Activation of the PIK3CA/AKT Pathway Suppresses Senescence Induced by an Activated RAS Oncogene to Promote Tumorigenesis

Drexel University College of Medicine, Philadelphia, PA 19129, USA
Molecular cell (Impact Factor: 14.02). 04/2011; 42(1):36-49. DOI: 10.1016/j.molcel.2011.02.020
Source: PubMed
ABSTRACT
Mutations in both RAS and the PTEN/PIK3CA/AKT signaling module are found in the same human tumors. PIK3CA and AKT are downstream effectors of RAS, and the selective advantage conferred by mutation of two genes in the same pathway is unclear. Based on a comparative molecular analysis, we show that activated PIK3CA/AKT is a weaker inducer of senescence than is activated RAS. Moreover, concurrent activation of RAS and PIK3CA/AKT impairs RAS-induced senescence. In vivo, bypass of RAS-induced senescence by activated PIK3CA/AKT correlates with accelerated tumorigenesis. Thus, not all oncogenes are equally potent inducers of senescence, and, paradoxically, a weak inducer of senescence (PIK3CA/AKT) can be dominant over a strong inducer of senescence (RAS). For tumor growth, one selective advantage of concurrent mutation of RAS and PTEN/PIK3CA/AKT is suppression of RAS-induced senescence. Evidence is presented that this new understanding can be exploited in rational development and targeted application of prosenescence cancer therapies.

Full-text

Available from: Indrani Manoharan, May 12, 2014
Molecular Cell
Article
Activation of the PIK3CA/AKT Pathway
Suppresses Senescence Induced by an
Activated RAS Oncogene to Promote Tumorigenesis
Alyssa L. Kennedy,
3,4,6
Jennifer P. Morton,
2,6
Indrani Manoharan,
1
David M. Nelson,
1
Nigel B. Jamieson,
1,5
Jeff S. Pawlikowski,
1
Tony McBryan,
1
Brendan Doyle,
2,7
Colin McKay,
5
Karin A. Oien,
1
Greg H. Enders,
4
Rugang Zhang,
4
Owen J. Sansom,
2,
*
and Peter D. Adams
1,
*
1
CR-UK Beatson Labs, University of Glasgow, Glasgow G61 1BD, UK
2
Beatson Institute for Cancer Research, Glasgow G61 1BD, UK
3
Drexel University College of Medicine, Philadelphia, PA 19129, USA
4
Fox Chase Cancer Center, Philadelphia, PA 19111, USA
5
West of Scotland Pancreatic Unit, University Department of Surgery, Glasgow Royal Infirmary, Glasgow G31 2ER, UK
6
These authors contributed equally to this manuscript
7
Department of Pathology, Coombe Women and Infant’s University Hospital, Dublin 8, Ireland
*Correspondence: o.sansom@beatson.gla.ac.uk (O.J.S.), p.adams@beatson.gla.ac.uk (P.D.A.)
DOI 10.1016/j.molcel.2011.02.020
SUMMARY
Mutations in both RAS and the PTEN/PIK3CA/AKT
signaling module are found in the same human
tumors. PIK3CA and AKT are downstream effectors
of RAS, and the selective advantage conferred by
mutation of two genes in the same pathway is
unclear. Based on a comparative molecular analysis,
we show that activated PIK3CA/AKT is a weaker
inducer of senescence than is activated RAS. More-
over, concurrent activation of RAS and PIK3CA/AKT
impairs RAS-induced senescence. In vivo, bypass
of RAS-induced senescence by activat ed PIK3CA/
AKT correlates with accelerated tumorigenesis.
Thus, not all oncogenes are equally potent inducers
of senescence, and, paradoxically, a weak inducer
of senescence (PIK3CA/AKT) can be dominant over
a strong inducer of senes cence (RAS). For tumor
growth, one selective advantage of concurrent muta-
tion of RAS and PTEN/PIK3CA/AKT is suppression of
RAS-induced senescence. Evidenc e is presented
that this new understanding can be exploited in
rational development and targeted application of
prosenescence cance r therapies.
INTRODUCTION
Different human cancers frequently arise due to genetic and
epigenetic alterations in the same relatively small number of
cancer pathways. Commonly mutated pathways include the
receptor tyrosine kinase (RTK)-RAS-BRAF growth factor
signaling pathway and the ARF-MDM2-p53 and p16-cyclin
D1-pRB tumor suppressor pathways (Yeang et al., 2008).
Although these same pathways are commonly deregulated in
different tumor types, the specific gene that is altered often
varies between tumors. For example, approximately 70% of
melanomas harbor mutations in BRAF, with most of the
remainder containing mutations in N-RAS (Brose et al., 2002;
Davies et al., 2002; Pollock and Meltzer, 2002). In most cases,
mutations in N-RAS and BRAF are mutually exclusive, presum-
ably because there is no selective advantage for a tumor cell
to alter both genes, since they act in the same linear signaling
pathway.
However, the genetics of human cancers is not always this
simple. An important effector of RAS is the lipid kinase, PIK3CA,
and its downstream effector, protein kinase AKT (hereafter
referred to as the PIK3CA/AKT signaling module) (Shaw and
Cantley, 2006). PIK3CA/AKT is also negatively regulated by the
lipid phosphatase PTEN, which is itself frequently mutated in
human cancers. Surprisingly, mutations in both RAS and the
PTEN/PIK3CA/AKT signaling axis can be found in the same
tumors. For example, Vogelstein and coworkers recently re-
ported that approximately 24% of human colon cancers harbor
mutations in both K-RAS and PIK3CA (Parsons et al., 2005).
Mutations in RAS genes and PIK3CA also co-occur in endome-
trial and thyroid cancer and acute lymphoblastic leukemia (ALL)
(Yeang et al., 2008). Some pancreatic cancers contain K-RAS
mutations and amplification of AKT2 (Tuveson and Hingorani,
2005). Since PIK3CA/AKT is an effector of RAS, the specific
selective advantage conferred by simultaneous mutation of
two genes in the same pathway is unclear. In this manuscript,
we set out to understand the molecular basis of the selective
advantage conferred by concurrent mutation of RAS and
PIK3CA/AKT in human tumors.
Oncogene-induced cellular senescence (OIS) is a permanent
cell growth arrest caused by an activated oncogene within
a primary untransformed cell (Adams, 2009). Although onco-
genes are best known for their ability to drive transformation,
a single oncogene in a primary cell often activates senescence
as a tumor suppression mechanism. Activation of senescence
depends on the pRB and p53 tumor suppressor pathways.
