termpaper eto

Polytechnic University of the philippinesCOLLEGE OF SCIENCE

DEPARTMENT OF BIOLOGY

Chaperon-Mediated Autophagy in Cancer Biology

TERM PAPER

Diana Amor

Jaguio, Nelly MarizPineda, Aljohn

3/10/2012


Cancer

What is cancer?

Cancer is an invasive distensions spreading crab-like as described by

Hippocrates. From the word crab; “karkinos” in Greek and cancer in Latin; came the

name of the disease and the name of its inducing agents, carcinogens. Cellular

biologists had identified cancer with abnormal cell growth in the mid nineteenth century

(Auyang).

Cancer is a potentially fatal disease caused mainly by environmental factors that

mutate genes encoding critical cell-regulatory proteins. The resultant aberrant cell

behavior leads to expansive masses of abnormal cells that destroy surrounding normal

tissue and can spread to vital organs resulting in disseminated disease, commonly a

harbinger of imminent patient death (Alison, 2001; Auyang). The cancerous cells may

occur in liquids, as in leukemia. Mostly, however, occur in solid tumors that originally

appear in various tissues in various parts of the body. By their original locations they

are classified into various types of cancer, such as lung, colon, breast, or prostate

cancer (Auyang).

Although cancer is an ancient disease that afflicts humans and other animals, its

prominence in the Western world rose from the nineteenth century to become “a

disease of civilization (Auyang).” Cancer is primarily a disease of elders; its risk

increases roughly as the fourth power of age (Alison, 2001; Auyang). And it is the

Polytechnic University of the Philippines College of Science Department of BiologyAmor Jaguio Pineda Page 1BS Biology 4-2


second-largest common disease—malignant tumors. Coronary disease and cancer

together are responsible for over 80% of all deaths in industrialized countries (Rath,

2001).

The overall 5-year survival rate of many cancers, including liver, lung, pancreas,

bone, and advanced breast cancer, has not increased in the past 30 years. On a

worldwide basis, cancer is fast replacing heart disease as the number one cause of

death in adults (Lam, 2003).

Incidences of cancer keep increasing on a global scale (Rath, 2001). Since 1950,

the overall cancer incidence, particularly in America, has increased by 44%, with breast

cancer and male colon cancer up by 60% and prostate cancer up by 100%. 44% of

Americans living today are expected to develop cancer (Lam, 2003). That is why

Americans have poured roughly $200 billion, in inflation-adjusted dollars, into cancer

research and cancer drug development between 1971 and 2004. Almost one-half of the

bills went to several government agencies, the balance to philanthropies and

pharmaceutical companies (Auyang).

In 1986, the director of National Cancer Institute predicted the eradication of

cancer by 2000. Reality was not anywhere close. In 2004, a new director envisioned

“the elimination of the suffering and death due to cancer by 2015.” The World Health



Organization estimated that if unchecked, annual global cancer deaths could rise to 15

million by 2020 (Auyang).

What causes cancer?

Cancers evolve through a complicated web of multiple causes and that it is not

only pointless, but also counterproductive, to attempt to assign certain exposures a

certain role in causing cancer (Clapp et. al., 2005).

In 1981, Doll and Peto produced a summary table that estimated that 2% of

cancer deaths were due to pollution and 4% to occupation, with ranges of acceptable

estimates of less than 1% to 5% for the pollution contribution and 2 to 8% for the

occupation contribution. In this same table, they estimate that the proportion of cancer

deaths due to tobacco is 30% and to diet, 35%. A variety of other factors, including

alcohol, food additives, reproduction and sexual behavior, industrial products,

medicines, geophysical factors, and infection are ascribed percentages. The sum of the

individual percentages is 97%, with a final category of “unknown” with no percentage

(Clapp et. al., 2005).

