Identification of PHLPP1 as a Tumor Suppressor
Reveals the Role of Feedback Activation
in PTEN-Mutant Prostate Cancer Progression
Muhan Chen,1Christopher P. Pratt,1,8Martha E. Zeeman,1,8Nikolaus Schultz,2Barry S. Taylor,2Audrey O’Neill,3
Mireia Castillo-Martin,4Dawid G. Nowak,1Adam Naguib,1Danielle M. Grace,1Jernej Murn,1Nick Navin,5
Gurinder S. Atwal,1Chris Sander,2William L. Gerald,6,9Carlos Cordon-Cardo,4Alexandra C. Newton,3Brett S. Carver,7
and Lloyd C. Trotman1,*
1Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA
2Computational Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA
3Department of Pharmacology, University of California San Diego, La Jolla, CA 92093, USA
4Department of Pathology, Columbia University, New York, NY 10032, USA
5Department of Genetics, Department of Bioinformatics and Computational Biology, MD Anderson Cancer Center, Houston, TX 77030, USA
6Department of Pathology, Human Oncology and Pathogenesis Program
7Department of Surgery, Division of Urology, Human Oncology and Pathogenesis Program
Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA
8These authors contributed equally to this work
Hyperactivation of the PI 3-kinase/AKT pathway is a driving force of many cancers. Here we identify the
AKT-inactivating phosphatase PHLPP1 as a prostate tumor suppressor. We show that Phlpp1-loss causes
neoplasia and, on partial Pten-loss, carcinoma in mouse prostate. This genetic setting initially triggers a
growth suppressive response via p53 and the Phlpp2 ortholog, and reveals spontaneous Trp53 inactivation
as a condition for full-blown disease. Surprisingly, the codeletion of PTEN and PHLPP1 in patient samples
is highly restricted to metastatic disease and tightly correlated to deletion of TP53 and PHLPP2. These data
With an annual average of 200,000 cases in the United States
(US) alone, cancer of the prostate (CaP) is the most commonly
diagnosed malignancy in western men and the second most
common cause of US male cancer deaths (American Cancer
Society, 2009). Due to effective CaP screening programs, an
increasing number of men are diagnosed and treated for clini-
cally localized CaP. However, the majority of these will not
develop life-threatening disease. Therefore, identification of
men who will suffer disease recurrence constitutes the great
challenge for CaP therapy (Shariat et al., 2008).
Recent advances in whole genome analysis are affording us
with a look at the bulk of alterations that occur in cancer tissue
and in commonly used cancer cell lines. In prostate, several
such whole genome efforts have identified and validated
commonly observed events, such as PTEN deletions, ERG
fusion genes and chromosome 8 aberrations (Lapointe et al.,
2007; Saramaki and Visakorpi, 2007). Furthermore, comparative
copy number analysis of metastatic CaP has demonstrated how
this technology can be used to trace the process of disease
dissemination (Liu et al., 2009). Most recently, the integration
of gene copy number, gene mutation, and transcriptome anal-
ysis has provided a comprehensive look at the nature of the
Excessive AKT activity triggers p53-dependent growth arrest in mouse prostate. However, it has remained ill-defined if and
barrier against prostate cancer progression. It antagonizes the codeletion of the AKT suppressors PTEN and PHLPP1,
whose deletions are mutually exclusive in primary cancers. Because rapamycin strongly inhibits the feedback activation
of p53 and Phlpp2, our data call for checking the status of this fail-safe response before patients receive mTorc1-targeting
therapy. Collectively, our findings identify the PHLPP proteins as key players in prostate cancer and reveal the tightly
orchestrated nature of tumor suppressor activity in this disease.
Cancer Cell 20, 173–186, August 16, 2011 ª2011 Elsevier Inc. 173
changes that separate indolent from aggressive disease,
because the extent of copy number alteration could be linked
to a patient’s risk of disease recurrence after prostatectomy
(Taylor et al., 2010). Collectively, these studies demonstrate
that advances in comprehensive cancer analysis could soon
afford us with the catalog of changes associated with lethal
disease progression in a patient—a prerequisite for the goal of
effective patient-based target therapy (Schreiber et al., 2010).
However, extraction of the relevant alterations that constitute
a driving force for disease still poses a major challenge (Chin
et al., 2011). Aberrant PI 3-kinase pathway signaling is common
in CaPand itsspecific targeting holds greattherapeutic potential
(Majumder and Sellers, 2005; Taylor et al., 2010; Wong et al.,
2010). Therefore, it is paramount to understand which pathway
players, together or alone, can be regarded as sentinels of
pathway deregulation when they are found to be mutated.
Modeling the relevance of disease associated genes in geneti-
for establishing causality in cancer (Frese and Tuveson, 2007).
Research using Pten mutant GEM models of CaP has revealed
that genetic context dictates disease outcome through both
extent and cellular distribution of Akt activity (Carver et al.,
2009b; Di Cristofano et al., 2001; Majumder et al., 2003; Trotman
alteration events that cooperate to enhance AKT/PI 3-kinase
signaling in prostate cancer. PTEN prevents activation of AKT
by dephosphorylating the membrane phospholipid PIP3(Mae-
hama and Dixon, 1998). Thus, loss of PTEN results in increased
AKT recruitment to the plasma membrane (PM), where it is acti-
vated by PDK-1 kinase through phosphorylation on the threo-
nine-308 (Thr308) residue. The second Akt activating kinase
consists of the mTOR complex 2 (mTORC2), which phosphory-
lates serine-473 (Ser473) on AKT (Sarbassov et al., 2005).
In vitro results have led to the conclusion that dual activation of
AKT is essential for activity, a notion that has been challenged
by recent results obtained in vivo using genetic interference with
Recently, PHLPP1 and PHLPP2 have been identified through
a search for genes that combine a phosphatase with a PH
domain, reasoning that such a design would counteract PH
domain containing kinases like AKT. Indeed the PHLPP1/2
In addition to their roles in growth control, they have been
implicated in memory formation and maintenance of circadian
rhythms in mice (Masubuchi et al., 2010; Shimizu et al., 2010).
Recent genetic studies in mice have shown that neoplasia and
cancer in Pten-deficient prostate depend on Akt activation on
Ser473 by mTorc2 (Guertin et al., 2009). Because PHLPP1/2
are the two known phosphatases to specifically revert this
activation, we sought to determine the relevance of PHLPP1
as a tumor suppressor by combining GEM modeling with patient
whole genome analysis.