Many studies have demonstrated the role of OIS as an in vivo
tumor suppression mechanism. For example, many benign
36 Molecular Cell 42, 36–49, April 8, 2011 ª2011 Elsevier Inc.
Page 1
neoplasms harboring activated oncogenes contain senescent
cells (Braig et al., 2005; Chen et al., 2005; Collado et al., 2005;
Courtois-Cox et al., 2006; Michaloglou et al., 2005). In a number
of mouse models, inactivation of the senescence program allows
progression of such benign precursor lesions to full-blown malig-
nant cancers (Braig et al., 2005; Chen et al., 2005; Dankort et al.,
2007; Ha et al., 2007; Sarkisian et al., 2007; Sun et al., 2007).
Underscoring the ability of senescence to block tumor growth,
its reactivation in murine tumors is associated with tumor regres-
sion (Ventura et al., 2007; Xue et al., 2007).
In addition to proliferation arrest, cell senescence is associ-
ated with many other phenotypes and depends on activation
of various signaling and effector pathways. In the nucleus of
senescent cells, activated DNA damage signaling pathways,
reflected in a focal distribution of DNA damage sensing proteins,
gH2AX and 53BP1, are instrumental in driving senescence
(d’Adda di Fagagna, 2008). Also, formation of specialized
domains of facultative heterochromatin, called senescence-
associated heterochromatin foci (SAHF), is thought to silence
proliferation-promoting genes such as cyclin A2, thereby
contributing to a more permanent cell-cycle arrest (Narita
et al., 2003). Formation of SAHF depends on a complex of
histone chaperones, HIRA/UBN1/ASF1a (Banumathy et al.,
2009; Zhang et al., 2005). In turn, function of this chaperone
complex in senescent cells depends on phosphorylation of
HIRA by GSK3b and recruitment of HIRA to a subnuclear organ-
elle, the PML body (Ye et al., 2007). Notably, GSK3b has also
been shown to be an important inducer of senescence in other
contexts (Kortlever et al., 2006; Liu et al., 2008; Zmijewski and
Jope, 2004).
Senescent cells also upregulate autophagy (Gamerdinger
et al., 2009; Young et al., 2009), an organelle recycling process,
and this might contribute to remodeling of senescent cells and
provide the raw materials for altered biosynthetic processes.
Prominently, senescent cells show a marked change in their
secretory program (Coppe et al., 2008). Upregulated genes
whose products are secreted from senescent cells include cyto-
kines and chemokines, such as IL6 and IL8, as well as extracel-
lular proteases, such as matrix metalloproteinases (MMPs)
(Acosta et al., 2008; Kuilman et al., 2008; Xue et al., 2007). Secre-
tion of these extracellular signaling molecules, collectively
referred to as the senescence secretome, may facilitate clear-
ance of senescent cells by the immune system, and so limit
tumor growth.
Given the apparent potency of OIS in tumor suppression, it is
not surprising that many oncogenes have been reported to
induce OIS. However, previous studies do not present a clear
picture regarding the ability of activated PIK3CA/AKT to induce
senescence (see the Discussion). In this study, by profiling the
full spectrum of phenotypes that constitute the senescent state,
we show that activation of the PIK3CA/AKT pathway is a poor
inducer of senescence, compared to activated RAS. This mani-
fests as an inefficient proliferation arrest, a deficient senescence
secretome, weak DNA damage signaling and autophagy, and no
detectable SAHF. Remarkably, we find that, when both path-
ways are activated, the senescence-impaired PIK3CA/AKT
phenotype is in some respects dominant over RAS-induced
senescence. The dominance of PIK3CA/AKT depends on the
ability of this pathway to intersect and counteract downstream
effectors of RAS-induced senescence, such as GSK3b and likely
mTOR. The significance of GSK3b in human cancer is under-
scored by the demonstration that a high level of phosphorylated
GSK3b is a predictor of poor survival in human pancreatic
cancer. In a mouse model of pancreatic carcinogenesis, genetic
inactivation of PTEN, an inhibitor of PIK3CA/AKT, leads to
bypass of RAS-induced proliferation arrest (with features of
senescence) and accelerated formation of pancreatic ductal
adenocarcinoma (PDAC). Together, these results indicate that
activation of the PIK3CA/AKT pathway cooperates with activa-
tion of RAS in tumorigenesis through its ability to suppress
RAS-induced senescence.
RESULTS
Activation of PIK3CA/AKT Fails to Induce a Robust
Senescence Program
We set out to compare the spectrum of senescence phenotypes
induced by activated RAS and PIK3CA/AKT. Human BJ fibro-
blasts immortalized with hTERT (BJ-hTERT) were infected with
a control retrovirus or viruses encoding activated H-RAS
(RASG12V) or activated myristoylated AKT1 (mAKT1), or an
shRNA (shPTEN) to knock down the PIK3CA pathway inhibitor,
PTEN. As expected, cells infected with activated RAS assumed
a flattened vacuolated morphology, characteristic of senes-
cence induced by this oncogene (Figure 1A). Compared to
RASG12V-infected cells, mAKT1 and shPTEN-transduced fibro-
blasts were less vacuolated, but did become larger and flatter.
However,
activated AKT1 and shPTEN were both weaker
inducers of proliferation arrest (Figure 1B and see Figure S1A
available online). Consistent with this, cells expressing mAKT1
expressed reduced amounts of cyclin A and exhibited some
biochemical changes consistent with senescence, such as
dephosphorylation of pRB and upregulation of p53 and
p21CIP1 (Figure 1C and Figure S1B). But mAKT1 tended to be
less efficient in these respects than RASG12V (Figure 1C and
Figure S1B), and after passaging at least a proportion of
mAKT1-expressing cells did resume growth (data not shown).
Similarly, shPTEN failed to arrest colony outgrowth after
infection and drug selection (Figure S1C). In line with these
observations, only activated RAS upregulated expression of
p16INK4a, an activator of the p16-cyclin D1-pRB tumor
suppressor pathway and key effector of senescence-associated
proliferation arrest (Figure S1D). Our results suggest that
activation of PIK3CA/AKT signaling can induce some features
of senescence, but is markedly less potent in this regard than
is activated RAS.
