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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