Phlpp1 Is a Tumor Suppressor in Mouse Prostate
By crossing Pten+/?with Phlpp1?/?mice we generated a cohort
containing >400 mice of six genotypes (note that Pten?/?mice
are embryonic lethal). As shown in Figure 1A, complete loss of
Phlpp1 in Pten+/?mice caused strong reduction of overall life-
span. Pathology analysis of these cohorts revealed acceleration
of the polyclonal autoimmune disorder, which constitutes the
major cause of death in our Pten+/?animals (Di Cristofano
et al., 1999) (not shown). Loss of Phlpp1 on its own also signifi-
cantly reduced overall lifespan, presumably due to the observed
to determine the tumor suppressor role of Phlpp1 in greater
detail. We focused on the role of Phlpp1 in prostate tumorigen-
esis, a tissue that is very sensitive to the degree of Akt pathway
activation (Trotman et al., 2003).
As shown in Figures 1B and 1C (bottom panel), prostates of
Pten+/?;Phlpp1?/?animals revealed regions of high grade pros-
tatic intraepithelial neoplasia (HGPIN), which is characterized by
an intraglandular proliferation of crowding cells with atypia,
enlarged nuclei and prominent nucleoli, and invasive adenocar-
cinoma (characterized by the proliferation of atypical cells that
break the basal membrane and invade through the prostatic
stroma) at full penetrance with onset at 8 months (see also
summary in Figure S1A available online). In contrast, prostates
of Pten+/?mice only suffered hyperplasia (characterized by the
proliferation of luminal with no cytological atypia) and HGPIN
with onset after 8 months (Figure S1A), as previously published
(Di Cristofano et al., 2001; Podsypanina et al., 1999; Trotman
et al., 2003). Although the Pten+/?;Phlpp1+/?mice did not
develop foci of invasive adenocarcinoma (Figure 1B), they
showed hyperplasia and low grade PIN, starting as early as
4 months and HGPIN at 8 and 12 months (see Figure S1A and
Figure 2C). Collectively, these data were consistent with
a gene dose-to-effect relation of progression previously identi-
fied for Pten alone (Trotman et al., 2003). Importantly, we found
that Phlpp1-loss on its own triggered HGPIN at full penetrance
within 9 months of age (Figure 1C and Figure S1A) and one
case of invasive adenocarcinoma at 12 months (Figure 1B and
Figure S1A). This was confirmed by immunohistochemistry
(IHC) for the basal cell marker cytokeratin 5, which is lost in the
invasive malignant glands (Figure S1B, left panels, red arrows).
Furthermore by staining early neoplasia for p63, we found no
expansion of basal cells (Figure S1B, right panels).
Pten-alteration in mouse prostate leads to increased prolifera-
tion, so we assessed Ki-67 staining over time. As shown
and quantified in Figure 1D, the Pten+/?and Phlpp1?/?tissue
regions (4 months) and in the neoplasms at 8 months and
12 months. In contrast, we observed a sharp increase in prolifer-
ation upon combined loss but only after 4 months, thus quantita-
tively demonstrating that the two genes synergize in a time-
average lesion sizes of the five cohort arms at three time points.
As shown (Figure S1C), combined gene loss strongly cooperated
to produce disproportionately larger lesions over time. Moreover,
we found that changes in apoptotic rates could not account
for tumorigenesis as they slightly increased (not decreased) in
Phlpp1- and compound mutants, nor could we detect inflamma-
tory responses as a cause of prostate malignancy (Figure S1D).
Collectively, our results demonstrate that Phlpp1 acts as a tumor
suppressor in mouse prostate, and that its loss is synergistic
with partial Pten-loss, but in a time-dependent manner.
PHLPP1 Is a Prostate Tumor Suppressor
174 Cancer Cell 20, 173–186, August 16, 2011 ª2011 Elsevier Inc.
Figure 1. Phlpp1 Is a Tumor Suppressor and Dictates Carcinogenesis in Pten Heterozygous Mice
(A) Kaplan-Meier plot for overall survival. Number of mice (n) and statistical significance (p) is given for comparison between Pten+/?and Pten+/?;Phlpp1?/?mice
and between WT and Phlpp1?/?mice. Numbers in cohort arms are: WT (98), Phlpp1+/?(97), Phlpp1?/?(49), Pten+/?(45), Pten+/?;Phlpp1+/?(81), and Pten+/?;
Phlpp1?/?(41) for a total of 411 animals.
(B) Kaplan-Meier plot for invasive-free survival showing complete penetrance of prostate cancer in Pten+/?;Phlpp1?/?animals. P value for Pten+/?;Phlpp1?/?
of 37 animals (ns, not significant).
(C) Microscopic analysis of 8-month prostate lesions reveals high grade PIN in Phlpp1?/?and adenocarcinoma in Pten+/?;Phlpp1?/?prostates, as indicated.
Insert shows high magnification. Scale bar represents 100 mm. (See also Figures S1A and S1B).
(D) Analysis of cell proliferation in prostate at 8months using Ki-67 immunohistochemistry (left panels;scale bar represents 50 mm) reveals increased proliferation
in compound mutant. Progression of disease over time as shown by average Ki-67 positive cells per 100 in neoplastic foci (graph, see also Experimental
Procedures). P values are <0.0001 and <0.004 for comparison of compound mutant prostates (Pten+/?;Phlpp1?/?) with Phlpp1?/?or Pten+/?, respectively, at
8 months and p < 0.0006 and < 0.003, respectively at 12 months. Error bars represent standard deviation (SD). See also Figures S1C and S1D.
PHLPP1 Is a Prostate Tumor Suppressor
Cancer Cell 20, 173–186, August 16, 2011 ª2011 Elsevier Inc. 175
Figure 2. Phlpp1-Loss in Prostate Triggers Akt Ser473 Activation and Localization to Plasma Membrane
(A) Western blotting of Akt activation in 8-month-old prostates and quantification of pAkt Thr308:Akt and pAkt Ser473:Akt ratios reveals strong phosphorylation
on the hydrophobic Ser473 motif. See also Figures S2A–S2D.
(B) Western blotting and quantification of Erk and Mek kinase activation (asterisksdenote actin leftover staining on blot) reveals no activation of this signaling axis
in Pten/Phlpp1 mutant prostate.
(C) Time course of signal activation by IHC staining for pAkt Ser473 demonstrates activation and strong membrane localization in compound mutant genotypes
after 4 months of age. Scale bar represents 100 mm.
(D) PHLPP1-IHC staining in human prostate cancer sample reveals membrane localization in epithelium. Scale bar represents 50 mm.
(E) Examples of differential PHLPP1 levels in human prostate cancers as detected by PHLPP1-IHC staining of the tumor tissue microarray. Scale bar represents
PHLPP1 Is a Prostate Tumor Suppressor
176 Cancer Cell 20, 173–186, August 16, 2011 ª2011 Elsevier Inc.
Phlpp1 Blocks Akt Activation at the Epithelial Plasma
Because Phlpp1 has been shown to directly dephosphorylate
Ser473 of Akt (Gao et al., 2005), we sought to determine the
phosphorylation of Akt (pAkt) in prostates of our mice. As shown
in Figure 2A, loss of Phlpp1 affected pAkt activation on Ser473.
We have been unable to detect significant above background
phosphorylation on Thr308 in Phlpp1?/?prostates. Although
these data are consistent with the published Ser473 specificity
vation of Akt on Thr308 in Phlpp1?/?prostate (see below). In the
context of Pten-heterozygosity, Phlpp1-loss also increased
on Thr308 (see Figure 2A and Figures S2A and S2B for double
heterozygous prostate and further quantification, respectively).
PHLPP1 has also been implicated in MAP-kinase and PKC
signaling (Gao et al., 2008; Shimizu et al., 2003), yet in the pros-
tates of compound mutant mice we did not find an increase in
MAP-kinase pathway signaling (Figure 2B) and no consistent
effects on PKC-b levels (not shown) but confirmed Gsk3-b,
4Ebp1, and S6 phosphorylation, whereas Foxo3a and Pras40
were not detectably affected (Figure S2C). Moreover, loss of
Phlpp1 had no general effect on Pten-levels (See Figure 2A,
top panels). However, microscopic analysis revealed the pres-
ence of some glands with spontaneously reduced Pten protein
and correlating pAkt Ser473 activation in the Pten+/?;Phlpp1?/?
lesions (Figure S2D). In the absence of an equally effective IHC
antibody for pAkt Thr308 activation, we assume that this reduc-
tion in Pten levels is contributing to the observed activation of
pAkt Thr308 above background levels in the Pten+/?;Phlpp1?/?
tumors (Figure 2A, last lane).
We have previously shown that the cellular localization of
activated Akt greatly varies in different CaP models: strong PM
association is seen in the conditional Pten null model (Trotman
et al., 2003), whereas tumors in Pten+/?;Pml?/?animals show
strong nuclear pAkt localization and activity (Trotman et al.,
2006) causing female sterility via Foxo3a inhibition (Castrillon
et al., 2003; Trotman et al., 2006). The membrane-associated
pAkt activation after complete Pten-loss correlates with senes-
cence arrest in vitro and in vivo (Alimonti et al., 2010; Chen
et al., 2005). Using immunohistochemistry time course analysis
ground cytoplasmic pAkt Ser473 localization in Pten+/?pros-
tates. In Phlpp1?/?prostate, we observed a similar stain, also
at all time points, confirming comparable overall Akt activation
levels seen in Figure 2A. At 4 months, the compound mutant
prostates revealed no major difference compared to the single
mutant mutant tissues. However, a striking shift of pAkt signal
to the PM was observed in 8-month and 12-month compound
mutant prostates. At these two time points, we found strong
ure 2C, two right most columns, 8 months and 12 months) and
number (not shown). Moreover, we found that these pAkt-foci
typically correlated with patches of slightly reduced Pten protein
Together, our findings are consistent with a Phlpp1-dose
dependent phenotype astheactivation foci were moredominant
in Pten+/?;Phlpp1?/?than in double heterozygous mice. There-
fore, the degree of Phlpp1 activity in the prostate epithelium
controls pAkt concentrations at the PM after partial Pten-loss.
Finally, we determined if this pAkt localization was due to the
cellular distribution of Phlpp1 itself. As shown (Figure S2E),
the Phlpp1 protein has several domains that could direct it to
the PM. Using a human PHLPP1 antibody, we confirmed strong
membrane localization of PHLPP1 in human prostate epithelium
(Figure 2D) and readily identified differential PHLPP1 expression
levels on a human tumor tissue microarray (Figure 2E). Thus, we
antagonize Ser473-activation of Akt and that its loss, especially
after Pten deregulation, can direct pAkt signaling to the plasma
Phlpp1-Loss Triggers p53 Activation in Pten+/–Prostate
The above data revealed focal full-blown pathway activation that
occurred only after a certain latency and in compound mutants,
consistent with the delayed onset maximal proliferation ob-
served in the double mutant genotypes quantified in Figure 1D.
Because complete Pten-loss triggers PM Akt activation and
p53-mediated senescence (Alimonti et al., 2010; Chen et al.,
2005; Kim et al., 2007) (as recently reviewed by Collado and
Serrano ), we next determined if inactivation of Phlpp1 in
Pten+/?mice could affect p53. As shown in Figure 3A, Phlpp1?/?
and also Pten+/?;Phlpp1?/?prostates showed elevated p53
levels at 8 months. This increase was consistently observed in
p53 protein whereas p53 transcript levels remained the same
(Figure S3A). However, we failed to detect a corresponding
increase in p21 transcription. On the contrary, we found lower
p21 transcription in these same prostate samples (Figure 3B)
suggesting spontaneous p53 loss-of-function.
To explore the state and activity of p53 during the neoplastic
process in more detail, we studied prostates of wild-type (WT)
and Pten+/?;Phlpp1?/?mice at 4 and 6 months of age and found
Yet, the Pten+/?;Phlpp1?/?prostate did show a p21 response at
4 months relative to WT prostate, which was absent from the
6 month time point, suggesting that p53 was transiently active.
Next, we validated this p53 activation and inactivation scenario
between 4 and 6 month samples by transcriptome analysis for
p53 activation signatures in the same two compound mutant
samples shown in Figure 3C (see Experimental Procedures).
As shown in Figure S3B, we found p53 target gene upregulation
(including p21) at 4 months. This, however, was absent from the
6-month-old prostate sample, confirming lack of p53 activity at
this time point, in spite of high protein levels in the same sample.