In light of these provocative differences between activated
RAS and PIK3CA/AKT, we investigated the status of other
molecular markers of senescence in mAKT1 and RASG12V-
transduced cells. Induction of senescence by activated RAS
has been shown previously to depend on RAS-induced hyperre-
plication or unscheduled DNA synthesis, and subsequent DNA
damage (d’Adda di Fagagna, 2008). We monitored oncogene-
induced DNA damage in mAKT1 and RASG12V-transduced
cells by examining two commonly used markers of DNA
damage, gH2AX and 53BP1. Cells transduced with RASG12V,
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PIK3CA/AKT Suppresses RAS-Induced Senescence
Molecular Cell 42, 36–49, April 8, 2011 ª2011 Elsevier Inc. 37
Page 2
as expected, had an increase in DNA damage over control cells.
However, transduction of activated AKT1 did not lead to an
increase in DNA damage, as judged by either gH2AX or 53BP1
(Figures 1D and 1E and Figures S1E and S1F). When we
examined levels of gH2AX by western blotting, we observed
consistent results (Figure S1G). Thus, analysis of DNA damage
signals support the notion that activated AKT1, compared to
RASG12V, does not induce the full senescence program.
A
RASG12V Con mAKT1 shPTEN2
B
Passages
0
2
4
6
8
10
12
1 2 3 4
Control
RASG12V
mAKT1
shCon
shPTEN1
shPTEN2
Cumulative PD
actin
p21CIP1
p53
Cyclin A
pRBpS780
pRBpS807/811
pRB
C
mAKT1
Con
RAS
G12V
DAPI H2AX
D
0
10
20
Percent H2AX +
E
F
EBSS
Con
mAKT1 RASG12V
AKT1
RAS
Figure 1. Inactivation of PTEN and Activation of AKT1 Fail to Induce Robust Growth Arrest
(A) BJ-hTERT fibroblasts were transduced with either control, mAKT1, RasG12V retroviruses, or a lentivirus encoding a short hairpin to PTEN. Cells were drug
selected and bright field images taken 7 days later.
(B) Growth curves of cells from (A).
(C) IMR90 fibroblasts were transduced with control, mAKT1, or RASG12V. Cells were drug selected for 7 days and lysates prepared and western blotted.
(D) Cells from (C) were fixed and stained for gH2AX.
(E) Percent cells from (D) containing at least 20 foci of gH2AX. Mean of three experiments with standard deviation.
(F) IMR90 cells were cotransduced with EGFP-LC3 and control, RASG12V, or mAKT1. As a positive control for autophagosome formation, EGFP-LC3-expressing
cells were treated with Earle’s Balanced Salt Solution (EBSS) for 1 hr.
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In RASG12V-infected cells, induction of autophagy is also
important for onset of senescence (Gamerdinger et al., 2009;
Young et al., 2009). To compare autophagy in RASG12V and
mAKT1-infected cells, we introduced either oncogene together
with GFP-LC3, a fluorescent fusion protein that is incorporated
into autophagosomes (Klionsky et al., 2008). As shown previ-
ously, activated RAS induced formation of autophagosomes,
reflected in a punctate distribution of GFP-LC3 in the cytoplasm
(Figure 1F). However, by this measure, activated AKT1 failed to
induce autophagy. These results also support the notion that,
compared to activated RAS, activated AKT1 does not induce
a robust senescence program.
Next, we compared the ability of activated RAS, AKT1, and
shPTEN to induce senescence-associated chromatin changes,
manifest as SAHF and recruitment of the HIRA histone chap-
erone to PML bodies (Narita et al., 2003; Zhang et al., 2005).
SAHF can be visualized by conventional epifluorescence
microscopy as punctate domains of DAPI-stained chromatin
that stain with specific heterochromatin proteins, such as
histone variant macroH2A. We observed characteristic mac-
roH2A-containing SAHF in cells transduced with activated
RAS (and an activated mutant of one of its effectors, BRAF
[BRAFV600E]), but not in activated AKT1- or shPTEN-trans-
duced cells (Figures 2A and 2B and Figure S2 ). Consistent
with this, activated RAS and BRAF also triggered HIRA’s reloc-
alization to PML bodies, whereas activated AKT1 did not
(Figures 2A and 2C). Rather, activated AKT1-infected cells
were much like control, lacking both HIRA foci and SAHF.
Finally, we compared induction of the senescence secretome
by activated RAS and AKT1, by quantitative PCR. Activated
RAS robustly increased expression of IL6, IL8, MMP1, and
MMP8, as expected. However, activated AKT1 was unable to
achieve this (Figure 2D). To confirm and extend these findings,
we performed a gene expression microarray of cells infected
with activated RAS, activated AKT1, or control. Gene Ontology
(GO) classification of genes induced by RASG12V compared
to control showed that the top-ranked GO term was ‘inflamma-
tion.’ Specific genes in this group upregulated by RASG12V
included IL8, CXCL2, and IL1a. This GO group as a whole was
not significantly altered by mAKT1, and, typically, individual
genes in this group were not upregulated by this oncogene (Fig-
ure 2E). In sum, by several measures, namely proliferation arrest,
DNA damage signaling, autophagy, activation of HIRA, forma-
tion of SAHF, and upregulation of the secretome, activated
PIK3CA/AKT signaling fails to induce a senescence program
as robust as that induced by activated RAS.
Activated AKT1 Antagonizes RAS-Induced Senescence
Knowing that some human tumors contain mutations in both
RAS and the PTEN/PIK3CA/AKT axis (Parsons et al., 2005; Tuve-
son and Hingorani, 2005; Yeang et al., 2008), we wanted to know
whether the senescence program of cells containing activated
RAS and AKT1 was more or less robust than cells containing acti-
vated RAS alone. To do this, we transduced IMR90 fibroblasts
with each oncogene alone, or both activated AKT1 and RAS
together, and scored markers of senescence. First, we asked
whether activated AKT1 is able to suppress RASG12V-induced
upregulation of p16INK4a. As shown previously ( Figure S1D),
activated RAS caused upregulation of p16INK4a, whereas
activated mAKT1 did not. Coinfection of RASG12V and mAKT1
showed that activated AKT1 suppressed RASG12V-induced
upregulation of p16INK4a (Figure S3A). Next, we looked at
recruitment of HIRA to PML bodies and formation of SAHF.
