Energy Sensing and Metabolism in Cancer

Boyi Gan

MD Anderson Cancer Center

The distinguishing features of living organisms

1. A high degree of chemical complexity and microscopic organization.

2. Systems for extracting, transforming, and using energy from the environment.

3. Defined functions for each of an organism’s components and regulated interactions among them.

4. Mechanisms for sensing and responding to alterations in their surroundings.

5. A capacity for precise self-replication and self-assembly.

(Lehninger Principles of Biochemistry, 6th edition)

1.How cells sense and respond energy and nutrient availability?2.How cancer cells adapt to survive and grow under metabolic stress?

Biosynthesis

Muscle contraction

Membranetransport

Cellularmovement

ATP: the “molecular unit of currency”

of intracellular energy transfer

Energy Expenditure

Energy Intake Energy Balance

Obesity DiabetesAging Cancer

heat

• Obesity is the second greatest risk factor for cancer development in USA (only after tobacco use).

Energy Metabolism and Cancer Development

• Deregulated energy metabolism is an emerging hallmark of cancer.

(Molecular Biology of the Cell, 6th edition; Hallmarks of cancer: the next generation. Hanahan, Weinberg, Cell, 2011; The biology of cancer, 2nd edition, Weinberg, 2014)

• Our understanding of energy metabolism in cancer has been translated into cancer detection (FDG-PET) and treatment (metformin, the widely used drug to treat diabetes, is associated with decreased tumor incidence and mortality.

Energy Sensing and Metabolism

Cancer

Regulation of energy sensor AMPK by lncRNA NBR2.

Energy stress-induced lncRNA FLINC1 regulates energy metabolism and tumor suppression.

Glutamate/cystine antiporter SLC7A11 regulates glucose dependency in cancer cells.

1.How normal/cancer cells sense energy availability?

2.How cancer cells adapt to survive and grow under energy stress?

3.How to translate our understanding of energy metabolism in cancer into novel cancer therapeutics?

Research Questions:

Research Topic:

Presentation Outline:

AMPK-mediated Energy Sensing Signaling

AMP/ATP

AMPKα

LKB1

β γ

ATP-generating catabolic processes

ATP-consuming anabolic processes

Restoring energy balance

TSC1-TSC2

mTORC1

Protein Synthesis,Cell Growth/Size Increase

Peutz Jeghers Syndrome, non-small cell lung cancer, pancreatic cancers, melanomas, cervical cancer

Tuberous Sclerosis, LAM, kidney and bladder cancer

Tumor SuppressionStem Cell Homeostasis

Energy Stress

AMPK-mediated Energy Sensing Signaling

AMP/ATP

AMPKα

LKB1

β γ

TSC1-TSC2

mTORC1

Protein Synthesis,Cell Growth/Size Increase

Tumor SuppressionStem Cell Homeostasis

Energy Stress

TSC is required for HSC maintenance in vivo (Gan et al, 2008, PNAS)

LKB1 is required for HSC maintenance in vivo (Gan et al, 2010, Nature)

NBR2(Liu et al, 2006, NCB)

Glucose starvation induces lncRNA NBR2 expression

NBR2 (neighbor of BRCA1 gene 2) for further study

218 bp

Genomic duplication and alteration

BRCA1: protein-coding gene, regulate DNA damage response and genome integrity

NBR2: long non-coding RNA, non-coding gene

BRCA1P1: BRCA1 pseudogene, non-coding gene

NBR1: protein-coding gene, function as autophagy receptor

LKB1-AMPK-dependent induction of NBR2 by energy stressA

B

C

Lkb1 deficient

p-AMPK

ACC

AMPK

p-ACC

p-Raptor

Raptor

Ctrl shGlucose

(mM): 25 0 25 0 25 0sh1 sh2

NBR2 depletion attenuates energy stress-induced AMPK activation and mTORC1 inactivation

p-S6K

S6K

p-S6S6

NBR2 regulates cell proliferation, apoptosis, and autophagy downstream of AMPK under energy stress.Overexpression of NBR2 activates AMPK

25 0 25 025 0

AMPKα

pAMPK

NBR2 interacts with AMPKα

Glucose(mM):

β1AMPKβ

AMPKγ1

β2

(Using biotinylated RNA, precipitated with streptavidin beads)

NBR2 promotes AMPK kinase activity

NBR2

A

B

(Liu X, et al, Gan B, Nature Cell Biology, 2016)

NBR2: A lncRNA switch for AMPK activation

Biguanides (Metformin/Phenformin) as anti-cancer drugs

• Metformin was originally developed from natural compounds found in the plant known as French lilac.

• Biguanides (Metformin/Phenformin) are inhibitors of mitochondrial respiratory chain complex I, and can decrease blood glucose levels. Metformin is most widely used drug to treat diabetes.