We also noted elevated transcripts of the prostate senescence
associated SerpinE1/Pai-1 gene (Chen et al., 2005) at 6 months
(see Figure S3B) and validated elevated Pai-1 transcripts by
qPCR (Figure S3C, 6 months). However, we found Pai-1 strongly
reduced in the 8-month-old double mutant prostate (Figure S3C,
8 months), similar to the loss of Cdkn1a mRNA shown in Fig-
ure 3Batthat timepoint. Insum,(inspite ofa 4–6weekvariability
between animals of the same genotype), we found that p53 was
transiently activated, peaking at around 4 months of age just
before overt focal lesions are forming.
ing neoplastic glands harbored mutant, inactive p53, we turned
to sequencing the above prostate samples for Trp53-mutations
at the level of the cDNA transcripts, which were abundant based
PHLPP1 Is a Prostate Tumor Suppressor
Cancer Cell 20, 173–186, August 16, 2011 ª2011 Elsevier Inc. 177
on our western blotting results. As summarized in Figure 3D, we
successfully isolated mutant forms of p53 (that were conserved
in human and confirmed to be functionally inactivating; see
Experimental Procedures) from three of five Pten+/?;Phlpp1?/?
blotting and sequencing correlations). This was neither seen in
the WT or Pten+/?control prostates nor in the primary Phlpp1?/?
MEFs (see Experimental Procedures).
Collectively, these data suggested that increased p53 activity
could be observed at ?4 months and was then lost at the
6-month and 8-month time points. They were also consistent
with the notion that compound mutant prostates were under
pressure to mutate p53. Paradoxically, these findings suggested
that, at 4 months, p53 activation (as revealed by transcription
profiling) and inactivating mutation could occur in the same
tissue and might give rise to tissue heterogeneity. Because this
notion was consistent with the focal nature of tumorigenesis
that we had already observed, we next explored the histological
features of this process in more detail.
p53 Inactivation Drives Compound Mutant Escape from
Based on the above findings we sought to visualize the molec-
ular events in early Pten+/?;Phlpp1?/?prostate at the micro-
scopic level. We therefore tested the 4-month-old compound
and Figure S3B, 4 months) and also harbored a p53 mutation
(Figure 3D, 4 months) for features of cellular senescence. Senes-
cence associated b-galactosidase (SABG) staining revealed
the presence of distinct positive blue areas in neoplastic glands,
absent from the normal surrounding and from WT glands, and
intriguingly also absent from the dysplastic core of the gland
(Figure 4A). To confirm that this differential staining was due to
molecular differences we tested pAkt activation by immunofluo-
rescence and found an inverse staining pattern for pAkt (Ser473)
activation in an adjacent section of the same gland (Figure 4B,
area 1 versus 2). Moreover the SABG positive peripheral area 1
showed few Ki-67 positive cells compared to the pAkt positive
luminal area 2, consistent with cellular senescence (Figure 4B).
Thus, our data suggested that tumorigenesis in the Pten+/?;
Phlpp1?/?model requires the inactivation of p53 to escape
from senescence (through spontaneous or pre-existing alter-
ations). We found that Phlpp1?/?
primary MEFs arrested in a growth assay when compared to
WT MEFs (see Figure 4C). Indeed, we found increased p53 in
these cells, correlating to pathway activation. On infection of
these single and double mutant MEFs with a virus-based anti-
p53 short hairpin (sh-p53 with Gfp marker) and Gfp-based
tracking of the infected population we found that these cells
underwent clonal expansion (see Figure 4D and Figure S3E)
Figure 3. p53 Activation and Mutation Occurs in Pten/Phlpp1 Mutants
(A) Western blotting and quantification of p53 shows increase in p53 levels in the Phlpp1 mutant prostates at 8 months of age. See also Figure S3A.
(B) RT-qPCR of p21waf1transcripts extracted from the same lysates as shown in (A) reveals lack of p21 activation in spite of p53 protein increase in the Phlpp1
mutant tissues. Bars represent different primer pairs. Error bars are SD of triplicates.
(C) Western blotting of p53 and p21 induction in prostates from indicated ages and genotypes confirms loss of p53 activity at 6 months. See also Figure S3B and
(D) Summary of p53 inactivating mutations identified in above mouse prostates of indicated genotypes and ages. Asterisks denote that analyses were derived
from identical samples (see also Experimental Procedures).
PHLPP1 Is a Prostate Tumor Suppressor
178 Cancer Cell 20, 173–186, August 16, 2011 ª2011 Elsevier Inc.
after 12 days postinfection (without antibiotic selection; see
Experimental Procedures) and we confirmed these results after
deriving the stable anti-p53 hairpin expressing MEFs (Figure 4E).
Of these, only (Pten/) Phlpp1 mutant cells could form colonies in
agar (Figure S3G). We confirmed Akt-dependence of these cells
using an AKT-inhibitory drug resulting in growth suppression,
decreased cell viability, and increased apoptosis (Figures S3H
Figure 4. p53 Inactivation and Escape from Senescence
(A) Hematoxylin and eosin (left) and corresponding adjacent section staining for senescence-associated b-galactosidase (SABG, right) in 4-month-old prostates
asterisks denote basal lamina. Scale bar represents 200 mm.
(B) Immunofluorescence on adjacent sections of Pten+/?;Phlpp1-/-prostate gland from (A) reveals strong pAkt Ser473 activation (top) and proliferation as
measured by Ki-67 (bottom) in the SABG-negative luminal area 2. In contrast, the SABG-positive area 1 shows few Ki-67 positive cells, and weak pAkt. Red
asterisks denote basal lamina. Scale bar represents 100 mm.
(C) Growth curve of WT, Phlpp1?/?, and Pten+/?;Phlpp1?/?MEFs reveals suppressed proliferation in compound mutant genotypes. Error bars are SD of
(D) p53-knockdown bypasses growth suppression in the Pten+/?;Phlpp1?/?and Phlpp1?/?mutant primary MEFs leading to clonal outgrowth of the sh-p53
positive cells (see also Figure S3E). Genotypes of primary MEFs are WT (blue), Pten+/?;Phlpp1?/?(brown), Phlpp1?/?(red). Hatched lines denote control
shRNA-luciferase infections (same color code).
(E) Growth curves of Phlpp1?/?;shp53, Pten+/?;Phlpp1?/?;shp53, and shp53 MEFs confirm results from (D) after selection of stably transduced cells. Error bars
are SD of triplicates.
PHLPP1 Is a Prostate Tumor Suppressor
Cancer Cell 20, 173–186, August 16, 2011 ª2011 Elsevier Inc. 179
These data demonstrated that loss of p53 in a (Pten/) Phlpp1
mutant setting could switch cells from arrest into proliferation.