Cancer is a complex genetic disease that is caused primarily by environmental

factors. The cancer-causing agents (carcinogens) can be present in food and water, in

the air, and in chemicals and sunlight that people are exposed to. More significantly, a

globalization of unhealthy lifestyles; particularly cigarette smoking and the adoption of



many features of the modern world Western diet (Alison, 2001; Auyang). The other

factors include emotional stress, infections, lack of oxygen, poor nutrition, genetic

mutation and environmental pollutants (Alison, 2001; Lam, 2003).

Only about one percent of cancers are unmistakably inherited. Strong genetic

dispositions contribute to a small portion of adult cancers; actually, it contributes

significantly 5-13 percent in cancer incidences (Clapp et. al., 2005; Auyang). Hormone

production during reproductive cycles and other internal factors can also contribute

(Auyang; Lam, 2003).

Inherited genetic defects account for some rare childhood cancers. Variations in

genetic predisposition partly explain why some people are more susceptible than others

are to a particular environmental carcinogen. Many genes involved are not cancer

genes; they do not themselves induce cancer. Rather, they code for enzymes with vital

normal functions, mainly to metabolize chemicals, breaking them down for excretion

(Auyang).

The vast majority of cancers are attributable to what people eat and inhale, how

they behave, their working conditions, viruses and bacteria, and natural and artificial

radiation and chemicals (Clapp et. al., 2005; Auyang); actually, Tobacco use and diet

each account for about 30% of new cancer cases, with infection associated with a



further 15%; thus, much of cancer is preventable (Alison, 2001). These are usually

called “environmental” risk factors for cancer (Auyang).

However, Rath said in his book, the origin of disease can be considered from two

cellular aspects: The lack of biological fuel needed by the cell’s power plants, the

mitochondria, or a failure in the function of the nucleus, the metabolic control center of

the cell; programming error causes uncontrolled “cell multiplication,” and at the same

time programming error causes a “disruption of the organization of the surrounding

connective tissue,” which enables the diseased cells to spread (2001).

How does it spread in the body?

Cancer does not develop overnight, instead often evolving over many years

(Alison, 2001; Auyang) with detectable premalignant lesions presaging the development

of full-blown malignancy (Alison, 2001).

Cancer cells move through the body with the presence of the cells that are

capable of temporarily dissolving the surrounding tissue—the collagen and elastic fibers

-so it can make its way through. For this purpose the cells use enzymes that can

temporarily digest and weaken the connective fibers surrounding them. All forms of

cancer spread with the help of the tissue-dissolving mechanism. The toxins entering the

body from the diet, such as pesticides and preservatives, are the most common cause

of liver cancer. Also, all pharmaceutical drugs have to be detoxified in the liver.



Research has established that the more enzymes a cancer cell produces, the more

aggressively the cancer develops. The faster the cancer can spread through a body, the

shorter the life expectancy of the patient if the mechanism is not stopped (Rath, 2001).

The collagen-dissolving mechanism also plays a major role in the spread of

cancer and the growth of secondary tumors in other organs or parts of the body

(metastasis). Small blood vessels provide oxygen and nutrients to tumor cells. The walls

of these blood capillaries are not obstacles for a cancer cell. With the help of collagen-

digesting enzymes, a cancer cell can “eat” its way into the lumen of the small blood

vessel and into the blood stream. The blood can then carry away cancer cells, by which

they can spread and invade other organs (Rath, 2001).

According to Lam, it is the body terrain that determines how the cancer is

expressed. The root of cancer therefore lies in the progress of growth and metastasis,

and not the tissue in which the tumor was first detected (2003).

Metastasis is the process where cancer cells invading the surrounding tissues,

entering the blood stream, spreading and establishing colonies in distant parts of the

body (Auyang). Tumors not only invade surrounding tissue, but are able to colonize

other, often vital, organs, in this process. Widespread metastatic disease is usually a

harbinger of imminent patient death (Alison, 2001).



What are the kinds of cancer?

