mtDNA) and NuclearGenome (nDNA)

Mitochondrial genes

Genes encoded Total number of mtDNAMitochondrial complex in mtDNA mtDNA genes and nDNA genes

Complex I NADHdehydrogenase

7 ND1, ND2, ND3, ND4,ND4L, ND5, ND6

43

Complex II FADdehydrogenase

0 — 4

Complex III cytochromeb-c1

1 Cytb 11

Complex IVcytochrome oxidase

3 COX1, COX2, COX3 13

Complex V ATPsynthase

2 ATP6, ATP8 16

Total 13 87

III, IV, and V) defects might have substantial physiologic effects, for example maylead to a dysregulation of oxidative phosphorylation, which in turn allows robustproduction of the carcinogenic ROS. Growing molecular evidence suggests that can-cer cells exhibit increased intrinsic oxidative stress, due in part to oncogenic stim-ulation, increased metabolic activity, and mitochondrial malfunction, which turnson the vicious circle of oncogenesis [13]. Given the critical role of mitochondriain apoptosis, it is conceivable that mutations in mtDNA in cancer cells could alsosignificantly affect the cellular apoptotic response to anticancer agents and promotemultidrug resistance [14]. At the same time, other studies have shown that mousecells carrying nuclei from tumors expressed tumorigenicity, whereas cell with non-cancerous nuclei and mitochondria from cancer cells did not [15]. Thus at leastfor some cancer cells, the mitochondria do not seem to have a profound effect onthe cancerous state of the cell. On the other hand, this does not exclude their rolein tumor growth and progression, and this has been experimentally demonstrated[16, 17].

Interest in potential therapeutic agents that could act on the mitochondria intumor cells has fueled an explosion in the research on mitochondria and mtDNAin recent years, and although the role of mitochondria in tumorigenesis remainsunclear, numerous publications emphasize the impact of mtDNA mutations in theprocess of carcinogenesis [3, 4, 9]. Reports on mtDNA mutations in cancer pub-lished to date present an unclear view of overwhelming mutation/polymorphismdata with doubtful clinical correlations. Moreover, much of the literature demon-strating mutations—whether heteroplasmic or homoplasmic—has been burdenedwith technical and conceptual errors that many times reduce the finding of mtDNAmutations in tumors to the level of technical artifacts [18, 19]. Thus far, a lim-ited number of mtDNA mutations are known that can be classified as pathologicmarkers. These include a 4977-bp deletion in Hurthle cell thyroid carcinoma;


mutations of complex I and IV in thyroid carcinoma arising from A cells; anda 264-bp deletion (3323–3588) in renal cell carcinoma. Further analysis revealedthat accumulation of mtDNA mutations is correlated with tumor aggressiveness andmultidrug resistance or with poor prognosis and acute symptoms and signs [1, 4,11]. The increasing ease with which the mitochondrial genome can be analyzed hashelped to recognize mtDNA mutations as a frequent event in the carcinogenic pro-cess. However, it is difficult to estimate the true prevalence of mtDNA-related can-cer owing to many clinical presentations and the involvement of numerous causativemutations.

3 Mitochondrial Genome Instability in Cancer

Mitochondrial genome instability (mtGI) in cancer can be defined as the occur-rence of mutations in tumor cells that are not found in the normal cells of the sameindividual [20]. The mtDNA mutation rate is 100-fold higher than in the nucleargenome [4, 11, 20], and several reasons are invoked to explain this fact, whichinclude (a) mtDNA is less protected by proteins, (b) it is physically associated withthe mitochondrial inner membrane where damaging ROS are generated, and (c) itappears to have less efficient repair mechanisms than does the nucleus, as subunit �of mitochondrial polymerase � has only limited 3′–5′ exonuclease activity [4, 20].Also, malfunction of mismatch repair (MMR) and slipped strand mispairing (SSM)should be considered as mtGI factors [20]. Mitochondrial DNA mutations arise notonly as a consequence of carcinogenetic stress but also as a result of polymerasegamma errors (T to C or G to A transitions) and are accumulated in daughter cellsas consequence of genetic drift [21].

One of the intrinsic factors responsible for mtDNA mutation generation are ROSproduced by the OXPHOS system located in the inner membrane. Because the mito-chondrial respiratory chain is a major source of ROS generation in the cells and thenaked mtDNA molecule is in close proximity to the source of ROS, the vulnerabilityof the mtDNA to ROS-mediated damage appears to be a mechanism to amplify ROSin already stressed cancer cells. Damaged, misfolded, or unfolded OXPHOS pro-teins in turn promote even more pronounced ROS production, which drives a viciouscircle of cell transformation [3, 4, 11, 20]. The proposal that mtDNA mutations andrespiratory dysfunction may be linked directly to carcinogenesis via apoptotic orROS-mediated pathways is challenging but urgently needs experimental proof incancer cells, preferably in specimens from cancer patients. To date, ROS overpro-duction has been demonstrated only in cells harboring mtDNA mutations responsi-ble for mitochondrial diseases, the most common being NARP (neurogenic muscleweakness, ataxia and retinitis pigmentosa), MELAS (mitochondrial encephalomy-opathy lactic acidosis, and strokelike episodes), MERRF (myoclonic epilepsy andragged-red fibers), LHON (Leber hereditary optic neuropathy), and KSS (Kearns-Sayre syndrome) [22].

Moreover, it is worth emphasizing that some somatic mutations found in cancercells are sometimes identical to those found in mitochondrial disease [23–25], which


offers the possibility of extrapolating mitochondrial disease defects into cancer cells.Nevertheless, further in vivo experiments are indispensable to validate the proposedhypothesis.

4 Homoplasmy and Heteroplasmy in Cancer Cells

Cells are polyploid with respect to mtDNA: most mammalian cells contain hun-dreds of mitochondria and subsequently mtDNA copies. If in a given individual,all mtDNA copies are identical, a condition known as homoplasmy arises, but ifmutations occur, are amplified and coexist with wild-type mtDNA, this is desig-nated heteroplasmy. At cell division, mitochondria and their genomes are randomlydistributed to daughter cells, and hence, starting from a heteroplasmic situation,homoplasmy and different levels of heteroplasmy may be found in daughter celllineages [2, 20, 26]. In cancer cells, mutated mtDNA can be found as homo- orheteroplasmic [4], but many reports show that mtDNA mutations in cancer cellsare homoplasmic. This raises the question of mechanisms responsible for normal tomutated mtDNA shift in the process of carcinogenesis [20, 26]. At least two scenar-ios are probable that drive this change. First of all, mutant mitochondria that losea certain mtDNA fragment by deletion can be considered to replicate more rapidlythan normal ones, resulting in an advantage in intracellular mitochondrial competi-tion (replicative advantage). If the competition is intense (high rate of proliferation),heteroplasmic cells possessing both types of mitochondria give rise to homoplasmicdaughter cells including mutant mitochondria only, with high probability. Accordingto mathematical models, the rate of switching from wild type to mutated mitochon-drial DNA is affected by factors including the intensity of intracellular competi-tion and the effective population size [27]. Researchers in several laboratories whoreported a high frequency of homoplasmic mitochondrial DNA mutations in humantumors proposed not only replicative advantage for mutated mtDNA copies [28] butalso an advantage in growth and/or tumorigenic properties for a cell containing cer-tain mtDNA mutations [24]. Polyak et al. [28] have proposed that mitochondrialgenomes replicate more rapidly to balance OXPHOS failure and reduced ATP pro-duction. On the other hand, it cannot be excluded that homoplasmy arises entirely bychance in tumor progenitor cells, without any physiologic advantage or tumorigenicrequirement. Through extensive computer modeling, it has been demonstrated thatthere is sufficient opportunity for a tumor progenitor cell to achieve homoplasmythrough unbiased mtDNA replication and sorting during cell division [29]. Com-puter modeling has shown that random drift is also a sufficient mechanism to explainthe homoplasmic nature of cancer cells [4]. Relaxed replication phenomenon anal-ysis in normal cells has proved that replicative or metabolic advantage is not indis-pensable for homoplasmy to arise [4, 24, 26]. Although a role for other mechanismsis not excluded, random processes are sufficient to explain the incidence of homo-plasmic mtDNA mutations in human tumors. Depending on how the mutant copiesare distributed between the stem and transition cells, the mutants are depleted orenriched, and the number of mutants in the lineage fluctuates as a random walk.


On average, only approximately 70 generations are required for a mutation that isdestined to become homoplasmic. The number of 70 generations is small comparedwith the number of cell divisions that a tumor progenitor cell is expected to undergo[28]. To summarize, whereas it cannot be excluded that selection occurs in a subsetof tumors, there is no need to invoke a physiologic advantage or a role for mito-chondrial mutations in tumorigenesis to explain the existence of homoplasmy or itsobserved frequency [29]. However, a mathematical model cannot prove or disprovethat a phenomenon is occurring in growing cells, as it is based on assumptions anddoes not constitute experimental proof.

5 Conclusion

Since the initial publications by Warburg [7] more than 50 years ago, a number ofcancer-related mitochondrial defects have been identified and described in the liter-ature. Growing evidence suggests that cancer cells exhibit increased intrinsic mito-chondrial stress, due in part to oncogenic stimulation, increased metabolic activity,and mitochondria-nucleus signaling malfunction [30]. Despite the increased identi-fication of signatures of mtDNA damage in transformed cells, the phenotypic effectsof these genetic changes remain to be established. While there are many reports ofthese phenomena, the mechanisms responsible for the initiation and evolution ofmtDNA mutations and their roles in the development of cancer, drug resistance,and disease progression still remain to be elucidated [9]. Research into the identi-fication of altered expression patterns of mitochondrial proteins in cancer cells hasbeen made possible by the relatively recent development of mitochondrial func-tional proteomics. The potential of this field may be realized in the identifica-tion of new markers and risk assessment as well as therapeutic targets [14, 30].Despite the difficulties with mitochondrial proteomics, it is likely that the combi-nation of the mitochondrial genetic and the proteomic approaches will provide aneffective weapon in the fight against cancer. It is hoped that this strategy will pro-vide specific genetic markers and protein profiles that will provide early detection,risk assessment, and new targets for treatment. The recently initiated generation ofmouse models for mtDNA-linked diseases may help to solve some of those openquestions.

There are more than 400 publications in Medline reporting mitochondrial DNAmutations in cancer. Recently, a whole issue of Oncogene was devoted to this prob-lem. What, if any, is the take-home message about the role of mtDNA mutations incancer?

First of all, the literature is not entirely reliable. There are reports of data anal-ysis that essentially denies any role for mtDNA mutations in tumors [18]. Thereare problems with some of the mtDNA mutation papers, one of them being thatmtDNA haplogroups differ by specific polymorphisms throughout the mitochon-drial genome, and are the results of mutations in distinct maternal lineages, as mito-chondria are inherited exclusively in the maternal line [3, 31]. Thus the comparisonof any mitochondrial sequence with the Cambridge reference sequence (CRS) is


simply a way of determining the differences between one sequence and another—and the results may have nothing to do with tumors and all to do with the fact thatthe sample and the CRS may belong to different haplogroups of mtDNA [6]. More-over, in most cases the analyzed groups of samples are small, for all sorts of reasons,and conclusions cannot be drawn as to the “most common mitochondrial mutation”in a particular type of tumor on the basis of 10 or even 20 or 30 samples as thepopulation of analyzed samples is not significant for statistical analysis. Thirdly,data get misquoted; for example, a paper quotes another one out of context as to thefact that a certain mitochondrial variant predisposes to a certain cancer even thoughthe original authors did not claim this and had no reason to do so. And finally, theeasily accessible mitochondrial databases are rarely used to see if something thatis suspected of being important in cancer is not simply a common polymorphismfound in many populations. The use of mtDBase [32] and MITOMAP [6] should beobligatory for any papers dealing with mtDNA variants.

During the past 2 years, there have been increasing numbers of complete genomesequences compared with appropriate control tissues and not with the CRS. Brandonet al. [33] have performed a comprehensive analysis of the literature and showedthat most of the encountered mutations are population polymorphisms, and theyhave put forth the attractive hypothesis that what happens in tumors is a phe-nomenon of selection that also took place during human adaptation to various envi-ronments [3]—though here it is the tumor cells and not the human beings whohave adapted to special circumstances. Thus most mutations in tumors would notbe dramatic changes profoundly affecting respiration, but small changes facilitatinggrowth under the conditions of relative hypoxia and crowding [33].

Some mutations—fairly rare—are mutations that profoundly affect cellular res-piration; not many of these have been observed. Petros et al. [16] and Shidara et al.[17] have shown that tumors containing mitochondrial DNA with the pathogenicNARP mutation at position A8993G in ATP6 grow faster and are more resistant toapoptosis than are tumors with the same type of cells but with wild-type mtDNA.This was accepted as evidence that the severe mitochondrial mutations in tumorsmay play a role of facilitating growth and decreasing susceptibility to apoptosisunder the exacting conditions of tumor growth. However, Salas et al. [18] have sug-gested that as this particular mutation has not been found in tumors, this experimentdoes not prove anything. On the other hand, the data presented there are quite con-vincing as the growth/apoptosis perturbations can be abrogated by expression of anunmutated gene (expressed from a gene introduced into the nucleus, as it is not yetpossible to introduce genes into human mitochondria) [17]. Brandon et al. [33] havesuggested that such severe mutations arise in early stages of tumor development andare successively lost. Recently, Gasparre et al. [34] have shown that in oncocytictumor, such mutations (defined as disruptive; i.e., nonsense or frameshift) occur inabout one quarter of the tumors, and they all occur in complex I genes. However—and this would be in agreement with Brandon et al. [33]—cell cultures grown fromthese tumors lose the severe mutations [34].

Zhou et al. [35] have demonstrated for the first time that a ND2 (again com-plex I) mutation found in a tumor (human squamous cancer of the head and neck)


can increase growth of cells in culture, thus showing that growth can depend onthe expression of a mutated mitochondrial gene. In this case, they also used agene introduced into the nucleus. Moreover, they showed that ROS production wasincreased [35].

Thus there certainly are mutations in mtDNA in cancer cells and they do affectthe growth properties of these cells. It should be borne in mind, however, that themutations that are observed in most tumors are likely to be ones that have onlyslight adaptive effects on their growth, and the dramatic mutations are rare. Wecertainly have spent a lot of time looking for them and essentially found onlyone [23].

Another problem important both for scientists and for epidemiologists is thequestion whether some of us are more predisposed to certain types of tumors. Mito-chondrial DNAs form haplogroups, and the differences between them believed untilrecently to be completely neutral and insignificant may contribute to the frequencyof some diseases. Again there is a problem with the data. Statistics is a double-edged sword; if the sample sizes are too small, many associations are uncertain.Samuels et al. [31] have recently shown that large cohorts are required for studiesof associations of diseases with mitochondrial haplogroups. Studies with 500 con-trols and 500 cases would not always reliably show a relative risk of about 1.75 fora common haplogroup; larger sizes would be required for less common ones [31].Rarely are the analyzed groups even one tenth of this size. Thus far, two studies ofcancer—haplogroup associations seem quite robust—a study by Booker et al. [36]showing increased relative risk for haplogroup U for prostate cancer and renal car-cinoma (though this has again been criticized by Salas et al. [18]), and a study of theG10398A polymorphism in ND3 in haplogroup N has shown that it is associatedwith an increased breast cancer risk [37].

Increased relative risks for certain haplogroups and certain types of tumors mightbe of interest to health care providers as predictors of cancer development. Whatcould the rationale be for this? Wallace [3] has postulated that the differences inmitochondrial DNA sequence transferred to the function of the electron transportchain in mitochondria could give important differences in, for example, ROS pro-duction, which could affect how susceptible the cells would be to damage. Themain problem with this hypothesis is that no differences have thus far been shownbetween various normal types of cells in ROS production for humans, though a num-ber of pathogenic mutations have been shown to affect ROS production. Recently,Moreno-Loshuertos et al. [38] have shown differences between various mouse mito-chondrial haplotypes both in respiration and in free radical production, though thishas aroused some discussion [39].

As the techniques of mutation analysis improve and more data become available,the role of mitochondrial mutations and haplogroup associations should be eluci-dated in the near future. However, they are unlikely to be specific markers for anytype of tumor, and there will probably not be very strong associations between hap-logroups and tumor occurrence. Finally, the importance of the highest standards inmtDNA analysis to produce reliable results in the many investigations in carcino-genesis should be emphasized [18, 19].


Acknowledgment This work was partly supported by an intramural grant from The Faculty ofBiology, University of Warsaw and grant NN 401 2327 33 from the Ministry of Science and HigherEducation.

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Cellular Respiration and Tumor SuppressorGenes

Luis F. Gonzalez-Cuyar, Fabio Tavora, Iusta Caminha, George Perry,Mark A. Smith, and Rudy J. Castellani

Abstract More than 70 years have passed since Dr. Otto Warburg first documentedthat cancer cells relied primarily on aerobic glycolysis. However, it was not untilthe late-1980s and 1990s when cellular respiration, cellular oxygen sensors, andhypoxia were convincingly related to tumorigenesis and tumor progression. Withthe discovery of hypoxia inducible factor (HIF-1) and its target genes, the rela-tionship that was once weak has become very strong. The expression of the HIF-1molecule has been proved to occur by hypoxic stress and confers an adaptationadvantage, upregulating genes involved in angiogenesis and glycolysis among oth-ers. It has been reported that HIF-1 is under the control of tumor suppressor genessuch as the von Hippel–Lindau (pVHL) protein, which, under normoxia, ubiquiti-nates HIF-1 for proteosomal degradation. Additionally, the tumor suppressor genep53 has been reported to be stabilized by HIF-1 and inhibits Mdm2-dependentdegradation. Recently, it was reported that synthesis of cytochrome c oxidase, aregulator of the cytochrome c oxidase pathway, is a p53 target. Moreover, interac-tions of pVHL and p53 stabilize p53 and permits p53-mediated apoptosis. The aimof this chapter is to outline how the cellular microenvironment affects cellular respi-ration and perhaps induces tumorigenesis, as well as to discuss ways in which tumorsuppressor genes are also involved.

Keywords Cellular respiration · Hypoxia inducible factor · p53 · Tumor suppressor· Von Hippel–Lindau

1 Introduction

For more than 70 years, the notion that cancer cells rely preferentially on glycoly-sis, a phenomenon identified as the Warburg effect [1], has been in existence, butonly over the past 15 or so years have major advances been made in elucidating

R.J. Castellani (B)Department of Pathology, School of Medicine, University of Maryland, 22 South Greene Street,Baltimore, MD, 21201e-mail: [email protected]


131

132 L.F. Gonzalez-Cuyar et al.

the role of oxygen and cellular respiration in neoplasia. A number of specific andwell-characterized genes are now known to be involved, directly and indirectly,as well as a number of different well-characterized molecular cascades. Promi-nent among these is hypoxia inducible transcription factor (HIF-1), which upreg-ulates the transcription of several target genes involved in cellular adaptation to thetumoral microenvironment (i.e., hypoglycemia, acidosis, and low oxygen tensions)and angiogenesis. Additionally, HIF-1 is under the posttranscriptional control ofpVHL that ubiquitinates HIF-1 and tags it for proteosomal degradation.

Two tumor suppressor genes have been identified in connection with changes inthe respiratory metabolism in cancer: TP53 and the VHL tumor suppressor gene.TP53 is the most common mutated gene in human tumors. Whereas the majorityof the studies on p53 in past decades have dealt with its role in cell division anddeath/apoptosis, some evidence now points toward its involvement also with cellu-lar metabolism. Matoba et al. [2] recently found that the gene that encodes synthe-sis of cytochrome c oxidase 2 (SCO2) is upregulated in a p53-dependent manner.SCO2 may be a transcriptional target for p53 [3], and this relation may explainhow inactivation of p53, as seen with somatic and familial mutations, may pro-mote oncogenesis by decreasing cellular dependence on oxygen [2]. In fact, otherstudies have linked p53 in the control of cell glycolysis by influence of down-stream molecules. For example, Kondoh et al. [4] showed that phosphoglyceratemutase, another glycolytic enzyme, is also controlled by p53 by posttranscriptionalactivity. Activation of the Akt kinase pathway is influenced by p53 and may alsoplay a role in switching the cell metabolism from oxidative phosphorylation toglycolysis [5].

