Nutrient and Growth Factor Control of Cell
Cycle Progression and Survival in Cancer Cells
Bio 714
Cell Biology
Feb, 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 Apoptosis
avoid default cell death programs
IV Acquire Immortality - telomerase expression
overcome cell senescence - cancer stem cells?
V Stimulate Angiogenesis
provide 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 Apoptosis
avoid default cell death programs
IV Acquire Immortality - telomerase expression
overcome cell senescence - cancer stem cells?
V Stimulate Angiogenesis
provide nutrition
VI Acquire the ability to migrate and invade
gain access to circulation (metastasis)
VII Breakdown Caretaker Function
genomic instability needed for many mutations
Regulation of Cell Cycle Progression
G0
(Quiescence)
Growth Factor Signals
Restriction
Point
G1-pm
G1-ps
Cell Growth
Checkpoint
(mTOR)
Tyrosine kinases
Ras/Raf/MEK/MAPK
S
Gatekeepers
G2
Myc
SV40 Early Region
(Suppression of
p53, Rb and PP2A)
M
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)
Conventional View of Cell Cycle
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
Zetterberg and colleagues have mapped the
Restriction Point to a site ~ 3.5 hr after
mitosis - where cyclin D is elevated
Figure 8.8 The Biology of Cancer (© Garland Science 2007)
From: Weinberg, The Biology of Cancer, 2007
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 activated
Rapamycin 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
Protozoans & Metazoans are… different!
Vander Heiden MG, Cantley LC, Thompson CB. Science (2009)
Restriction Point (R)
G0
G0
G1
R
S
- GF
Restriction Point: Point in G1 after which cells no longer need GF permissive
signals to divide.
Cell Cycle: START vs. R
Yeast
Mammalian
Restriction
Point
Cooper GM. The Cell: A Molecular Approach (2000)
Pyronnet S, Sonenberg N
Curr. Opin. Genetics Dev. (2001)
Mammalian Restriction Point is Analogous to START in Yeast
• Lodish H, et al. Molecular Cell Biology (2008)
What is a Restriction Point ?
3.5 hr
+ GF
M
- GF
G1
G0
S
R
Based on Zetterberg and Larsson, PNAS (1985)
Competence
Factor
(PDGF)
Progression
Factor (IGF1)
Based on Pledger and Stiles, PNAS (1979)
Where is the Restriction Point ?
Dowdy SF, Curr. Opin. Gen Dev (2002) Weinberg RA, Biology of Cancer (2006)
Boonstra J, Adv Enzyme Regul (2007)
Zetterberg A, Exp Cell Res (2005)
Lodish H, Molecular Cell Biology (2008)
http://medicinembbs.blogspot.com/
2011_10_01_archive.html
… and where is the Nutrient Sensing ?
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
Rb
G1/S checkpoint
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
Rb KO
p53
Rb
G1/S checkpoint
All G1 checkpoints
Myc
PTEN KO
Gene expression
mTORC1
Cell Growth checkpoint (?)
Cell Growth checkpoint (?)
Foster DA, Yellen P, Xu L, Saqcena M. Genes & Cancer (2011)
GF / nutrient deprivation mediate cell cycle arrest
Immortalized primary cell line (BJ hTERT)
GF / nutrient deprivation mediate cell cycle arrest
Sequential Blocking Experiments:
1st Block
2nd
Block
= No thymidine incorporation
+ 3H-Thymidine
1st Block
= Thymidine incorporation
2nd
Block
+ 3H-Thymidine
R & Nutrient Sensing Checkpoints are distinguishable
First Block: -EAA
3H-TdR
Inc. (%Ctrl)
First Block: -GF
First Block: -Q
First Block: +Rapa.
Second Block Conditions
A temporal
relationship can be
established
whereby the GFdependent 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, determine
whether 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 evolutionarily
equivalent 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
MCF 7
MDA MB 231
% Cells
-GF -EAA -Q +Rapa.
Ctrl
-GF -EAA -Q +Rapa.
Ctrl
-GF -EAA -Q +Rapa.
# Cells
Ctrl
Panc-1
C
% Cells
B
% Cells
A
Ctrl
-GF
-EAA
MCF 7
-Q
+Rapa.
Ctrl
-GF
-EAA
-Q
MDA MB 231
+Rapa.
Ctrl
-GF
-EAA
Panc-1
-Q
+Rapa.
Metabolic Dereguation in Cancer: The Warburg Effect
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/ottowarburg-cura-cancro.html
Glutamine supports anaplerosis
Glucose
~90%
Pyruvate
Lipid Synthesis
NAD
Lactate
NADPH
Glutamate
Glutamine
Based on: DeBerardinis RJ, et al. PNAS (2011)
Glutamine is “conditionally essential”
Gao P, et al. Nature (2009)
Glutamine in cell culture
•
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)
Genetic mutations determine differential glutamine
sensitivity displayed by cancer cells
A
B
Mutant K-Ras
WT K-Ras
Glutamine deprivation causes loss of cell proliferation
Dual inhibition of K-Ras/MAPK and PI3K/mTOR pathway restores
glutamine-mediated G1 cell cycle arrest
% Non-viable cells
Glutamine deprivation creates synthetic lethality for cytotoxic drugs – Capecitabine
and Paclitaxel
Cape.
Pacli.
C+P
Ctrl
Cape.
Pacli.
C+P
Ctrl
Cape.
Pacli.
C+P
Ctrl
Cape.
Pacli.
C+P
% Non-viable cells
Ctrl
AOA and EGCG inhibit glutamine utilization
Aminotransferase inhibitor
Amino-oxyacetate (AOA)
α-KG + Asp
GOT
Glutamate dehydrogenase inhibitor
Epigallocatechin gallate (EGCG)
Glu +
Glu
Gln
% Cells
GDH
Blocking glutamine utilization creates synthetic lethality to cell cycle phase-specific
cytotoxic drugs in K-Ras mutant cancer cells
AOA
Conclusions
•
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).
Complementing oncogenic alterations dysregulate Restriction Point
and Cell Growth checkpoints
Restriction Point
Cell Growth Checkpoint
mTOR Signals
Growth Factor Signals
Ras
Insulin/IGF1
PI3K
PIP3
PIP2
PTEN
mTORC2
PDK1
Raf
Akt
Ser473
T308
TSC1/2
Mek
Amino
acids
Rheb
PLD1
MAPK
FKBP38
mTORC1
S6K
TGF-
Myc
Cyclin D
Cyclin E
AMP
LKB1
AMPK
Energy
status
Conclusions
The GF-dependent R can be distinguished from late G1 metabolic
checkpoints 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
Growth Factor
Signals
Cell Growth
Checkpoint
(START)
Restriction
Point
G1-pm
G1-ps
Nutritional
Sufficiency
Amino acids
Fatty acids
Energy
ATP
RalA
O2
Vps34
Cell
Size
Nutritional
Sufficiency
S
Cyclin D-CDK4/6
Cyclin E-CDK2
G0
Cyclin A-CDK2
Rheb
PLD
mTOR
TGF-
Commitment
Cell Growth