1. Restriction Point G0 TGF-  and Cell Cycle Progression Cyclin D CDK4/6 Cyclin E CDK2 TGF-  Cell Growth Checkpoint G1-pm SG1-psG2M

2. Effect of rapamycin on cell cycle progression in MDA-MB-231 cells G1 S G2/M Sub genomic G1 S G2/M Rapamycin induces primarily G1 arrest in the presence of serum - and apoptosis in the absence of serum

3. Can TGF-  suppress rapamycin-induced apoptosis? TGF-  is sufficient to suppress rapamycin-induced apoptosis Is TGF-  necessary for serum to suppress rapamycin- induced apoptosis?

4. Is TGF-  in serum necessary for serum to suppress rapamycin-induced apoptosis TGF-  is necessary for serum to suppress rapamycin-induced apoptosis

5. Summary: Rapamycin induces apoptosis in MDA-MB-231 cells in the absence of serum In the presence of serum, rapamycin induces G1 arrest TGF-  is sufficient to suppress rapamycin-induced apoptosis in the absence of serum TGF-  present in serum is necessary for serum to suppress rapamycin-induced apoptosis Question: Why does rapamycin induce apoptosis when TGF-  is absent?

6. TGF-  suppresses G1 Cell Cycle Progression Restriction Point G0 Cyclin D CDK4/6 Cyclin E CDK2 TGF-  Cell Growth Checkpoint G1-pm SG1-psG2M

7. TGF-  mTOR mTOR suppresses TGF-  -induced G1 Cell Cycle Arrest Nutrients Restriction Point G0 Cyclin D CDK4/6 Cyclin E CDK2 Cell Growth Checkpoint G1-pm SG1-psG2M

8. TGF-  mTOR Rapamycin Rapamycin reverses the mTOR suppression of TGF-  signaling and cells arrest in G1 in a TGF-  -dependent mechanism Restriction Point G0 Cyclin D CDK4/6 Cyclin E CDK2 Cell Growth Checkpoint G1-pm SG1-psG2M

9. If TGF-  signaling is suppressed or defective, there is no G1 arrest with rapamycin treatment - and now the cells die in the presence of rapamycin - Why? TGF-  mTOR Rapamycin Restriction Point G0 Cyclin D CDK4/6 Cyclin E CDK2 Cell Growth Checkpoint G1-pm SG1-psG2M X

10. Hypothesis: There is a critical requirement for mTOR in S- phase. Therefore, allowing cells into S-phase in the presence of rapamycin (ie w/o mTOR) could result in apoptosis TGF-  mTOR Rapamycin Restriction Point G0 Cyclin D CDK4/6 Cyclin E CDK2 Cell Growth Checkpoint G1-pm SG1-psG2M

11. If hypothesis is correct, then blocking cells in S-phase - in the presence of serum/TGF-  - should result in apoptosis. This is because cells have passed the putative “ Cell Growth Checkpoint ” and need mTOR signals to facilitate cell cycle progression through S TGF-  mTOR Rapamycin Restriction Point G0 Cyclin D CDK4/6 Cyclin E CDK2 Cell Growth Checkpoint G1-pm SG1-psG2M Aphidicolin Synchronizes Cells in early S

12. Blocking cells in S-phase with aphidicolin sensitizes cells to rapamycin In the presence of serum/TGF-  - if cells are allowed to enter S-phase, then the lack of mTORC1 signals to 4E-BP1 tells the cell that nutrients are in short supply and that replicating the genome is probably a bad career move! The cells then do the honorable thing – and commit suicide

13. IMPLICATION: Cancer cells with defective TGF-  signaling could be selectively killed by rapamycin in the presence of either serum or TGF-  Importantly: Many cancers have defects in TGF-  signaling – especially Smad4 - that is critical for suppression of G1 cell cycle progression

14. Cancer cells with defective TGF-  signaling are Selectively killed by rapamycin in the presence of serum Colon (Smad4) Breast (Smad4) Breast (PKCδ) Breast (No TGF-  defect) MDA-MB-231

15. Summary: 1)If TGF-  is present, rapamycin induces cell cycle arrest in G1 - by increasing TGF-  signaling 2)In the absence of TGF-  signaling, rapamycin does not arrest cells in late G1 and they progress through the remainder of G1 into S-phase 3)However, if cells progress into S-phase in the presence of rapamycin, they undergo apoptosis rather than arrest - because of an apparent stringent requirement for mTOR during S-phase S Cell Growth Checkpoint mTOR TGF-  G1 p27 Cyclin D-CDK4/6 Rapamycin Cyclin E-CDK2 Survival Signals PLD PI3K Rapamycin induces arrestRapamycin induces apoptosis Nutrients T Growth Factors

