defective tgf- signaling creates a synthetic lethality for suppression of mtor
TRANSCRIPT
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-pm SG1-ps G2 M
Regulation of Cell Cycle Progression
Cell GrowthCheckpoint(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-pm SG1-ps G2 M
Cell GrowthCheckpoint(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
CH2 CH2 CH2
C=O C=O
(CH2)n (CH2)n
CH3 CH3
PLDOH
H+
Hydrolysis
Phosphatidylcholine
H
O
O=P O-
O
CH2 CH2 CH2
C=O C=O
(CH2)n (CH2)n
CH3 CH3
+Ch OH
Phosphatidic acid
Phospholipase D
Ch
O
O=P O-
O
CH2 CH2 CH2
C=O C=O
(CH2)n (CH2)n
CH3 CH3
OH
CH2
CH3
+PLD
Transphosphatidylation
Phospholipase D
Phosphatidylcholine
CH3
CH2
O
O=P O-
O
CH2 CH2 CH2
C=O C=O
(CH2)n (CH2)n
CH3 CH3
+Ch OH
Phosphatidyl-ethanol
Regulators of PLD1Rho family GTPases (Rho, Rac, Cdc42)
Ral GTPase
Arf family GTPases
Rheb GTPase
PKC
Phosphatidylinositol-4,5-bis-phosphate (PIP2)
Vps34 (PI-3-P)
Regulators of PLD2Constitutively active in vitro
Fatty acids
PIP2
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-B
tOH
Contr
ol
iso-B
tOH
2-B
tOH
t-BtO
H
PLD provides a survival signal in 786-O renal cancer cells
Actin
Parp
Cl. Parp
Control
PLD1 siR
NA
PLD2 siR
NA
PLD1+2 siR
NA
PLD1 siR
NA
PLD2 siR
NA
PLD1+2 siR
NA
Mock
Mock
FBS + - + - - - + + +
PLD1
PLD2
Toschi et al. Oncogene. 2008
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
mTORVesicle
formation
EndocytosisExocytosis
PI(4)PNADPHoxidase
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 callinga 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-pm SG1-ps G2 M
Cell GrowthCheckpoint(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 DCDK4/6
Cyclin ECDK2
TGF-
Cell GrowthCheckpoint
G1-pm SG1-ps G2 M
Effect of rapamycin on cell cycle progression in
MDA-MB-231 cells
G1 S G2/M G1 S G2/M
Sub genomicG1 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 DCDK4/6
Cyclin ECDK2
TGF-
Cell GrowthCheckpoint
G1-pm SG1-ps G2 M
TGF- mTOR
mTOR suppresses TGF--induced G1 Cell Cycle Arrest
Nutrients
Restriction Point
G0
Cyclin DCDK4/6
Cyclin ECDK2
Cell GrowthCheckpoint
G1-pm SG1-ps G2 M
TGF- mTOR Rapamycin
Rapamycin reverses the mTOR suppression of TGF- signalingand cells arrest in G1 in a TGF--dependent mechanism
Restriction Point
G0
Cyclin DCDK4/6
Cyclin ECDK2
Cell GrowthCheckpoint
G1-pm SG1-ps G2 M
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 DCDK4/6
Cyclin ECDK2
Cell GrowthCheckpoint
G1-pm SG1-ps G2 M
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 DCDK4/6
Cyclin ECDK2
Cell GrowthCheckpoint
G1-pm SG1-ps G2 M
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 DCDK4/6
Cyclin ECDK2
Cell GrowthCheckpoint
G1-pm SG1-ps G2 M
Aphidicolin Synchronizes Cells in early S
Blocking cells in S-phase with aphidicolinsensitizes 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 GrowthCheckpoint
mTOR
TGF-
G1
p27
Cyclin D-CDK4/6
Rapamycin
Cyclin E-CDK2
SurvivalSignals
PLD
PI3K
Rapamycin induces arrest Rapamycin induces apoptosis
Nutrients
T
GrowthFactors
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