• Retrospective analyses, clinical trials and many functional studies support the beneficial effect of biguanides in cancer prevention and treatment.

• At least three mechanisms have been proposed to explain the antitumor effects of biguanides.

(from Hardie DG, 2013, Cancer Cell)

05

101520253035

Ctrl shNBR2 sh1NBR2 sh2

PhenforminControl

Apo

ptos

is(%

)

***

Ctrl sh

Phenformin

NBR2 sh1

NBR2 sh2

-Actin

- + - + - +Cleaved

PARP

Biguanides induce NBR2 expression and NBR2 regulates cancer cell sensitivity to biguanides

02468

10121416 Control

Phenformin

SLR20 BT549 HEK-293T

Rel

ativ

e N

BR

2 le

vels

786-O MDA-MB-231

*

**

**

**

**

BA

NBR2 does not regulate biguanide-induced AMPK activation

BA

NBR2 regulates biguanide-induced GLUT1 expression and glucose uptake

A B

Glucose Starvation

AMPK

mTORC1 activation,Cell Proliferation,Tumor Development

NBR2

LKB1-AMPK

Differential effects of NBR2 under glucose starvation and biguanide treatment

?

?

(Liu X, et al, Gan B, Nature Cell Biology, 2016)

Biguanide treatment

NBR2

GLUT1

Glucose uptake,Cancer cell survival

(Liu X, Gan B, Cell Cycle, 2016)

MetabolicStress

TFs (metabolic stress response)

RNA sequencing

Computational Analysis

TCGA Analysis

MolecularMechanism

lncRNAs

MetabolismFunctionValidation Clinic

FoxO transcription factors: at the crossroad of cancer and metabolism

LncRNA

Our previous studies showed FoxOs play critical roles in mediating energy stress response, drug resistance, and tumor suppression in renal cancer (Gan B, et al, Cancer Cell, 2010; Lin A, et al, Gan B, Oncogene, 2013; Lin, et al, Gan B, Cancer Research, 2014; Dai F, et al, Gan B, PNAS, 2017).

(From Salih DA, Brunet A, Curr Opin Cell Biol. 2008)

TCGA datasets

FoxO(TA)ERT2RCC cells

4OHT 12 hrtreatment

-

+

Analysis focusing on lincRNAs

FILNC1 (FoxO-induced long noncoding RNA #1)

FILNC1

Identification of FoxO-induced lincRNAs

RNA sequencing

FILNC1 is a FoxO target.FILNC1 is induced by energy stress in a FoxO-dependent manner

shLuc sh1 sh2

1% Glucose medium

FILNC1 KD does not affect cell cycle.

FILNC1 deficiency inhibits energy stress-induced apoptosis and promotes renal tumor development

A B

C

FILNC1 is down-regulated and its low expression correlates with poor clinic outcome in renal cancers

Real-time PCR validation

A B

Clinical outcome analysis

CTCGA analysis

FILNC1 deficiency promotes glucose uptake and lactate production

FILNC1 KD does not affect Myc mRNA level.

Myc largely mediates the biological effects afforded by FILNC1deficiency under energy stress

FILNC1 deficiency increases Myc protein level

Beads Antisense SenseIGF2BP1 0 0 1AUF1 0 1 3SART3 0 0 5

Mass spectrometry for FILNC1

AUF1 is an (A+U)-rich elements (AREs)-binding protein, and can binds to AREs within 3’ untranslated region (UTR) of Myc mRNA and promotes Myc translation without affecting Myc mRNA level.

FILNC1 interacts with AUF1 under energy stress and sequesters AUF1 from binding to Myc mRNA

CB

Energy stress-induced lncRNA FILNC1 inhibits Myc-mediated energy metabolism and renal tumor suppression

(Xiao Z, et al, Gan B, Nature Communications, 2017)

Potential function of LncRNAs in cancer metabolism

Adaptive Response

AMP/ATP

Energy Stress

Suicidal Response(AMPK activation) (FoxO activation)

Cell Death

(Short-term and mild stress)

(Long-term and severe stress)

(Gan et al, Cancer Cell, 2010; Lin et al, Oncogene, 2013; Lin et al, Cancer Research, 2014; Liu X, et al, Nature Cell Biology, 2016; Dai et al, PNAS, 2017; Xiao Z, et al, Nature Communications, 2017)

0h 4h 8h (-Glc)

SLC7A11 **

SLC7A11

Vinculin

0 6 8-Glc (h) 0 6 12

786-O

P-AMPK

AMPK

UMRC6

SLC7A11

BJNCI-H226

0 2 0 12-Glc (h) 0 12 0 12

U2OSSLR20

Vinculin

0 12 0 12 0 12

RCC4

0 12

**

UMRC-6

Glucose starvation induces the expression of SLC7A11

31

Glucose(Glc)

Glycolysis

Pyruvate(Pyr) Pyr

(Glu) Glutamate(Glu)