Note that consistent with the previous reports on senescence
after Pten-loss (Alimonti et al., 2010; Chen et al., 2005), we found
no phospho-Ser15 phosphorylation in the increased p53 of
Pten+/?;Phlpp1?/?prostate (Figure S3F) nor changes in phos-
phorylation on a panel of other p53 modification sites (see
Experimental Procedures) in Phlpp1 knockdown cells (not
To summarize, our findings revealed that loss of Phlpp1 in
the context of partial Pten-loss causes p53 activation and
cellular senescence in the prostate and in MEFs. Although
this fail-safe response can delay disease progression, we did
observe that it is invariably overcome in the prostates of our
mice. Our data thus suggested a general conceptual paradigm
of triggering and breaking senescence as a condition for Pten/
Phlpp1 mutant prostate tumorigenesis. However, in contrast to
the natural human disease, every prostate cell in our engi-
neered model suffers Pten- and Phlpp1-loss, thus increasing
the likelihood of spontaneous escape from arrest through either
pre-existing or spontaneous p53-mutations. To test if and at
what stage breaking of the p53 senescence response is impor-
tant in the human disease, we validated this genetic progres-
sion scheme in a comprehensive human prostate cancer
PHLPP1 Is a Tumor Suppressor in Human Prostate
To validate our findings in human prostate cancer, we studied
a data set of 218 tumor samples (181 primary and 37 metastatic
CaP specimens) from patients with clinical and pathologic anno-
tation (Taylor et al., 2010). Of these, 128 samples were also
profiled with gene expression arrays (see Experimental Proce-
dures). We found that PHLPP1 (18q21) was indeed lost at the
22 of 29 samples exhibited broad heterozygous loss of PHLPP1
that often included the recently validated prostate cancer gene
SMAD4 (Ding et al., 2011). For comparison, 38 and 37 samples
had either broad heterozygous or focal homozygous deletions
involving PTEN or TP53, respectively. In addition, PHLPP2 (on
chromosome 16), the paralog of PHLPP1, was found in a region
of frequent broad heterozygous loss (Figure S4). Moreover,
these genes were often lost in the samples from metastatic sites
(Figure 5A, bottom).
We then tested for more specific PHLPP1 alteration, first at
the transcript level. As shown in Figure 5B (top), we found
that genomic loss was significantly associated with reduced
PHLPP1 expression levels using statistical variance testing
(p value < 0.001; see Experimental Procedures). We also identi-
fied eight human tumors in which PHLPP1 was apparently
diploid, but harbored reduced transcript expression levels (>2
standard deviations below the mean expression of 29 normal
Figure 5. PHLPP1 Is a Tumor Suppressor of Human Prostate Cancer
(A) Upper panel: copy number loss of PTEN, PHLPP1, TP53, and PHLPP2 in the data set of 218 human prostate cancer samples (see Experimental Procedures).
Lower panel: deletion frequency of above genes in the 181 primary tumors and 37 metastases. See also Figure S4.
(B) PHLPP1 copy number alterations correlate with gene expression profiling (top). Boxes contain expression values of 50% of cases, bars show the remaining
50% except for rare outliers. A bold line indicates the median expression value. Z-scores are relative to expression in normal prostate. Bottom graph: overview of
alteration frequencies in 79 samples that were triple-analyzed at copy number, RNA expression and protein level for PTEN and PHLPP1.
(C) Breakdown of alterations in samples with low PTEN (top) and PHLPP1 (bottom) protein levels. Protein reduction (scored low or absent) in cancers is observed
most frequently in spite of the presence of both gene copies and normal RNA levels.
PHLPP1 Is a Prostate Tumor Suppressor
180 Cancer Cell 20, 173–186, August 16, 2011 ª2011 Elsevier Inc.
adjacent prostate samples). Similarly, we found statistically
significant association between PTEN expression levels and its
copy number (Figure S5A). To further explore the extent of
croarrays (TMAs) for PTEN and PHLPP1 proteins in the patients.
As shown in Figure 5B (bottom), our analysis revealed that low or
absent levels of PTEN or PHLPP1 were three to five times more
frequent than alteration at either DNA or RNA level. We also
found some overlap between PTEN and PHLPP1 protein loss
(p = 0.017). By integrating the TMA data with genomic and
expression analysis on 79 samples we could demonstrate that
the vast majority of PTEN and PHLPP1 protein loss happens
in cases with normal DNA and/or expression of the genes
Thus, both PTEN and PHLPP1 are frequently and specifically
targeted at the translational or posttranslational level. Note
that candidate exon resequencing of 80 tumors from this set
[Taylor et al., 2010]). We therefore chose not to resequence the
full data set (no PHLPP1 mutations were found in nine samples).
Our analysis confirmed that PHLPP1 is frequently deleted in
primary and advanced disease and also altered at the RNA-
and, in particular, at the protein-level.
Codeletion of PHLPP1 and PTEN Is Strongly Associated
with Metastatic Disease
Through mouse modeling we have discovered a relationship
between triggering of a p53-fail-safe response by combined
lossof Pten and Phlpp1 on the one hand,and breaking it through
loss of p53-function on the other. If, as previously suspected
(Chen et al., 2005), senescence arrest after complete PTEN/
PHLPP1-loss would suppress the early, still benign stages of
disease, then the escaping prostate cancers would frequently
present with triple alteration of PTEN, PHLPP1, and TP53.
Thus, we analyzed the human data set for coalteration of
As shown (Figure 6A), primary samples showed no significant
association between loss of PHLPP1 and PTEN– thisoccurred in
only one of the 181 samples, which had no deletion of TP53. In
contrast, we found statistically significant codeletion of PTEN
and PHLPP1 in metastatic samples. When testing for concomi-
statistically significant association (p value < 2.6e-6; see also
Figure 6A legend) and frequent broad 16q codeletion events
that harbor the PHLPP2 locus. Because the metastatic lesions
harbor a greater number of alterations than the primary samples,
we performed two independent statistical analyses. First, we
determined the genome-wide deletions, which are associated
with PTEN-loss in metastatic samples as a function of the asso-
ciation’s p value. At p % 0.01 (Figure 6C, left plot) we find that
PHLPP1 is in one of only 17 deletions containing 69 genes that
are codeleted with PTEN (p = 0.008). At higher p value (%0.05)
significant, consistent with the recent discovery of Pten/Smad4
cooperation in mouse (Ding et al., 2011). We indeed observed
activation of Tgf-b signaling in the Phlpp1-deficient mouse
prostate (Figure S3D) and the human data showed significant
association with further indicated events that were recently
validated to strongly cooperate with loss of Pten in mouse
(Carver et al., 2009a; Chen et al., 2005; King et al., 2009). Also,
combined PTEN-PHLPP1 loss correlates significantly with loss
of TP53 (Figure S5D).