Cancer can either ‘benign’ or ‘malignant’. Benign tumours are generally slow-

growing expansive masses that compress rather than invade surrounding tissue. As

such they generally pose little threat, except when growing in a confined space like the

skull, and can usually be readily excised. However, many so-called benign tumours

have malignant potential, notably those occurring in the large intestine, and these

should be removed before malignancy develops. Malignant tumors are usually rapidly

growing, invading surrounding tissue and, most significantly, colonizing distant organs.

The ability of tumour cells to detach from the original mass (the primary tumour) and set

up a metastasis (secondary tumour) discontinuous with the primary is unequivocal proof

of malignancy. Tumours are also classified according to their tissue of origin;

recognition of the parent tissue in a lymph node metastasis could establish the location

of a hitherto undiagnosed primary tumour (Alison, 2001).

Cancers that are not inherited are called “sporadic.” This means not that they

have no genetic component but that their genetics occurs not in germ cells but in

somatic cells, which constitute the bulk of our body. Some somatic cells, such as

muscle cells or neurons in the brain, stop dividing upon maturity. They can grow bigger

in size or establish more connections, but their numbers do not multiply. Cancer

seldom if ever appears in such non-dividing cells. It appears in tissues where cells die

and are replenished by new cell divisions (Auyang).



What are the cures for cancer?

According to Lam in 2003, there is no “single cure” for cancer and none is

proposed. For the past 50 years, war on cancer has been fought with three tools –

surgery (cut), radiation therapy (burn), and chemotherapy (poison). Localized tumors

can be removed by surgery or irradiation with high survival rates (Auyang). Benign

tumours can normally be removed by surgery. Malignant solid tumors will, if possible, be

surgically resected, probably followed and even preceded by other treatment modalities.

If the tumour is amenable to surgery, then surgery is the single most effective tool in the

anticancer armamentarium. Targeted radiotherapy is another option, as are

combinations of anticancer drugs (Alison, 2001).

Most conventional anticancer drugs have been designed with deoxyribonucleic

acid (DNA) synthesis as their target. Therein lies the problem, in that tumor cells are not

the only proliferating cells in the body; cells that line the alimentary tract, bone marrow

cells that generate red blood cells and cells to fight infection, and epidermal cells

including those that generate hair are all highly proliferative. Thus, patients with cancer

receiving chemotherapy commonly su er unwanted (hair loss) and sometimesff

potentially life-threatening side e ects that limit treatment (Alison, 2001). ff

At a cellular level cancer is a very rare disease given that an individual has many

millions of cells, so normally the repair and/or elimination mechanisms of damaged cells

must be very e cient, asking to have a ‘caretaker’ function. To account for the multipleffi



mutations in cancer cells, attention has become focused on the mechanisms of DNA

metabolism that maintain genome integrity, looking for the so-called ‘mutator

phenotype’. If the mechanisms of DNA repair are faulty, this leads to ‘genetic instability’,

facilitating an increased rate of alterations in the genome (Alison, 2001).

However, naturally oriented physicians think of the body as a closed internal

ecosystem, and believe that it is the dysfunction of this ecosystem that is primarily

responsible for the development of cancer. According to him, no treatment,

conventional or otherwise, can completely eliminate all cancer cells. The reason is

simple. Cancer is a systemic disease, and there are simply too many cancerous or pro-

cancerous cells within the ecosystem of the body. Cancer is not a localized problem but

a whole-body phenomenon of metastatic growth. Its growth process is affected by

biological conditions. He then, therefore proposed fights against cancer by optimizing

the internal terrain and enabling the patient’s internal system to destroy the tumor. It

enhances the patient’s health so that cancer cells cannot grow and multiply (Lam,

2003).

Rath proposed a way of preventing the spread of the cancer cells. He said that

the nature itself provides us with two large groups of molecules that can block collagen

digestion and its dissolving actions which lead to the spread of cancer. The first group is

the body’s intrinsic enzymatic block that can stop the action of collagen-digesting

enzymes in a few moments. The second group is the enzyme- blocking substances that



come from our diet or as dietary supplement. The most important one in this group is

the natural amino acid L-lysine. When lysine is supplied in a sufficient amount as a

dietary supplement; it can block the anchor sites in the connective tissue that collagen-

digesting enzymes use to attach themselves to the tissue. In this way lysine prevents

these enzymes from uncontrollably disintegrating connective tissue (2001).