TCA Cycle

α‐ketoglutarate(α‐KG)

(Glu)

Cystine

Cystine

System Xc‐

32

Glutamine(Gln)

SLC7A11 (xCT)(catalytic subunit)

SLC3A2(regulatory subunit)

CysteineGlutathione

ROS

H2DCFDA (ROS)

+Glc+Glc, NAC‐Glc‐Glc, NAC

UMRC6

+Glc

‐Glc

Con NAC

Hypothesis: glucose starvation‐induced SLC7A11 serves as an adaptive response to promote survival under metabolic stress

(NAC: N‐acetylcysteine)

33

Glucose starvation

ROS

Cell death

NAC

SLC7A11

GSH

(Adaptive response)

SLC7A11

Vinculin

High SLC7A11 expression correlates with increased sensitivity to glucose starvation‐induced cell death in cancer cells

34

SLC7A11high

SLC7A11low

***

**

**

***

sh S

LC-2

sh S

LC-1

shC

on

SLC7A11

Vinculin

UMRC6

sh S

LC-2

sh S

LC-1

shC

on

SLC7A11

Vinculin

NCI-H226

+Glc

-Glc

shCon shSLC-1 shSLC-2

SLC7A11 sensitizes cancer cell to glucose starvation‐induced cell death 

35

Pharmacological inhibition of SLC7A11 by sulfasalazine has similar effect.

SLC7A11

Vinculin

786-O

EV OE

SLC7A11

Vinculin

RCC4

EV OE

+Glc

-Glc

EV SLC7A11

0 8 12 24 36 480

20

40

60

80

‐Glc (h)

Cel

l Dea

th(P

I Sta

inin

g, %

)

RCC4

EVSLC7A11-WT

****

Vinculin

NRF2

SLC7A11

sgC

on

sgN

RF2

-1sg

NR

F2-2

sgC

on

sgN

RF2

-1sg

NR

F2-2

+ Glc - Glc

UMRC6

sgC

on

sgAT

F4-1

sgAT

F4-2

sgC

on

sgAT

F4-1

sgAT

F4-2

+ Glc - Glc

Vinculin

ATF4

SLC7A11

UMRC6

ATF4 and NRF2 promotes glucose starvation‐induced cell death through SLC7A11

36

Glc

sgCon

sgNRF2-1

sgNRF2-2

(+) (-)

sgATF4-1

sgATF4-2

sgCon

Glc (+) (-)

SLC7A11 regulation of glutamate efflux underlies SLC7A11‐mediated increased sensitivity to glucose starvation 

37

******* ****

****

***

***

****

****AOA, EGCG

Inhibition of glutamate conversion to aKG by AOA or EGCG restores glucose dependency in SLC7A11-deficient cells.

Glucose

TCA Cycle

Glutamate

Glutamine

SLC7A11

Cystine

Glutamate

Glucose

TCA Cycle

Glutamate

Glutamine

SLC7A11

Cystine

Glutamate

Glucose

TCA Cycle

Glutamate

Glutamine

SLC7A11

Cystine

Glutamate

Glucose

TCA Cycle

Glutamate

Glutamine

SLC7A11

Cystine

Glutamate

High glucose / Low SLC7A11 Low glucose / Low SLC7A11

High glucose / High SLC7A11 Low glucose / High SLC7A11

38

Model

SLC7A11 limits metabolic flexibility and enhances cancer cell dependency on glucose by exporting glutamate.Suggest to use glycolysis inhibitors to target the metabolic vulnerability in tumors with high SLC7A11 expression (such as Keap1 or NRF2 mutant tumors).

(Koppula P, Zhang Y, et al, Gan B, 2017, JBC)

Energy Sensing and Metabolism

Cancer

Regulation of energy sensor AMPK by lncRNA NBR2. (Liu X, et al, NCB, 2016; Liu X, et al, Cell Cycle, 2016)

Energy stress-induced lncRNA FLINC1 regulates energy metabolism and tumor suppression. (Xiao Z, et al, Nature Communications, 2017)

Glutamate/cystine antiporter SLC7A11 regulates glucose dependency in cancer cells. (Koppula, JBC, 2017)

1.How normal/cancer cells sense energy availability?

2.How cancer cells adapt to survive and grow under energy stress?

3.How to translate our understanding of energy metabolism in cancer into novel cancer therapeutics?

Research Questions:

Research Topic:

Presentation Outline:

Acknowledgement• Li Zhuang• Hyemin Lee• ZhenDong Xiao• Xiaowen Liu• Yilei Zhang• RajKumar Yadav• Anoop chauhan• Pranovi Koppula

Current Lab Members:

Funding:

• Xifeng Wu • Christopher Wood• Junjie Chen• Han Liang• Hui-kuan Lin• Deepak Nagrath• Wei Li

Collaborators: