Nutrient and Growth Factor Control of Cell Cycle Progression and Survival in Cancer Cells
Bio 714Cell BiologyFeb, 2014
Hurdles in Progression to Cancer
I Constitutive Growth Factor Signaling avoid quiescence - cell cycle exit into G0
II Suppression of “Gatekeeper Functions”to get past cell cycle checkpoints
III Suppression of Apoptosisavoid default cell death programs
IV Acquire Immortality - telomerase expressionovercome cell senescence - cancer stem cells?
V Stimulate Angiogenesisprovide nutrition
VI Acquire the ability to migrate and invade gain access to circulation (metastasis)
VII Breakdown Caretaker Function genomic instability needed for many mutations
Cell Cycle Control and Cancer
I Constitutive Growth Factor Signaling avoid quiescence - cell cycle exit into G0
II Suppression of “Gatekeeper Functions”to get past cell cycle checkpoints
III Suppression of Apoptosisavoid default cell death programs
IV Acquire Immortality - telomerase expressionovercome cell senescence - cancer stem cells?
V Stimulate Angiogenesisprovide nutrition
VI Acquire the ability to migrate and invade gain access to circulation (metastasis)
VII Breakdown Caretaker Function genomic instability needed for many mutations
(Quiescence)
Gatekeepers Myc SV40 Early Region (Suppression of p53, Rb and PP2A)
Restriction Point
Growth Factor Signals Tyrosine kinases Ras/Raf/MEK/MAPK
G0
G1-pm SG1-ps G2 M
Regulation of Cell Cycle Progression
Cell GrowthCheckpoint(mTOR)
Cooperating Oncogenes and Survival Signals in Tumorigenesis
• Weinberg and colleagues demonstrated that signaling oncogenes (Ras) cooperate with gatekeeper override oncogenes (Myc) to transform primary rodent cells - and with SV40 early region genes to transform human cells
• Activated Ras by itself - induces apoptosis
• Thus, Myc suppresses the apoptosis induced by activated Ras
• Signals that lead to elevated Myc provide a “Survival Signal”
Foster DA, Yellen P, Xu L, Saqcena M. Genes & Cancer (2011)
Figure 8.8 The Biology of Cancer (© Garland Science 2007)
Conventional View of Cell Cycle
Zetterberg and colleagues have mapped the Restriction Point to a site ~ 3.5 hr after mitosis - where cyclin D is elevated
Points:
The Restriction Point, originally characterized by Arthur Pardee, is a point in G1 where cells no longer require growth factors and commit to completing the cell cycle
In the absence of growth factors, cells exit the cell cycle into quiescence or G0
Leland Hartwell described a site in the Yeast cell cycle called START that is late in G1 - where cells evaluate whether there is sufficient nutrition to complete cell division
In some texts, the Restricition Point is referred to as the mammalian equivalent of START - and located near the site where cyclin E is activatedRapamycin treatment results in the activation of TGF- signaling and arrest at the cyclin E site - that can be clearly distinguished both temporally and genetically from the growth factor-dependent Restriction Point
From: Weinberg, The Biology of Cancer, 2007
Protozoans & Metazoans are… different!
Vander Heiden MG, Cantley LC, Thompson CB. Science (2009)
Restriction Point: Point in G1 after which cells no longer need GF permissive signals to divide.
Restriction Point (R)
G0
G1 S
G0
R - GF
Mammalian Restriction Point is Analogous to START in Yeast
• Lodish H, et al. Molecular Cell Biology (2008)
Yeast Mammalian
Cooper GM. The Cell: A Molecular Approach (2000)
RestrictionPoint
Pyronnet S, Sonenberg NCurr. Opin. Genetics Dev. (2001)
Cell Cycle: START vs. R
Based on Zetterberg and Larsson, PNAS (1985)
Progression Factor (IGF-
1)
Competence Factor
(PDGF)
Based on Pledger and Stiles, PNAS (1979)
What is a Restriction Point ?
G0
G1 S
R
- GF
M
3.5 hr + GF
Where is the Restriction Point ?
… and where is the Nutrient Sensing ?
Dowdy SF, Curr. Opin. Gen Dev (2002) Weinberg RA, Biology of Cancer (2006) Lodish H, Molecular Cell Biology (2008)
Boonstra J, Adv Enzyme Regul (2007) Zetterberg A, Exp Cell Res (2005) http://medicinembbs.blogspot.com/2011_10_01_archive.html
Genetic requirements for the transformation of human cells (I)(Hahn et al., Nature 400:464, 1999; MCB 22;2111, 2002)
Genetic effect Molecular Target Cell cycle target
Ras Growth factor signals Restriction point
SV40 Large T p53 G1/S checkpointRb All G1 checkpoints
SV40 small t PP2A Cell Growth checkpoint (?)Genetic requirements for the transformation of human cells (II)(Boehm et al., MCB 25:6464, 2005)
Genetic effect Molecular Target Cell cycle target
Ras Growth factor signals Restriction point
p53 KO p53 G1/S checkpointRb KO Rb All G1 checkpoints
Myc Gene expression Cell Growth checkpoint (?)PTEN KO mTORC1 Cell Growth checkpoint (?)
Foster DA, Yellen P, Xu L, Saqcena M. Genes & Cancer (2011)
Immortalized primary cell line (BJ hTERT)
GF / nutrient deprivation mediate cell cycle arrest
GF / nutrient deprivation mediate cell cycle arrest
1st Block
2nd Block
+ 3H-Thymidine= No thymidine incorporation
1st Block
2nd Block + 3H-Thymidine
= Thymidine incorporation
Sequential Blocking Experiments:
R & Nutrient Sensing Checkpoints are distinguishable
3 H-T
dR
Inc.
(%
Ctr
l)
First Block: -GF
First Block: -Q
First Block: -EAA
First Block: +Rapa.
Second Block Conditions
A temporal relationship can be established whereby the GF-dependent R is upstream from sites that are sensitive to EAA, Q, and mTOR suppression
Temporal Mapping of Checkpoints from G0
Indicates that the mTOR control point is at least two hr downstream from the amino acid checkpoints – likely very close to the G1/S boundry
GF / Amino acid deprivation and mTOR inhibition impact differentially on
PI3K/mTOR signaling
GF / AA deprivation and mTOR inhibition: Cell cycle regulators
Summary
Data support a model where there is GF-dependent R where multi-cellular organisms determine whether it is appropriate for a cell to divide
During G1-ps, cells that have been given the green light to divide, determinewhether they have the means/raw materials to double the mass of a cell, Replicate its genome, and divide into two daughter cells
The late G1 “Metabolic Checkpoints” in late G1 collectively represent a“Cell Growth” checkpoint that responds to nutrients that is evolutionarilyequivalent to START in the yeast cell cycle – or mSTART
TOR/mTOR is likely the ultimate arbiter for determining nutrient sufficiency
Metabolic Checkpoints are Dysregulated in Cancer Cells
A
Ctrl -GF -EAA -Q +Rapa.
% C
ells
MCF 7
Ctrl -GF -EAA -Q +Rapa.
# C
ell
s
MCF 7
B
Ctrl -GF -EAA -Q +Rapa.
% C
ells
MDA MB 231
Ctrl -GF -EAA -Q +Rapa.
MDA MB 231
C
Ctrl -GF -EAA -Q +Rapa.
% C
ells
Panc-1
Ctrl -GF -EAA -Q +Rapa.
Panc-1
Preferential utilization of glucose through aerobic
glycolysis by cancer cells leading to lactate production,
independent of the oxygen availability.
Only 2 ATPs are formed from glucose to lactate as opposed
to 36 ATPs realized by engaging TCA cycle and
oxidative phosphorylation.
http://cronachedal900.blogspot.com/2012/12/otto-warburg-cura-cancro.html
Metabolic Dereguation in Cancer: The Warburg Effect
Lactate
~90%
Glutamine
Glutamate
NADPH
NAD
Glutamine supports anaplerosis
Glucose
Pyruvate
Lipid Synthesis
Based on: DeBerardinis RJ, et al. PNAS (2011)
Gao P, et al. Nature (2009)
Glutamine is “conditionally essential”
• Cancer cell propagation in mouse ascites.
• In 1950s, Harry Eagle* formulated a media which can support cell culture in vitro (DMEM).
• Key ingredient: Glutamine!
• “Added glutamine since serum had lots of glutaminase in it…” –Jim Darnell, Jr.
• Started adding excess glutamine (over 10-fold greater than any other amino acids), so that it is not a “growth-limiting” component of the medium.
*Eagle, H. Nutrition needs of mammalian cells in tissue culture. Science (1955)
Glutamine in cell culture
A
B
Genetic mutations determine differential glutamine sensitivity displayed by cancer
cells
Mu
tan
t K
-Ras
WT
K-R
asGlutamine deprivation causes loss of cell
proliferation
Dual inhibition of K-Ras/MAPK and PI3K/mTOR pathway restores glutamine-mediated G1 cell cycle
arrest
Ctrl Cape. Pacli. C+P Ctrl Cape. Pacli. C+P
% N
on
-via
ble
cel
ls
Ctrl Cape. Pacli. C+P Ctrl Cape. Pacli. C+P
% N
on
-via
ble
cel
ls
Glutamine deprivation creates synthetic lethality for cytotoxic drugs – Capecitabine and Paclitaxel
AOA and EGCG inhibit glutamine utilization
Gln
Glu
GDH
Glu +
α-KG + Asp
GOT
% C
ells
Aminotransferase inhibitor Amino-oxyacetate (AOA)
Glutamate dehydrogenase inhibitorEpigallocatechin gallate (EGCG)
Blocking glutamine utilization creates synthetic lethality to cell cycle phase-specific cytotoxic drugs in K-Ras mutant
cancer cells
AOA
• Amino acids and mTOR mediate distinct late-G1 metabolic checkpoints.
• Cancer cells with K-Ras mutation override the amino acid-mediated G1 cell cycle checkpoints and these cells arrest in S and G2/M.
• Thus, the aberrant response to amino acid deprivation could prove to be an Achilles’ heel in K-Ras mutant cancer cells – by sensitizing cancer cells to agents that kill cells in S-phase.
• Ras is mutated in ~30% of all cancers, occurs in over 90% of pancreatic cancers , which has 5-year survival rate of less than 5%. However, it is considered “therapeutically undruggable” owing in part to its extremely high affinity with GTP (in the picomolar range).
Conclusions
Ras
Raf
Mek
MAPK
Cyclin D
Restriction Point Cell Growth CheckpointGrowth Factor Signals
PTEN
mTORC1
Rheb
TSC1/2
Akt
PDK1mTORC2
Ser473 T308
S6K
FKBP38
Myc
PLD1
Insulin/IGF1
TGF-
Cyclin E
mTOR Signals
PIP2
PI3K
AMPK
LKB1
Energystatus
Aminoacids
PIP3
AMP
Complementing oncogenic alterations dysregulate Restriction Pointand Cell Growth checkpoints
Conclusions
The GF-dependent R can be distinguished from late G1 metaboliccheckpoints and mTOR
The G1 metabolic checkpoints – like R – are dysregulated in human cancer cells
Cooperating genetic alterations in cancer cells disable both R and the late metabolic checkpoints that collectively may represent a “Cell Growth” checkpoint with mTOR as the final arbitor
Surprisingly, mTOR, which is widely known to be regulated by amino acids, blocked cell cycle progression well downstream of the amino acid sites
It is hypothesized that other nutrient inputs – such as glucose and phosphatidic acid (lipids) may be required for complete activation of mTOR and progression into S-phase
Restriction Point
G1-pm G1-ps S
Growth FactorSignals
NutritionalSufficiencyAmino acidsFatty acidsEnergyATPO2
Cyclin D-CDK4/6
Cyclin E-CDK2
Cyclin A-CDK2
Cell GrowthCheckpoint
(START)
mTOR
TGF-
NutritionalSufficiency
Cell GrowthCommitmentCellSize
PLD
Rheb
G0
RalA
Vps34