Lam said that, the use of non-toxic natural therapies has achieved huge

successes over the past few decades. Extensive studies have proven them to have an

edge over conventional therapies success rate of natural treatment is so much better

than for many conventional cancer treatments (2003).

More than cures, scientists are cautiously optimistic about the possibility of

improving early detection and prevention of cancer. Cancer takes several steps and a

long time to develop. Its long latent period gives many opportunities to catch cells in

their early stages of mutation and intervene to stop cancer progression. For instance,

the pap smear followed by surgical removal of detected lesions have reduced death rate

of cervical cancer by almost 80 percent. To extend the success in cervical cancer to

cancer in general, scientists strive to identify biological markers that can finger incipient

cancerous cells and predict whether they will evolve to significant cancer (Auyang).

Alison said that, no individual can guarantee not to contract the disease, but it is

so strongly linked to diet and lifestyle that there are plenty of positive steps that can be



taken to reduce the chances: eat more fruit and vegetables, reduce the intake of red

meat and definitely do not smoke. Carcinogens interact with the individual’s constitution,

both inherited and acquired, determining vulnerability to cancer induction (2001).

Chaperone-Mediated Autophagy

Chaperone-mediated autophagy (CMA) is an intracellular catabolic pathway that

mediates the degradation of a selective subset of cytosolic proteins in lysosomes.

Autophagy or self-eating is broadly used to designate the lysosomal delivery and

degradation of intracellular components (Yang and Klionsky, 2010). Various types of

autophagy co-exist in almost all cells, and they can be differentiated by the mechanisms

that mediate the delivery of cargo to lysosomes. Macroautophagy and microautophagy

are kinds of the autophagic process, in which entire regions of cytosol or selective

cytosolic components are sequestered in vesicular compartments. Lysosomal enzymes

can gain access to the enclosed cargo through direct fusion of the vesicles with

lysosome, or by internalization of cargo-containing vesicles that form at the lysosomal

membrane. A third form of autophagy, solely dedicated to degradation of soluble

proteins can also be detected in most cell types in mammals. This autophagic process,

known as chaperone-mediated autophagy, differs from the other forms of autophagy in

both the way in which cargo proteins are recognized for lysosomal delivery and the way

in which these proteins reach the lysosomal lumen (Dice, 2007; Cuervo, 2010).


http://jcs.biologists.org/content/124/4/495#ref-8




CMA is a process activated during long term starvation in which cells selectively

degrade proteins in order to recycle their amino acids or use them for energy. During

nutrient deprivation, substrates that contain a consensus motif related to KFERQ are

recognized by a chaperone-cochaperone complex containing the heat shock cognate

protein of 70 kDa (hsc70). Once this chaperone-cochaperone complex binds the

substrate, it docks on the lysosomal membrane via a receptor known as the lysosomal

associated membrane protein 2a or lamp2a. The substrate then is unfolded,

presumably by the chaperone-cochaperone complex, translocated into the lumen with

the help of a lysosomal isoform of hsc70, and degraded. Like most organelle protein

import pathways, CMA is saturable as well as temperature-dependent. The substrates

for CMA also compete with one another for binding and import, which provides an

experimental method for discovering new substrates. There have been several

substrates identified for CMA including ribonuclease A and glyceraldehyde 3-phosphate

dehydrogenase (GAPDH).

The cytosolic chaperones and co-chaperones that participate in CMA are also

involved in other intracellular pathways this was according to Chiang et al., 1989.

According to Cuervoet al., (1997), the chaperones located in the lysosomal lumen or

associated to the lysosomal membrane – namely lys-Hsc70, membrane associated

Hsc70 and lys-Hsp90– appear to be exclusively dedicated to CMA. However, as these

are post-translational variations of cytosolic chaperones rather than independent gene




products, regulating their expression does not provide a mean for selectively affecting

CMA activity.