Compared to RASG12V alone, coexpression of activated AKT1
and RAS decreased both SAHF formation and HIRA foci (Figures
3A–3D). Activated RAS and AKT1 were both efficiently
expressed in all infections (Figure 3B). Significantly, we also
observed that activated BRAF is a more potent inducer of
SAHF than is activated RAS (Figure 3E). This is consistent with
the ability of RAS, but not BRAF, to activate AKT1 (Figure S3B)
(Shaw and Cantley, 2006), which in turn is able to antagonize
SAHF formation. Finally, we examined indicators of autophagy
in single or double oncogene-infected cells. Consistent with acti-
vated RAS-induced upregulation of autophagy described previ-
ously and demonstrated in Figure 1F, activated RAS caused
accumulation of LC3-II, the lipidated form of the protein that is
incorporated into autophagosomes and which characteristically
migrates faster in SDS-PAGE (Klionsky et al., 2008)(Figure 3F). In
contrast, cells transduced with both RASG12V and
mAKT1
showed decreased LC3-II and an increased level of
p62, a protein whose accumulation is indicative of decreased
autophagy (Klionsky et al., 2008). These experiments indicate
that the combination of activated AKT1 and RAS in cells results
in a less complete senescence program than does activated
RAS alone.
Mechanism of Antagonism of Senescence
by Activated AKT1
We next wanted to know the mechanism by which activated
AKT1 antagonizes aspects of RASG12V-induced senescence.
Since AKT1 activates mTOR and mTOR is a potent inhibitor
of autophagy (He and Klionsky, 2009), we hypothesized that
activated AKT1 suppresses RASG12V-induced autophagy by
activation of mTOR. Consistent with this idea, in the presence
of activated RAS, activated AKT1 activated mTOR, as judged
by phosphorylation of mTOR substrates, 4EBP1 and p70S6K
(Figure 4A). With respect to SAHF, we previously showed that
activated RAS induces HIRA localization to PML bodies and
formation of SAHF through its ability to activate GSK3b (Ye
et al., 2007). In contrast, AKT is known to directly inhibit
GSK3b through inhibitory phosphorylation on serine 9 (Cross
et al., 1995). Therefore, we hypothesized that mAKT1’s ability
to block RASG12V-induced SAHF formation might depend on
its ability to phosphorylate and inhibit GSK3b. Consistent with
this idea, in cells coexpressing activated RAS and AKT1, GSK3b
was heavily phosphorylated on serine 9 (GSK3bpS9) (Figure 4B).
This indicates that RASG12V-induced activation of GSK3b is
overridden by mAKT1-induced inhibition of GSK3b. To test our
hypothesis further, we expressed activated AKT1 with or without
a nonphosphorylatable mutant of GSK3b (GSK3bS9A), and
found that, even in the presence of activated AKT1, GSK3bS9A
was able to induce both localization of HIRA to PML bodies and
SAHF formation ( Figures 4C–4E). We verified appropriate
expression of GSK3bS9A and activated AKT1 by western blotting
(Figure 4D). These results are consistent with the notion that
activated AKT1 suppresses HIRA activation and formation of
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PIK3CA/AKT Suppresses RAS-Induced Senescence
Molecular Cell 42, 36–49, April 8, 2011 ª2011 Elsevier Inc. 39
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SAHF, at least in part, through phosphorylation and inhibition of
GSK3b. Underscoring the importance of AKT1-mediated GSK3b
phosphorylation in human cancer, we found that in a pancreatic
cancer tissue microarray (TMA) the level of GSK3bpS9 corre-
lated with poor patient survival, independent of tumor size, tumor
grade, perineural invasion, resection margin involvement, and
lymph node status (Figures 5A and 5B). Phosphorylation
and activation of AKT1 and its downstream effector, mTOR,
and combined phosphorylation and activation of AKT1 and
mTOR similarly correlated with poor disease outcome (Figures
5C and 5D, Figure S4, and Tables S1–S5), also emphasizing
the significance of activated AKT1 in this disease.
0
2
4
6
8
Con
RASG12V
mAKT1
IL6
IL8 MMP1 MMP3
D
DAPI PML HIRA
Con
BRAF
V600E
RAS
G12V
mAKT1 shPTEN2
A
Fold increase in expression
0
20
40
60
Percent cells with HIRA foci Percent cells with SAHF
0
10
20
30
40
B
C
E
Figure 2. AKT Activation Fails to Induce SAHF or the Senescence Secretome
(A) IMR90 fibroblasts were transduced with BRAFV600E, RASG12V, mAKT1, or a short hairpin that targets PTEN. Cells were drug selected, fixed, and stained for
SAHF, PML, or HIRA foci.
(B and C) One hundred cells from (A) were scored for HIRA foci or SAHF. Mean of three experiments with standard deviation.
(D) RNA was harvested from mAKT1, RASG12V, or control cells and assayed for expression of IL-6, IL-8, MMP-1, and MMP-3 by quantitative RT-PCR.
(E) Expression profiling was performed on control, RASG12V, or mAKT1-transduced IMR90. Heat map of significantly upregulated (green) and downregulated
(red) genes with GO classification ‘inflammation’ is shown.
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AKT
RAS
-actin
p62
LC3
DAPI
HIRA
0
10
20
30
40
50
60
Percent cells with SAHF
AB
CD
EF
RAS
AKT1
-actin
0
10
20
30
40
Percent cells with HIRA foci
Percent cells with SAHF
0
10
20
30
Figure 3. Activation of AKT Antagonizes RASG12V-Induced SAHF Formation and Autophagy
(A) IMR90 fibroblasts were transduced with either control, mAKT1, RASG12V, or both mAKT1 and RASG12V and double drug selected for 7 days. Cells were then
fixed and stained for HIRA foci and SAHF.
(B) Expression of transduced proteins and phosphorylation of AKT (AKTpS473) was assayed by western blotting.
(C and D) One hundred cells from (A) were scored for HIRA foci or SAHF. Mean of three experiments with standard deviation.
(E) IMR90 cells were transduced with RASG12V or BRAFV600E and scored for SAHF formation at 5 days post drug selection. Mean of three experiments with
standard deviation.
(F) Western blotting of cell lysates from (A) with indicated antibodies. The arrow marks the cleaved lipidated form of LC3, LC3-II.