The pVHL protein is a positive regulator of p53, and germ-line or somatic muta-tions on the VHL gene are related to tumors such as renal cell carcinomas, heman-gioblastomas, and pheochromocytomas. It has been postulated that VHL proteininteracts with HIF-1 tagging it for proteosomal degradation [6]. In the absence ofoxygen under normal circumstances or when pVHL is mutated, the HIF-1 tran-scription factor is inactivated with direct action upon molecules such as vascularendothelial growth factor (VEGF), culminating in angiogenesis and, thus, cancerprogression [7].

In this chapter, we will discuss the role of tumor suppressor genes in themetabolism of cells, more specifically in the various components of cellular res-piration, interactions with HIF-1, and how interactions of the gene products willpromote oncogenesis, cell survival, and cancer progression through upregulationof transcription of genes involved in angiogenesis, resistance to apoptosis, andmetastasis.

2 Otto Warburg and Cellular Respiration

Otto Heinrich Warburg (1883–1970), a German biochemist, made major contribu-tions to the study of respiration and neoplasia. He hypothesized early on that as can-cer cells have an abnormal growth rate, the means by which the required energy is

Respiration and Tumor Suppressor Genes 133

obtained may be central to the pathogenesis of neoplasia [1, 8]. Results from earlierworks demonstrating that rates of respiration of the sea urchin egg after fertiliza-tion increase up to sixfold motivated him to study cancer cells in a similar manner[1, 8]. As he measured the rates of respiration in transplantable cancer cells from theFlexner-Jobling rat carcinoma, he concluded that there was no increase in oxygenconsumption and lactate was being produced by glycolysis, even in the presence ofoxygen [1, 8]. Warburg concluded that contrary to cells in normal tissues, cancercells could not suppress glycolysis in the presence of oxygen. Interestingly, he alsodescribed aerobic glycolysis in tissues exposed to ethylcarbilamine.

Warburg further demonstrated that although there was no significant increment inlevels of cellular respiration or ATP production of cancer cells, anaerobic glycolysisprovided a major energy source. He hypothesized that when normal cells becomecancerous that metabolic activity shifts in favor of glycolysis. This switch in hisopinion was secondary to a “dedifferentiation” or “reorientation of gene expression”that diverted the baseline energy production to growth.

After a paper by Goldblatt and Cameron [9] citing induction of fibrosarcomafrom fibroblasts exposed to intermittent hypoxia, he demonstrated that embry-onic cells exposed to hypoxia reverted to glycolysis and that normal metabolism,as in cancer, was irreversible, even in the presence of oxygen [1, 8]. Addition-ally he pointed out that subsequent animal inoculation led to the development ofcancers.

In 1956, Warburg described that irreversible damage to the cellular respirationcan be achieved by low oxygen tensions, respiratory poisons such as arsenic, hydro-gen sulfide, and urethane, and x-rays [1, 8].

In an excerpt from a 1967 paper entitled “The prime cause and prevention ofcancer,” he simplified his conclusions:

Cancer, above all other diseases, has countless secondary causes. Almost anything can causecancer. But, even for cancer, there is only one prime cause. Summarized in a few words, theprime cause of cancer is the replacement of the respiration of oxygen in normal body cellsby a fermentation of sugar.

Although his work was not studied for close to 70 years, Warburg’s conclusionson the rates of respiration are still valid with respect to tumorigenesis research andcancer therapeutics.

3 Molecular Homeostasis and Genetic Instability: GeneticInstability and Tumorigenesis

There is a consensus pertaining to the biological definition of life, which is sum-marized in seven categories: homeostasis, organization, metabolism, growth, adap-tation, response to stimuli, and reproduction. Mammals, as the highest life form,have an exquisite intrinsic balance of all of these functions at the molecular level[10–12]. Hanahan and Weinberg [13] described six essential characteristics dictat-ing tumorigenesis and progression, which are somewhat analogous to the above:


self-sufficiency of growth signals, insensitivity to growth-inhibitory signals, evasionof programmed cell death, limitless replicative potential, sustained angiogenesis,and tissue invasion and metastases. Huang et al. [14] proposed that tumor devel-opment is composed of expansion and progression preceded by genetic adaptation(glycolysis, angiogenesis) and genetic alteration (malignant selection of character-istics), respectively.

Decades of scientific research in the area of tumorigenesis and neoplasia haveyielded an insurmountable amount of information as to how all of these essen-tial characteristics of life are deranged in neoplasia [14–16]. Current thought sug-gests that cancer is a genetic disease that originates as a stepwise process in whichnormal cells go through several premalignant phenotypes by acquiring cumulativegenetic alterations each conferring a more malignant phenotype leading eventu-ally to progression and metastases [13]. Alterations in the genetic composition ofcells either by nucleotide mutations or chromosomal aberrations are regarded asthe initiator of neoplasia, while accumulation of these lead to progression, inva-sion, and metastases [14, 17]. The process by which clones are selected thus fol-lows selection for mutations that confer a survival advantage in a hostile tumoralmicroenvironment [14, 17].

A central question with respect to oncogenesis is that of etiology. Although thisremains an open question, current thought generally suggests that adverse condi-tions in the tumor microenvironment contribute to genetic instability and inductionof mutagenesis by direct DNA damage and damage to DNA repair systems [18, 19].According to some, intermittent hypoxia/reperfusion leads to generation of reac-tive oxygen species (ROS), leading in turn to the formation of 8-hydroxyguanineand thymine glycols, which lead to transversions [18–20]. Apart from such oxida-tive damage, aberrant DNA synthesis as seen by amplification as well as increasedincidence of point mutations contribute to further progression [18, 19].

4 Hypoxia

Hypoxia is among the most studied stressors in solid tumors, the central theme beingthe shift from aerobic respiration to glycolysis due to limited availability of oxygenand nutrients. Hypoxia occurs in tumor tissue that is between 100 and 200 �m froma functional blood supply [17, 21, 22]. Normal tissue displays an oxygen diffusiongradient of 400 �m from a blood supply, but significant hypoxia is seen in tumorcells even directly adjacent to a capillary with a mean oxygen tension of 2%, andcells located 200 �m from capillaries showed 0.02% [22, 23]. In addition, studieshave suggested that hypoxia is an independent prognostic indicator.

Reynolds et al. [20] demonstrated a fivefold increase in mutations in both tumorsand cells cultured in hypoxia. Hypoxia has also been implicated as a stimulus forp53 activation and accumulation [24]. Hypoxia seems to not only induce the geneticinstability that accounts for cumulative genetic aberration but also selects for malig-nant clones. It has been suggested that hypoxia-induced apoptosis can be mediated


by HIF-1 upregulation of p53-induced apoptosis. Therefore, to an extent, HIF-1selects for tumor clones that have lost p53 and thus are less sensitive to hypoxia andhypoglycemia.

5 HRE and HIF-1

In 1992, Semenza and Wang [12] described a 50-nt region located 116-nt 3′ of thepolyadenylation site at the flanking sequence of the human erythropoietin (EPO)gene that was able to dramatically increase transcription in the presence of lowoxygen tensions. The region known as the hypoxia responsive element (HRE)increased the transcription of EPO up to 7-fold in cells cultured at 2% O2 andup to 20-fold in cells cultured at 1% O2 [12]. Also it was described that muta-tions within the 4–12 nt and 19–23 nt would not produce the desired increase intranscription, thus it was hypothesized that these sites were crucial for binding oftranscription factors [12]. Concordantly with manipulation of these mutation sites,researchers were able to discover HIF-1, which binds to the HRE at the 1–18 ntregion, thus increasing the transcription of the desired gene under hypoxic butnot normoxic conditions [12]. Because maximal levels of EPO mRNA after theinduction by hypoxia takes about 2 to 4 hours, this suggested that maximization ofgene products involving HRE in the face of hypoxia required de novo synthesis ofproteins.

Further molecular characterization of HIF-1 revealed that it is a heterodimerictranscription factor composed of two subunits, HIF-1� and HIF-1�, which is alsoknown as the aryl hydrocarbon nuclear translocator (ARNT) [11, 12, 25–28]. Theseproteins have a basic helix-loop-helix (bHLH) configuration with a Per, Ahr/ARNTdomain (PAS) [11, 12, 25–28]. Whereas the former facilitates the nuclear dimer-ization of the two subunits, the latter is required for DNA binding [25, 26, 29, 30].Thus far, three isoforms have been described for each subunit, HIF-1, 2, 3�, andARNT1, ARNT2, ARNT3, respectively [28, 29, 31–33]. It is important to note thatthe ARNT is constitutively transcribed, whereas the transcription of the � subunitis stimulated by low oxygen tensions as well as several oncogenes [29, 31]. Whendimerized, the subunits bind to the cis-acting HRE in the RCGTG conserved motifmediated by Ser22, Ala25, Arg30 [28, 29, 31–33]. Subsequently, increased tran-scription of the gene products involved in cellular adaptations to hypoxia such asVEGF and EPO are attained [12, 28, 29, 31–35].

The N-terminal region of the HIF-1� molecule is necessary for dimerization,whereas the C-terminus is required for nuclear localization, transactivation, andstabilization of the protein. The molecule also contains an oxygen dependentdegradation (ODD) domain in the region of residues 401–603, which in additioncontains two proline residues, 401 and 564, which are essential for degradationunder normoxic conditions [31]. There are two areas of nuclear localization signals(NLS), one on the N-terminal (residues 17–33) and one on the C-terminal (residues718–721) [31, 36]. Additionally, HIF-1� contains two transactivation domains


(TAD) at the C-terminal 531–575 and 786–826 [31, 33]. The area between thesetwo nucleotides contains a domain that inhibits transactivation.

Approximately 53% of malignant tumors express HIF-1 by immunohistochem-istry including colon, breast, gliomas, prostate, renal cell carcinomas, and glioblas-tomas when compared with the respective normal tissue. Low-grade gliomas havelittle or no staining with HIF-1. Glioblastomas show intense staining, particularlyin the pseudopalisading cells and in the neighboring areas of necrosis. Apparently,malignant gliomas take advantage of the HIF-1 activation by upregulation of plateletderived growth factor (PDGF) and VEGF, which appear directly proportional. Todate, approximately 30 target genes that are transactivated by HIF-1 have been iden-tified as downstream targets of HIF-1 including genes involved in glycolysis, glu-cose transport, angiogenesis, vascular remodeling and tone, erythropoiesis, and ironmetabolism [10, 15].

6 Oxygen Sensors

Under normoxic conditions, both subunits of HIF-1, HIF-1� and HIF-1�, are tran-scribed into primary transcripts and exported to the cytoplasm where they are trans-lated. Whereas constitutively expressed HIF-1� is stable under both normoxic andhypoxic conditions, HIF-1� is unstable under normoxic conditions. In the presenceof oxygen, it is hydroxylated at two proline residues located on amino acid 401 andamino acid 564, and then tagged by pVHL, forming part of the ubiquinone-ligasecomplex [25, 29, 37–40], rendering it susceptible to proteosomal degradation. It isimportant to note that these proline hydroxylases that account for the protein’s post-transcriptional modification are oxygen dependent, therefore under hypoxic condi-tions the proline residues will not be hydroxylated so the protein will be translocatedto the nucleus where it would dimerize with its counterpart [25, 29, 37–40]. Afterdimerization, the molecule interacts with several HREs located upstream of geneproducts involved in cellular adaptations to hypoxia, acidosis, and hypoglycemia.

It has also been suggested that under hypoxic conditions, the pVHL–HIF-1complex still forms but with ineffective degradation of HIF-1� [41]. Also, in thepresence of iron or desferrioxamine, the pVHL–HIF-1 complex is not formed,thus demonstrating another way of HIF-1 stabilization [42]. However, it has beenreported that this type of induction can be reversed by cyclohexamide, showing theneed of de novo protein synthesis [43]. Additionally, in wild-type pVHL cell lines,investigators described that the HIF-1 DNA binding doublet contains pVHL, thuspointing out that there is pVHL–HIF-1 complex formation under normoxia [41].

7 Tumor Microenvironment

The tumor microenvironment is fundamentally hostile for normal cellularmetabolism, being characterized by hypoxia, hypoglycemia, and acidosis, therebyenhancing the selection process for malignant clones. In this regard, it is not


surprising that hypoxia has been established as an independent adverse prognos-tic factor for a variety of tumor types. Moreover, aberrant tumor vasculature maycontribute to the low oxygen tension, and this lack of adequate perfusion, coupledwith cellular overpopulation, leads to decreased cellular “resources” and increasedneed for the cells to adapt.

As mentioned above, the hostile microenvironment at the epicenter of the tumoraffects a shift from oxidative metabolism to glycolytic pathways. HREs have beenidentified in a variety of genes involved in this shift. These include EPO and VEGF,as well as several of the enzymes required for glycolysis such as glyceraldehydephosphate dehydrogenase, enolase, phosphofructokinase, phosphoglycerate kinase,and pyruvate kinase, as well as other genes such as transferrin, transferrin receptor,IGF binding proteins, lactate dehydrogenase, and glucose transporters (GLT-1 andGLT-3).

Some authors have hypothesized that products of glycolytic metabolism, such aspyruvate, can further stabilize the HIF-1� molecule by substituting 2-oxoglutaratefrom the proline hydroxylase and thus inhibit the hydroxylation of HIF-1� at theODD domain [38]. Both lactate and carbonic anhydrases 9 and 12 may furtherfoster an acidic environment contributing to tumorigenesis. These carbonic anhy-drases appear to be also responsive to HIF-1–HRE induction, as they are notnormally expressed in normoxic cells. Moreover, tumors that have a faulty gly-colytic pathway, secondary to increased mutations, still have an acidic environ-ment [44], which further suggests the role of these anhydrases in promoting a tumormicroenvironment.

8 Cellular Respiration and Tumor Suppressor Genes

8.1 p53

p53 is a tumor suppressor gene located on 17p13.1 and encodes a 393-amino-acidDNA- binding protein that is mutated in more than 50% of human tumors andthat is responsible for inhibiting uncontrolled cellular proliferation and invasion[2, 45]. p53 induces cell cycle arrest and apoptosis by enhanced transcription oftarget genes involved in every step of the apoptosis signaling pathway in responseto DNA damage by various stimuli [3, 45–49]. p53 is activated by hypoxia, DNAdamage, expression of certain oncogenes, and cytotoxic stimuli and, as with alltumor suppressor genes, inactivation is accomplished by loss in both alleles, usu-ally by missense mutations [50, 51]. In the absence of cellular stress, the p53 geneproduct is ubiquitinated and tagged for proteosomal degradation by the ubiquitinligase Mdm2 [24, 45]. Graber et al. [52] also described that the tumor expressingwild-type p53 underwent apoptosis in hypoxic areas whereas the mutant p53 cellswould show resistance to hypoxia-induced apoptosis, giving the p53-deficient cellsa selective advantage over their wild-type p53 counterparts [24, 52]. Under hypoxiaand addition of HIF-1 mimetics such as CoCl2 and DFX [53], it has been demon-strated that HIF-1 and p53 associate leading to p53 stabilization [24, 54] Moreover,


it was reported by Carmeliet et al. [55] that accumulation of HIF-1 was inducedby anoxia and hypoglycemia only in the presence of HIF-1. However, others havereported that accumulation of p53 is induced by the usual HIF-1 stimuli, even incells that are HIF-1 deficient [24, 56].

p53 is the most common mutated gene in cancer cells, and it has been demon-strated that it can also affect metabolism [2, 3]. p53 has been demonstrated to havea role in cellular respiration, and it has a direct impact in cellular respiration bycontributing to the assembly of cytochrome c oxidase complex [2]. In mice, lossof p53 resulted in decreased oxygen consumption, increased lactic acid production,but comparable ATP production [2]. However, p53-dependent mice had a markeddecreased in stamina, which suggests that p53 plays a crucial role in adequate ATPproduction in sustained physical exercise [2,3]. SCO2 is a transcription target ofp53, and p53-deficient cells have decreased levels of SCO2 and no aerobic respi-ration. After reintroduction of SCO2 in p53 null cells, cells are then driven intoglycolysis due to increased ATP requirements. Matoba et al. [2] described SCO2as the first direct target that modulates energy metabolism [2, 3]. SCO2 is requiredfor adequate assembly of a critical component of the respiratory chain cytochromec oxidase. Thus, the loss of p53 and the subsequent decrease in SCO2 might bethe switch that leads cells to use a glycolytic pathway therefore giving the cell thecapability to adapt to increased energy requirements.

p53-SCO2 connects tumor suppressors to cellular respiration and to metabolism,which correlates with the previous views on how cellular respiration and tumori-genesis intersect. Mutations in p53 render cells less sensitive to apoptosis inducedby hypoxic states, and tumors that lack p53 appear to be more resistant to radi-ation and chemotherapy [49]. Approximately 90% of mutations in p53 are pointmutations of residues affecting the DNA binding domain [49, 57–59]. Addi-tionally, p53 posttranscriptionally controls the level of phosphoglycerate mutase(PGM) [4].

Mechanistically, there are many schools of thought. For example, some find thathypoxia induces the accumulation of p53 not the downstream upregulation of itstarget genes [60]. On the other hand, others suggest that, under prolonged hypoxia,HIF-1 and p53 form a complex with Mdm2 therefore inhibiting HIF-1 downstreamgene activation [61].

8.2 von Hippel–Lindau Syndrome

The von Hippel–Lindau syndrome takes its name from German ophthalmologistEugen von Hippel and Swedish neuropathologist Arvid Lindau who, in the early19th century, described a syndrome of angiomas in the retina and the cerebellum,respectively. The VHL gene is located at 3p16-15 and is ubiquitously expressed.It consists of three exons that are transcribed into two transcripts that in turn aretranslated into three proteins [62]. The first transcript contains all three exons and


is translated into two proteins, VHL30 and VHL19 containing 213 and 160 aminoacids, respectively, both of which are active in tumor suppression [63]. The secondtranscript contains exons 1 and 3 and is hypothesized to have decreased tumor sup-pressor activity [64–66]. The gene product consists of a 30-kDa protein that has an� subunit, comprising residues 155–213, and a � subunit that includes the first 154residues [67, 68].

VHL exon 2 encodes for the � domain, and exon 3 encodes for the � domain[69] The � domain is necessary for conjugation with HIF-1 and fibronectin andapparently mediates nuclear cytoplasmic shuttling of pVHL independent of bothO2 and HIF-1. The � domain is encoded by exon 3 and is required for the for-mation of the BC–cullin-2 complex, E3 ligase activity. Several mutations of theVHL gene account for three main phenotypes of this familial syndrome. The mostcommon phenotype includes cerebellar hemangioblastomas and clear cell renal cellcarcinoma with or without adrenal pheochromocytomas. It is often considered anautosomal dominant disease because although both VHL copies need to be mutatedaccording to the Knudson two-hit hypothesis, with one inherited mutant copy ofVHL, the second mutation is almost guaranteed [64, 70–72]

Previously, we mentioned that pVHL interacts with HIF-1� under normoxia afterhydroxylation of two proline residues of the latter by oxygen-dependent prolinehydroxylases, which targets the protein for proteosomal degradation [16, 29, 71,73, 74]. Elongin C recruits an E3 ubiquitin ligase complex that in turn consists of anElongin B, CUL2, and RBX1. Additionally, an E2 ubiquitin-conjugating enzymeattaches to this complex and, later, mediates the ubiquitination of HIF-1. The �subunit will recognize HIF-1-OH.

