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Transcript
Bio 714 Foster TGF-beta/cell cycle material 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? TGF- and Cell Cycle Progression G0 Restriction Point G1-pm G1-ps S Cell Growth Checkpoint Cyclin D CDK4/6 Cyclin E CDK2 TGF- G2 M Effect of rapamycin on cell cycle progression in MDA-MB-231 cells G1 S G2/M G1 S G2/M G1 S G2/M Sub genomic Rapamycin induces primarily G1 arrest in the presence of serum - and apoptosis in the absence of serum Can TGF- suppress rapamycin-induced apoptosis? TGF- is sufficient to suppress rapamycin-induced apoptosis Is TGF- necessary for serum to suppress rapamycininduced apoptosis? Is TGF- in serum necessary for serum to suppress rapamycin-induced apoptosis % Non-Viable Cells Figure 3A 100 % Non-Viable 50 Cells Series1 0 1 - 2 - + 3 + Rap - + + + TGF- -Ab - - - + Serum 4 Cl PARP actin TGF- is necessary for serum to suppress rapamycin-induced apoptosis 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? G0 Restriction Point G1-pm G1-ps S G2 Cell Growth Checkpoint Cyclin D CDK4/6 Cyclin E CDK2 TGF- TGF- suppresses G1 Cell Cycle Progression M G0 Restriction Point G1-pm G1-ps S G2 M Cell Growth Checkpoint Cyclin E CDK2 Cyclin D CDK4/6 TGF- mTOR mTOR suppresses TGF--induced G1 Cell Cycle Arrest Nutrients G0 Restriction Point G1-pm G1-ps S G2 M Cell Growth Checkpoint Cyclin E CDK2 Cyclin D CDK4/6 TGF- mTOR Rapamycin Rapamycin reverses the mTOR suppression of TGF- signaling and cells arrest in G1 in a TGF--dependent mechanism G0 Restriction Point G1-pm G1-ps S G2 M Cell Growth Checkpoint Cyclin D CDK4/6 Cyclin E CDK2 X TGF- mTOR Rapamycin 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? G0 Restriction Point G1-pm G1-ps S G2 M Cell Growth Checkpoint Cyclin E CDK2 Cyclin D CDK4/6 TGF- mTOR Rapamycin Hypothesis: There is a critical requirement for mTOR in Sphase. Therefore, allowing cells into S-phase in the presence of rapamycin (ie w/o mTOR) could result in apoptosis G0 Aphidicolin Restriction Point G1-pm Synchronizes Cells in early S G1-ps S G2 M Cell Growth Checkpoint Cyclin E CDK2 Cyclin D CDK4/6 TGF- mTOR Rapamycin 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 Blocking cells in S-phase with aphidicolin sensitizes cells to rapamycin % Non-Viable Cells Figure 6A 100 % Non-Viable 50 Cells Series1 0 Rap Aph Cl PARP actin 1 - 2 + 3 - 4 - + + + 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 Cancer cells with defective TGF- signaling are Selectively killed by rapamycin in the presence of serum Breast (Smad4) Breast (No TGF- defect) MDA-MB-231 Breast (PKCδ) 100 SW480 50 Series1 % Non Viable Cells % Non Viable Cells Colon (Smad4) 100 BT-549 50 Series1 0 0 Serum + - - + - Serum + - - + - Rap - - + + + Rap - - + + + TGF- - - - - + TGF- - - - - + Cl PARP actin Cl PARP actin 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 Cell Growth Checkpoint Rapamycin induces arrest Rapamycin induces apoptosis G1 S Nutrients Cyclin D-CDK4/6 Cyclin E-CDK2 p27 Rapamycin PLD mTOR TGF- Survival Signals PI3K Growth Factors 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 Complementary Signals promote G1 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 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 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 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 Rb p53 Rb G1/S checkpoint All G1 checkpoints Myc PTEN Gene expression mTORC1 Cell Growth checkpoint (?) Cell Growth checkpoint (?) Restriction Point Cell Growth Checkpoint Growth Factor Signals Ras mTOR Signals 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 Cell Growth Checkpoint Rapamycin induces arrest Rapamycin induces apoptosis G1 S Cyclin D-CDK4/6 Nutrients Cyclin E-CDK2 p27 Rapamycin PLD mTOR TGF- PI3K Survival Signals Growth Factors