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AKT Pathway Activation Antagonizes RAS-Induced
Proliferation Arrest, with Features of Senescence,
to Drive Tumorigenesis in the Mouse Pancreas
We next wanted to test whether activation of PIK3CA/AKT
signaling is able to suppress activated RAS-induced senescence
and accelerate tumor formation in vivo. To do this, we utilized
a mouse model in which expression of activated RAS is restricted
to the cells of the pancreas, by virtue of a conditional RAS allele
(K-RASG12D) at its normal genomic locus that can be acti-
vated by Cre-mediated recombination, and pancreas-specific
C
DAPI PML HIRA
Con mAKT1
GSK3
S9A
mAKT1/
GSK3
S9A
B
GSK3 pS9
RAS
AKT1
actin
GSK3
AKT1
-actin
DE
Combined % HIRA/SAHF foci
0
20
40
60
x
x
p<0.05
actin
4EBP1
p70S6K
phosphop70S6K
AKT1
RAS
0 6 12
Hrs + rapamycin
A
4EBP1
phospho4EBP1
Figure 4. mAKT1 Counters Effects of RASG12V on mTOR and GSK3b
(A) IMR90 cells were transduced with control, RASG12V, mAKT1, or both RASG12V and mAKT1. Cells were double drug selected and lysates prepared and
western blotted. Uninfected cells were treated with 1 nM rapamycin to define unphospho and phospho-4EBP1.
(B) Western blotting of cells from (A).
(C) IMR90 cells were transduced with control, GSK3bS9A, mAKT1, or both GSK3bS9A and mAKT. Cells were fixed and stained for PML, HIRA foci, or SAHF.
(D) Expression of proteins in (C) was verified by western blotting.
(E) One hundred cells from (C) were scored for both HIRA foci and SAHF and a combined score plotted. The horizontal line inside the box is the median (50th
percentile); the box itself encompasses the 25th and 75th percentiles (Inter Quartile Range [IQR]); the whiskers are the most extreme data points within 1.5 3 IQR;
crosses outside the whiskers are outliers.
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expression of Cre recombinase under control of a PDX1
promoter (Hingorani et al., 2003). These PDX1-Cre/RASG12D
animals develop normally but develop benign precursor lesions
termed pancreatic intraepithelial neoplasms (PanINs) that can,
with long latency, progress to form PDAC. As shown previously
(Morton et al., 2010b), these neoplastic lesions stain positively
for markers of senescence, including SA b-gal and expression
of p53 and p21CIP1 (Figures 6A and 6B). Conversely, they
largely lack markers of proliferation, namely Ki67, MCM2 expres-
sion, and incorporation of BrdU (Figures 6A and 6B). To test the
A
Low GSK3 pS9 High GSK3 pS9
0 25 50
75
100
125
Survival (months)
0
0.2
0.4
0.6
0.8
1.0
Cumulative proportion surviving
B
High GSK3 pS9
Low GSK3
pS9
D
C
0 25 50
75
100
125
Survival (months)
0
0.2
0.4
0.6
0.8
1.0
Cumulative proportion surviving
High AKTpS473/high mTORpS2448
Low AKTpS473/low mTORpS2448
0
5
10
15
20
25
30
35
40
45
Figure 5. GSK3bps9, AKTpS47 3, and mTORpS2448 Phosphorylation Correlates with Poor Overall Survival in Human Pancreatic Cancer
(A) Representative ‘high’ (top panel) and ‘low’ (bottom panel) scoring images of GSK3bps9 immunohistochemical staining from a human pancreatic adeno-
carcinoma TMA.
(B) Kaplan-Meier curve representing patient survival per unit time. Survival is shown for patients with tumors with GSK3bps9 > 100 (high) and < 100 (low).
(C) Kaplan-Meier curve for patients with low AKT1pS473/low mTORpS2448 and high AKT1pS473/high mTORpS2448.
(D) Mean survival of indicated groups of patients. For each phospho-epitope in (B)–(D), the high staining group comprised 24 patients, and the low staining group
comprised 18 patients.
Molecular Cell
PIK3CA/AKT Suppresses RAS-Induced Senescence
Molecular Cell 42, 36–49, April 8, 2011 ª2011 Elsevier Inc. 43
Page 8
impact of PIK3CA/AKT pathway activation on this activated
RAS-induced in vivo senescence-like state, the PDX1-Cre/
RASG12D animals were crossed to animals that have one or
both PTEN alleles flanked by Cre recombination sites (Suzuki
et al., 2001), to drive simultaneous activation of RAS and partial
or biallelic inactivation of PTEN in the pancreas (PDX1-CRE/
RASG12D/PTEN). Significantly, complete inactivation of PTEN
in the mouse pancreas does not induce senescence (Stanger
et al., 2005)(Figure S5A). Comparing PanINs in the pancreata
of 6-week-old PDX1-Cre/RASG12D and PDX1-Cre/RASG12D/
PTEN animals, we found that inactivation of PTEN largely abol-
ished expression of senescence markers, p53, p21, and SA
b-gal (Figures 6A and 6B). Consistent with the idea that inactiva-
tion of PTEN facilitates a complete bypass of the senescence-
like state, we found the PanINs of the PDX1-Cre/RASG12D/
PTEN animals to be highly proliferative, as measured by an
increase in immunohistochemical staining of Ki67, MCM2, and
incoporation of BrdU (Figures 6A and 6B). Senescence bypass
was associated with phosphorylation of GSK3 on serine 9,
similar to the in vitro model (Figures 4B and 6A). In line with
this senescence-like state being a potent tumor suppression
mechanism in this in vivo model, expression of activated RAS
and concurrent inactivation of PTEN resulted in rapid progres-
sion of PanINs into PDAC (Figure 6C and Figure S5B), as
reported recently (Hill et al., 2010). Previously, we have reported
that inactivation of p21CIP1 accelerates tumorigenesis in this
model, likely through inactivation of senescence (Morton et al.,
2010a). Significantly, deficiency of p21CIP1 did not further
accelerate tumorigenesis in PDX1-Cre/RASG12D/PTEN
fl/+
animals (Figure 6D), indicating that loss of p21CIP1 and PTEN
accelerate PDAC via the same pathway, further implicating
loss of PTEN in abrogation of senescence in this model.
IHC analysis of PTEN indicated that tumors arising from PDX1-
Cre/RASG12D/PTEN
fl/+
mice had lost the second allele of PTEN
(Figure S5C). Also, the effects of PTEN disruption were more
marked when both, rather than one, alleles of PTEN were engi-
neered for inactivation in the pancreas ( Figure 6C and Fig-
ure S5B). Loss of two alleles of PTEN led to an incredibly lethal
acceleration of tumorigenesis, leading invariably to rapid death
and a mean survival of 15 days (Figure 6C and Figure S5B). In
these mice, almost the entire pancreas was replaced by
neoplastic tissue, with very little normal tissue remaining.
Neoplastic tissue contained widespread mitoses, including
some aberrant figures (Figure S5D). In areas, there was loss of
the normal pancreatic architecture with angulated glands, indi-
cating invasive carcinoma (Figure S5D). Tumors in these mice
were large and exhibited a high proliferative index, as judged
by Ki67 and BrdU incorporation (Figures S5E and S5F). These
observations suggest that the tumor suppressor function of
PTEN in this model conforms to the Knudson ‘two-hit’ paradigm
for tumor suppressors.