Under hypoxic conditions, the hydroxylation of HIF-1 and pVHL-ligase complexrecognition does not take place, and this leads to dimerization and nuclear translo-cation of both HIF-1 subunits with subsequent binding to HREs and transcriptionalupregulation of target genes. Taking into consideration that HIF-1 mediates angio-genesis by transcriptional upregulation of vascular endothelial growth factor recep-tor (VGFR) among others and that pVHL is necessary for HIF-1 degradation, it isno surprise that two carcinomas with known VHL mutations, such as CCRCC andcerebellar hemangioblastomas, have such a prominent and characteristic vascularnetwork [41, 72]. Several studies point to the fact that pVHL inactivation has onlybeen linked to renal cell carcinoma (RCC) and hemangioblastoma (HB). In thesetumors, the cellular composition varies from VHL–/– to VHL+/− whereas endothe-lial cells are VHL+/− and VHL−/− [72].

Immunocytochemistry shows cellular and vascular proliferation seen by theautocrine-acting TGF-� and paracrine-acting VEGF, PDGF, respectively, bothdownstream genes upregulated by HIF-1 [72]. Furthermore, it has been suggestedthat the pVHL has more targets, for example the pheochromocytoma-only vari-ant (2C), which might retain its ability to ubiquitinate HIF-1 [37, 67, 68, 71, 72,75, 76]. Not surprisingly, tumors notorious for their prominent vasculature are partof the VHL syndrome, perhaps due to ineffective ubiquitination of HIF-1� leadingto upregulation of VEGF and other angiogneic gene products.


pVHL has also been reported to control the extracellular matrix (ECM) by con-trolling fibronectin deposition [77, 78]. In pVHL-deficient cells, the extracellulardeposition seen possibly contributes to increased migration and perhaps increasestheir metastatic potential [78]. Fibronectin 1 expression is not affected by hypoxia,suggesting a HFI-1–independent mode of regulation by pVHL [62]. Thus, there aretumor suppressor pathways of pVHL that are independent of HIF-1 [62, 79, 80].Additionally, it has been described that pVHL also controls ECM degradation regu-lating metalloproteinases 2 and 9, their inhibitors, and the urokinase type plasmino-gen activator system [62, 81–83].

Other genes regulated by the HIF-1/pVHL complex include aminopetidase A,collagen type IV, alfa1, cyclin G2, DEC1/Straqw, endothelin 1, low-density lipopro-tein receptor-related protein-1, and MIC/CD99 [79, 80]. In fact, the total number ofgenes that pVHL targets, either directly or independently of HIF-1, is likely to beof the order of hundreds [80]. Mutations in VHL will stabilize HIF-1 and contributeto tumorigenesis and progression [69]. Independently of HIF-1, through inadequatedeposition of ECM proteins, mutations in VHL might select for tumor clones witha phenotype that can more readily invade and metastasize.

8.3 Interactions Between p53 and VHL

Apart from their independent contributions to tumor suppression, it has recentlybeen described that the gene products of both genes can also work with each otherfor their common goal. In 2006, Roe and colleagues [84] showed that p53 binds topVHL at the latter’s � domain and thus competes for this site with Elongin C. As wementioned before, the domain of pVHL is the one responsible for the recognitionand subsequent proteosomal degradation of HIF-1�. The pVHL-p53 complex leadsinhibition of the Mdm2-mediated ubiquitination of p53, consequently leading to p53stabilization and upregulated downstream transcription of proapoptotic p21 and Baxpromoters [62]. Additionally, it has been shown that it increases the acetylation andassociation with p300 under genotoxic stress and thus p53 transcriptional activityand p53 mediated cell cycle arrest [62, 84, 85].

9 Conclusion

Conditions in the cellular microenvironment, such as hypoxia, not only lead togenetic instability but also lead to the activation of molecular mechanisms deter-mined to confer a survival advantage by phenotypic adaptation. Mechanisms suchas the HIF-1 cascade permit the cells to attain these capabilities by activating down-stream genes. Limited data exists on how the tumor suppressor genes potentiallyaffect cellular respiration. This aspect perhaps will be a potential target for cancertherapy in the future.


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Uncoupling Cellular Respiration: A Link toCancer Cell Metabolism and Immune Privilege

M. Karen Newell, Elizabeth M. Villalobos-Menuey, Marilyn Burnett,and Robert E. Camley

Abstract Epidemiologic evidence strongly correlates suppression of maturation ofthe immune system to incidences of cancer. The effectiveness of chemotherapeuticagents significantly depends upon the ability of the cancer cells to express (cos-timulatory) molecules on their surface that can be recognized and engaged by theirsurrogate ligands of the immune system, thereby resulting in immune-directed cellkilling. Alterations in the metabolic strategies of cancer cells (such as: the rate ofglucose utilization, glucose or fatty acid oxidation, the magnitude of the membranepotential and the pH gradient across the mitochondrial membrane, and/or changes inuncoupling protein levels) can modulate the expression of some cell surface “deathreceptors”; thereby conferring resistance to certain cell death-inducing stimuli.

Keywords Cancer cell metabolism · Fas · Drug resistance · Uncoupling protein ·Metabolic strategy · Metabolic modulators · Mitochondria · Cellular respiration ·Immune privilege

1 History

Cancer cells exhibit distinct metabolic pathways to meet their energy demands. OttoWarburg postulated that there must be some sort of deficit or abnormality in whathas long been described as the most energy-efficient strategy that a cell can use—mitochondrial respiration. The purpose of this chapter is to propose an alternateexplanation and to present a hypothesis that can be rigorously tested.

M.K. Newell (B)CU Institute of Bioenergetics and Immunology, University of Colorado at Colorado Springs,Colorado Springs, CO 80918e-mail: [email protected]: [email protected]


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146 M.K. Newell et al.

2 An Overview of Cellular Metabolism

Every cell in the body uses carbohydrates, protein, or fat in different proportionsto ensure that the cell has sufficient energy to perform its normal function. Thecell’s choice of fuel (i.e., the cell’s metabolic strategy) changes depending on itsactivation or differentiation state as well as its environment. For example, a cellthat is dividing has different energy demands than does one that is nondividing and,thus, may employ an alternative metabolic strategy. Another example would be thechange in strategy for a cell that has been damaged by infection or stress.

The majority of nondividing cells in the body use carbons derived from carbohy-drate or glucose to efficiently produce the cell’s ultimate energy unit of exchange,adenosine triphosphate (ATP). The process begins with the binding of glucose tothe glucose receptor/transporter and transport into the cytosol, where the moleculebecomes phosphorylated by hexokinase. Once phosphorylated, the molecule contin-ues to be processed into two molecules of pyruvate. Pyruvate then enters the Krebscycle, a process that occurs in the mitochondria where ultimately the production ofNADH and FADH2 provide reducing equivalents that flow through the four com-plexes of the electron transport chain, eventually creating a proton gradient betweenthe inner mitochondrial membrane and the matrix. In concert, the electrochemicalgradient drives the synthesis of adenosine diphosphate (ADP) to ATP as a result ofthe electrochemical gradient across the inner mitochondrial membrane.

3 Tumor-Specific Metabolic Pathways

Rapidly dividing tumor cells display a characteristic metabolic strategy that dis-tinguishes the tumor cell from its normal, nondividing counterpart cell of the tis-sues. The key difference is that the rate of glycolysis is much greater than therate of glucose breakdown in a normal cell under resting conditions. Glycolysisis an oxygen-independent process of splitting the six-carbon glucose molecules andthereby providing energy in the form of ATP to the cell. Aerobic cells use the gly-colysis pathway as the initial pathway that ultimately involves oxygen consumptionand complete oxidation of the carbons derived from carbohydrates. From a series of10 reactions, the first five of which are energy “investments” and the second five ofwhich are energy generating, the final product is pyruvate [1].

The fate of the pyruvate molecules produced by glycolysis largely depends on theoxidative state of the cell in question. Glycolysis needs a pool of cytosolic NAD+,derived from the oxidation of NADH, to proceed. In the presence of oxygen (aerobicglycolysis), the NADH is oxidized in the mitochondria. If the rate of glycolysisin the cell is faster than the rate at which the NADH can be oxidized by electrontransport in the mitochondria, the excess NADH must then be used to drive thereduction of another substrate to maintain cellular redox balance. Pyruvate itself issuch a substrate, and by the activity of the enzyme lactate dehydrogenase, pyruvateis reduced to lactic acid [1].

Uncoupling Cellular Respiration 147

Glycolysis, whether aerobic or anaerobic, releases only a very small percent ofthe stored energy in the glucose molecule. The evolution of aerobic metabolismmade possible a more energy-efficient use of the stored energy in glucose and inother carbon-containing fuels. Pasteur was the first to observe that when cells metab-olizing glucose anaerobically were exposed to oxygen, the rate of glycolysis sloweddramatically (a phenomenon known as the Pasteur effect). This effect likely occursbecause it is far more energy efficient to completely oxidize glucose than via justthe initial glycolytic steps alone. Much of the control over these processes is deter-mined, not surprisingly, by the energy needs of the cell [1].

4 Mitochondrial Metabolism

Pyruvate derived from carbohydrate is one of the three suppliers of acetyl-CoAfor oxidation in the Krebs cycle. The conversion of pyruvate to acetyl-CoA is cat-alyzed by the enzyme pyruvate dehydrogenase. The glycolytic product, pyruvate, istransported from the cytosol to the mitochondria. Once there, the Ca2+-dependentenzyme pyruvate dehydrogenase catalyzes its production to acetyl-CoA and in thatmanner feeds the glucose-derived carbons to the Krebs cycle ultimately providingthe reducing equivalents necessary for electron transport, oxidative phosphoryla-tion, and the very efficient enzymatic production of ATP. The activity of pyruvatedehydrogenase requires a high mitochondrial membrane potential across the innermitochondrial membrane [1].

When pyruvate supplies are high or when there is insufficient Ca2+ to acti-vate pyruvate dehydrogenase, the source of the acetyl-CoA switches from glucose-derived pyruvate to the acetyl-CoA produced by fatty acid oxidation. The source offatty acid can be endogenous or exogenous, composed of short-, medium-, or long-chain fatty acids. When long-chain fatty acids are used, the process that providesacetyl-CoA is beta-oxidation. A key enzyme in the transport of fatty acids is carni-tine palmitoyl transferase (either I or II depending on the tissue of origin). Tumorcells in general and drug-resistant tumor cells in particular, as well as other cells thatremain “immune privileged,” have a flexible capacity for the ready switch to fattyacid oxidation as a result of mitochondrial expression of key players, including mito-chondrial uncoupling proteins that uncouple glycolysis from oxidative phosphory-lation. This process results in a lower mitochondrial membrane potential, decreasedsensitivity to free radical damage, and insensitivity to apoptosis inducing stimuli,such as chemotherapy or radiation [1].

5 The Immune Response in Cancer

Our work has addressed the possibility that the immune system monitors themetabolic state and degree of damage that characterizes all cells. We have foundthat, though perhaps preferentially destroying those in an excessively damaged state(i.e., stressed/damaged cells or infected cells), nonetheless, tumor cells often survivethis surveillance by changing their metabolic strategy to one that is protective [24].


When tumor cells are treated with traditional chemotherapeutic drugs or radiation,the damage from these treatments causes cell surface changes allowing the cell tobe recognized once again by the immune system. Drug-resistant cell variants dis-play a characteristic metabolic state that masks them from the immune system (evenafter treatment with chemotherapeutic drugs or radiation) and that confers upon thetumor cell profound protection from further insults [2].

We have focused on understanding the characteristic metabolic states that protectabnormally proliferating cells. Cancer cells, particularly multidrug-resistant vari-ants, have a strategy for survival that involves selective fatty acid oxidation in themitochondria. We have identified several drug candidates that block the activityof these proteins. Preliminary studies indicate that these compounds are capableof interfering with the metabolic strategy of most tumor cells and have strikingtherapeutic activity in tumor-bearing mice when used alone or in conjunction withstandard chemotherapy. We are currently investigating several novel drugs designedto target metabolic strategies used by tumor cells, in particular multidrug-resistanttumors, and other diseases characterized by abnormally proliferating cells.

Attempts to trigger an effective immune response against growing cancer cellshave been a priority in a large body of research. This is not surprising given theresults that tumor allografts are routinely rejected by an effective immune response,the observation that children and the elderly (considered somewhat immunocom-promised) have increased frequencies of tumors relative to the general population,and the increased incidence of tumors in immunocompromised hosts. The problemshave been to understand enough about how the immune system detects abnormalproliferation and, likely more importantly, how an immune response can distinguishself from non-self, leading to the question of whether tumor cells are foreign or self.Clearly, in many instances there are associations with viral infections, although inother cases there are no clear links between a pathogen and tumorigenesis.

6 Fundamental Questions

Every year at least 6.2 million people die worldwide from cancer [3]. The develop-ment of drug resistance attenuates the effectiveness of chemotherapy in many cases.In this chapter, we explore whether chemotherapy can be enhanced by increasingthe response of the immune system to the tumor cells and by making drug-resistanttumor cells more visible to the immune system.

Specifically, we ask the following questions:1. Do effective chemotherapeutics sensitize tumors to immune destruction by

increasing cell surface expression of costimulatory molecules?2. Can this effect be enhanced by other treatments?3. Is drug resistance, in part, due to differences in metabolic behavior between drug-

sensitive and drug-resistant cells? Using this idea, can we create new methods fortreating drug-resistant tumors?


7 The Immune System and Cancer

Extraordinary advances in our understanding of the immune system have been madein the past 100 years, especially since the discovery of the T cell and the B cell[4–6]. Naıve T cells require two signals for activation. These are recognition ofantigens in major histocompatibility complex (MHC)-encoded molecules [4] anda costimulation signal [7–11] provided by the B7/CD28 family members or othercostimulatory molecules such as Fas (CD95) [12]. Previously activated T cells canbe reactivated by costimulation alone [13, 14]. In the absence of activation, T cellsdisregard the tissue. If a T cell is activated, the consequences can be (1) destructionof the damaged cells or (2) repair of damaged cells by promoting regeneration eitherdirectly or indirectly.

There is substantial evidence that the immune system plays an extensive role insuppressing cancer. However, it is also clear that the ability of the immune systemto control cancer is not perfect.

Researchers have tried to stimulate the immune system as a therapeutic strategyagainst cancer for many years [15, 16]. These attempts have generally been ineffec-tive, although there have been some recent successes [17]. There are many reasonsfor this variability. These include (1) the inability to activate T cells that can destroythe tumor due to the absence of signal one (recognition of the appropriate tumorantigen); (2) the presence of a signal two that results in the production of the wrongcytokines by T cells, which may lead to the growth of tumors, or (3) the failure ofactivated T cells to kill cancerous cells [18].

In contrast with therapies that concentrate primarily on activating the immunesystem, we will explore the idea that effective chemotherapeutics work in concertwith the immune system and derive some of their effectiveness from the immuneresponse. This means that by tuning the immune response, we may be able toincrease effectiveness in cancer therapy.

Cellular metabolism may play a dominant role in modulating the intercellular com-munication between a cancer cell and a T cell and thereby plays a dominant role inT-cell activation. In particular, we have observed a significant correlation betweencellular metabolism and the expression of cell-surface Fas, one of the key costimu-latory molecules. Fas is expressed on most rapidly dividing and self-renewing cells,including a wide variety of tumor cells [19]. Whereas Fas is widely known as a “deathreceptor” and can induce programmed cell death when engaged by Fas ligand (FasL,CD95L) or anti-Fas antibodies [20, 21], paradoxically, Fas engagement can also resultin accelerated proliferation of T lymphocytes and some tumor cells [22, 23].

Cell surface Fas expression is upregulated in response to increasing glucose con-centrations in vitro in transformed cell lines and in freshly isolated cells from avariety of tissue origins [19]. Obviously, glucose is a key source of fuel, and theavailability of glucose drives metabolic activity. A further example of the connec-tion between cellular metabolism and Fas is found in the work of Bhushan et al. [2]who showed that drug-resistant tumor cells fail to express cell surface Fas and thework of Harper et al. [24] who showed that drug-resistant tumor cells also demon-strated a unique metabolic strategy, quite different from drug-sensitive tumor cells.


8 Drug Treatment and Fas-Induced Tumor Cell Death

Anticancer agents may work to promote the death of tumor cells in multiple ways.First, these agents may work by direct cytolysis requiring active participation ofthe tumor cell in the death process [25]. Second, chemotherapeutics may promotethe ability of the tumor cell to be recognized by cells of the immune system andto be killed by immune-directed cell death [26]. Third, these agents may work to“rewire” the death-inducing receptor/ligand pairs, which include Fas and FasL [23,25, 26]. The second and third possibilities are not mutually exclusive and may workin concert to result in tumor cell death (see Table 1).

Drug resistance is the leading cause of death in cancer [27]. Mechanisms thathave been suggested to account for drug resistance include overexpression of amultidrug-resistance transporter (pgp -1) [27], failure to express death-inducingreceptors [27, 28], and our recent description of a metabolic strategy that may pro-vide protection from a variety of stresses [24].

Our hypothesis is that mitochondrial metabolism in tumor cells significantlyinfluences susceptibility or resistance to Fas-dependent/anticancer agent–inducedtumor cell death. Our preliminary work provided the framework for the hypothe-sis that metabolic strategy confers resistance or susceptibility to apoptotic death oftumor cells. We suggest that treatment of some cancer cells by chemotherapeuticsdoes indeed produce substantially enhanced costimulatory signals and that the treat-ment of cancer cells by chemotherapeutics and immune system involvement resultsin substantially higher tumor cell death rates in the measured cases.

Figure 1 shows that treatment of L1210 tumor cells (a mouse leukemic cellline [29]) cultured for 24 hours with methotrexate induces significant increases incell surface Fas. The lower concentration of 10–8 M is physiologically relevant tochemotherapy because this is the upper limit of drug serum levels in humans. Ourresults here are consistent with some of our previous published work where weshowed that a different costimulatory signal (B7.1) also increased in response tomethotrexate [2].

Figure 2 shows that different chemotherapeutic agents induce an increase in Fas,but that different drugs can produce increases of varying amounts. This leads to thetantalizing possibility that drug effectiveness could be correlated with levels of Fas

Table 1 Percent Death of L1210 cells with or without a 96-Hour Incubation with 10–8 MMethotrexate and with or without an Exposure to Anti-Fas Antibodies∗

Percent death L1210 cells (%)

Anti-Fas exposure No methotrexate With methotrexate

No anti-Fas 1 4.72Anti-Fas coated plates 7 79.98

∗The results indicate that methotrexate sensitize leukemic cells to Fas-induceddeath.


Fig. 1 Expression of cellsurface Fas as a function oftreatment with a typicaloncolytic agent. There is asignificant increase in cellsurface Fas as a result of drugtreatment. The level of cellsurface Fas was determinedusing fluorochrome-conjugated anti-Fasantibodies and flowcytometry. The Fas levels aremeasured relative to a controlfor staining artifacts

expression and that this could be used to predict effective targeting of drugs viaFas-induced destruction.

