1. Bio 714 Foster TGF-beta/cell cycle material

2. Points: Suppression of either PLD or mTOR in the absence of serum results in apoptosis Importantly, suppression of PLD or mTOR does not induce apoptosis in the presence of serum Conclusion There is a factor(s) in serum that prevents apoptosis in cells in response to the suppression of PLD or mTOR Point: Danielpour and colleagues showed that mTOR suppresses TGF-  signaling (Song et al., EMBO J, 25:58, 2006). Question: Is TGF-  the factor in serum that prevents rapamycin-induced apoptosis in MDA-MB-231 cells?

3. Restriction Point G0 TGF-  and Cell Cycle Progression Cyclin D CDK4/6 Cyclin E CDK2 TGF-  Cell Growth Checkpoint G1-pmSG1-psG2M

4. 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

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

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

7. 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?

8. TGF-  suppresses G1 Cell Cycle Progression Restriction Point G0 Cyclin D CDK4/6 Cyclin E CDK2 TGF-  Cell Growth Checkpoint G1-pmSG1-psG2M

9. 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-pmSG1-psG2M

10. 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-pmSG1-psG2M

11. 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-pmSG1-psG2M X

12. 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-pmSG1-psG2M

13. 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-pmSG1-psG2M Aphidicolin Synchronizes Cells in early S

14. Blocking cells in S-phase with aphidicolin sensitizes cells to rapamycin

15. 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

16. 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

17. 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

18. Implications: Rapamycin and rapamycin analogues have been largely disappointing in clinical trials 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 Alternatively Strategies that suppress mTOR could be combined with strategies that suppress TGF-  signaling - leading to regression rather than just suppressing cell division

19. (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)

20. 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

21. 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

22. 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 p53p53G1/S checkpoint RbRbAll G1 checkpoints MycGene expressionCell Growth checkpoint (?) PTENmTORC1Cell Growth checkpoint (?)

23. Ras Raf Mek MAPK Cyclin D Restriction PointCell 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

24. 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