16. Implications: In addition to the rapamycin concentration issues – another complication is that in most cancers, rapamycin is cytostatic rather than cytotoxic Therefore - Defective TGF-  signaling may be an Achilles heel for strategies that target mTOR - especially in colon and pancreatic cancers where defects in TGF-  signaling are common Defective TGF-  signaling creates a “Synthetic Lethality” for strategies that suppress the phosphorylation of 4E-BP1 by mTORC1 Alternatively Strategies that suppress mTOR could be combined with strategies that suppress TGF-  signaling – creating a synthetic lethal situation

17. (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-pmSG1-psG2M Complementary Signals promote G1 Cell Cycle Progression Cell Growth Checkpoint (mTOR)

18. Restriction Point G1-pmG1-ps S Growth Factor Signals Nutritional Sufficiency Amino acids Fatty acids Energy ATP O 2 Cyclin D-CDK4/6 Cyclin E-CDK2 Cyclin A-CDK2 Cell Growth Checkpoint (START) mTOR TGF-  Nutritional Sufficiency Cell Growth Commitment Cell Size PLD Rheb G0 RalA Vps34

19. 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 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 From: Weinberg, The Biology of Cancer, 2007

20. Genetic requirements for the transformation of human cells (I) (Hahn et al., Nature 400:464, 1999; MCB 22;2111, 2002) Genetic effectMolecular TargetCell cycle target Ras Growth factor signals Restriction point SV40 Large Tp53G1/S checkpoint RbAll G1 checkpoints SV40 small tPP2ACell Growth checkpoint (?) Genetic requirements for the transformation of human cells (II) (Boehm et al., MCB 25:6464, 2005) Genetic effectMolecular TargetCell cycle target Ras Growth factor signals Restriction point p53 KO p53G1/S checkpoint Rb KORbAll G1 checkpoints MycGene expressionCell Growth checkpoint (?) PTEN KOmTORC1Cell Growth checkpoint (?)

21. Ras Raf Mek MAPK Cyclin D Restriction PointCell Growth Checkpoint Growth Factor Signals PTEN mTORC1 Rheb TSC1/2 Akt PDK1 mTORC2 Ser473T308 S6K FKBP38 Myc PLD1 Insulin/IGF1 TGF-  Cyclin E mTOR Signals PIP2 PI3K AMPK LKB1 Energy status Amino acids PIP3 AMP

22. Growth factor, essential amino acid, glutamine, and suppression of mTOR block G1 cells cycle progression at distinguishable sites in G1 A temporal relationship can be established whereby the GF- dependent R is upstream from sites that are sensitive to EAA, Q, and mTOR suppression

23. Temporal mapping of G1 cell cycle checkpoints Cells require 12- 16 hr to enter S-phase after GF deprivation or mTOR suppression Cells require 20+ hr to enter S-phase after amino acid deprivation For cells arrested in G0 by GF deprivation, amino acid deprivation blocks entry into S- phase for 12-14 hr For cells arrested in G0 by GF deprivation, mTOR suppression blocks entry into S- phase for 16-18 hr

24. Blocking G1 cell cycle progression by growth factor and nutrient deprivation, and suppression of mTOR Different blocking conditions have differential effects on cell cycle regulators and on autophagy – notably for EAA and Q P-Akt T308 P-Akt S473 P-S6K T389 P-4EBP1 LC3-II Akt S6K 4EBP1 Actin A p85 -EAA -GF -Q CM +Rapa. p70 CM (hrs.) -GF -EAA -Q +Rapa. D1 11 0257 17 14 20 Cyclin D D2/D3 11 0257 17 14 20 Pan-Rb 11 0257 17 14 20 P-Rb S807/811 11 0257 17 14 20 p21 BD CE CM (hrs.) -GF -EAA -Q +Rapa.

25. Summary Data supports 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 TOR/mTOR is likely the ultimate arbiter for determining nutrient sufficiency

26. Ras Raf Mek MAPK Cyclin D Restriction Point Cell Growth Checkpoint Growth Factor Signals PTEN mTORC1 Rheb TSC1/2 Akt PDK1 mTORC2 Ser473T308 S6K FKBP38 Myc PLD1 Insulin/IGF1 TGF-  Cyclin E mTOR Signals PIP2 PI3K AMPK LKB1 Energy status Amino acids PIP3 AMP Complementing oncogenic alterations dysregulate Restriction Point and Cell Growth checkpoints

27. Dysregulated metabolic checkpoints in cancer cells In response to amino acid deprivation, MDA-MB-231 breast and Panc1 pancreatic cells arrest in S-phase and G2/M

28. 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