We have concentrated on Fas as an important example of the costimulatory sig-nal; however, there are a number of other cell surface molecules that also play a rolein T-cell activation. In Fig. 3 , we show that B7.2 levels in HL60 (human leukemiccells) also increase after treatment with 10–8 M Adriamycin. HL60 is a human cell

Fig. 2 Flow cytometry data showing increasing levels of cell surface Fas in L1210 cells as a resultof treatment with different chemotherapeutic agents. The horizontal axis shows intensity of thefluorescence, and the vertical axis shows the number of cells at each intensity. Peaks that appearfarther to the right have higher levels of Fas per cell


Fig. 3 Expression of the cellsurface costimulatorymolecule B7.2 as a functionof treatment with a typicaloncolytic agent. The level ofcell surface B7.2 wasdetermined usingfluorochrome-conjugatedanti-B7.2 antibodies and flowcytometry. The B7.2 levelsare measured relative to acontrol for staining artifacts[31]

line, and the drug is different than that in previous figures. This implies that theincrease in the costimulatory signal as a result of drug treatment may be a generalphenomenon.

The data above strongly support the connection between the efficacy of drugtreatment and an increase in costimulatory immune signals. However, it is importantto know if this increase actually results in an increased immune-mediated death ratefor the tumor cells. In contrast with treatment with anti-Fas or methotrexate alone,anti-Fas produces a striking increase in percent death for L1210 cells that have beencultured in methotrexate. Therefore, drug treatments result in increased expressionof costimulatory signals and this, in turn, plays an important role in successful tumorcell treatment.

Our second question concerned whether the treatment of tumor cells bychemotherapeutics supplemented with agents that promote reactive oxygen inter-mediates result in increased susceptibility to immune-mediated cell death.

As we saw from the data above and from the work of others [28], chemothera-peutics cause an increase in the expression of costimulatory molecules. There areother treatments that can also produce a similar increase. For example, Fig. 4 showsthe effects of culturing two tumor cell lines in subcytoxic concentrations of H2O2.

The H2O2 treatment results in a threefold increase in the costimulatory moleculeB7.2. The increase from treatment with H2O2 is also significantly larger that theincrease resulting from treatment with Adriamycin alone (Fig. 3).

Experiments with other cell types show that the expression of other costimulatorymolecules can also be substantially changed by the H2O2 treatment. For example,Fig. 5 shows that treatment of JB6 cells (a mouse fibroblast tumor cell line) withH2O2 causes up to a fourfold increase in cell surface Fas expression. Clearly what


Fig. 4 Relative B7.2 levelsfor two different tumor celllines with and withoutaddition of subcytotoxiclevels of H2O2. Obviously,the addition of H2O2

increases the expression ofcell surface costimulatorymolecules significantly

is needed is to look at the effectiveness of combined approaches, for example, usingboth chemotherapeutics and H2O2.

Our third question dealt with whether drug resistance can be overcome by treat-ments that promote immune involvement in the destruction of tumor cells andinvolved the hypothesis that uncoupling proteins regulate drug resistance by chang-ing the metabolic state of a cell.

Fig. 5 Relative cell surfaceFas expression in JB6 cells asa function of time afterexposure to subcytotoxiclevels of H2O2. We see,again, that the levels ofcostimulatory moleculesincrease substantially afterexposure to H2O2


Fig. 6 A scatter diagram for drug-sensitive (left panel) and drug-resistant (right panel) tumor cells.The horizontal axis is a measure of the change in pH across the inner mitochondrial membrane.The vertical axis measures mitochondrial membrane potential. The results show that mitochondrialmembrane potential and pH difference is lower for the majority of drug-resistant cells. The dataare obtained from flow cytometry using a JC-1 dye

First we show that drug-sensitive cancer cells and drug-resistant tumor cells havevery different metabolic properties. As is well-known, cell metabolism is largelygoverned by the action of the mitochondria. This, in turn, depends on some keyparameters that include the potential difference across the inner mitochondrial mem-brane and the change in acidity across the same membrane. Figure 6 shows a scatterdiagram for drug-sensitive and drug-resistant tumor cells indicating the range ofboth of these parameters. The abscissa is a measure of the change in pH acrossthe inner mitochondrial membrane. The ordinate measures mitochondrial mem-brane potential. We see that the drug-resistant cells, in general, have a lower mem-brane potential, and the pH gradient is also significantly reduced for these cells.These data have been substantiated and extended to other cell lines (including HL60and HL60/MDR) by other measurements [24]. This provides strong evidence thatdrug resistance is correlated with altered metabolic behavior.

A second example of how drug-resistant cells exhibit a different metabolicbehavior compared with drug-sensitive cells is provided by the rate of mitochon-drial oleate consumption in these two different types of cells [24]. Figure 7 showsthat the drug-resistant cells typically have higher levels of oleate consumption, andas glucose becomes limited, the rate of consumption increases dramatically in thedrug-resistant cells only. This argues that the ability to ”burn fat” is substantiallydifferent between the two types of cells.


Fig. 7 Rates of fatty acid (oleate) oxidation for drug-sensitive and drug-resistant cells. The oxida-tion rates for drug-resistant cells are generally higher than those for drug-sensitive cells, and thisbecomes more pronounced at low glucose levels [24]

An important question is whether this metabolic difference leads to differences inthe way tumor cells present themselves to the immune system. In Fig. 8 , we presentevidence that drug-sensitive tumor cells (those in the left panels) typically displayrelatively high levels of cell surface Fas, one of the requirements for Fas-induceddeath [2, 30]. In contrast, the drug-resistant cells (seen in the right panels) showvirtually no cell surface Fas. These data suggest that the differences in metabolicbehavior are correlated with expression of cell surface Fas. Clearly, it is not knownif this is a cause-and-effect behavior or if it is mere correlation. Nonetheless, thepossibility of a causal relationship between cellular metabolism and the expressionof cell surface Fas may offer an intriguing tool for making tumor cells visible to theimmune system.

Treatment of drug-resistant tumor cells with chemotherapeutic agents does notincrease expression of cell surface Fas or other costimulatory molecules. Thisbehavior is illustrated in Fig. 9 below. This is in stark contrast with the behaviorof drug-sensitive tumor cells (see Fig. 2, Fig. 3, and Fig. 4), which do show substan-tial increases in Fas with chemotherapeutic treatments.

The results above suggest that one possible reason that chemotherapy is inef-fective for drug-resistant cells is that the costimulatory signal is absent. This nat-urally leads to the question of whether costimulation, particularly in drug-resistantcells, could be increased by other treatments. In Fig. 10 , we show a summary ofexperiments on three different (relatively) drug-resistant human cell lines. We see


Fig. 8 Levels of cell surface Fas expression on drug-sensitive (left panels) and drug-resistant(right panels) cells as measured by flow cytometry. The horizontal axis shows intensity of thefluorescence, and the vertical axis shows the number of cells at each level of fluorescence intensity.Peaks that appear farther to the right have higher levels of Fas per cell. Note that the drug-sensitivecells have significantly higher levels of cell surface Fas

that treatment with subcytotoxic H2O2 increases B7.2 expression in all cases andthat it is increased by a factor of 2 to 3 in some cases. This increase is, in fact, verysimilar to that for the drug-sensitive cells (see Fig. 4 for example).

We have suggested that differences in metabolic behavior correlate with differ-ences in sensitivity to drugs [24]. One possible mechanism that would account forthis could be the activity of the uncoupling protein UCP2. These proteins havebeen shown to produce a number of metabolic changes that are consistent withthe metabolic behavior of drug-resistant cells. We measured the presence of UCP2in mitochondria of drug-resistant and drug-sensitive cells by isolating mitochon-dria and using antibodies to UCP2 in Western blot analysis (Fig. 11 ). Significantlyhigher levels of UCP2 were detected consistently in the resistant cell lines (lanes 4and 5). These results provide the rationale for an investigation of the role of uncou-pling proteins as a part of the mechanism for drug resistance. If this mechanismwere confirmed, this would create a new approach for treatments of drug-resistantcancers.


Fig. 9 Levels of cell surfaceFas expression ondrug-sensitive L1210 (upperpanel) and drug-resistantL1210/DDP (lower panel)cells as measured by flowcytometry. Cells werecultured in the presence ofchemotherapeutic agentsovernight. The drug-sensitivecells show increases in Fas asa result of the drug. Incontrast, the drug-resistantcells are unchanged withtreatment. The horizontal axisshows intensity of thefluorescence, and the verticalaxis shows the number ofcells at each intensity. Peaksthat appear farther to the righthave higher levels of Fas percell. Note that thedrug-sensitive cells havesignificantly higher levels ofcell surface Fas

Fig. 10 Relative expression ofthe costimulatory molecule B7.2on three different drug-resistanthuman cell lines with and withouttreatment with subcytotoxicamounts of H2O2. In contrastwith treatments withchemotherapeutics alone, we seehere a substantial increase in thecostimulatory signal, which isachieved by adding the H2O2


Fig. 11 Western blot analysis forUCP2 of mitochondrial extractsfrom drug-sensitive L1210 (lanes2 and 3) and drug-resistantL1210/DDP (lanes 4 and 5) tumorcells. UCP2 levels aresubstantially larger in thedrug-resistant cells [24]

9 Therapeutic Possibilities

As an example of using a metabolic approach to provide therapeutic benefit, we willdiscuss the performance of one compound, dichloroacetate, in detail. There are twooverall goals: first, to slow or inhibit high-rate glycolysis, and the second, to blockfatty acid oxidation at several key enzymatic steps.

We and others have identified 2-deoxyglucose (2DG) as a key regulator of high-rate glycolysis. However, our work has demonstrated that for most rapidly dividingtumor cells, treatment with 2DG alone does not cause tumor cell death. In fact, interms of percent viability, treatment with 2DG results in improved percent viabilityfor the vast majority of tumors. The mechanism appears to be that the compoundresults in growth arrest of the cells. In combination with traditional chemotherapeu-tic agents, the addition of 2DG increases the oncolytic activity of the therapeutic.Though beneficial, this approach leaves open the likely possibility that treatmentwith 2DG and chemotherapeutic agents such as Taxol (paclitaxel) or Adriamycinwill eventually result in drug resistance, the leading cause of death due to cancer.

Targeting metabolism as a chemotherapeutic approach is a major focus of ourwork, and we focus on the inhibition of tumor cell–specific fatty acid oxidation.Toward that end, we have used the several fatty acid oxidation (FAO) inhibitors.We have extensive data showing the effectiveness of this approach in inducing theapoptotic death of a wide variety of tumor types.

We note the listing of dichloroacetate (DCA) in each of these two categories ofmetabolic modifications: as a glycolytic inhibitor as well as a fatty acid oxidationinhibitor. With metabolic strategies, one must consider that cells have feedback strate-gies that communicate when energy demands are met or when energy demands areunmet. The cell will respond accordingly with either catabolic or anabolic pathwaysto provide optimization of energy expenditure. Because DCA is similar in structureto pyruvate, the molecule can act as a negative regulator of glycolytic processes.In addition, the structure of DCA is known to fit the negative regulatory bindingpocket of pyruvate dehydrogenase kinase, an enzyme that increases under condi-tions of starvation promoting fatty acid oxidation when glucose or carbohydrate


stores are limited. Thus, DCA will act as a negative regulator of fatty acid oxida-tion, a key strategy of drug-resistant tumor cells. This selective interference causescells that are normally selectively nonapoptotic to now die from apoptotic cell death.While theoretically promising, the effects of DCA when used in combination with cur-rent “standard of care” chemotherapeutics need to be rigorously tested for potentiallyharmful or toxic side effects.

In summary, the technique of metabolic disruption shows promise for creatingtherapeutic strategies that may help combat cancer or other diseases. These strate-gies have the potential of changing the way the immune system interacts with tumorsand other metabolically altered tissues.

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How Cancer Cells Escape Death

Erica Werner

Abstract Programmed cell death, or apoptosis, is a cell-autonomous mechanismto restrict deviation from normal cellular function. Central to this mechanism areBcl-2 proteins, which monitor normal cellular function to define apoptotic fateby surveying, integrating, and decoding multiple survival and proapoptotic signalsfrom the intra and extra cellular environment. Escape from these restrictive mecha-nisms is essential to accommodate the cellular changes driving tumor establishmentand development. To do so, many of the factors promoting tumorigenesis, such asgenetic lesions, hypoxia, p53 mutations and oncogene activation, gain control overthe main apoptotic pathway to promote cell survival. For this reason, the apoptoticpathway is the focus of remarkable interest for targeted cancer therapy development.However, as apoptosis evasion is a tumor promoting factor itself, the results of firsttherapeutic targeting attempts reveal that these targets pose drawbacks similar tothe exhibited by classic oncogenic targets, including the rapid development of resis-tance through high mutation rate, intra and inter tumor heterogeneity and narroweffective range.

In addition to tumor promoting pathways, apoptosis evasion is fostered by factorssecondary to transformation and tumor development. These factors, such as changesin the interaction with the extracellular environment and metabolism, have receivedless attention because of their underestimated role in transformation but might proveto be fruitful avenues for intervention as they are common to many tissues and affectapoptotic fate under the influence of reduced oncogenic pressure. The significanceof these factors with respect to apoptosis evasion and their potential impact for ther-apy development are discussed.

Keywords Apoptosis · Bcl-2 · Mitochondria · Prosurvival · Environment ·Glycolysis

E. Werner (B)Department of Cell Biology, School of Medicine, Emory University, Atlanta, GA 30322e-mail: [email protected]


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1 Introduction

Death is universal and certain. At the cellular level, this fate is controlled by apopto-sis, a genetic program present in every cell type of organisms, including cancer cells.Evasion of this program is deemed key to carcinogenesis and tumor progression.Thus, extraordinary efforts have been committed to understand how cancer cellsevade the apoptotic pathway and thereby influence disease prognosis and targetedtherapy design. Although past and current studies have consistently revealed the sig-nificant role of apoptosis evasion in the process of carcinogenesis, correlations ofdisease progression and prognosis with the expression of apoptosis-related markershave been inconsistent, and therapies targeting effectors of the apoptotic pathwayhave been of limited efficacy [1]. This suggests that additional cell autonomous orextracellular mechanisms are likely contributing to apoptotic outcome. This will bethe major focus in this chapter.

The apoptotic pathway is a surveillance mechanism monitoring cell activity andimposing strict limits to permissible cellular function. These limits are constantlychallenged during cancer initiation and progression. Early deregulated prolifera-tion demands multifaceted changes encompassing intracellular signaling pathways,metabolism, nutrient and energy requirements, and tissue architecture, which triggerintracellular and extracellular proapoptotic signals in normal cells. At later stagesof tumor development, cancer expansion and metastasis adds a further cohort ofapoptosis-inducing signals, generated in opposition to changes in the function ofmultiple cell types and altered environments.

In contrast with mutations in oncogene and tumor suppressor genes, evasion ofthe apoptotic pathway does not initiate and drive carcinogenesis per se, but is instru-mental for tumor cells to successfully thrive despite harboring alterations that wouldnot be tolerated in normal circumstances. The mechanisms driving tumor progres-sion are integrated with apoptosis resistance acquisition, at least at three levels, soas to ensure successful apoptosis evasion:

Somatic mutations, promoter methylation, chromosomal translocations and dele-tions associated with the inherent genetic instability of cancer cells alter the expres-sion of oncogenes as well as those of key apoptotic players. Even though thishas been the most explored mechanism to explain the increased apoptosis resis-tance of tumor cells [1], correlations in most cases are difficult to generalize andin many cases are nonconfirmatory. One interpretation of this outcome could bethat activity rather than expression levels are consequential for carcinogenesis, asthe apoptotic players are mainly regulated through posttranslational mechanisms,such as phosphorylation or ubiquitin-targeted proteasome-dependent degradation.Another reason may be associated with the difficulty to evaluate whether the iden-tified changes in expression are indeed a causal factor in disease progression or aremerely coincidental.

A second factor promoting apoptosis resistance is an overall shift in the natureof signals surveyed by the apoptotic machinery, reinforcing prosurvival signalswhile interfering with proapoptotic signals (see Fig. 1). This altered input is thedirect result of two concurrent processes; one leading to independence from an

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Fig. 1 Apoptosis input is balanced by prosurvival (gray) and proapoptotic (black) signals. Thesesignals control the activity (crossed arrows represent inhibitory signals, and pointed arrows re-present activation; dashed lines represent loss of that particular connection) of antiapoptotic Bcl-2members (round boxes) and proapoptotic Bcl-2 family members (square boxes). (A) In normalcells, the extracellular environment through growth factors, cell adhesion, and cytoskeleton orga-nization restrains activity of proapoptotic Bcl-2 family members. P53 responds to endogenousstress providing proapoptotic signals. (B) During carcinogenesis, control of the apoptotic path-way is taken over by oncogene-activated kinases, prosurvival cell-surface interactions, glycolysisactivity, and loss of p53 function

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environment rich in proapoptotic signals and the other altering the intracellular sig-nal transduction network through oncogene activity and tumor suppressor silencing.The marked influence of these factors on apoptotic fate becomes apparent as apop-tosis sensitivity can be restored by normalizing cancer cell communication with theextracellular environment or by neutralizing oncogene activity (see Section 3).

A third, far less studied factor affecting apoptotic fate is the characteristic alteredmetabolism associated with transformed and tumor cells. Mitochondria are a cen-tral piece in the apoptotic pathway, but are a control center for normal and tumormetabolism as well. The importance of metabolism in carcinogenesis is gainingrecognition and promises a source of potentially viable therapeutic approaches, asmany of the involved pathways control enzymatic activities suitable for pharmaco-logic inhibition are linked to apoptosis.

Because of space constrains, in this chapter I will only outline the essential pla-yers and concepts necessary to provide a conceptual framework illustrated by a fewexamples and will cite only select key reviews to refer the reader to further detailsin each section.

2 The Bare Pathway to Death

The essential apoptotic pathway responds to specific triggers, such as proapoptoticextracellular ligands, cellular stressors, or deprivation of a survival signal. Theseelicitors activate an intracellular proteases cascade in charge of cleaving multipletargets to cause cell death. The resulting cell debris, organelle and nucleus fragmentsare packed into apoptotic bodies to be cleanly disposed by professional phagocytesand/or neighboring cells thereby averting inflammatory responses [2].

At the center of the apoptotic pathway is a regulatory step dictated by the protein-protein interaction of the Bcl-2 family members, controlled by proapoptotic andprosurvival signals generated in the extracellular environment (extrinsic pathway)and inside the cell (intrinsic pathway). If the activity balance of Bcl-2 proteins isinclined toward cell death, proapoptotic members redistribute to mitochondria andcause outer membrane permeability, with the consequent release of proteins con-tained in the mitochondrial intermembrane space [3]. The proteins released into thecytosol activate caspases and other regulators/effectors of cell breakdown.

2.1 The Family of Bcl-2 Proteins: Initiators of the IntrinsicPathway

Cellular stress signals such as growth factor deprivation, DNA damage, oncogeneactivation, or hypoxia trigger apoptosis by regulating the activity of members of theBcl-2 protein family. Each member shares a protein-protein interaction domain withhomology to Bcl-2 (BH domains) and is grouped according to its role in apoptosis:multidomain antiapoptotic (Bcl-2, Bcl-xL, Bcl-w, Mcl-1, and Bfl-1/A1), multido-main proapoptotic (Bax and Bak), and BH3-only proapoptotic (Bid, Bim, Bad, Bik,


Noxa, Puma, Bmf, and HRK). Tissue-specific expression and posttranslational re-gulation contribute to establish an activity balance of these proteins, which mediatesstimuli-selective and tissue-specific induction of apoptosis. The signals that influ-ence apoptosis converge to control the activity of proapoptotic Bax and Bak, asgenetic deletion of both proteins renders cells unable to undergo apoptosis via theintrinsic pathway [4]. When the balance favors activity of proapoptotic members,Bax translocates from the cytosol to the mitochondrial outer membrane, where itpolymerizes with mitochondria-localized Bax to form a pore. Bcl-2 family mem-bers modulate this step by two alternative but nonexclusive mechanisms. Some sig-nals induce apoptosis by activating proapoptotic BH3-only proteins, which bind toand neutralize selectively antiapoptotic Bcl-2 family members, releasing Bak/Bax tooligomerize. For example, proapoptotic Bad mediates induction of apoptosis by thismechanism [5]. Other proapoptotic BH3-only proteins such as Bid and Bim com-pete with antiapoptotic members but also have the potential to interact directly withBax/Bak for activation. In both scenarios, the emerging consensus is that severalof the antiapoptotic factors need to be simultaneously neutralized by proapoptoticfactors for apoptosis to proceed, requiring the cooperation of multiple membersof the family acting as sensitizers or de-repressors. Thus the current view is thatbecause proapoptotic BH3-only members present a selective pattern of interactionwith antiapoptotic Bcl-2 family members, the right combination of antiapoptotic andproapoptotic members has to be engaged to either halt or unleash apoptosis. Theseinteractions between Bcl-2 family members are further regulated by phosphoryla-tion status and/or targeted degradation (see Fig. 1 and below and Ref. 3).