Next, we performed an unbiased determination of significant
codeletions in metastases. To this end, we carried out a false
discovery rate analysis by first performing a Monte Carlo permu-
tation test of the significance of each possible codeleted pair of
genes in the metastatic genomes (total of >3000 genes giving
5 million pairs). The result is the histogram of p values depicted
in Figure S5C. As shown, the vast majority of these pairs are
statistically consistent with absence of correlation: their p values
form a constant plateau in the graph. In contrast, there is
a number of true positives, i.e., gene pairs that are significantly
correlated, statistically. These are exhibited by the overrepre-
sentation of low p-values to the left side of the graph (see also
Experimental Procedures). This small subset of significantly
correlated genes contains all the pertinent gene pairs formed
by loss of PTEN, PHLPP1, PHLPP2, and TP53. Considering
that these are physically independent genes, they are statisti-
cally likely to be biologically linked.
To summarize, our two additional analyses confirm that the
metastatic samples show a statistically significant enrichment
of codeletion of these genes, whereas in contrast, the primary
samples do not. When we analyzed the heat-map of inactivation
of these genes among only metastatic samples (shown in Fig-
ure 6B), we indeed observed that codeletion of PTEN, PHLPP1,
PHLPP2, and TP53 clustered together. These data are consis-
tent with a prostate cancer progression model, where loss of
p53 is a prerequisite for the combined loss of PTEN and the
PHLPP genes, which is found in metastasis.
Taken together, our analysis suggests that (i), PHLPP1 is
a tumor suppressor in human prostate cancer; (ii), strong activa-
tion of AKT signaling through loss of PTEN and PHLPP1 is
associated with the loss of TP53; and (iii), the coincidence of
these three deletion events is frequent in metastatic samples
but absent from primary tumors. These data are thus consistent
with the notion that the p53-response acts as a barrier to pros-
tate cancer progression, and not initiation. However, we note
these three human deletion events occur.
To test if the latter finding could be of clinical value, we
analyzed if combined alteration of PTEN and PHLPP1 at RNA
or protein level could predict disease recurrence after prostatec-
tomy. PTEN/PHLPP1 protein levels did not correlate with
levels of PTEN and PHLPP1 in prostatectomies (Figure 6D),
we did observe a significant difference between the time to
biochemical relapse in PTEN/PHLPP1 low mRNA samples and
those with at least one normal transcript level (p = 4e-5). This
outcome analysis was similar to using the histopathology based
Gleason score as predictor in this data set (Figure 6D). These
results suggest that combining pathology-based information
with the PTEN/PHLPP1 transcript signature could identify
patients that would benefit from PI 3-kinase pathway therapy.
Note that only three (of 37) samples with low PTEN/PHLPP1
transcripts had Gleason scores of 7. Thus, we could not test if
the transcript levels could significantly stratify this important
patient group. When performing this analysis with other pairs
that are associated with low PTEN expression including cancer
PHLPP1 Is a Prostate Tumor Suppressor
Cancer Cell 20, 173–186, August 16, 2011 ª2011 Elsevier Inc. 181
genes near the PHLPP1 locus (Figure S5B, bottom), the PTEN/
SMAD4 combination also stood out with significant predictive
power (Figure S5E) consistent with recent findings (Ding et al.,
Phlpp2 and p53 Show a Concerted Response
to Pten/Phlpp1 Status
Finally, we asked why late stage samples consistently showed
loss of the PHLPP2 gene (Figure 6B, bottom) when PTEN/
PHLPP1 deletion could already drive the pathway to prostate
cancer, especially after loss of p53. To this end, we studied
Phlpp2 regulation in the genetically defined setting of primary
WT, Phlpp1?/?, and Pten+/?;Phlpp1?/?double mutant MEFs.
As shown in Figure 7A, both Phlpp1 mutant settings resulted in
increased Phlpp2 as well as increased p53 levels. To test if this
response was elicited by AKT activation, we used NIH 3T3 cells
stably transfected with constitutively active AKT (via myristoyla-
tion, myr-AKT; Figure 7B). This experiment confirmed AKT-
dependent increase in Phlpp2 and p53 levels. Importantly,
a myr-AKT plasmid that was targeted exclusively to the cyto-
plasm (myr-AKT-NES; see Figure S6A for localization) was also
able to induce the two proteins consistent with a Phlpp2 and
p53 response triggered by cytoplasmic AKT. To confirm that
Phlpp2 and p53 levels are coupled to Pten and Phlpp1 levels
we used RNAi in this cell line. As shown (Figure 7C and Fig-
ure S6B), combined Pten/Phlpp1 knockdown resulted in
increased Phlpp2 and p53 levels, confirming a putative negative
feedback mechanism. Next, we tested this response in
a controlled setting with a human PTEN knockout cell line, the
HCT116 PTEN+/+and PTEN?/?system (Kim et al., 2007). Fig-
ure S6C shows that the PTEN-deficient cells display activation
of pAKT and p53, as published (Kim et al., 2007). At the same
Figure 6. Codeletion of PHLPP1 and PTEN Is Restricted to the Metastatic Samples
(A) Codeletion analysis of PTEN, PHLPP1, and TP53 in primary and metastatic samples shows frequent codeletion in metastatic samples. The p values for the
significance of association between both PTEN and PHLPP1 loss of any kind with primary (asterisk) or metastatic (double asterisk) samples are indicated. A triple
asterisk denotes the p value for the association of PTEN, PHLPP1, and TP53 triple-loss among all samples (no triple deletion occurred in primary samples). See
also Figures S5A and S5B.
and amplification is indicated. See also Figure S5C.
(C) Circos plot of genome-wide codeletion events in PTEN mutant metastatic samples at p % 0.01. PHLPP1 (with two next neighbors, p = 0.008) is in one of
17 PTEN-associated deletion regions that harbor a total of 69 genes. Right panel: at p % 0.05 a greater number of deletions are associated with PTEN-loss. See
also Figure S5D.
(D) Kaplan-Meier outcome analysis for relapse after radical prostatectomy based on expression levels of PTEN and PHLPP1 mRNA. The same analysis based on
Gleason-scoring is shown for reference. Patients with Gleason score 6 (asterisks) and 7 (arrowheads) are shown. See also Figure S5E.
