Upload
others
View
6
Download
0
Embed Size (px)
Citation preview
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: