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Transcript
Defective TGF- signaling creates a
synthetic lethality for suppression of mTOR
Regulation of 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
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)
Regulation of Cell Cycle Progression
G0
(Quiescence)
Restriction
Point
Growth Factor Signals
G1-pm G1-ps
Cell Growth
Checkpoint
(mTOR)
Hypothesis:
Tyrosine kinases
Ras/Raf/MEK/MAPK
S
G2 M
Gatekeepers
Myc
SV40 Early Region
(Suppression of
p53, Rb and PP2A)
Elevated PLD activity provides gatekeeper
overrides for progression through G1-ps;
and cooperates with growth factor signals that
promote passage through the Restriction Point
PLD
Phospholipase D
Hydrolysis
Ch
H
O
O
O=P O-
O=P OO
CH2
CH2
C=O
C=O
OH
CH2 + H
PLD
O
CH2
CH2
C=O
C=O
CH2
(CH2)n (CH2)n
(CH2)n (CH2)n
CH3
CH3
Phosphatidylcholine
CH3
CH3
Phosphatidic acid
+
Ch OH
Ch
Phospholipase D
CH3
Transphosphatidylation
CH2
O
O
O=P O-
O=P O-
O
O
CH2
CH2
C=O
C=O
OH
PLD
CH2 + CH
2
CH2
CH2
CH3
C=O
C=O
CH2
(CH2)n (CH2)n
(CH2)n (CH2)n
CH3
CH3
Phosphatidylcholine
CH3
CH3
Phosphatidyl-ethanol
+
Ch OH
Regulators of PLD1
Rho 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 PLD2
Constitutively 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
45%
% Apoptotic Cells
40%
35%
PARP
30%
25%
20%
15%
10%
5%
0%
Controll 1-BtOH
iso- 2-BtOH t-BtOH
BtOH
(Zhong et al.. BBRC 302, 615, 2003)
PLD provides a survival signal in 786-O renal
cancer cells
FBS
+
-
+
-
-
-
+
+
+
Parp
Cl. Parp
PLD1
PLD2
Actin
Toschi et al. Oncogene. 2008
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?
Targets of Phosphatidic Acid
PLD
PA
Ras-GAP
PI(4,5)P2
Rho/Arf-GAP
PI(4)P5-kinase
PI(4)P
NADPH
oxidase
Raf
mTOR
Vesicle
formation
MEK
MAP Kinase
Survival
Endocytosis
Exocytosis
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
G0
(Quiescence)
PLD and mTOR are required
for progression through
G1-ps - at what we are calling
a Cell Growth Checkpoint
Restriction
Point
G1-pm G1-ps
Growth Factor Signals
Tyrosine kinases
Ras/Raf/MEK/MAPK
S
Cell Growth
Checkpoint
(mTOR)
G2
Gatekeepers
Myc
SV40 Early Region
(Suppression of
p53, Rb and PP2A)
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?
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
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!
% Non-Viable Cells
Figure 6A
100
% Non-Viable
50
Cells
Series1
0
Rap
Aph
Cl PARP
actin
1
-
2
+
3
-
4
-
+
+
+
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
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
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