1. Nutrient and Growth Factor Control of Cell Cycle Progression and Survival in Cancer Cells 
Bio 714
Cell Biology
Feb, 2014

2. 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

3. 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

4. Regulation of Cell Cycle Progression
(Quiescence)
Growth Factor Signals
Tyrosine kinases
Ras/Raf/MEK/MAPK
Restriction
Point
G1-pm
G1-ps S G2 M
Gatekeepers
Myc
SV40 Early Region
(Suppression of
p53, Rb and PP2A)
Cell Growth
Checkpoint
(mTOR)

5. 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”

6. Foster DA, Yellen P, Xu L, Saqcena M. Genes & Cancer (2011)

7. Conventional View of Cell Cycle Points:
Figure 8.8 The Biology of Cancer (© Garland Science 2007)
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
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
From: Weinberg, The Biology of Cancer, 2007
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

8. Protozoans & Metazoans are… different!
Vander Heiden MG, Cantley LC, Thompson CB. Science (2009)

9. G1 S Restriction Point (R)
- GF
Restriction Point: Point in G1 after which cells no longer need GF permissive signals to divide.

10. Mammalian Restriction Point is Analogous to START in Yeast
Cell Cycle: START vs. R
Yeast
Mammalian
Restriction
Point
Pyronnet S, Sonenberg N
Curr. Opin. Genetics Dev. (2001)
Cooper GM. The Cell: A Molecular Approach (2000)
Mammalian Restriction Point is Analogous to START in Yeast
Lodish H, et al. Molecular Cell Biology (2008)

11. What is a Restriction Point ?
3.5 hr
+ GF R
- GF G0 M G1 S
Competence Factor (PDGF)
Progression Factor (IGF-1)
Based on Zetterberg and Larsson, PNAS (1985)
Based on Pledger and Stiles, PNAS (1979)

12. Where is the Restriction Point ?
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)
2011_10_01_archive.html
… and where is the Nutrient Sensing ?

14. 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 checkpoint
Rb 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 checkpoint
Rb KO Rb All G1 checkpoints
Myc Gene expression Cell Growth checkpoint (?)
PTEN KO mTORC1 Cell Growth checkpoint (?)

15. Foster DA, Yellen P, Xu L, Saqcena M. Genes & Cancer (2011)

16. GF / nutrient deprivation mediate cell cycle arrest
Immortalized primary cell line (BJ hTERT)

17. GF / nutrient deprivation mediate cell cycle arrest

18. Sequential Blocking Experiments:
1st Block
= No thymidine incorporation
2nd Block
+ 3H-Thymidine
1st Block
= Thymidine incorporation
2nd Block
+ 3H-Thymidine

19. R & Nutrient Sensing Checkpoints are distinguishable
First Block: -GF
First Block: -EAA
A temporal relationship can be established whereby the GF-dependent R is upstream from sites that are sensitive to EAA, Q, and mTOR suppression
3H-TdR Inc. (%Ctrl)
First Block: -Q
First Block: +Rapa.
Second Block Conditions

20. 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

21. GF / Amino acid deprivation and mTOR inhibition impact differentially on PI3K/mTOR signaling

22. GF / AA deprivation and mTOR inhibition: Cell cycle regulators

23. 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

24. Metabolic Checkpoints are Dysregulated in Cancer Cells
Ctrl -GF -EAA -Q +Rapa.
% Cells
MCF 7
Ctrl GF -EAA Q +Rapa.
# Cells B
Ctrl -GF -EAA -Q +Rapa.
% Cells
MDA MB 231
Ctrl GF -EAA Q +Rapa. C
Ctrl -GF -EAA -Q +Rapa.
% Cells
Panc-1
Ctrl GF -EAA Q +Rapa.

25. 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.

26. Glutamine supports anaplerosis
Glucose
~90%
Pyruvate
Lipid Synthesis
NAD
Lactate
NADPH
Glutamate
Glutamine
Based on: DeBerardinis RJ, et al. PNAS (2011)

27. Glutamine is “conditionally essential”
Gao P, et al. Nature (2009)

28. 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)

29. Genetic mutations determine differential glutamine sensitivity displayed by cancer cells

30. Glutamine deprivation causes loss of cell proliferation
Mutant K-Ras
WT K-Ras

31. Dual inhibition of K-Ras/MAPK and PI3K/mTOR pathway restores glutamine-mediated G1 cell cycle arrest

32. Glutamine deprivation creates synthetic lethality for cytotoxic drugs – Capecitabine and Paclitaxel
Ctrl Cape. Pacli C+P
% Non-viable cells
Ctrl Cape. Pacli C+P
Ctrl Cape. Pacli C+P
% Non-viable cells

33. AOA and EGCG inhibit glutamine utilization
Aminotransferase inhibitor
Amino-oxyacetate (AOA)
Glutamate dehydrogenase inhibitor
Epigallocatechin gallate (EGCG)
Glu +
α-KG + Asp
GOT
% Cells
Gln
Glu
GDH

34. Blocking glutamine utilization creates synthetic lethality to cell cycle phase-specific cytotoxic drugs in K-Ras mutant cancer cells
AOA

35. 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).

36. Complementing oncogenic alterations dysregulate Restriction Point
and Cell Growth checkpoints
Restriction Point
Cell Growth Checkpoint
Growth Factor Signals
PTEN
mTORC1
Rheb
TSC1/2
Akt
PDK1
mTORC2
Ser473
T308
S6K
FKBP38
Myc
PLD1
Insulin/IGF1
TGF-
Cyclin E
mTOR Signals
PIP2
PI3K
AMPK
LKB1
Energy
status
Amino
acids
PIP3
AMP
Ras
Raf
Mek
MAPK
Cyclin D

37. 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

38. G1-pm G1-ps S Cell Size G0 PLD mTOR Growth Factor Signals Nutritional
Sufficiency
Cell Growth
Checkpoint
(START)
Restriction
Point
G1-pm
G1-ps S
Nutritional
Sufficiency
Amino acids
Fatty acids
Energy
ATP O2
Cyclin D-CDK4/6
Cyclin E-CDK2 G0
Cyclin A-CDK2
Rheb
RalA
PLD
mTOR
Vps34
TGF-
Cell
Size
Commitment
Cell Growth