2.2 Mitochondrial Outer Membrane Permeability

Once Bak/Bax become activated, they increase mitochondrial outer membrane per-meability to release proteins contained in the intermembrane space. This eventcauses mitochondrial membrane potential dissipation and matrix swelling, compro-mising mitochondrial function and structure. This is the mechanistically least under-stood step in the pathway, as the exact identity of the pore releasing the proteins isstill a matter of debate [6]. A candidate participant and modulator is the permeabilitytransition pore (PTP), a multiprotein complex spanning the outer and inner mito-chondrial membrane formed by voltage-dependent anion channel (VDAC), ade-nine nucleotide translocase, benzodiazepine receptor, cyclophilin D, hexokinase,and other proteins. This pore mediates the exchange of ATP/ADP and other solutesin the normal state. Its participation in apoptosis is supported by the effects of phar-macologic inhibitors and activators for the different components and thus constitutesan attractive target for therapy bypassing Bcl-2–dependent regulation [7]. However,these inhibitors do not abolish all pathways conducive to apoptosis, and apopto-sis still occurs in models with genetic deficiencies of these proteins, suggesting thatthis complex regulates other pores releasing the apoptotic effectors or that—in addi-tion to the PTP—there are multiple mechanisms for proapoptotic effectors release.Regardless of the mechanism, however, once the mitochondrial membrane becomes

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permeable, the cell is committed to die even if downstream caspase activity is abro-gated with pharmacologic inhibitors [8].

Proteins residing in the intermembrane space such as cytochrome c, Smac/DIABLO, apoptosis-inducing factor (AIF), Endo G, and Omi/HtrA2 are releasedsimultaneously and shortly after Bax/Bak translocation. Free in the cytoplasm,cytochrome c binds Apaf-1, causing a conformational change facilitating ATP/dATPexchange and oligomerization followed by recruitment of caspase 9. This multipro-tein complex, the apoptosome, is formed to activate the initiator caspase 9, whichcleaves and activates the downstream effector caspases 3 and 7. Smac/DIABLOand Omi/HtrA2 are proteases that inactivate the endogenous cytosolic inhibitorsIAPs (inhibitors of apoptosis) to facilitate the activity of the newly formed apopto-some [9].

AIF as well as Endo G translocate to the nucleus and participate in chromatincondensation and lysis [2]. The extent of their contribution to the decision toundergo apoptosis is still unclear, but they are significant contributors to biochemi-cal and morphologic manifestations of apoptosis. AIF is a flavoprotein participatingin normal mitochondrial bioenergetic and redox metabolism, thus, its release con-tributes to mitochondrial dysfunction and to apoptosis irreversibility [10].

2.3 Caspases Are the Executors of Apoptosis

Active cell dismantling is brought about by proteases termed caspases, which use anucleophilic cysteine in the active site to cleave immediately distal to an aspartate-containing motif present in specific substrates. Among more than 14 members ofthis family in mammals, only 7 play known roles in apoptosis, while many partici-pate in physiologic functions unrelated to apoptosis. All caspases exist as zymogensin the cytosol and are grouped according to their mechanism of activation. The ini-tiator or apical caspases (caspase 2, 8, 9, and 10) are autocatalytically activated byproximity after associating through their N-terminal domain to a multiprotein acti-vating complex incrementing their catalytic activity by several orders of magnitude.Effector caspases (3, 6, and 7) are activated by initiator caspases–mediated cleavagein a 20- to 30-amino-acid linker sequence [11].

A further level of regulation comes from the activity of cytosolic IAPs, whichneutralize caspase activity. Discovered for their ability to suppress apoptosisin baculovirus-infected cells, the IAP proteins are regulated by ubiquitylation-mediated proteasome degradation [12].

2.4 Changes in Apoptotic Players Associated with Cancer

Unlike oncogenic mutations, changes in the apoptotic pathway are generally insuf-ficient to initiate the carcinogenesis process but play a significant role in tumorpromotion. Overexpression of antiapoptotic Bcl-2 or Bcl-xL is observed in multiplecancers and accounts for acquired chemoresistance [3]. Bcl-2 was first identifiedas an oncogene at a chromosomal breakpoint of t(14:18) in human follicular B-celllymphoma. Unexpectedly, this oncogene localized to the mitochondrion and did not


induce cell proliferation. Accordingly, when Bcl-2 is overexpressed as a transgenein murine models of carcinogenesis, it is not sufficient to elicit oncogenesis but pro-motes clonogenic survival and growth of the primary tumor when expressed togetherwith an archetypal oncogene. Bcl-2 coexpression with Myc drives tumorigenesis inlymphocytes and in mammary gland. Further studies conditioning Bcl-2 expressionin a double transgenic mouse together with Myc shows that switching Bcl-2 expres-sion off does not alter the onset of Myc-induced leukemia but results in tumor remis-sion and prolonged mice survival. These experiments highlight the tumor-promotingfunction of antiapoptotic factors such as Bcl-2 while simultaneously revealing thepersistence and continuous pressure of proapoptotic signals acting on a tumor.

Contrary to expectations, deletion of apoptosome components does not con-tribute to oncogenic transformation. This is evidenced by experiments where theselective deficiency of Apaf-1 or caspase 9 does not enhance lymphomagenesis norcontributes to fibroblast transformation [13]. Analysis of tumor samples reveal thatalterations in molecules regulating the apoptosome function are more frequent [9].From these studies stands out a correlation between the increased expression intumors of caspases inhibitors (IAPs), especially survivin, with poor disease progno-sis. Enhanced survival capabilities become significant at advanced stages of tumorprogression during invasion and metastasis. In fact, isolated prostate tumor cells cir-culating in the bloodstream in animals bearing an orthotopic xenograft of prostatecarcinoma cells showed increased resistance to anoikis, a special type of cell death(see Section 3.1) due to induction of the caspase inhibitors XIAP, cIAP2, and sur-vivin, correlating with enhanced metastatic potential [14]. Survivin is a member ofthe AIF protein family that interferes with caspase activity and is one of the mostcommonly expressed proteins in transformed cell lines and human tumors, whileabsent in normal tissues [15]. Moreover, it is a poor prognosis factor correlatingwith aggressiveness and invasive behavior. Survivin is a target of p53 transcriptionalrepression (see Section 4.1), therefore it is found upregulated in tumors bearing p53mutations. Targeting survivin for therapeutic purposes, however, is problematic dueto the essential role it plays in mitosis.

3 Deceiving the Surveillance Mechanism by UpregulatingSurvival Signals

The activity of Bcl-2 family members is regulated by information collected from theintracellular and extracellular milieu, both of which undergo significant alterationsduring carcinogenesis.

3.1 Evading Restrictions Imposed by Cell-Cell Interactionsand Tissue Architecture

Multicellular organisms have a number of signaling mechanisms geared towardensuring systematic cell proliferation and differentiation during development andlimited clonal expansion of somatic cells during growth and cell renewal in the

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adult organism. Furthermore, proper tissue organization is maintained by the spa-tially restricted availability of proliferation and prosurvival signals from the base-ment membrane and by repressive proapoptotic signals from neighboring cells.

Repressive signals from adjacent cells actively promote apoptosis directing theselective death of cell populations to organize organs and tissues during develop-ment, as is the case for eliminating abnormal, misplaced, or harmful cells duringlymphocyte T and B development or for neuronal pruning during development. Inthe adult state, controlled apoptosis actively maintains tissue structures. For exam-ple, in vivo and in vitro experiments show that apoptosis actively maintains thelumen in the mammary gland acini through a signal that promotes proapoptoticBim activity. A lumen is maintained even when disorganization is experimentallyintroduced by forced expression of proliferation inducing proteins leading to abnor-mal cell multiplication in the acini. Lost of structure by lumen repopulation canoccur only when survival signals are delivered either by the coexpression of anti-apoptotic proteins or by an oncogene conferring both prosurvival and proliferativesignals [16]. Thus, these experiments illustrate the existence of geometric cues builtin the organization of tissues, which need to be surmounted very early in tumordevelopment.

Cell surface molecules involved in adhesion communicate these controlling sig-nals, acting upon later stages in tumor development as well. In multistage cancermodels such as chemically induced squamous cell carcinoma, expression of sur-vivin as a transgene in skin opposes apoptotic signaling from surrounding cells,thereby significantly reducing the spontaneous regression rate of benign lesions[17]. The intercellular adhesion molecule E-cadherin mediates tissue architecturerestrictive signals that are active during cancer initiation as well as during metastasis,as downregulation of E-cadherin expression correlates with skin carcinoma progres-sion. Accordingly, restitution of E-cadherin expression in transformed keratinocytesreduces in vivo tumor formation potential [18]. E-cadherin silencing in mammaryepithelial cells, accelerates invasive disease and metastasis formation in transgenicmice models, forming tumors resembling invasive lobular carcinoma [19].

An additional source of restrictive signals is the obligatory epithelial cell con-tact with the basement membrane, which delivers localized proliferation and pro-survival signals, actively opposing apoptosis [20]. When epithelial cells detach andlose contact, they undergo a special form of cell death designated anoikis. Thismechanism maintains digestive epithelia homeostasis by inducing death and shed-ding of enterocytes at the intestinal villi tip. In contrast, it is not observed in highlymotile cells such as fibroblasts, where interference with integrin-matrix interactionstriggers instead a tissue repair and remodeling phenotype characterized by the acti-vation of NF-�B and matrix metalloproteinase release [21].

Cell detachment causes integrin disengagement from the extracellular matrix,focal adhesion complex disassembly, and cytoskeleton disorganization, triggeringmultiple proapoptotic signals. Interference with �1 integrin binding to the extracel-lular matrix using function blocking antibodies is sufficient to induce keratinocyteapoptosis characterized by caspase 8 activation and proapoptotic Bid translo-cation to mitochondria [22]. Disassembly of focal adhesion complexes disrupts


prosurvival signaling mediated by FAK, SRC, and PI3K-AKT kinases, all recruitedand activated by organized focal adhesion complexes in substrate-attached cells.Interference with FAK activity induces rapid Bax translocation to mitochondriain mammary gland epithelial cells [23]. The disorganization of microfilament andmicrotubule cytoskeleton during cell detachment also releases proapoptotic BH3-only proteins Bmf and Bim that neutralize Bcl-2 antiapoptotic activity. ProapoptoticBmf is indirectly associated to the actin cytoskeleton by binding to dynein lightchain 2 complexed to myosin V. Proapoptotic Bim associates indirectly to tubulinthrough dynein light chain 1 sequestered by associated dynein motor complex (seeRef. 24 and references therein). Resistance to anoikis is a key factor to cell survivalduring invasion, metastasis, and growth in new environments at distal places fromthe primary tumor. Consequently with their prominent role in anoikis prevention,suppression of FAK activity diminishes resistance to anoikis as well as metastaticpotential [25].

An additional strategy used by tumor cells to escape apoptosis is to alter the cellsurface repertoire of receptors. The apoptotic balance is modulated either by silen-cing cell surface molecules mediating proapoptotic signals in non-transformed cellsand/or by inducing new molecules promoting cell survival. The receptor for netrin-1, NHC5 or “deleted in colon cancer,” induces apoptosis when unengaged, thus itsexpression is reduced in most colorectal cancer types and many other tumor types[26]. Because survival signaling is integrin-selective, squamous cell skin carcinomasswitch the expression of integrin subunit, repressing the anoikis-promoting alphavbeta5 and inducing alphav beta6, which renders these cells protected by increasingAKT activation [27]. However, in leukemias the significance of cell adhesion andthe environment in the control of apoptosis is indisputably most evident: These nor-mally nonadherent cells exhibit a much higher sensitivity to apoptosis when testedseparated from their environment in vitro than when tested in vivo [28]. In fact,�1 integrin–dependent adhesion of leukemia cells in vitro is sufficient to induceproapoptotic Bim destabilization and confer resistance to apoptosis. A role for theenvironment is further supported by experiments showing that multiple myelomacells become significantly more resistant to chemotherapeutic drugs when adheringto bone marrow stroma [29].

3.2 Autonomous Survival Signals Acquired Through OncogeneActivation and Anti-oncogene Silencing

The remarkable effects of oncogene-targeted therapeutic agents used in mousetumor models reveal the dominant influence of an activated oncogene on tumorphenotype and survival. This has led to the concept of “oncogene addiction” [30].Most oncogene-activated and tumor suppressor–silenced pathways drive prolifera-tion and promote cell survival by increasing the activity of ERK, PI3K, and AKTkinases [31]. The activation of these kinases downstream of Src, Abl, and EGFRincreases prosurvival signaling [30]. Mutations in the effectors of these pathwayseither increase their catalytic activity or silence the activity of negative regulators.

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These kinases have a profound effect on the function of the apoptotic cascade byregulating the expression, function, and stability of Bcl2 family members throughtranscriptional and posttranslational mechanisms. ERK induces the expression ofantiapoptotic proteins Bcl-2, Bcl-xL, and Mcl-1, while MAPK-dependent phospho-rylation stabilizes Bcl-2 and causes proapoptotic Bim degradation [32].

AKT is the active kinase most frequently found in tumors and mediates prosur-vival signals from many oncogenes and growth factors [33]. AKT exerts a dominantprosurvival activity through multiple mechanisms. AKT phosphorylates the BH3domain of proapoptotic Bad, interfering with apoptotic function and promotingsequestration by cytosolic 14-3-3 proteins [34]. In addition, AKT regulates glucosemetabolism thereby amplifying prosurvival signals (see Section 4.2).

4 Survival Effects of Metabolic Changes in Cancer Cells

Unrestricted proliferation amid accumulated mutations drive the accrual of pro-found alterations in cell metabolism and function, in addition to signal transduction.Furthermore, metabolic changes are advanced by stress that follows local hypoxiaand metabolite accumulation imposed by the substantial and rapid increment in cellmass. There is mounting evidence to suggest that these abnormalities, which wouldbe a source of stress and proapoptotic signals in normal cells, are not only opposedby tumors but also are key in fostering the acquisition of survival capabilities andare a determinant factor in tumor promotion and expansion [35].

4.1 p53

When normal cells experience alterations in the cell cycle and metabolism, such asreactive oxygen species, hypoxia, double-strand DNA breaks, and abnormal proli-feration rate, p53 tumor suppressor is activated to induce growth arrest and/or apop-tosis to ensure DNA repair in order to proceed with cell replication. P53 inducesapoptosis by multiple mechanisms. It promotes apoptosis through the transcrip-tional induction of the proapoptotic BH3-only proteins Bad, Bax, Puma, and Noxa.In a second mechanism, p53 activates Bax directly, releasing proapoptotic BH3-only proteins sequestered by Bcl-X and promoting Bak oligomerization by displac-ing bound antiapoptotic Mcl-1. P53 activation can be detected in pre-neoplasticlesions, including colon adenomas, breast carcinomas in situ, and lung hyper-plasias in response to replicative stress resulting from deregulated cell proliferationand to double-strand DNA breaks, but preceding any signs of genomic instabil-ity or genetic alterations in the p53 pathway [36]. This observation indicates thatp53 is activated very early in carcinogenesis by oncogenic stress and acts as anapoptosis-promoter and a barrier for tumor development. This barrier, however,is early averted by inactivating or attenuating p53, as mutations can be detectedin early dysplastic lesions of non–small cell lung carcinomas as well as in mosthuman cancers, thereby driving tumor development [37]. Experiments in models


of myc-driven lymphomas show that the apoptosis promoting activity is sufficientfor effective p53-dependent tumor suppression, while growth arrest function is dis-pensable [38]. Restitution of p53 expression in advanced tumors causes significanttumor regression by inducing apoptosis in lymphomas and senescence in sarco-mas and liver carcinomas [39]. Thus, these experiments underscore the significantrole of p53 in suppressing tumor development through the control of the apoptoticpathway.

4.2 Glycolysis

A switch to glycolysis as an alternative or additional source for ATP is essentialto the cancer phenotype. In normal cells, p53 imposes a limitation on the gly-colytic rate by a two-pronged mechanism [40]. P53 induces the expression of acytochrome oxidase regulatory subunit, synthesis of cytochrome C oxidase, increa-sing mitochondrial respiration, and induces the expression of Tigar, which controlsfructose-2,6-biphosphate intracellular levels, inhibiting glycolysis at the level of6-phosphofructo-1-kinase activity. During transformation, glycolysis increases byseveral mechanisms in addition to loss of p53 control. The levels of fructose-2,6-biphosphate are regulated by several kinases, including AKT, which phosphorylatesand activates 6-phosphofructo-2 kinase to increase glycolysis. Local hypoxic con-ditions, as well as alterations in metabolism resulting from accrued mutations andfrom oncogene activation, such as Bcr-Abl, Myc, and K-Ras, can trigger a switch toglycolysis [41].

Glucose utilization is a survival factor per se, conferring resistance to apoptosisproportional to the glycolytic rate [42]. Glycolysis inhibition in Myc-transformedcells precipitates apoptosis, whereas normal cells respond with cell cycle arrest [43].Although the mechanism is not entirely clear yet, and glycolysis could modulateseveral steps in the apoptotic pathway, most of the evidence points to an alterationof mitochondrial function downstream of the Bcl-2 regulatory step. Tumor cellsincrease their glycolytic rate by promoting hexokinase localization to the mitochon-drial outer membrane and binding to VDAC, where hexokinase uses ATP producedby oxidative phosphorylation to catalyze the first step of glycolysis [41]. Recon-stitution of hexokinase binding to VDAC in liposomes shows that this event alsopromotes pore closure, thus this event could regulate apoptosis by modulating themitochondrial outer membrane permeability. Consistent with this possibility, forceddisruption of hexokinase association to mitochondria causes cytochrome c releaseand apoptosis by a mechanism independent of Bax/Bak and Bcl-2. Conversely,forced expression of mitochondria-binding hexokinase neutralizes the ability ofactivated proapoptotic Bid and Bax to induce apoptosis. AKT prosurvival activi-ties include the upregulation of glucose transporters and maintenance of hexokinasebinding and activity at mitochondria [41]. The resulting upregulation of glucosemetabolism accounts for most of AKT antiapoptotic function, because this activitycan be completely abolished by a hexokinase inhibitor [44].

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The progressive metabolic dependence of tumors on glycolysis for ATP gene-ration is currently being explored for therapy purposes, revealing glycolysis as aninfluential control point for apoptosis. The use of glycolysis inhibitors in tumor celllines induces apoptosis even in multidrug-resistant cells and are most effective incells with respiratory deficiencies or in those exposed to hypoxia [41, 45].