As expected, tumors that resulted from inactivation of
PTEN exhibited a strongly activated AKT signaling pathway, as
shown by immunohistochemical staining for activated phospho-
serine 473 AKT (Figure 7A and data shown). Consistent with
inactivation of PTEN and activation of AKT driving tumorigenesis
through inactivation of GSK3b and activation of mTOR, tumors
from PDX1-Cre/RASG12D/PTEN mice stained strongly for
phosphoserine 9 GSK3b and phospho-mTOR (Figure 7A).
Moreover, treatment of PDX1-Cre/RASG12D/PTEN
fl/+
mice
with rapamycin, a potent inhibitor of mTOR, restored cell senes-
cence, as measured by proliferation arrest (BrdU) and p53 and
p21 expression (Figure 7B and Figure S6). Taken together, these
in vivo data support our hypothesis that inactivation of PTEN and
activation of AKT and its downstream effector, mTOR, are
capable of antagonizing activated RAS-induced proliferation
arrest (with features of senescence) leading to rapid acceleration
of tumorigenesis.
DISCUSSION
Previous studies do not present a clear picture regarding the
ability of activated PIK3CA/AKT to induce senescence. Some
reports have indicated that activation of the PIK3CA/AKT
pathway does induce senescence (Chen et al., 2005; Majumder
et al., 2008; Miyauchi et al., 2004; Nogueira et al., 2008; Oyama
et al., 2007). Other reports have concluded that PIK3CA/AKT
activity is a weak inducer of senescence (Lin et al., 1998), is
downregulated in senescence (Courtois-Cox et al., 2006; Young
et al., 2009), and can antagonize senescence (Courtois-Cox
et al., 2006; Kortlever et al., 2006; Tresini et al., 1998). A recent
report on PTEN loss-induced senescence (PICS) supports our
finding that senescence induced by PIK3CA/AKT activation is
not associated with activation of DNA damage signaling, but
did not examine chromatin changes, autophagy, and the senes-
cence secretome (Alimonti et al., 2010). In this study, by directly
comparing activated RAS and PIK3CA/AKT, we find that the
latter is not an efficient inducer of senescence. Specifically, we
show that inactivation of PTEN and activation of AKT1 is impaired
in its ability to induce senescence, as recorded by multiple effec-
tors of senescence, including upregulation of p16, induction of
DNA damage, recruitment of HIRA to PML bodies, formation of
SAHF, and upregulation of autophagy. Importantly, we also
show that activation of PIK3CA/AKT is deficient in its ability to
drive two functional outputs of the senescence program that
are central to senescence-mediated tumor suppression, namely
upregulation of the senescence secretome and efficient prolifer-
ation arrest. Most importantly, concurrent activation of both RAS
and PIK3CA/AKT impairs RAS-induced senescence, both
in vitro and in vivo.
Activated PIK3CA/AKT suppresses senescence induced by
activated RAS through multiple routes. First, activated AKT1
reversed the upregulation of p16INK4a induced by activated
RAS. Second, GSK3b kinase is another key nodal point at which
signals from activated RAS and PIK3CA/AKT interact. We and
others have previously shown that activation of GSK3b kinase
contributes to onset of senescence ( Kortlever et al., 2006; Liu
et al., 2008; Ye et al., 2007; Zmijewski and Jope, 2004). Specif-
ically, we showed that activation of GSK3b phosphorylates the
HIRA histone chaperone, thereby localizing this protein to PML
bodies and instigating the formation of SAHF (Ye et al., 2007).
Here we present evidence that activated PIK3CA/AKT
suppresses RASG12V-induced HIRA relocalization and forma-
tion of SAHF through its ability to phosphorylate and inhibit
GS3Kb. The significance of the PIK3CA/AKT-GSK3b signaling
axis in human cancer is underscored by our finding that a high
Molecular Cell
PIK3CA/AKT Suppresses RAS-Induced Senescence
44 Molecular Cell 42, 36–49, April 8, 2011 ª2011 Elsevier Inc.
Page 9
A
RASG12D
NInaP
RASG12D/Pten
fl/+
NInaP
p21 GSK3 pS9 Ki67 MCM2 SA -gal
p53 p21
BrdU
/sllec evitisop tnecreP
lesion
B
Survival (%)
0
20
40
60
80
100
0 50 100 150 200
Survival (%)
0
20
40
60
80
100
0 50 100 150 200
C
D
RASG12D
PTENfl/fl
RASG12D/+ PTENfl/fl
RASG12D/+ PTENfl/+
RASG12D/+ PTENfl/+
RASG12D/+ PTENfl/+ p21+/-
Figure 6. Inactivation of PTEN Abrogates a Senescence-like State in a Mouse Model of Pancreatic Cancer
(A) Immunohistochemical staining of PanIN from pancreata of mice of indicated genotype. Markers of senescence include SA b-gal and p21; markers of
proliferation include Ki67 and MCM2.
(B) Quantitation of p53, p21, and BrdU from (A). Box plots are as in Figure 4E.
(C and D) Kaplan-Meier curve showing percentage of animals of indicated genotype surviving per unit time.
Molecular Cell
PIK3CA/AKT Suppresses RAS-Induced Senescence
Molecular Cell 42, 36–49, April 8, 2011 ª2011 Elsevier Inc. 45
Page 10
RASG12DPten
lf/lf
CADP
H & E GSK3 ps9 AKT1ps473 Phos-mTOR
A
D21GSAR
CADP
B
vehicle rapamycin
p21
p53
BrdU
vehicle rapamycin
vehicle rapamycin
vehicle rapamycin
Figure 7. Rapamycin Reactivates Senescence in PDAC Harboring Activated PIK3CA/AKT
(A) Immunohistochemical staining of AKT pathway activation in pancreata of RASG12D/PTEN
fl/fl
mice or RASG12D mice.
(B) RASG12D/PTENfl/+ mice were treated with rapamycin for 7 days and then pancreata harvested and stained for p53, p21, and BrdU. Box plots are as in
Figure 4E.
Molecular Cell
PIK3CA/AKT Suppresses RAS-Induced Senescence
46 Molecular Cell 42, 36–49, April 8, 2011 ª2011 Elsevier Inc.
Page 11
level of AKTpS473 (with or without high mTORpS2448) or
GSK3bpS9 is a predictor of poor survival in human pancreatic
cancer, independent of other common prognostic indicators.
