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

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)


Restriction Point


G0

G1-pm SG1-ps G2 M


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)


Restriction Point


G0

G1-pm SG1-ps G2 M


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-


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


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


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


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


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


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


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

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

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