4.3 Hypoxia

Hypoxia can impose stress on cancer cells due to their reduced access to nutri-ents and oxygen. However, it can also provide a selective pressure in tumors for theexpansion of variants that have lost their apoptotic potential. Cellular oxygen tensionis sensed by prolyl and asparaginyl hydroxylases, which at normal oxygen levelstarget hypoxia inducible factor (HIF) for von Hippel–Lindau dependent degrada-tion. When oxygen tension decreases, HIF accumulates and is able to function as atranscription factor, inducing the expression of genes involved in both proapoptoticand antiapoptotic pathways, resulting in the removal of cells that have retained theirapoptotic potential and proliferation of cells that have lost their apoptotic poten-tial [46]. HIF-� induces cell death through a p53-dependent mechanism and bythe direct transcriptional induction of proapoptotic BH3-only proteins Noxa, Bnip3,and Nix [47]. Bnip3 mediates a distinctive form of apoptosis, which is cytochromec, AIF, and caspase independent, but involves mitochondrial dysfunction, a mecha-nism compatible with mitochondrial permeability transition and resembling necro-sis in some aspects [48]. Consistent with this finding, high expression levels of theproapoptotic factors Bnip3 as well as Nix are found in necrotic areas of tumors andcorrelate with poor prognosis in cervical cancer, non–small cell lung carcinomas,and pancreatic cancer.

Tumor hypoxia is an additional selective pressure for p53 inactivation and otherchanges leading to apoptosis resistant cells, including Bnip3 silencing. Pancreaticadenocarcinomas switch off the expression of Bnip3 late in progression by pro-moter hypermethylation. Additionally, hypoxia is a selective pressure at later stagesof tumorigenesis by promoting apoptosis resistance through antiapoptotic Mcl-1expression and thereby promoting the selection of highly metastatic Lewis lung car-cinoma cells [49].

4.4 Tumor Acidosis

The consequences of increased glucose utilization are lactate production and localacidification, therefore this will be an attribute of all glycolytic tumors. Relativelylittle is known about the molecular effects of environmental acidosis on tumorphysiology. Some evidence suggests that local acidosis could be a barrier to tumorgrowth in early carcinogenesis. In vitro studies reveal that acidosis can cause growtharrest and can alter sensitivity to chemotherapeutic drugs [50]. Environmental acido-sis induces p53-dependent cell death in colon adenocarcinoma cells in vitro. Other


evidence suggests that acidosis exacerbates hypoxia-mediated proapoptotic signals.This has been observed in normal cardiomyocytes under ischemia, where hypoxiainduces the expression of proapoptotic Bnip3 while acidosis is necessary to accu-mulate Bnip3 protein in the mitochondria and activate cell death [51].

At later stages of tumor development though, acidosis follows the pattern of allthe other factors reviewed here thus far, correlating with tumor aggressiveness andthe acquisition of proinvasive properties during, for example, the transition fromcolon adenomas to invasive cancer and from carcinoma in situ to invasive breastcancer [52]. Human melanoma cells cultured at low pH exhibit increased invasioncapabilities in vitro and in vivo. Thus, it is foreseeable that acidosis could be a factoraffecting tumor progression. It remains uncertain whether acidosis alters the apop-totic pathway in tumor cells as a factor independent of glycolysis.

4.5 Mitochondria

The pathways leading to cell death or survival converge at the mitochondria forintegration, interpretation, and amplification. Even though mitochondria are at thecenter of apoptosis, very little is understood on how mitochondrial function affectsapoptosis and vice versa. Most tumors harbor some level of mutations in mitochon-drial DNA, many of them affecting oxidative phosphorylation [53]. Nevertheless,the role of mitochondrial respiration on apoptosis remains controversial. Some stud-ies indicate that proper Bak-Bax function is dependent on a functional respiratorychain. Bax function is completely impaired in yeast mutants in any component of therespiratory chain. These studies contrast with other studies showing that ho0 cellsmaintain the capacity to undergo apoptosis in vitro and in vivo, as well as sensitivityto doxorubicin and growth factor withdrawal [54]. The impact of mitochondrial res-piration on apoptosis could be indirect. Acute mitochondrial respiration deficiencyinduced by culturing cells in EtBr or treatment with respiratory chain inhibitors issufficient to increase the glycolytic rate, to facilitate cell growth in hypoxic condi-tions, and to reduce susceptibility to several chemotherapeutic agents. This pheno-type induced by reducing mitochondrial respiration correlates with increased AKTactivity and can be reversed with inhibitors for this kinase. Thus, this work pro-vides an alternative interpretation to experiments examining respiratory chain func-tion requirement in apoptosis, in addition to suggest that resistance could emergefrom a switch to glycolysis rather than from a decrease in respiratory chain functionper se [55].

Apoptosis can affect mitochondrial function as well. Many experiments hint toa role for apoptotic players in the control of mitochondrial function. In addition tocytochrome c participation in the respiratory chain, Bcl-x regulates outer membranepermeability to anions and inhibits apoptosis by facilitating ADP transport requiredto maintain membrane potential. AIF has NADH-dependent oxidase activity, whichis required not only for complex I activity but also for anchorage-independentgrowth and tumor formation. Other mitochondrial changes remain poorly stu-died, although they may be of potential therapeutic interest. Mitochondria from

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transformed cells and tumors exhibit a higher membrane potential than do normalcells, correlating with growth rate. Mitochondria isolated from tumors have a higherrelative intrinsic resistance to mitochondrial outer membrane permeability. Theseproperties have been exploited to concentrate toxic molecules with a delocalizedpositive charge in mitochondria and promote apoptosis and mitochondrial dysfunc-tion in transformed cell lines and tumors [56].

5 Concluding Remarks: Implications for Therapy

The wide recognition of the important role played by apoptosis evasion in car-cinogenesis has fueled the development of therapies targeted at overcoming thisresistance and thereby increasing cell susceptibility to chemotherapy [57]. Afterpromising results in preclinical studies, approaches targeting the apoptotic path-way are currently being tested in clinical studies. The results observed illustratethe advantages and problems associated with these strategies. Initial studies withG3139/Oblimersen, an antisense oligonucleotide directed at interfering with anti-apoptotic Bcl-2 expression, demonstrated a complete lack of effect as a singletherapeutic agent. In combination with fludarabine and cyclophosphamide, whichaffect DNA metabolism, however, it caused a limited increase in drug sensitivity inrelapsed chronic lymphocytic leukemia and advanced melanoma patients. Becausedrugs directed at proteins involved in apoptosis target a ubiquitous pathway, theyhave toxic effects in normal cells, causing neutropenia and thrombocytopenia, aswell as disrupting angiogenesis by altering endothelial cell function [58].

The limited efficacy of Bcl-2 targeting could be explained by several factors.One possibility is that the results may not correlate with delivery and expressionknock-down efficiency. Another significant factor conferring resistance could be thepresence of other functionally compensating antiapoptotic family members. Thus,rather than disrupting the function of a single member of the family, interfering withBH-3 domain activity widens the inhibitory range of the potential drug. The drugABT-737 is a potent BH3 peptidomimetic, antagonizing the activity of the antiapop-totic factors Bcl-2, Bcl-xL, and Bcl-w. However, this drug has proved ineffectivewhen Bcl-2 is phosphorylated or when antiapoptotic Mcl-1 is present [59].

An alternative strategy is to bypass Bcl-2–dependent regulatory mechanism andtarget mitochondria directly to trigger apoptosis and release intermembrane effec-tors. Using this approach, the use of a Smac/Diablo mimetic to activate caspasesdirectly has been successful in preclinical studies, overcoming resistance inducedby increased Bcl-2 expression, autocrine growth factors, and adhesion in multiplemyeloma [60].

Combination therapies would benefit altogether by considering environmentaland bioenergetic factors affecting the apoptotic pathway, offering additional targetsand aiding in proapoptotic therapy selectivity. In the case of ABT-737, therapeuticefficacy could be improved by using glucose metabolism inhibitors, which wouldcause antiapoptotic Mcl-1 destabilization, as turnover and activity is controlledby glucose-dependent inhibition of GSK-3 activity [41]. Furthermore, the tumor


characteristic alterations in metabolism could impart selectivity to apoptosis-targeted therapy. Inhibition of glycolysis causes massive apoptosis accentuated intumors with mitochondrial respiration deficiency and in hypoxic cells [45]. Reduc-tion of mitochondrial respiration with As2O3 increases drug-sensitivity as well.Furthermore, local acidosis could be exploited to activate chemotherapeutic drugslocally [50]. Thalidomide, a drug known for immunomodulatory effects, disruptstumor homeostasis and interaction with the environment. It successfully inducedapoptosis in refractory multiple myeloma cells and decreased the resistance tochemotherapeutic drugs in vitro, preventing adhesion to bone marrow cells [61].In prostate cancer, this drug restores interactions with the environment by increa-sing E-selectin expression [62]. Given these results, thalidomide is currently beingtested in phase II trials for refractory multiple myeloma and solid tumors.

Therapies aimed at modifying the cellular metabolism in order to increase sus-ceptibility to apoptosis address tumor heterogeneity, an additional issue arising inclinical circumstances and poorly reproduced by mouse models. This is a signifi-cant source of variability, accounting in large part for the disappointing inconsis-tency observed in the results obtained from preclinical and clinical studies, as wellas the difficulties in correlating disease prognosis with apoptotic marker expressionin tumor samples. Tumor models of clonal origin, where oncogenesis is driven byeither the expression of a single oncogene in transgenic mice or by the graft of acell line originally isolated as a clone from a tumor, are ideal models to dissectthe role of particular components of a pathway, regulating factors in specific stagesof disease, for evaluation of possible therapeutic targets. However, tumor samplesfrom patients show high heterogeneity in expression levels of apoptosis markers indifferent areas of the same tumor, coexisting with areas of high apoptotic index.Single cell profiling reveals that heterogeneity determines differential response tocytokines and drugs and has been proposed to be a key factor for promoting diseaseprogression in esophageal cancer. Thus, because drugs modulating cell metabolismdo not target specific oncogenic pathways or mutations, they are expected to be moreeffective in overcoming tumor heterogeneity as well as in targeting a wider range oftumors.

Acknowledgments This work was supported by an award from the American Heart Associationand by grant number K22CA127136 from the National Cancer Institute.

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Index

AAbate, C., 64Abbey, C. K., 173Abu-Hamad, S., 12Acetoin, 106, 107Acin-Perez, R., 128Acker, T., 135Aconitase in Krebs cycle, 4Adams, J. M., 36, 164, 166Adebanjo, O. A., 63Adida, C., 167Agani, F., 82Ahern, K. G., 106, 113Ahn, H. J., 172Ainscow, E. K., 115Akimoto, M., 59, 123Akt/PBK (protein kinase B)

pathway, 8Alam, N. A., 25, 59Alarcon, R., 138Alazard, N., 58, 62Alderson, M. R., 149Aldolase (ALD), 84, 85Alessi, D. R., 12Allan, S., 94Allen, S. M., 168Almodovar, C. R., 114Alonso, A. M., 6, 10Alonso, C., 30Alquier, T., 12Altenberg, B., 6Altieri, D. C., 167Ambrosini, G., 167Amir, S., 136Amstad, P., 64Amuthan, G., 48–50, 58, 61–63, 67, 119Ananadatheerthavarada, H. K., 48–50, 58,

61–63, 67Anbazhagan, R., 62

Anderson, S., 121Andersson, U., 61Angiogenesis, 37–39Antiapoptotic genes, 66–67Antioxidant

antioxidant response element(ARE), 38

defense pathway, 65enzyme levels, 105

Aoyama, N., 7Apoptosis, 36, 47, 162–163

Bcl-2 family proteins, 164–165caspases and, 166

Appel, L. J., 2Apse, K., 97Armour, S. M., 24Arnold, R. S., 35, 38Arora-Kuruganti, P., 63Aryl hydrocarbon nuclear translocator

(ARNT), 135Asham, A. M., 134Ashley, S. W., 169Aslan, M., 27Astuti, D., 23, 59, 136Attardi, G., 60Atzei, P., 168Augenlicht, L. H., 126Avadhani, N. G., 48–50, 58, 61–63, 67, 119Azoulay-Zohar, H., 12

BBabior, B. M., 20Bach, D., 66Bader, S., 139Bae, S. H., 24, 80, 81Bae, Y. S., 34Baeuerle, P. A., 64Baggett, B. K., 8Baggetto, L. G., 9, 106, 107

179

180 Index

Baldi, P., 25, 27, 47, 127Bale, A. E., 46Ballard, J. W. O., 108Bamezai, R. N., 128Bandelt, H. J., 22, 47, 126–128Bankier, A. T., 121Baranov, E., 171Barrell, B. G., 121Barrett, J. N., 60, 61Bartkova, J., 170Bartnik, E., 121, 123, 126Bassik, M. C., 165Battersby, B. J., 128Bauer, C., 78Bauer, D. E., 8, 79, 113Baumann, A. K., 9, 58, 64, 127Bayascas, J. R., 12Baysal, B. E., 9, 20, 23, 59Beach, D., 94, 95Beasley, N. J., 137Bellamy, W. T., 29Bell, E. L., 38Benit, P., 24, 25Benizri, E., 77Bensaad, K., 9Ben-Shlomo, I., 115, 116Berardi, M. J., 174Berchner-Pfannschmidt, U., 75, 77Berezovskaya, O., 167Berman, S. B., 47Bernard, D., 132Bernet, A., 169Berra, E., 77Bhat, A. K., 128Bhat-Nakshatri, P., 64Bhujwalla, Z. M., 137Bhushan, A., 148Bianchi, M. S., 124, 125Bianchini, F., 107Bianchi, N. O., 124, 125Bindra, R. S., 134Birch-Machin, M., 123Birnbaum, M. J., 7Birrer, M. J., 64Bishopric, N. H., 114, 173Bissell, M. J., 168Biswas, G., 48–50, 58, 61–63, 67, 119Bi, X., 11Blachly-Dyson, E., 11Blanco-Rivero, A., 2, 9–11Blasco, M. A., 94Bluyssen, H. A., 140Boatright, K. M., 166

Bodyak, N. D., 57, 58, 125, 126Bond, J. D., 63Bonhoeffer, S., 2, 5, 9Bonicalzi, M. E., 139Bonni, A., 41Bonod-Bidaud, C., 169Bonora, E., 127Booker, L. M., 128Borisenko,G. G., 107Borowsky, A. D., 173Bottoni, P., 50, 51Bouchier-Hayes, L., 165Boudreau, N., 168Bouillaud, F., 21Boulianne, G. L., 96Bourgeron, T., 22, 25Bove, K., 38Bowden, G. T., 57, 64, 65Boyer, P. D., 3Bracale, R., 107Braithwaite, K. L., 92Brand, M. D., 115Brandon, M., 25, 27, 47, 127Brands, M., 2Braunstein, L. D., 97Breitschopf, K., 170Brenner, C., 165Bretscher, P. A., 149Briehl, M. M., 29Briere, J. J., 12, 20, 23–25, 166Brigelius-Flohe, R., 64Briggs, J., 140Briones, P., 66Bromley, L., 63Brown, D. T., 124Brown, S. J., 46Brundtland, G. H., 148Brune, B., 138Brune, B., 79Brunelle, J. K., 38Brunet, A., 41Bryk, J., 128Buecher, T., 22Bulavin, D. V., 11Bunn, H. F., 136Burdon, R. H., 63Buricchi, F., 37Burk, D., 5Burne, J. F., 173Bustamante, E., 8, 27Butow, R. A., 67, 68Butts, B., 65Buzzai, M., 8, 79

Index 181

CCaballero, O. L., 57Caenorhabditis elegans and mev1 mutant, 23Cairns, R. A., 10Calcium signaling pathway, 62, 63Calderwood, S. K., 48Calle, E. E., 107Camenisch, G., 138Cameron, G., 133Camley, R. E., 152Campanella, C., 48Campisi, J., 95, 108Cancer, 46

apoptotic pathway in, 166–167cells, 145

Achilles’ heel of, 29AKT activation in, 78–79bioenergetic signature of, 9HIF-1α expression in, 88Notch-3 and carbonic anhydrase IX

(CA-IX), 42PTENoxidative inactivation and AKT

signaling pathway, 41retrograde signaling, 60–67Warburg effect in, 8, 46Warburg phenotype of, 10–11

COXI mtDNA mutations, 58drug treatment and Fas-induced tumor cell

death, 150–157fumarate hydratase (FH) mutations, 88G3139/Oblimersen studies with, 174HKII transcription, 83homoplasmy and heteroplasmy, 125immune

response in, 147–148system and, 149

metabolic changes in cell, survival effectsglycolysis, 171–172hypoxia, 172mitochondria and, 173–174p53 tumor suppressor, 170–171tumor acidosis, 172–173

mitochondrial genome instability (mtGI)in, 124–125

OXPHOSdefects in, 56dysfunction and, 66

Smac/Diablo mimetic use in, 174–175surveillance mechanism

cell-cell interactions and tissuearchitecture, 167–169

oncogene activation and anti-oncogenesilencing, 169–170

thalidomide drug for, 175therapeutic possibilities, 158–159

Cande, C., 12, 20, 166Cano, A., 168Cappello, F., 48Carcinogenesis

aerobic respiration and energy generation,104

calorie restriction inhibitionenergy expenditure and intake, 108metabolic disorders, 107UDPGlcNAc decreased levels, 109

chronic infection andsignaling molecules, 110

diversion of pyruvate mitochondriarespiration (PDH) and fermentation

(PDC), 106TCA cycle, 106

electron transport modulationNADH/cyt-C electron transport

pathway, 110glycolysis involvement in, 93immortalization and transformation, 91–92induced apoptosis, 111–112metabolic modulation of, 103organelle and cell membrane

perturbations, 107OXPHOS phenotype and apoptosis

ATP synthase and suppression of, 104reactive oxygen species and

cell-signaling pathways, 105mitogenic signals, 105

Warburg model, 113–115Cardile, A., 107Cardiolipin (CL), 107Carew, J. S., 9, 120, 122–125, 173Carling, D., 12Carmeliet, P., 138Carney, D., 126Carothers, A. D., 126, 128Carotid body PGL, 23Carracedo, A., 22, 47, 126–128Carter, M. J., 107Casado, E., 9–11Casimiro, M. C., 11Caspases and cellular proteins, 36Castilla, J., 30C2C12 rhabdomyosarcoma, ryanodine

receptor-1 (RyR-1) and-2 (RyR-2)genes, 63

Cellular respirationtumor suppressor genes

p53, 137–138

182 Index

Cellular respiration (cont.)von Hippel–Lindau syndrome, 138–140

and Warburg hypothesis, 132–133Cepeda, V., 30Cerutti, P., 64Cerutti, P. A., 63Chan, D. C., 66Chandel, N. S., 173Chandra, D., 48Chang, G. W., 136Chang, H. J., 10Chang, I., 11Chang, T. S., 34Chan, T. L., 57Chauhan, D., 174Chemodectoma, see Carotid body PGLChemotherapeutic-resistant tumor cells

sequester cytosolic calcium(Cacyt), 48

Chen, E. I., 52Chen, G., 6, 10Cheng, G., 38Cheng, M. L., 97Cheng, W. C., 47Chen, H., 66Chen, X., 22Chen, Z., 11Chen, Z. J., 65, 110Cheung, A. N., 57Cheung, H. S., 61Chiarle, R., 23Chiarugi, P., 37Chieco, P., 140Chilov, D., 138Chinnery, P. F., 124–126, 128Chipuk, J. E., 171Chi, S. M., 135Chittenden, T., 170Cho, E. J., 140Choi, J., 100Choi, K. Y., 107Chomyn, A., 66Chretien, D., 22, 25Chu, G., 42Chun, Y. S., 171Chu, S. C., 41Cinalli, R. M., 8Ciocca, D. R., 48Cisplatin-resistant ovarian carcinoma, 38Cizeau, J., 172Clayton, D. A., 22Clear cell renal carcinoma (CCRC) cells, 81Clifford, S. C., 136, 139