Moreover, Cuervo and Dice (1996) determined that LAMP-2A, the membrane

protein that acts as a receptor for the CMA substrates has often been manipulated to

regulate CMA activity. In fact, levels of LAMP-2A at the lysosomal membrane directly

determine rates of CMA activity, because substrate binding to the cytosolic tail of

LAMP-2A is a limiting step in CMA. Eskelinenet al., (2005) added that LAMP-2A is a

spliced variant of a single Lamp2 gene, which also encodes two other variants, LAMP-

2B and LAMP-2C with identical luminal regions but different transmembrane and

cytosolic tails. Substrate binding to the LAMP-2A cytosolic tail does not occur at the

KFERQ-targeting region and a designated LAMP-2A-binding motif in the substrate has

not been identified yet. However, the fact that substrate binding requires the four

positive charges in the LAMP-2A cytosolic tail suggests that electrostatic interactions,

rather than specific amino acid residues, mediate substrate binding.

The function of LAMP-2A extends beyond that of a receptor as this protein is also

an essential component of the CMA translocation complex (Bandyopadhyay et al.,

2008). Binding of substrate proteins to LAMP-2A monomers drives its organization into

a 700 kDamultimeric complex at the lysosomal membrane. The motif present in the

transmembrane region of LAMP-2A is important for multimerization. We have shown





that mutations that prevent multimerization abolish substrate translocation but not

substrate binding to LAMP-2A (Bandyopadhyay et al., 2008).

Although, in contrast to other translocation systems, luminal chaperones do not

form part of a stable translocon unit in CMA, they are still essential for substrate uptake.

In fact, a form of hsc70 resident in the lysosomal lumen is needed for complete

translocation of substrate proteins into lysosomes. Incubation of cultured fibroblasts with

blocking antibodies against hsc70 that reach the lysosomal lumen through endocytosis

exerts a strong inhibitor effect on CMA. Furthermore, levels of lys-hsc70 have helped

identify subgroups of lysosomes that manifest different ability to perform CMA. Only

those lysosomes containing hsc70 in their lumen are competent for uptake of CMA

substrates. Interestingly, the percentage of hsc70-containing lysosomes, which is no

more than 40% under resting conditions, escalates to 80% in liver under conditions in

which CMA is up-regulated, such as during prolonged starvation or mild oxidative

stress. This increase in the amount of lysosomes competent for CMA is a consequence,

at least in part, of changes in the luminal acidification in these organelles. Thus, lys-

hsc70 is stable in the lysosomal lumen at a pH of around 5.2, but a slight increase in the

lysosomal pH is enough to destabilize this protein and make it amenable to degradation

by the abundant lysosomal protease. Many factors could contribute to transient changes

in lysosomal pH and the subsequent destabilization of hsc70. Among them, we have

recently identified that fusion of lysosomes with autophagosomes when

macroautophagy is maximally activated contributes to a dissipation of pH enough to




render hsc70 unstable, and results in decreased CMA activity. The pH dependence of

lys-hsc70 may thus constitute a novel regulatory node in the cross-talk between

macroautophagy and CMA.

The study of CMA in particular cell types and the characterization of the

degradation of specific cellular proteins by CMA are behind the cell type–specific

functions recently proposed for this pathway. CMA activity has been linked to the

regulation of cellular proliferation in tubular kidney cells through the degradation of Pax-

2. Levels of this transcription factor, an essential regulator of kidney cell proliferation

and differentiation, are controlled through its degradation by CMA. Consequently,

changes in CMA activity may modulate kidney organogenesis and growth. Similarly, a

role for CMA in antigen presentation has been proposed in dendritic cells.

