Defective TGF-  signaling creates a synthetic lethality for suppression of mTOR

(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 Regulation of Cell Cycle Progression Cell Growth Checkpoint (mTOR)

Survival Signals Generated by Phospholipase D History: Phospholipase D activity is elevated in cells transformed by v-Src (Song et al., MCB 11:4903, 1991) Phospholipase D cooperates with elevated tyrosine kinase expression to transform rat fibroblasts (Lu et al., MCB 20:462, 2000) Phospholipase D suppresses apoptosis induced by over-expressed Raf (Joseph et al., Oncogene 21:3651, 2002) Phospholipase D suppresses both p53 expression and PP2A activity (Hui et al., MCB 24:5677, 2004; Hui et al., JBC 280, 35829, 2005) Phospholipase D is required for the phosphorylation (suppression) of Rb (Gadir et al., Cell Cycle 62840,2007) Phospholipase D stimulates Myc stabilization in breast cancer cells (Rodrik et al., MCB 25, 7917, 2005; FEBS Lett 580:5647, 2006)

(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 Cell Growth Checkpoint (mTOR) Hypothesis: Elevated PLD activity provides gatekeeper overrides for progression through G1-ps; and cooperates with growth factor signals that promote passage through the Restriction Point Regulation of Cell Cycle Progression PLD

Ch O O=P O - O CH 2 CH 2 CH 2 C=O C=O (CH 2 ) n CH 3 CH 3 PLD OHHOHH + Hydrolysis Phosphatidylcholine H O O=P O - O CH 2 CH 2 CH 2 C=O C=O (CH 2 ) n CH 3 CH 3 + Ch OH Phosphatidic acid Phospholipase D

Ch O O=P O - O CH 2 CH 2 CH 2 C=O C=O (CH 2 ) n CH 3 CH 3 OH CH 2 CH 3 + PLD Transphosphatidylation Phospholipase D Phosphatidylcholine CH 3 CH 2 O O=P O - O CH 2 CH 2 CH 2 C=O C=O (CH 2 ) n CH 3 CH 3 + Ch OH Phosphatidyl-ethanol

Regulators of PLD1 Rho family GTPases (Rho, Rac, Cdc42) Ral GTPase Arf family GTPases Rheb GTPase PKC  Phosphatidylinositol-4,5-bis-phosphate (PIP 2 ) Vps34 (PI-3-P) Regulators of PLD2 Constitutively active in vitro Fatty acids PIP 2 Vps34? PLD1?

Phospholipase D activity is elevated in human cancer and cancer cell lines Breast (PLD1) ( Noh et al. Cancer Lett.161:207, 2000) Kidney (PLD2) ( Zhao et al. BBRC 278:140, 2000) Gastric (?) ( Uchida et al. Anticancer Res. 19:671, 1999) Colorectal (PLD2) ( Yamada et al. J. Mol. Med. 81:126, 2003) Lung (?) (Zheng et al., JBC 281:15862, 2006) Bladder (?) (Zheng et al., JBC 281:15862, 2006) Pancreatic (?) (Our unpublished results)

Blocking PLD-mediated PA Synthesis Induces Apoptosis In MDA-MB-231 Cells Deprived of Serum (Zhong et al.. BBRC 302, 615, 2003) PARP 1-BtOH Control iso-BtOH 2-BtOH t-BtOH

PLD provides a survival signal in 786-O renal cancer cells Actin Parp Cl. Parp Control PLD1 siRNAPLD2 siRNA PLD1+2 siRNA PLD1 siRNAPLD2 siRNA PLD1+2 siRNA Mock FBS PLD1 PLD2 Toschi et al. Oncogene Cl. Parp

Conclusion Elevated PLD activity in human cancer cells provides a survival signal that prevents apoptosis induced by the stress of serum withdrawal Question How does elevated PLD activity generate survival signals in these cells?

Raf Rho/Arf-GAP Ras-GAP PI(4,5)P2 PI(4)P5-kinase MEK MAP Kinase mTOR Vesicle formation Endocytosis Exocytosis PI(4)P NADPH oxidase Targets of Phosphatidic Acid PLD PA Survival mTOR

mTOR (Mammalian Target of Rapamycin) Regulator of cell proliferation and cell growth Responds to nutrients (amino acids, glucose, lipids?) Regulates initiation of protein synthesis - including Myc Inhibited by rapamycin There are two mTOR complexes - mTORC1 and mTORC2 Phosphatidic acid (PA), the product of PLD, interacts with mTOR competitively with rapamycin How does PA impact on mTOR? How does rapamycin work?

mTOR and PLD are part of a signaling network that responds to nutrients, energy, and insulin/IGF1 PLD and mTOR are required for the survival of cancer cells - especially when deprived of serum PLD and mTOR are required for progression through G1-ps - at what we are calling a Cell Growth Checkpoint (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 Cell Growth Checkpoint (mTOR)

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?

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

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

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

Is TGF-  in serum necessary for serum to suppress rapamycin-induced apoptosis 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?

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

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

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

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

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

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

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

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 Colon (Smad4) Breast (Smad4) Breast (PKCδ) Breast (No TGF-  defect) MDA-MB-231

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

Implication: Defects in G1 cell cycle progression can create a “Synthetic Lethality” by allowing cells into S-phase where they are more susceptible to apoptotic insult