Cockman, M. E., 139Cohen, A., 110Cohen, C., 7Cohn, M., 149Colburn, N. H., 65Coller, H. A., 57, 58, 125, 126Colombini, M., 173Combs, C. A., 42Connor, K. M., 37, 38, 40Contractor, R., 174Copeland, W. C., 121, 122, 124Corral-Debrinski, M., 22Corso, A., 175Cory, S., 164, 166Coughlin, S. S., 107Couplan, E., 21Cozzi, V., 107Crawford, D., 64Crisp, W., 152Crosby, M. E., 134Cucuianu, A., 109Cuezva, J. M., 2, 6, 7, 9–12Curran, T., 64Cycloheximide (CHX), 36Cytochrome c oxidase (COX), 9Czarnecka, A. M., 48, 121, 123, 126

DDachs, G. U., 8Dalgard, C. L., 8Dallol, A., 23, 59Dalton, W. S., 169Dang, C. V., 7, 10, 46, 92, 134, 171Dang, G., 7Danial, N. N., 12, 164, 166Darin, N., 22Darvishi, K., 128Dasgupta, J., 37Dayan, F., 172Debatin, K. M., 30Decorps, M., 7de Heredia, M. L., 6, 7, 9–11Dellian, M., 134Delsite, R., 62De, Marzo, A. M., 80Deml, E., 9Demont, J., 9, 10, 58, 59Denko, N. C., 10, 134de Paulsen, N., 139De Pinho, R. A., 94Derksen, P. W., 168Desbaillets, I., 138Desbarats, J., 149

Index 183

Desvergne, B., 51Dey, R., 11, 67Diala, E. S., 66Dickinson, D., 96Dictyostelium discoideum and phosphoinosi-

tide (3, 4, 5) 3-phosphate(PtdIns[3, 4, 5]P3), 40

Dietrich, A., 22Dimmeler, S., 170Ditsworth, D., 113Dolde, C., 7, 93Domann, F. E., 64Domann, F. E. Jr., 64Don, A. S., 123Dong, Z., 64, 174Dor, Y., 138Douglas-Jones, A., 61Drago, I., 62Droge, W., 47Drosophila simulans, 108Dubik, D., 172Dujon, B., 22Dukes, I. D., 115Du, M., 10, 113Dunn, G. P., 110Dunning, S. P., 136Du, X., 113Duxbury, M. S., 169

EEarl, A., 66Ebert, B. L., 8Edelstein, D., 113Edens, W. A., 38Efstathiou, E., 175Egea, G., 11Electron transport chain (ETC)

cell death and proliferation, 28in mammalian cell, 56modulation, 110–111

El Ghouzzi, V., 25Elia, A. J., 96Elson, J., 125Elstrom, R. L., 8, 79Endo, T., 61Endothelial PAS-domain protein

1 (EPAS1), 79Energy-deprived HepG2 hepatoma cells,

mitochondrial biogenesis andOXPHOS, 108

Enolase (ENO), 85Enriquez, J. A., 119, 121, 125Epstein, C. B., 68, 137

Erlacher, M., 137Ernest, I., 135Erythropoietin (EPO) and VEGF, role in

hypoxia, 80E3 ubiquitin ligases and HIF-1α, 78Eukaryotic peroxiredoxins (PRXs), 35Evan, G. I., 150Evans, I. H., 66

FFabregat, I., 10–12Faddy, H. M., 62Faller, D. V., 135Fang, H. M., 8, 133, 135, 136Fang, W. G., 135Fan, M., 65Fantin, V. R., 46, 49, 51, 174Favier, J., 23–25Felty, Q., 50Fernandez, A., 135Fernandez, P. L., 6, 9, 11Fernandez-Silva, P., 119, 121, 125, 128Ferrans, V. J., 34Ferrell, R. E., 9, 20, 23, 59Ferretti, E., 175Fibrate/thiazolidinedione-induced

differentiation, 51Finch, J. S., 64Finkel, T., 34, 42Fink, K., 124Firth, J. D., 8Flier, J. S., 7Fliss, M. S., 57Flohe, L., 64Floodgate hypothesis, 35Florell, S. R., 168Floyd, R. A., 35Foekens, J. A., 140Foker, J., 11Folkman, J., 134Fontana, L., 10Fontana, L. A., 134Foo, T. W., 11Forbes, R. A., 82, 136Forkhead transcription factors (FoxO), 41–42Fornace, A. J. Jr., 11Fox, C. J., 8, 170, 171Franc, B., 59Francis, M. J., 63Frataxin and Fe-S cluster biogenesis, 11Freedman, B. D., 63Freedman, D. A., 137French, S., 42

184 Index

Fridman, J. S., 171Fuertes, M. A., 30Fuhrer, J. P., 66Fulda, S., 30

GGabrielson, E., 62Gajewski, C. D., 64Gajowniczek, P., 169Galluzzi, L., 30Gamallo, C., 168Gambhir, S. S., 7GAPDH overexpression and human prostate

adenocarcinoma cells (cell lineLNCap), 85

Garber, K., 46, 137Garca-Garca, E., 6, 7, 9, 10Garrido, C., 20Gasparre, G., 127Gassmann, M., 78, 138Gatenby, R. A., 7, 46, 50, 93, 115Gatter, K. C., 8Gauthier-Rouviere, C., 135Gebbink, M. F., 140Genes encoding complex II mutations, tumor

and cancer formation, 23Geromel, V., 22, 25Giacomello, M., 62Giardia lamblia, 4Giardina, B., 50Giatromanolaki, A., 8Gil, J., 96, 97, 132Gill, V., 63Gillies, R. J., 7, 8, 46, 50, 93, 115Gilmore, A. P., 169Gimenez-Roqueplo, A. P., 23, 24Ginkgo biloba, proapoptotic molecules

expression, 37Ginouves, A., 77Giorgio, M., 42Giovannini, C., 42Glazer, P. M., 134Gleadle, J. M., 8Glinskii, A. B., 167Glucose-6-phosphate dehydrogenase (G6PD)

activity on cell proliferation, 97Glucose phosphate isomerase (GPI), 83Glucose transporters (GLUT1 to GLUT5), 87Glyceraldehyde phosphate dehydrogenase

(GAPDH), 85Glycolysis, 2

aldolase (ALD), 84–85apoptotic pathways and, 114

ATP levels and OXPHOS, 111cell cycle and apoptotic pathway,

regulation, 112and cellular senescence, 94–95enolase (ENO), 85glucose phosphate isomerase (GPI), 83glyceraldehyde phosphate dehydrogenase

(GAPDH), 85glycolysis-dependent phenotype

of malignancies, 8hexokinases (HKs), 82–83hypoxia inducible factor 1 alpha

(HIF-1α), 8induction of hypoxia path by succinate

through stabilization of, 26hypoxic conditions and, 92–93lactate dehydrogenase and lactate carrier

MCT4, 86–87LMW-PTP activity and, 37malignant growth and, 5Nox1-dependent generation of H2O2, 382-OG dioxygenase, 75–76oncogene and, 93–94organisms life span and

calorie restriction, 98–99Sir2 as longevity gene in Yeast, 97–98

phosphofructokinase 1 (PFK-1), 84phosphoglycerate kinase (PGK), 85pyruvate dehydrogenase (PDH), 86pyruvate kinase (PK), 86as radical scavenger, 97regulatory mechanism of

p53 and, 99–100and PGM, 100–101

Sp1 and Bnip3, 106TIGAR acts as inhibitor of, 9

Glycolytic malignant cells, 106Gnarra, J., 139Gnarra, J. R., 139Goeddel, D. V., 36Goldberg, M. A., 136Goldblatt, H., 133Goldin, A., 150Golik, P., 121, 123, 126, 128Golstein, P., 20, 149Gomez, M., 168Gordan, J. D., 24, 113Gorgoglione, V., 110Goswami, P. C., 36Gottlieb, E., 47, 121Gottlieb, R. A., 20Gottlob, K., 171Goulet, R. J. Jr., 64

Index 185

Govindarajan, B., 7, 8Graeber, T. G., 137Gramlich, J. L., 41Gramm, C. F., 12Greco, M., 60Greenawalt, J. W., 57Greenblatt, M. S., 137Green, D. R., 4, 19, 20, 150, 171Greim, H., 9Greulich, K. O., 6Grimm, S., 165Gross, A., 11Groszer, M., 10Groulx, I., 139Grover-McKay, M., 7Guan, K. L., 12Guarente, L., 98Guppy, M., 5Gupta, A., 57, 64, 65Gutman, M., 22Gu, W., 132Gu, Y., 110Guyetant, S., 59Gyllensten, U., 127

HHabano, W., 9Habermacher, G. M., 128Haendeler, J., 170Hagler, J., 65Hale, W. T., 68Hammerman, P. S., 8, 170Hammond, E., 138Hanahan, D., 11, 92, 134Hanks, A. N., 168Hannan, R. L., 135Hannon, G. J., 94Haraguchi, K., 61Hardie, D. G., 12Harman, D., 94Harper, M-E., 149, 150, 154–158Harris, A. L., 8, 134, 140Harris, M. H., 11, 108, 171Hashida, M., 27Haspel, H. C., 7Hayflick, L., 94Hay, N., 171Heat shock proteins (HSPs), 48Heerdt, B. G., 126Heese, C., 9Helicobacter pylori, 109Helmlinger, G., 134Hendrix, M. J., 7

Hepatitis B and Epstein-Barr viruses, 109Herbert, J. M., 138Herman, J. G., 139Hernandez, O. M., 114, 173Herrero-Jimenez, P., 57, 58, 125, 126Herrmann, E. C., 9Herrmann, P. C., 9Herrnstadt, C., 47Hershman, J. M., 61Hervouet, E., 9, 10Hewel, J., 52Hexokinase II, 48Hexokinases (HKs), 82–83Hexosamine biosynthetic pathway (HBP)

nutrient uptake pathways, 105, 108O-GlcNAc glycosylation, 105–106

Hickey, M. M., 24Hickman, J. A., 51Higuchi, T., 9Hilliker, A. J., 96Hirabayashi, Y., 29Hirota, K., 132Hoberman, H. D., 9Hock, J. B., 104Hoffman, M. A., 139Hoffman, R. M., 171Hogg, P. J., 123Ho, H. Y., 97Hollstein, M., 137Hooper, L., 136Hopfl, G., 78Horejsi, Z., 170Horiuchi, S., 9Horton, R., 126, 128Horvath, J. C., 150Howell, N., 47Hruban, Z., 66HSP70 overexpression, 48Huang, C., 64Huang, L. E., 134Huang, P., 9, 120, 122–126, 171, 173Hudson, K. M., 134Human osteosarcoma cells, 104Human placenta choriocarcinoma cell line

BeWo (JCRB911) and GLUT1mRNA, 87

Human spontaneous cervical cancercells (SiHA) and GAPDHoverexpression, 85

Huntly, B. J., 42Hwang, P. M., 10Hypoxia, 38, 107, 109, 134–135

hypoxia response elements (HREs), 80

186 Index

Hypoxia (cont.)hypoxic cells, studies of GLUT mRNA

expression in, 87Hypoxia inducible factor 1 α (HIF-1 α)

in cancer, 80–81and glycolytic pathway, 81–82

ectopic expression of, 94glucose transporters and, 87–88HRE and, 135–136nonhypoxic stimuli, induction by, 80regulation, 75

acetyl transferase, 79MAPK and PI3K pathways, 78polyubiquitination, 77–78stabilization in normoxia condition,

76–77structure of, 75

transactivation domains and, 74TCA intermediates upregulation and

carcinogenesis, 88as therapeutic target, 88

IIchikawa, M., 59, 123Ido, Y., 113Igney, F. H., 162Immortalization, 92

enhanced glycolysissenescence and, 94–95

See also CarcinogenesisInghirami, G., 23Ingman, M., 127Ingram, V. M., 63Inoki, K., 12Irani, K., 57Iron sulfur (Fe-S) clusters, mitochondrial

enzymes, 4Isidoro, A., 6, 9–11Isken, F., 11Israelson, A., 12Itahana, K., 96Itahana, Y., 96Ito, H., 169Ivan, M., 139Ivanovska, I., 47Iyer, N. V., 82Izquierdo, J. M., 6, 11

JJaattela, M., 4Jacks, T., 137Jacobs, H. T., 22Jacobson, M. D., 173Jacques, C., 60, 61

Jagiello, G., 22Jain, R. K., 134Janes, S. M., 169Jazwinski, S. M., 68Jeong, J. W., 24, 80, 81Jessie, B. C., 128Jewell, U. R., 78Jiang, B. H., 135Jiang, J., 107Jiang, W. G., 61Jiao, J., 10Johnson, F. M., 121, 122, 124Johnson, J. H., 115Jones, R. G., 12Joo, E., 174June, C. H., 149Ju, X., 11

KKaaks, R., 107Kachhap, S., 10, 62, 127, 128Kadhom, N., 25Kaelin, W. G., 24, 139Kaelin, W. G. Jr., 23, 139Kagan, V. E., 107Kahn, B. B., 12Kanamori, T., 58, 123, 127Kandel, E., 171Kang, J. G., 10, 100, 132Kang, S. W., 34Kappler, J., 149Karin, M., 64, 65Karplus, P. A., 35Kaschten, B. J., 7Kataoka, T., 149Kearns-Sayre syndrome (KSS), 124Kennedy, S., 171Khaleque, M. A., 48Kheradmand, F., 168Khrapko, K., 57, 58, 125, 126Kilo, C., 113Kim, H., 140Kim, J. H., 41, 79Kim, J. W., 10, 46, 94Kim, J. Y., 11, 172Kim, K. W., 24, 80, 81Kim, M. K., 107Kim, S., 7Kim, S. H., 24, 79–81Kim, S. T., 140Kim, W. Y., 139Kim, Y. H., 11King, A., 47

Index 187

King, M. P., 173Kinzler, K. W., 137, 169Kirkinezos, I. G., 124Kispal, G., 11Klatt, P., 100Klausner, R. D., 4Klco, J., 139Klco, J. M., 139Klein-Szanto, A., 58, 61, 62, 119Klingenberg, M., 22Kniffin, C. L., 8Knudson, A. G. Jr., 139Koch, C. J., 134Koed, K., 170Kofler, B., 124Kohl, R., 138Kollipara, R., 42Kol, S., 115, 116Kondoh, H., 96, 97, 100, 132Kondo, K., 139Konopleva, M., 174Koocheckpour, S., 140Korsmeyer, S. J., 164–166, 174Koshikawa, N., 172Kotch, L. E., 82Koukourakis, M. I., 8Koumenis, C., 138Kourembanas, S., 135Kovacs, G., 57Ko, Y. H., 27, 48Krajewska, M., 6, 7, 9–11Krammer, P. H., 162Krebs cycle, 3, 107Krebs, H., 6, 133Krippner, A., 20Kroemer, G., 19, 20, 25, 30Kron, S. J., 11, 108Krueger, J. S., 52Kruse, J. P., 132Kubasiak, L. A., 114, 173Kubek, S., 12Kuchroo, V. K., 149Kuzmin, I., 140Kvietikova, I., 78Kwei, K. A., 65Kwong, J. Q., 67

LLactate dehydrogenase A (LDH-A)

expression, 52and lactate carrier MCT4, 86, 87

Lambeth, J. D., 38Lamb, N., 135

Landowski, T. H., 150, 169Lanier, L. L., 149Larochette, N., 30Larsson, N. G., 22Larsson, R., 64Lartigue, L., 165Latif, F., 23, 59, 139Laughner, E., 80Leber hereditary optic neuropathy

(LHON), 124Lechago, J., 7Lechago, L. V., 7Lechpammer, M., 139Ledbetter, J. A., 149Leder, P., 46, 49, 51, 174Lee, F. S., 65Lee, H., 106Lee, J. W., 24, 80, 81Lee, S., 139Lee, S. M., 140Lee, S. R., 34Lee, S. Y., 107Lee, T. Y., 11Lee, W. C., 107Lehninger, A. L., 10, 106Leon-Avila, G., 4Letai, A., 165Leukemia, 109Leung, S. W., 135Levine, A. J., 137, 170Levy, J. P., 64Lewis, B. C., 7, 93, 171Lichtenstein, A. H., 2Lien, A. D., 8Lightowlers, R. N., 22Li, G. X., 29Li, J. J., 65Lill, R., 4, 11Lim, A. L., 10Lin, A. W., 95Lin, Q., 11Lin, S. J., 98Lincoln, D. W., 38Lindsten, T., 165Linsley, P. S., 149Lipid signaling molecules and migratory

phenotype regulation, 40Liu, J., 23Liu, J. W., 48Liu, V. W., 57Liu, X., 168Liu, Y., 170Liu, Z., 64

188 Index

Li, X. D., 110Li, X. X., 173Li, Y., 57, 125, 126Li, Z., 11Ljungberg, B., 57Lleonart, M. E., 96, 97, 100, 132Lodish, H. F., 7Lofromento, N. E., 110Lolkema, M. P., 140Lonergan, K. M., 140Lopez de Heredia, M., 6, 11Lopez-Ros, F., 6, 7, 9, 10Lorenc, A., 128Lorenz, M., 96Los, M., 140Lott, M. T., 120, 127Lou, Y. R., 29Lowe, S. W., 95, 171Lucchesi, P. A., 63Lu, F. J., 97Lu, H., 8, 82, 136Luis, A. M., 11Lunghi, M., 175Luo, Y., 63Lutz, R. J., 170Lu, X., 138Lu, Y., 11Lu, Y. P., 29Lynch, M., 121

MMabjeesh, N. J., 136Macaulay, V., 22, 47, 126–128MacGregor, G. R., 165MacKenzie, E. D., 24, 25Maejima, C., 172Maher, E. R., 136Mahon, P. C., 132Majewski, N., 171Malchow, P., 170Malthiery, Y., 59Mammalian mitochondrial oxidative

phosphorylation (OXPHOS), 4, 55Bcl-2 expression and, 67defects in cancer

cellular adaptations and, 59–60functional significance of, 57–59

dysfunctionmtDNA mutations and, 58tumor-specific marker genes, 61–62

gene expression and transcription, 106mitochondrial-nuclear intergenomic

signaling in, 59

phenotype and apoptosis, 104representation of, 56

Manfredi, G., 67Manganese-dependent superoxide dismutase

(MnSOD) overexpression and cellproliferation, 28

Maniatis, T., 65Manka, D., 106Mansel, R. E., 61Mansouri, J., 41MAPK pathways, 64Maraver, A., 100Marconi, A., 168Marin-Hernandez, A., 46Marquardt, C., 11Marrack, P., 149Marsden, P. A., 135Marsden, V. S., 167Martin, D. A., 64Martindale, D., 149Martin, D. S., 171Martinez-Diez, M., 10–12Martinez, M., 6, 9, 11Martorana, G. E., 50, 51Marzulli, D., 110Matheu, A., 100Mathupala, S. P., 9, 27, 48Matoba, S., 10, 100, 132Matsumura, T., 113Matsuno-Yagi, A., 20Matsuyama, S., 11Matthew, C. K., 106, 113Mattiazzi, M., 64Mavligit, G. M., 149Ma, W., 10Maxwell, P. H., 8, 136, 140Mayr, J. A., 124Mazelin, L., 169Mazure, N. M., 172McAndrew, D., 62McCormick, F., 170McCurrach, M. E., 95McFate, T., 8McGoldrick, E. T., 173McKnight, S. L., 11McKusick, V. A., 8McQuillan, L. P., 135Mechta-Grigoriou, F., 80Meierhofer, D., 124Melamede, R. J., 152Melendez, J. A., 38, 40Melvin, R. G., 108Menon, S. G., 36

Index 189

Mertz, R. J., 115Merzak, A., 140Metcalfe, A. D., 169Metzen, E., 75, 77Miceli, M. V., 68Michael, E. M., 124Michalak, E., 137Michalik, L., 51Michel, G., 135Miething, C., 171Migliaccio, E., 42Millhorn, D. E., 106Minami, R., 7Minet, E., 135Mirebeau, D., 60, 61Mitochondria

activities in, 4and apoptosis, 29cancer, 20cell immune system, 110cellular respiration and dedifferentiation,