Macroautophagy has been shown to contribute to both the presentation of

endogenous peptides on major histocompatibility complex class II molecules, as well as

the presentation mediated by MHC class I molecules. Although relatively limited

information is available on the contribution of CMA to immunity, recent studies have

shown that reduction of LAMP-2A or hsc70 levels decreases presentation via MHC

class II. Interestingly, pharmacological inhibition of hsp90, which will reduce CMA

activity, also resulted in decreased antigen presentation in a second independent study.

Antigen processing and loading usually occurs in endosomes rather than in secondary

lysosomes, as the lower proteolytic capacity of the former compartment allows

preservation of the presenting peptides and their loading on MHC class II molecules.



However, to date, CMA has only been described to take place in secondary lysosomes.

Future studies are required to determine whether this hsc70-mediated presentation of

antigens takes place in late endosomes or in lysosomes.

Lysosomal storage disorders (LSDs) are a group of genetic diseases resulting

from loss of a specific lysosomal enzyme activity, and the consequent accumulation of

its substrates in the lysosomal compartment. The enzymatic impairment can result from

the malfunctioning of a specific enzyme in lysosomes, or from failure in its delivery to

this compartment. CMA, like any other type of autophagy, is likely to be indirectly

affected in these pathologies, because lysosomes are the final compartment for all

autophagic pathways. However, a direct connection to CMA has been recently

established with two different LSDs which includesgalactosialidosis and mucolipidosis

type IV. Patients with galactosialidosis lack cathepsin A, a protein that acts as

chaperone for different lysosomal enzymes, but that has also been recently shown to

participate in LAMP-2A turnover. The inability to properly degrade LAMP-2A in the cells

from these patients results in abnormally high rates of CMA. In the case of patients with

mucolipidosis type IV, who bear a mutation in the transient receptor potential mucolipin-

1, CMA activity decreases. The fact that hsc70 interacts with this receptor has led to the

proposition that altered docking of hsc70 at the lysosomal membrane could be behind

the observed decrease in CMA. However, another possible explanation that requires

further testing is that the small molecule channeling activity of this membrane protein is

required for CMA.



Although the classification as LSD has been traditionally restricted to defects in

enzymatic activity, recently, alterations in nonenzymaticlysosomal proteins, such as

membrane proteins, have also been shown to result in lysosome malfunctioning and

substrate accumulation. Among these new forms of LSD, of particular relevance for

CMA is Danon disease, a vacuolar myopathy that originates from a primary defect in

the lamp2 gene. Although, as indicated in previous sections, elimination of the three

protein variants of this gene will result in a complex phenotype, it is anticipated that

patients with Danon disease will also have reduced CMA activity.

Chaperone-Mediated Autophagy in Cancer Biology

Autophagy plays a crucial role in maintaining neuronal homeostasis through

clearance of defective organelles and unfolded/aggregating proteins. Knockout of

autophagy pathway genes leads to accumulation of poly-ubiquitinated protein

aggregates and can result in neurodegeneration, and motor and behavioral deficits in

mice. Also, autophagy interacts with other protein degradationand vesicular trafficking

pathways. While autophagy can at least partially substitute for reduced proteasomal

activity and vice versa, the disturbance of the endosomal-lysosomal system disrupts

autophagy and reduced autophagy impairs endosomal-lysosomal trafficking. It clears

neurotoxic proteins. The activation of this reduces the toxicity of aggregation prone

proteins, while inhibition of autophagy impairs their clearance and causes enhanced

cellular stress and neurodegeneration. This can also be a cellular death pathway, which



isactivated in neurons after acute injury and inhibition of autophagy under those

conditions can reduce neurodegeneration. However, autophagy is impaired in the final

stages of most neurodegenerative diseases.