Third, activated PIK3CA/AKT and activated RAS antagonize
each other through mTOR signaling. mTOR is well-documented
to be a potent repressor of autophagy (He and Klionsky, 2009).
While activated RAS inhibits mTOR activity to upregulate au-
tophagy and promote senescence (Young et al., 2009) (Figures
1F and 4A), activated AKT1 was able to activate mTOR even in
the presence of activated RAS, likely explaining the ability of
mAKT1 to inhibit RASG12V-induced autophagy. To affirm this
in vivo, in mice haboring activated RAS and activated PIK3CA/
AKT signaling, the potent mTOR inhibitor, rapamycin, reacti-
vated RAS-senescence. We conclude that activated PIK3CA/
AKT suppresses RAS-induced senescence through its ability
to intersect with and antagonize several outputs of chronic acti-
vated RAS, including upregulation of p16INK4a, activation of
GSK3b, and repression of mTOR. While activated PIK3CA/AKT
signaling is known to have many targets in the cell, TMA analysis
of human pancreatic cancer underscored GSK3b and mTOR as
important targets in this disease. Phosphorylation of all three
proteins was significantly directly correlated (Supplemental
Information), and high phosphorylation of each protein is a
predictor of poor patient survival. Thus, the PIK3CA/AKT/
GSK3b/mTOR axis is an important driver of disease outcome
in human pancreatic cancer.
Although activation of AKT1 impaired RASG12V-induced
senescence in vitro by at least three criteria (suppression of
p16INK4a, SAHF, and autophagy ), it did not completely abolish
activated RAS-induced senescence, as measured by prolifera-
tion arrest (Figure S3C). On the other hand, inactivation of
PTEN did bypass activated RAS-induced senescence-like arrest
in vivo (as measured by proliferation markers) and caused
a dramatic acceleration of tumorigenesis. There are several
possible explanations of this difference between the in vitro
and in vivo models, including differences between cell types,
use of RASG12V in vitro and RASG12D in vivo, and influence
of cellular microenvironment in vivo. It is also important to note
that in the mouse model, we cannot conclude that inactivation
of PTEN is sufficient to abrogate senescence in all of the
RASG12D-expressing cells. Rather, inactivation of PTEN might
weaken the senescence program enough to facilitate complete
escape from senescence, but only in cooperation with additional
acquired and selected mutations. Regardless of the correct
explanation, the in vitro and in vivo results are consistent in
showing that inactivated PTEN/activated AKT can antagonize
activated RAS-induced senescence and that in vivo this facili-
tates tumorigenesis.
Our results show that all oncogenes are not equal in their
abilities to induce senescence, and, surprisingly, a weak
inducer of senescence can be dominant over a strong. This
idea has important implications for understanding mechanisms
of oncogene cooperation. Concurrent mutations of RAS and
the PTEN/PIK3CA/AKT pathway have been described in
a number of human tumor types, including colon, endometrium,
and ALL (Parsons et al., 2005; Yeang et al., 2008). Concurrent
mutations are also probable in pancreatic cancer, as RAS
mutations are thought to occur in >90% of cases (Tuveson
and Hingorani, 2005) and functional inactivation of PTEN by
promoter methylation (Asano et al., 2004), decreased mRNA
levels (Ebert et al., 2002), loss of protein expression (Altomare
et al., 2002; Asano et al., 2004), or loss of heterozygosity
(Okami et al., 1998) has also been reported. Furthermore,
amplification or activation of AKT2 kinase, related to AKT1,
occurs in up to 60% of pancreatic cancers (Altomare et al.,
2002; Ruggeri et al., 1998; Schlieman et al., 2003), and AKT
is activated in pancreatic cancer based on IHC staining (Semba
et al., 2003). Most strikingly, approximately 75% of human
colon cancers that contain PIK3CA mutations also harbor
mutations in K-RAS (Parsons et al., 2005). In addition, activating
mutations of RAS and in the PTEN/PIK3CA/AKT pathway have
been shown to cooperatively drive tumorigenesis in mouse
models of glioblastoma, endometrium, thyroid, and pancreas
(Hill et al., 2010; Holland et al., 2000; Kim et al., 2010; Miller
et al., 2009) (this study). To date, the molecular basis of coop-
eration between these mutations in human tumors and mouse
models has been poorly understood. Here, we present
evidence from both in vitro and in vivo studies to indicate that
these mutations cooperate, at least in part, through the ability
of PTEN/PIK3CA/AKT mutations to suppress RAS-induced
senescence, thereby allowing for these oncogenic pathways
to cooperate in tumorigenesis. Importantly, this new mecha-
nistic understanding might be exploited as a prosenescence
cancer therapy. Rapamycin is a potent and specific inhibitor
of mTOR, is a key effector of activated PIK3CA/AKT signaling,
and is already used in the clinic. We found that rapamycin can
reactivate
senescence in mouse tumors harboring mutations in
both RAS and PTEN, pointing to possible therapeutic activity
(of rapamycin or similar drugs) against human tumors of this,
or equivalent, genotype.
EXPERIMENTAL PROCEDURES
Cell Culture
IMR90 and BJ (ATCC) cell lines were cultured according to ATCC guidelines
in low oxygen (2%) unless otherwise indicated. Fibroblasts were cultured in
Dulbecco’s modified Eagle’s medium supplemented with 20% fetal bovine
serum.
Immunofluorescence, SAHF, and SA b-Gal Staining
Two-color indirect immunofluorescence and SAHF assays were performed
as described previously (Ye et al., 2007; Zhang et al., 2005). SA b-gal
staining was performed as described previously (for in vitro studies) (Dimri
et al., 1995 ).
Genetically Modifi ed Mice
The Pdx1-Cre, LSL-K-RASG12D, and PTEN
flox
mice have been described
previously (Hingorani et al., 2003; Suzuki et al., 2001). The p21CIP1
/
mice
have been previously described (Deng et al., 1995). Conditional LSL-K-
RAS
G12D/+
mice were from Tyler Jacks via MMHCC.
For additional methods, see the Supplemental Information.
SUPPLEMENTAL INFORMATION
Supplemental Information includes six figures, five tables, and Supplemental
Experimental Procedures and can be found with this article at doi:10.1016/j.
molcel.2011.02.020.