PHLPP1 Is a Prostate Tumor Suppressor
182 Cancer Cell 20, 173–186, August 16, 2011 ª2011 Elsevier Inc.
time these PTEN null cells also exhibited strong PHLPP2 activa-
tion, consistent with our findings in primary and immortalized
To better understand these findings mechanistically, we
tested if the Phlpp2 protein response was caused by increased
transcript levels or protein stability in the NIH 3T3 Pten/Phlpp1
knockdown cells. However (see Figures S6D and S6E), we
found neither increased RNA levels and slightly decreased, not
increased Phlpp2 stability after Pten/Phlpp1 knockdown. This
transcript-independent principle was confirmed in the com-
pound mutant prostate, and we confirmed that reversal of this
surge by Phlpp2 knockdown results in Akt activation in
compound mutant MEFs (Figures S6F and S6G). Because we
found inactivation of the Tsc2-axis after Phlpp1-loss (Fig-
ure S6H), we tested if the response was mediated by translation
downstream of mTOR, as previously shown for p53 on loss of
Figure 7. Pten/Phlpp1 Codeletion Triggers Rapamycin-Sensitive p53 and Phlpp2 Activation
(A) A surge of steady state Phlpp2 and p53 levels is seen in the primary Phlpp1?/?and the Pten+/?;Phlpp1?/?mutant MEFs. See also Figures S6B, S6C, and S6F.
(B) Activated cytoplasmic AKT overexpression recapitulates the Phlpp2 and p53 activation from (A) in NIH 3T3 cells. See also Figure S6A.
(C) The Phlpp2/p53 response is also found after shRNA-mediated Phlpp1/Pten knockdown in NIH 3T3 cells. See also Figures S6D and S6G.
(D) The surge of Phlpp2/p53 in double mutant primary MEFs from (A) is blocked by rapamycin suggesting mTor-dependent translation (see also Figures S6D
(E) Phlpp2/p53 activation driven by myrAKT overexpression in NIH 3T3 cells is blunted by rapamycin.
(F) Phlpp2/p53 activation after Pten/Phlpp1 knockdown in NIH 3T3 cells is reverted by rapamycin. See also Figures S6D, S6E, S6H, and S6I).
PHLPP1 Is a Prostate Tumor Suppressor
Cancer Cell 20, 173–186, August 16, 2011 ª2011 Elsevier Inc. 183
Pten (Alimonti et al., 2010). Intriguingly, using the Pten+/?;
Phlpp1?/?primary MEFs (Figure 7D), addition of rapamycin
abrogated the Phlpp2 activation that persisted in the DMSO
control treatment. Similarly (see Figure 7E), rapamycin also
blunted the Phlpp2/p53 feedback in the NIH 3T3 cells, which
were stably transfected with myrAKT- or the anti-Pten/Phlpp1
RNAi-plasmids (Figures 7E and 7F). Finally, we confirmed that
genetic activation of mTorc1 by Tsc1-ablation also leads to a
strongPhlpp2 surgewithoutincreasing itstranscript(FigureS6I).
Taken together, our data show that in the genetically defined
context of Pten+/?;Phlpp1?/?primary MEFs, Phlpp2 positively
responds to pathway activation in an mTorc1-dependent
manner, similar to p53. These findings suggest that the high
codeletion rate of the PHLPP1/2 and TP53 genes with PTEN in
advanced cancer is required to break this feedback in late stage
disease. Furthermore, our results suggest that clinically relevant
pathway inhibitory drugs that target mTORC1 could negatively
affect the levels of PHLPP2 and p53 in patients.
Cancer researchers are facing a deluge of genome data. Turning
this powerful information into our advantage ultimately depends
on highly reliable systems for experimental validation. Our study
shows how data from large scale genomics can be put into
a meaningful biological context and transformed into actionable
information by comparison with results from hypothesis driven
research in genetically engineered mice.
First, wehaveidentified in PHLPP1, a‘‘druggable’’ suppressor
of prostatecancerprogression becauseits antagonist, mTORC2
can be pharmacologically inhibited. Most importantly, genetic
mTORC2-inactivation has no adverse effects on adult prostate
tissue (Guertin and Sabatini, 2009). The deletion involving
PHLPP1 (18q21) contains still other suspected and confirmed
tumor suppressors. The TGF-b effectors SMAD2, SMAD4,
SMAD7, as well as the DCC gene, are codeleted in the majority
of cases. The cooperation of Smad4 with complete Pten-loss
in prostate cancer has recently been shown in knockout mice
(Ding et al., 2011) and similarly, we find Smad-activation in
response to Phlpp1-loss in prostate. The codeletion of SMAD4
and PHLPP1 could thus conceivably exacerbate the conse-
quences of PTEN-loss in human prostate.
Second, our study reveals a progression principle of PTEN-
pathway driven aggressive prostate cancer. After its initial
discovery (Chen et al., 2005), the senescence response in pros-
lesions from becoming clinically relevant cancer (reviewed in
Narita and Lowe ). In contrast, our genomic analysis
reveals that strong activation of the pathway coincides with
p53-deletion in metastasis, not cancer. Therefore we propose
that primary prostate lesions must and do develop in PTEN
haploinsufficiency (75% show reduction in protein) in order to
fly below the radar of the p53 activation system unless a p53
alteration has already occurred.
Third, we find that low PTEN/PHLPP1 transcription correlates
with biochemical relapse in patients after prostate surgery. If
molecular information that might be used to stratify patients for
PI 3-Kinase inhibitor trials. Importantly, recent results (Carver
et al., 2011; Mulholland et al., 2011) have revealed that blockade
of AR- or PI 3-kinase signaling is mutually reinforcing, demon-
strating theneed forcombined therapeuticpathway inactivation.
Intriguingly, this research has shown that AR-mediated AKT-
inhibition is carried out by PHLPP1 activation because PHLPP1
is degraded by AR blockade, a mainstay of advanced prostate
cancer therapy. These results reinforce the crucial role of
PHLPP1 status in prostate cancer progression.
Finally,weidentifythePHLPP2protein aspartof acellautono-
mous fail-safe mechanism, which in concert with p53 responds
to excessive pathway signaling in prostate. The activation of
these responses cannot prevent tumorigenesis in our animal
system, yet they critically shape the disease time course in
amodelwhere,unlike inhuman,every prostatecellisengineered
to suffer Pten/Phlpp1-loss. The PHLPP2-mediated negative
pathway feedback represents another potential mechanism by
which mTORC1 activation inhibits AKT activity. Because the
pharmacological inhibition of mTorc1 is able to derail this re-
sponse, our data argue for checking this PHLPP2 activation in
Taken together, our results identify the critical role of the
PHLPP proteins in prostate cancer and suggest that defining
their status in relation to PTEN and p53 is important for under-
standing and combatting the disease.