Numerous observations suggest the existence of strong links between autophagy

and cancer. Several tumor suppressor genes stimulate autophagy, whereas oncogenes

are known that inhibit autophagy. The connection between both processes is probably

related to the overlap that exists between the pathways involved in regulation of

autophagy and tumorigenesis. Mutations that affect the function of mTOR or Beclin 1

have been identified in human cancers (Cao, 2007).The function of mTOR overlaps

with signaling pathways involved in tumorgenesis. Several tumor suppressor genes

have been shown being involved in the upstream inhibition of mTOR signaling and in

this way stimulate autophagy. Moreover, oncogene proteins are known that activate

mTOR. The importance of Beclin 1 in human cancers is illustrated by the fact that

mono-allelic deletions in Beclin 1 occur in a 40 – 75 % of cases of human breast,

ovarian and prostate cancer. Whether this only relates to the function of Beclin 1 in

autophagy or also to autophagy-independent functions of Beclin1 is not yet known

(Cecconi and Levine, 2008).

Selectively inhibited CMA by knocking down the LAMP-2A lysosomal receptor in

cultured cancer cell lines and in mice carrying human primary lung tumor xenograft s

using short hairpin RNAs (shRNAs). Although such an approach is the best available



method to impair CMA in tumor cells, it does raise an important cave at as it is formally

possible that LAMP-2A may possess additional functions that are separate from its role

in CMA. Nonetheless, even bearing this in mind, the results here are provocative

because they demonstrate that impaired CMA is sufficient to reduce tumor cell

proliferation rates both in vitro and in vivo. These effects are CMA-specific because

knockdown of a gene that is essential for macroautophagy (atg7) had little effect on

tumor growth. Inhibition of the CMA pathway also reduces the metastatic potential of the

tumor cells, which is extremely important from a clinical perspective given that

metastatic disease remains the principal cause of cancer mortality. In the real world,

most treatment is targeted to patients presenting with established cancers (Kon et al.,

2011) to ask whether an established tumor is affected when CMA is blocked by viral

delivery of shRNAs that target LAMP-2A. They found that the direct injection of

lentivirus encoding LAMP-2A shRNAs into tumor xenogra s caused marked regression

that was associated with increased tumor cell death as well as reduced staining for Ki-

67, which is a standard marker for tumor cell proliferation.

Thus, it appears that CMA is required for optimal tumor growth and metastasis to

distant sites and that targeting CMA in established tumors can induce the tumor cells to

slow their growth and undergo apoptosis leading to tumor regression. How does

blockade of the CMA pathway elicit such profound antitumor phenotypes. At least part

of the explanation is related to changes in cellular metabolism in CMA-defi cient cancer

cells. Bioenergetic assays indicated that inhibiting CMA produced a decrease in the

glucosedependent extracellular acidifi cation rate (ECAR), a finding consistent with



reduced glycolysis. In contrast, no changes in oxygen consumption or oxidative

metabolism were eadily evident in the CMA-deficient cancer cells. Increased aerobic

glycolysis is a well-known characteristic of tumor cells, commonly termed the “Warburg

effect”; moreover, abundant data indicate that reduced glycolytic metabolism, such as

observed here are CMA blockade, can profoundly attenuate both energy (adenosine

triphosphate) production and biosynthetic capacity, which are vital for cancer cell growth

and proliferation. Notably, this reduction in glycolysis in CMA-decient cancer cells was

associated with a decrease in several glycolytic enzymes. This result is somewhat

counterintuitive because these glycolytic enzymes possess KFERQ motifs and are

known substrates for CMA-mediated degradation; as a result, one would expect that

blocking CMA should result in an increase in these glycolytic enzymes, rather than the

decrease observed. The authors propose that this decrease might be at least partly due

to activation of the tumor suppressor p53 in CMA-defi cient cells, which results in the

transcriptional downregulation of multiple glycolytic enzymes. Nonetheless, because

CMA activity is upregulated in cancer cells independent of their p53 status, it is very

likely that CMA deficiency inhibits tumorigenesis by other mechanisms in addition to

p53-mediated suppression of glycolysis.