49–52DNA deletions and mutations, 110genome, 121–124H2O2-dependent transcription of MMP-1

transcription, 40–41H2O2, generation of, 37metabolism in, 147mitochondrial-derived ROS, 34

angiogenesis, 37–39cancer stem cells, 41–42and cell cycle, 35–36cell survival, 36–37invasion/migration, 39–41

mitochondrial DNA (mtDNA)mutations, 47, 57–58

mitochondrial encephalomyopathy lacticacidosis, and strokelike episodes(MELAS), 124

mitochondrial HSP-mediated, 51mitochondrial transcription factor A

(TFAM), 61optic atrophy 1 (Opa 1), 66outer membrane permeability, 165–166OXPHOS and respiration, 5–6, 10peroxisome proliferator activated receptor

(PPAR)-α–mediated effects inrodents, 51

p66shc regulation and, 42respiration and ROS production, 106specific phospholipid cardiolipin (CL)

complexation, 107superoxides role, 27

type II hexokinase, interaction between, 24voltage-dependent anion channel

(VDAC), 27Mitofusin 2 (Mfn 2) downregulation, 66Miyashita, T., 173Miyazaki, K., 172MnSOD-overexpressing MCF-7 cells, 65Modica-Napolitano, J. S., 47Mohyeldin, A., 8Molecular homeostasis and genetic instability,

133–134Montanaro, L., 140Monteith, G. R., 62Montoya, J., 119, 121, 125Mookerjee, B., 137Moorhead, P. S., 94Moraes, C. T., 11, 60, 61, 67, 124Moreno-Loshuertos, R., 128Moreno-Sanchez, R., 46Morris, H. P., 8, 57Morrissey, C., 140Mueckler, M. M., 7Muehlematter, D., 64Muhlenhoff, U., 4, 11Muller, U., 23, 59Munk Pedersen, I., 169Munnich, A., 22, 27Murad, E., 35Murrell, G. A., 63Muse, K. E., 65Myoclonic epilepsy and ragged-red fibers

(MERRF), 124

NNADPH oxidase family members (Nox1-5), 35Nagata, S., 149Nagy, A., 57Nakagawara, A., 172Nakamura, E., 23, 139Nakamura, S., 9Nakashima, Y., 96, 100Nakshatri, H., 64Nass, M. M., 66Navarro, P., 168Nefedova, Y., 169Negelein, E., 5, 20Neilson, A., 9Nejfelt, M. K., 135Nekhaeva, E., 57, 58, 125, 126Nelson, K. K., 38, 40, 41Nemoto, S., 34, 42Neri, P., 174Neumann, H. P., 9

190 Index

Neurogenic muscle weakness, ataxia andretinitis pigmentosa (NARP), 124

Newell, M. K., 149, 152Newmeyer, D. D., 165Niemann, S., 23, 59Niikura, M., 59, 123Nishikawa, M., 27Nisoli, E., 107Nocca, G., 49, 50Normoxia, 77

See also HypoxiaNuclear DNA mutations and mitochondrial

proteins, 47Nuclear respiratory factors 1 and 2 (NRF-1 and

NRF-2), 60Nyengaard, J. R., 113

OOakes, S. A., 48Oberley, L. W., 65Oberley, T. D., 65Oca-Cossio, J. A., 67Odstrcil, E. A., 11Oesterle, D., 9Ogunshola, O., 78Ohh, M., 138–140Ohta, K., 61Ohtani, N., 48Old, L. J., 110Onaya, T., 61Opferman, J. T., 48Oprysko, P. R., 134Orr, W. C., 96Orsini, F., 42Ortega, A. D., 2, 9–11Osmanian, C., 137Osthus, R. C., 7Ostronoff, L. K., 11Oxidative stress and senescence, 95–96Oxygen

homeostasis, 73sensors, 136

Ozben, T., 27

PPacker, L., 64Pagano, M., 23Paik, J. H., 42Paik, S. G., 106Palacios, C., 114Pandolfi, P. P., 91Pandolfi, S., 140Pang, S. F., 28Pan, Y., 134

Panza, C., 168Papandreou, I., 10Papa, S., 60Paragangliomas (PGLs), 23Parfait, B., 22Parkes, T. L., 96Park, J. A., 79Park, J. H., 172Park, J. Y., 10Park, S., 140Park, S. Y., 11Pasteur effect, 20Pasteur, L., 2Pastorino, J. G., 104Patel, L., 64Patino, W. D., 10, 100, 132Paulin, F. E., 9Paulus, P., 7Pawlu, C., 9Payne, G., 170Pecina, P., 9, 10Pecqueur, C., 21Peczkowska, M., 9Pedersen, P., 104Pedersen, P. L., 6–9, 27, 48, 57Pelicano, H., 10, 113, 126, 171, 172Penta, J. S., 121, 122, 124Perez, J. M., 30Peroxisome-proliferator activated γ coactivator

1 (PGC-1) gene family, 60Peskin, B. S., 107Petrelli, J., 107Petros, J. A., 9, 58, 64, 127Pette, D., 22Peutz-Jeghers syndrome, 12Pfeiffer, K., 20, 58, 59, 62Pfeiffer, T., 2, 5, 9Pfister, M. F., 11PGC-1 related coactivator (PRC), 61Pheochromocytomas (PHEOs), 23Phillips, J. P., 96Phosphoenolpyruvate carboxykinase

(PEPCK), 49Phosphofructokinase 1 (PFK-1), 84Phosphoglycerate kinase (PGK), 85Piana, G. L., 110Pich, S., 66PI3K/AKT signaling pathway and VEGF

expression, 78–79Pilcher, K., 11Pilkington, G. J., 140Pineda, G., 110Pinedo, C. M., 114

Index 191

Piva, R., 23Pizarro, A., 168Pizzo, P., 62Plas, D. R., 8, 12, 132, 171Plasminogen-activator inhibitor 1 (PAI-1), 61Plate, K. H., 135Platelet-derived growth factor (PDGF)-induced

mitogenic signaling, 35Podda, A., 23Polyak, K., 57, 125, 126Poole, L. B., 35Porcelli, A. M., 127Porin-like protein voltage-dependent anion

channel (PPVAC), 48Posener, K., 5Pouyssegur, J., 77, 80, 172Powis, G., 29Poyton, R. O., 104, 113Pozzan, T., 62Preston, G., 47Prosser, R., 22p53 tumor suppressor protein, 8–9

p53 tumor suppressor gene and organismallife span, 99–100

Pugh, C. W., 140Puigserver, P., 61Pyruvate dehydrogenase (PDH), 86Pyruvate kinase (PK), 86

QQuesada, N. M., 38Quevedo, C., 30Quintanilla, M., 168

RRabbitts, P. H., 92Racker, E., 9Raff, M. C., 173Raghunand, N., 7Rai, E., 128Ramanathan, A., 104, 113Ranganathan, A. C., 41Ratcliffe, P. J., 8, 136, 140Rathmell, J. C., 8, 171Rauscher, F. J., 64Ravi, R., 137Reactive oxygen species (ROS), 33

and carcinogenesis, 105dependent stabilization of HIF-1α, 39and redox signaling, 34–35related signaling pathways, 50ROS-mediated cell growth, molecular

mechanism(s), 64signal transducing molecule, 34

Rechcigl, M. Jr., 66Redondo A., 9–11Redox-based signaling, 35Redox signaling pathway, 63–65Reed, J., 169Reed, J. C., 4, 11, 20, 173, 174Regan, K. J., 38, 40Reichert, M., 172Remy, C., 7Respiration, 3Respiratory chain (RC)

biochemical and genetic features, 20cell proliferation and, 25genetic origin and organization, 21redox status, 22and tumors, 29–30

Retrograde signaling in cancer cellaltered antioxidant defense pathway, 65altered expression of OXPHOS regulatory

genes and subunits, 60–61altered mitochondrial morphology and,

65–66antiapoptotic genes activation, 66–67calcium signaling pathway activation,

62–63genes activation involved in tumor invasion

and progression, 61–62mechanism of, 67–68redox signaling pathway activation, 63–65

Reynier, P., 59Reynolds, T. Y., 134Reznick, R. M., 12Rhee, S. G., 34Rho GTPase-activating protein, 39Ricchetti, M., 22Rice-Evans, C., 63Rice, K., 137Richards, F. M., 51Richard, S. M., 124, 125Richhardt, N., 11Ricquier, D., 21Ridnour, L. A., 65Riedl, S. J., 166Rigo, P., 7Ristow, M., 2, 11Ritchie, J. M., 29Rivas, A. L., 114Roberts-Thomson, S. J., 62Robey, I. F., 8Robey, R. B., 171Rockwell, S., 134Rodien, P., 59Rodriguez-Enriquez, S., 46

192 Index

Roeder, L. M., 115, 116Roe, J. S., 140Roe, R., 135Roifman, C. M., 110Romer, L. H., 169Rosenberger, S. F., 57, 64, 65Rosen, O. M., 7Ross, A. J., 165Roth, J. C., 137Roth, P. H., 8, 133, 135, 136Rotig, A., 22Rouault, T. A., 4Roux, D., 77Rowan, A. J., 25, 59Roy, D., 50Rue, E., 135Rue, E. A., 135Ruiz, C. R., 114Ruiz-Pesini, E., 9, 58, 64, 120, 127Rustin, P., 22–25, 27Ryan, K. M., 138Ryu, J. H., 172

SSaavedra, E., 46Saccharomyces cerevisiae, 2, 106

arrest defective-1 (ARD1) in, 79Crabtree effect, 106

Sadlock, J., 22Sakamaki, T., 11Sakamoto, K., 12Sakashita, M., 7Sala, S., 2, 9–11Salas, A., 22, 47, 126–128Salinas, M., 11Saliou, C., 64Salvesen, G. S., 166Samuels, D. C., 124–126, 128Sanchez-Arago, M., 2, 6, 7, 9–11Sanchez, L. B., 4Sansone, P., 42, 140Santamaria, G., 10–12Santaren, J. F., 11Saretzki, G., 96Saridin, F., 168Savagner, F., 59–61Sawyer, D. B., 48Scarpulla, R. C., 106Scatena, R., 49–51Schade, A. L., 5Schagger, H., 20Scheid, A., 78Schimmer, A. D., 167

Schmeller, N., 124Schmid, T., 138Schmitt, C. A., 171Schneider, P., 149Schon, E. A., 22, 128Schreiber, R. D., 110Schreiber, S. L., 104, 113Schuler, M., 167Schulz, T. J., 11Schumacker, P. T., 173Schuster, S., 2, 5, 9Scorrano, L., 12, 48, 174Scott, C. L., 167Seftor, E. A., 7Sekido, Y., 139Sekito, T., 67, 68Selak, M. A., 9, 24, 25, 47Semenza, G. L., 8, 10, 75, 81, 92, 132–136,

135, 136, 139Senescence, 94

bypassing effect of enhanced glycolysis,96–97

oxidative stress and, 95–96Senoo-Matsuda, N., 23Serizawa, S., 11Serrano, M., 94, 95Serra, V., 96Seth, R. B., 110Shah, A., 7Sharma, R. I., 9Sharma, S., 128Sharma, S. V., 169Shaw, R. J., 12Shay, J. W., 94Sheldon, D. R., 150Shephard, H. M., 48–50, 58, 61–63, 67Sherr, C. J., 94Shidara, Y., 58, 123, 127Shi, H. H., 57Shi, J., 35Shim, H., 7, 93, 171Shim, Y. J., 107Shin, Y. K., 10Shi, X., 64Shi, Y., 166Shi, Y. H., 135Shoshan-Barmatz, V., 12Shoubridge, E., 128Shulman, G. I., 12Simonetti, S., 22Simoni, D., 150Simon, M. C., 24, 113, 134Simonnet, H., 58, 59, 62

Index 193

Sinclair, D. A., 98Singh, G., 120Singh, K. K., 9, 10, 47, 62Sinkovics, J. G., 150Sivridis, E., 8Sledge, G. W. Jr., 64Sligh, J. E., 8Smallwood, A. C., 139Smith, T. A., 9Sohal, R. S., 96Sole, P. D., 49, 50Sorcinelli, M. D., 165Soslau, G., 66Spector, M., 9Spencer, B., 115Sperl, W., 124Srivastava, S., 60, 61Steinbach, J. P., 172Stengel, P., 75, 77Stiehl, D. P., 80, 85Stiles, B., 10Storci, G., 42, 140Strasser, A., 137Streuli, C. H., 169Strompf, L., 23Subbaram, S., 37, 38, 40Succinate dehydrogenase (SDH)

deficiency and cancer, 88mutations, 23

Sugai, T., 9Suk, K., 172Sukosd, F., 57Sung, H. J., 10Sun, L., 110Sun, W., 127, 128Sun, Y., 61Supra, P., 172Swartzendruber, D., 152Swift, A. L., 9Swift, H., 66Symons, M., 168Sympson, C. J., 168Synthesis of Cytochrome c Oxidase

2 (SCO2), 10p 53 gene, 132

TTachibana, K., 11Tait, A. S., 8Takahashi, A., 48Takeda, K., 34Takenaga, K., 172Tang, D. G., 48

Tarassov, I., 22Taylor, R. W., 9Tchernyshyov, I., 10Tedesco, L., 107Tekaia, F., 22Tennant, D. A., 25Terashima, M., 9Teras, L. R., 107Testa-Parussini, R., 107Thierbach, R., 11Thilly, W. G., 57, 58, 125, 126Thioredoxin (TRX)

and cysteine, 74–75overexpression and cell proliferation, 28

Thomas, P. A., 7Thompson, A. M., 9Thompson, C. B., 8, 11, 12, 108, 113, 132,

165, 170, 171, 173Thornton, J., 67, 68Thun, M. J., 107Tian, W. N., 97Tiller, G. E., 8Timofeev, O., 11TNF-α/CHX–induced apoptosis, 36Tolomeo, M., 150Tomiyama, A., 11Tomlinson, I. P., 25, 47, 59Tomlinson, J. P., 121Tonello, C., 107Tonks, N. K., 35Tory, K., 139Tothova, Z., 42Tovar, J., 4TP53-induced glycolysis and apoptosis

regulator (TIGAR), 9, 100Traber, M. G., 64Transcription factor Ets-1, 38Trauger, R., 152Tremble, P., 168Troncoso, P., 175Tsao, T., 174Tschopp, J., 149Tsuda, M., 23Tsuruo, T., 149Tsuruta, A., 9Tu, B. P., 11Tumor

cell mitochondria, 66glucose-specific transporters (GLUTs)

induction, 7metastasis and, 39microenvironment for, 136–137PTEN suppressor, 40

194 Index

Tumor (cont.)superoxides role in, 27tumorigenesis

temporal and spatial effects in, 112–113tumor-specific metabolic pathways,

146–147VEGF role, 38

Turnbull, D. M., 9, 124, 125Tyner, S. D., 100Tyurina, Y. Y., 107Tyurin, V. A., 107Tzagoloff, A., 19, 21

UUesugi, N., 9UnCoupling proteins (UCP), 21–22

uncoupling protein 2 (UCP2) expression incolon cancers, 110

Usadel, H., 57Usher, P., 7

VVahsen, N., 12, 20, 166Vainio, H., 107van Beest, M., 140Vander Heiden, M. G., 11, 108, 171, 173Vande Velde, C., 172Vandromme. M., 135van Holde, K. E., 106, 113van Waveren, C., 61Vega, A., 22, 47, 126–128Velankar, M., 174Velours, J., 11Venditti, J. M., 150Venkatachalam, S., 100Verma, A., 8, 82, 136Vijayasarathy, C., 48–50, 58, 61–63, 67, 119Vijayvergiya, C., 64Villalobos-Menuey, E., 152Villalobos-Menuey, E. M., 155Villani, G., 60Villunger, A., 137Vincent, B. J., 8Vincenzoni, F., 50Voest, E. E., 140Voet, D., 146, 147Voet, J., 146, 147Vogelstein, B., 137, 169Voigt, A., 11Volmat, V., 77von Hippel–Lindau syndrome, 8, 138–140

VHL gene, mutation in, 81von Hippel–Lindau tumor suppressor

protein (pVHL), 75

von Kienlin, M., 7von Zglinicki, T., 96Vousden, K. H., 138

WWachsman, J. T., 121, 122, 124Waddle, J. A., 68Wahli, W., 51Walensky, L. D., 165Wallace, D. C., 25, 27, 47, 119–122,

120, 127, 128Walsh, S. A., 7Wan, X. S., 65Wang, C., 11, 104, 110, 113Wang, G. L., 8, 133, 135, 136Wang, J., 94Wang, S., 10Wang, T., 11Wang, X., 4Wang, Y., 94Wang, Z. Q., 113Warburg, O., 5, 6, 20, 56, 81, 120, 126,

131–133Warburg hypothesis

hypoxia inducible factor 1 alpha (HIF-1α)and, 8

model, 113–115principles of, 7

Warren, L., 66Watson, A., 51Watson, P. H., 137Watt, F. M., 169Waugh, T. A., 29Way, I. P., 169Webster, K. A., 114, 173Weiler, S., 165Weinberg, R. A., 11, 92, 134Weinhouse, S., 5Weller, M., 172Welsh, S. J., 8, 29Wenger, R. H., 78, 80, 85, 138Weng, Y., 139Wen, S., 175Werb, Z., 168Werner, E., 168Weydert, C. J., 29Whang, E. E., 169Whelan, M., 149Wiesener, M. S., 136Wilhelm, M., 57Wilkie, D., 66Willett-Brozick, J. E., 9, 20, 23, 59William, O., 108

Index 195

Williamson, J. R., 113Wind, F., 20Wolff, T., 9Wong, G. H. W., 36Woods, M., 5Wood, Z. A., 35Worley, J. F., 115Wright, W. E., 94Wu, H., 10Wu, L., 137Wu, M., 9Wu, X., 91Wu, Z., 61Wurster, R. D., 63Wykoff, C. C., 137, 140

XXia, Y., 57Xie, L. Y., 94Xue, W., 171Xue, Y., 65Xu, J. N., 28Xu, Q., 11, 140Xu, R. H., 10, 113, 171, 172Xu, R. K., 28

YYamagata, K., 58, 123, 127Yamakoshi, K., 48Yamauchi, A., 125Yang, H., 139Yang, M., 171Yang, Q. H., 28Yao, Y. G., 22, 47, 126–128Yasuda, K., 23Yauch, R. L., 140YC-1–treated Hep3B hepatoma cells, aldolase

and enolase mRNAs, 85Yeast, Sir2 as longevity gene in, 97–98

calorie restriction and, 98–99

Yee, A. J., 11Yen, P., 29Yoo, B. C., 10Yoon, B. I., 29Younes, M., 7Youn, H. D., 140Yuan, F., 134Yu, Z. X., 34

ZZaidi, M., 63Zamzami, N., 30Zandi, E., 64Zanssen, S., 128Zatyka, M., 140Zbinden, I., 64Zeamari, S., 140Zeiher, A. M., 170Zeitlin, B. D., 174Zeller, K. I., 94Zender, L., 171Zhang, S. Y., 58, 61, 62, 119Zhao, Y., 65Zheng, J. Z., 135Zhong, H., 80Zhou, J., 79, 138Zhou, S., 10, 127, 128Zhou, X. M., 170Zhou, Y., 172Zhu, H., 57, 125, 126Zhu, T., 12Ziegler, A., 7Zigmond, M. J., 41Zinner, M. J., 169Zong, W. X., 113, 165Zou, Y., 96Zummo, G., 48Zu, X. L., 5Zweier, J. L., 57

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