Molecular Cell
PIK3CA/AKT Suppresses RAS-Induced Senescence
Molecular Cell 42, 36–49, April 8, 2011 ª2011 Elsevier Inc. 47
Page 12
ACKNOWLEDGMENTS
We are indebted to Daniel Peeper, Masashi Narita, and Chris Sell for commu-
nication of results prior to publication; to Michael Bouchard, Jane Clifford,
Maureen Murphy, and Matt Kennedy for helpful discussions and advice; to
Stuart Pepper and the CR-UK Microarray Facility, Paterson Institute, for micro-
array analyses; to Colin Nixon for immunohistochemistry; and to Jane Hair for
curating the NHSGGC biorepository. The lab of P.D.A. is funded by CR-UK
(C10652/A10250). Work in the lab of G.H.E. was funded by NIH
R01GM062281. The lab of O.J.S. is funded by CR-UK. Thanks to the Scottish
Executive Chief Scientist Office (CSO) for funding for N.B.J., including the
production of tissue microarrays (CSO refence number 41175). Additional
support was from Think Pink Scotland.
Received: May 5, 2010
Revised: September 24, 2010
Accepted: January 27, 2011
Published: April 7, 2011
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Molecular Cell
PIK3CA/AKT Suppresses RAS-Induced Senescence
Molecular Cell 42, 36–49, April 8, 2011 ª2011 Elsevier Inc. 49
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    • "Activated RAS induces the formation of senescence-associated heterochromatin foci by activating GSK3β which phosphorylates the histone chaperone HIRA facilitating its localization to PML nuclear bodies [25]. AKT activation inhibits RAS-mediated oncogene-induced senescence in part through the inhibitory phosphorylation of GSK3β at serine 9 [26]. Thus, a molecular exclusion for this arm is concomitant abnormalities which result in AKT activation, such as PIK3CA mutation, PTEN loss or AKT mutation. "
    [Show abstract] [Hide abstract] ABSTRACT: Background The management of NSCLC has been transformed by stratified medicine. The National Lung Matrix Trial (NLMT) is a UK-wide study exploring the activity of rationally selected biomarker/targeted therapy combinations. Patients and methods The Cancer Research UK (CRUK) Stratified Medicine Programme 2 is undertaking the large volume national molecular pre-screening which integrates with the NLMT. At study initiation, there are eight drugs being used to target 18 molecular cohorts. The aim is to determine whether there is sufficient signal of activity in any drug–biomarker combination to warrant further investigation. A Bayesian adaptive design that gives a more realistic approach to decision making and flexibility to make conclusions without fixing the sample size was chosen. The screening platform is an adaptable 28-gene Nextera next-generation sequencing platform designed by Illumina, covering the range of molecular abnormalities being targeted. The adaptive design allows new biomarker–drug combination cohorts to be incorporated by substantial amendment. The pre-clinical justification for each biomarker–drug combination has been rigorously assessed creating molecular exclusion rules and a trumping strategy in patients harbouring concomitant actionable genetic abnormalities. Discrete routes of pathway activation or inactivation determined by cancer genome aberrations are treated as separate cohorts. Key translational analyses include the deep genomic analysis of pre- and post-treatment biopsies, the establishment of patient-derived xenograft models and longitudinal ctDNA collection, in order to define predictive biomarkers, mechanisms of resistance and early markers of response and relapse. Conclusion The SMP2 platform will provide large scale genetic screening to inform entry into the NLMT, a trial explicitly aimed at discovering novel actionable cohorts in NSCLC. Clinical Trial ISRCTN 38344105.
    Full-text · Article · Sep 2015 · Annals of Oncology
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    • "However, activated forms of Ras also promote oncogene induced senescence (Serrano et al., 1997; Cox and Der, 2003; Lowe et al., 2004 ). The detection of Rasinduced senescence in multiple cell culture systems (Serrano et al., 1997; Ferbeyre et al., 2002), in vivo mouse models (Collado et al., 2005; Morton et al., 2010; Kennedy et al., 2011), human rasopathies (Courtois-Cox et al., 2006 ), and premalignant activated Ras-containing human pancreatic tumors (Caldwell et al., 2012) confirms that the process is physiological (Dimauro and David, 2010; Kuilman et al., 2010). However, the mechanisms by which Ras drives senescence and how these mechanisms are subverted during the development of malignancy (Chen et al., 2005; Collado et al., 2005; Kuilman et al., 2010) remains poorly understood (Smiraglia and Nikiforov, 2012 ). "
    [Show abstract] [Hide abstract] ABSTRACT: The Ras oncoprotein is a key driver of cancer. However, Ras also provokes senescence, which serves as a major barrier to Ras-driven transformation. Ras senescence pathways remain poorly characterized. NORE1A is a novel Ras effector that serves as a tumor suppressor. It is frequently inactivated in tumors. We show that NORE1A is a powerful Ras senescence effector and that down-regulation of NORE1A suppresses senescence induction by Ras and enhances Ras transformation. We show that Ras induces the formation of a complex between NORE1A and the kinase HIPK2, enhancing HIPK2 association with p53. HIPK2 is a tumor suppressor that can induce either proapoptotic or prosenescent posttranslational modifications of p53. NORE1A acts to suppress its proapoptotic phosphorylation of p53 but enhance its prosenescent acetylation of p53. Thus, we identify a major new Ras signaling pathway that links Ras to the control of specific protein acetylation and show how NORE1A allows Ras to qualitatively modify p53 function to promote senescence. © 2015 Donninger et al.
    Full-text · Article · Mar 2015 · The Journal of Cell Biology
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    • "These experiments will be replicated in Protocol 2. To date, conflicting reports of Ras-associated senescence have been reported. Using a mouse model with conditional pancreatic expression of Kras, Kennedy et al. (2011) demonstrated that activated KrasG12D-induced senescence in pancreatic cells. However, using an intrahepatic injection system similar to Kang et al., Ho et al. (2012) did not detect positive markers for senescence in the livers of Nrasinjected wild-type mice as compared to controls. "
    [Show abstract] [Hide abstract] ABSTRACT: The Kang et al. (2011), published in Nature in 2011. The experiments that will be replicated are those reported in Figures 3B, 3C, 3E, and 4A. In these experiments, Kang et al. (2011) demonstrate the phenomenon of oncogene-induced cellular senescence and immune-mediated clearance of senescent cells after intrahepatic injection of NRAS (Figures 2I, 3B, 3C, and 3E). Additionally, Kang et al. (2011) show the specific necessity of CD4+ T cells for immunoclearance of senescent cells (Figure 4A). The Reproducibility Project: Cancer Biology is a collaboration between the eLife.
    Full-text · Article · Jan 2015 · eLife Sciences
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