Generationof Phlpp1 knockoutmice wasrecently described (Masubuchi et al..
2010). Phlpp1 null mice (129 Sv/C57BL6) were crossed with wild-type mice
(129 Sv/C57BL6, from our Pten+/?cohort) for two generations. Offspring were
intercrossed for six generations to obtain the study cohort of >400 animals.
For genotyping, PCR primers 50-TGGGAAGAACCTAGCTTGGAGG-30, 50-TTC
CATTTGTCACGTCCTGCAC-30, and 50- ACTCTACCAGCCCAAGGCCCGG-30
were used for Pten. Primers 50- TAGGAGAGACTAGTGACATC-30, 50-TGAGCT
TATACGCTGTGATGC-30, and 50-AGCCGATTGTCTGTTGTGC-30were used
for Phlpp1. Overall and disease-free survival curves were calculated by the
Kaplan-Meier method and log rank (Mantel-Cox) testing with Prism 5.0 soft-
wareforApple MacintoshOSX. Cohort sizes were411miceforoverallsurvival
(WT , Phlpp1+/?, Phlpp1?/?, Pten+/?, Pten+/?;Phlpp1+/?,
Pten+/?;Phlpp1?/?), and 37 animals for prostate cancer free survival (WT
, Pten+/?, Phlpp1?/?, Pten+/?;Phlpp1+/?, Pten+/?;Phlpp1?/?).
All work with animals was performed along the Cold Spring Harbor
Laboratory Institutional Animal Care and Use Committee- approved protocol
11-08-3 (‘‘Tumor suppressor mutation in mouse models for cancer [11-08-
3], Trotman, PI’’).
Prostates from dissected animals of all genotypes were homogenized and
protein was extracted simultaneously with DNA and RNA using the AllPrep
DNA/RNA/Protein kit (QIAGEN) per manufacturer’s instructions. Cells were
lysed in 50 nM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 0.1% NP-40, 1 mM
sodium ortho-vanadate (Na3VO4), 10 mM NaF, protease inhibitor cocktail
(Roche) and cleared by centrifugation; concentrations were determined by
Bio-Rad Protein Assay (Bio-Rad Laboratories). Resulting samples were taken
into a sodium dodecyl sulfate sample loading buffer followed by brief sonica-
tion and centrifugation, then the supernatant was collected for western blot-
ting. Prostate and MEFs were from at least two sets of mice of all genotypes
and samples were analyzed three times or more for confirmation.
Gene set or pathway analysis was done by using GAGE (Luo et al., 2009),
generally applicable gene set enrichment. The most differentially regulated
KEGG pathways and GO groups were selected with FDR q value < 0.1. In
PHLPP1 Is a Prostate Tumor Suppressor
184 Cancer Cell 20, 173–186, August 16, 2011 ª2011 Elsevier Inc.
significant KEGG pathways or GO groups, genes with above noise-level
expression changes are taken to be substantially perturbed. These data
were then row-/gene-wise normalized and visualized using heat maps.
Plasmids and Stable Lines
pCDNA3-Myr-HA-AKT plasmid was obtained from Addgene (originally gener-
ated by William Sellers, Addgene plasmid 9008). pCDNA3-Myr-HA-AKT-NES
was constructed by cloning synthetic oligos corresponding to the nuclear
export signal (NES) of the PKI protein to the 30end of Myr-HA-AKT. Plasmids
were transfected into NIH 3T3 cells using Lipofectamine 2000 (Invitrogen),
and selected with 800 mg/ml neomycin 48 hr posttransfection to generate
Human Prostate Cancer Data
For the analysis of human prostate tumors, 218 frozen prostate cancer spec-
imens and 149 matched normal tissue samples were procured from patients
treated by radical prostatectomy at Memorial Sloan-Kettering Cancer Center
(MSKCC). DNA from 194 tumor specimens were labeled and hybridized along
with either their matched normal tissue or a pool of reference normal to Agilent
244K array comparative genomic hybridization arrays (aCGH) using the
manufacturer’s protocol. Raw copy number profiles were normalized,
segmented with circular binary segmentation (Venkatraman and Olshen,
2007) and analyzed with RAE. Expression levels were determined for 128 of
these tumors and 29 normal prostate tissues using Affymetrix Human Exon
1.0 ST arrays and tested for their association with copy number status by
ANOVA testing as published (Taylor et al., 2010). For clinical evaluation, data
from this work was previously analyzed by the MSKCC Prostate Cancer
Oncogenome Group. Clinical and pathologic data from this patient cohort is
maintained in a prospective fashion on the MSKCC prostate cancer clinical
database and the published data on 181 primary and 37 metastatic tumors
(Taylor et al., 2010) have been visualized using the Nexus Copy Number
software v 5.1 (Biodiscovery). All analyses for this publication were performed
on de-identified patient data and material and thus qualified for exemption
from human subjects statements.
Microarray data generated in this study have been deposited in the Gene
Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) with the accession
Supplemental Information includes six figures and can be found with this
article online at doi:10.1016/j.ccr.2011.07.013.
We thank S. Lowe, S. Powers, M. Zhang, K. Maimer, J. Hicks, M. Spector, J.
Zuber, and W. Xue for discussion, help with analyses, and reagents, L. Bianco,
A. Nourjanova, K. Manova, and A. Barlas for help with histology procedures
and analysis, R. McCombie and S. Muller for help with sequencing, M. Ham-
mell, W. Luo, and C. Johns for discussion and help with RNA expression array
procedures. Thiswork was supportedby grants toL.C.T.from the Department
of the Army (W81XWH-09-1-0557), the Starr Foundation (I3-A154), the V
Foundation, the V Kann Rasmussen Foundation (VKRF), and the NIH
(1R01CA137050-01A2), as well as by the MSKCC Prostate SPORE and NIH
grant GM-43154 to A.C.N. L.C.T. is a Rita Allen Foundation Scholar and would
like to dedicate this work to the memory of William L. Gerald.
The authors declare that they have no competing financial interests.
Received: April 6, 2011
Revised: June 5, 2011
Accepted: July 27, 2011
Published: August 15, 2011
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