One of the main mechanisms by which macroautophagy potentially suppresses

tumor development is by eliminating stress-related molecules, such as p62/SQSTM1,

as well as oxidized, damaged proteins (Matthew, etal. 2009 ). This does not seem to

apply to CMA because there were no apparent differences in p62/SQSTM1 protein

levels and no increases in oxidized or aggregated proteins when the CMA pathway was



inhibited. Notably, the authors demonstrate that CMAdeficient cancer cells display a

compensatory increase in proteasomal degradation of CMA protein substrates, which

appears to be critical for maintaining protein quality control and mitigating the effects of

oxidative stress. Second, are the mechanisms underlying the antitumor effects of CMA

inhibition similar to those underlying the effects of macroautophagy inhibition. Despite

the compensatory increase in proteasomal degradation, an intriguing possibility is that

there is accumulation of specific target proteins containing the KFERQ motif, resulting in

the phenotypic changes observed in cells with a defective CMA pathway. In principle,

approximately one-third of the proteome can be targeted for CMA-mediated degradation

because about 30% of proteins contain the hsc70-targeting sequence (Arias and

Cuervo, 2011). However, the number of definitively identified CMA substrates is much

smaller than this. Hence, if currently unknown CMA substrates do indeed exist in cancer

cells, identifying these molecules will be key to understanding the precise mechanisms

through which CMA is mediating tumor development.

Most important of all, how do these findings relate to better treatments for cancer,

The data presented in the Kon et al. paper suggest that CMA inhibitors could be useful

for cancer therapy, as they should inhibit tumor growth and also reduce the ability of

tumor cells to metastasize. From a therapeutic standpoint, an important limitation is that

we do not currently have a feasible method to selectively inhibit CMA in patients (unlike

in the mouse xenograft model). Thus, we are unable to reliably reduce LAMP-2A protein

concentrations within tumor cells in people and, to date, we lack pharmacological



inhibitors that selectively block the CMA pathway. We do, however, have drugs that

more broadly inhibit all forms of lysosomal degradation. The most widely used of these

include chloroquine and hydroxychloroquine, which are being tested in a number of

clinical trials (more than 30 such trials are currently listed on the ClinicalTrials.gov Web

site) in combination with other anticancer drugs (Aramavadi, etal., 2011). The rationale

behind these trials is to block macroautophagy, but the question is whether CMA will

also be inhibited in these patients. Therefore, should there be any beneficial results

from these trials, we would need to consider whether it is CMA rather than

macroautophagy that is the critical target of chloroquine or hydroxychloroquine. Last,

but not least, like all anticancer strategies, it seems unlikely that all tumors will respond

equally well to CMA inhibition. Therefore, it will be critical to identify markers that

predict the dependence of tumors on CMA before blocking this pathway. In this regard,

the interconnections between macroautophagy and CMA will be important to uncover.

Remarkably, when tumor cells are transformed due to mutation of the small GTPase

Ras, this leads to a requirement for macroautophagy that has been proposed to

facilitate glycolysis (Lock, etal., 2011). Similarly, it has been suggested that the

presence of oncogenic Ras mutations makes tumor cells “addicted” to

(macro)autophagy (Guo, 2011). Accordingly, tumor types in which Ras mutations are

particularly common, such as pancreatic cancer, have been found to be particularly

sensitive to growth inhibition by chloroquine (Yang, 2011). These studies point to

specific patient subsets, such as those with tumors harboring Ras mutations, where

inhibition of macroautophagy may be particularly useful. Interestingly, several of the



lung cancer cell lines used by Kon et al. possess K-Ras mutations, immediately raising

the question of whether Ras-transformed cells are similarly addicted to CMA and

whether CMA inhibition, rather than macroautophagy inhibition, dictates the response to

chloroquine observed in the aforementioned studies. The answers to these and the

many other questions that arise from the exciting new study of Kon et al. will no doubt

keep cancer investigators working on autophagy very busy. However, what seems clear

is that it is unwise for cancer biologists to exclusively focus on macroautophagy in tumor

cells at the expense of CMA. If we continue to do so, we are certain to overlook some of

the most important aspects of autophagy in cancer biology.



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