MOLECULAR AND CELLULAR BIOLOGY, Jan. 2007, p. 662–677
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 27, No. 2
Activation of p53-Dependent Growth Suppression in Human Cells by
Mutations in PTEN or PIK3CA?
Jung-Sik Kim,1Carolyn Lee,1,2Challice L. Bonifant,1,2Habtom Ressom,1and Todd Waldman1*
Department of Oncology1and Tumor Biology Training Program,2Lombardi Comprehensive Cancer Center,
Georgetown University School of Medicine, Washington, D.C. 20057
Received 27 March 2006/Returned for modification 5 June 2006/Accepted 9 October 2006
In an effort to identify genes whose expression is regulated by activated phosphatidylinositol 3-kinase
(PI3K) signaling, we performed microarray analysis and subsequent quantitative reverse transcription-
PCR on an isogenic set of PTEN gene-targeted human cancer cells. Numerous p53 effectors were upregu-
lated following PTEN deletion, including p21, GDF15, PIG3, NOXA, and PLK2. Stable depletion of p53 led
to reversion of the gene expression program. Western blots revealed that p53 was stabilized in HCT116
PTEN?/?cells via an Akt1-dependent and p14ARF-independent mechanism. Stable depletion of PTEN in
untransformed human fibroblasts and epithelial cells also led to upregulation of p53 and senescence-like
growth arrest. Simultaneous depletion of p53 rescued this phenotype, enabling PTEN-depleted cells to
continue proliferating. Next, we tested whether oncogenic PIK3CA, like inactivated PTEN, could activate
p53. Retroviral expression of oncogenic human PIK3CA in MCF10A cells led to activation of p53 and
upregulation of p53-regulated genes. Stable depletion of p53 reversed these PIK3CA-induced expression
changes and synergized with oncogenic PIK3CA in inducing anchorage-independent growth. Finally,
targeted deletion of an endogenous allele of oncogenic, but not wild-type, PIK3CA in a human cancer cell
line led to a reduction in p53 levels and a decrease in the expression of p53-regulated genes. These studies
demonstrate that activation of PI3K signaling by mutations in PTEN or PIK3CA can lead to activation of
p53-mediated growth suppression in human cells, indicating that p53 can function as a brake on phos-
phatidylinositol (3,4,5)-triphosphate-induced mitogenesis during human cancer pathogenesis.
Inactivating mutations of the PTEN tumor suppressor gene
are found in a wide range of common human cancers, includ-
ing glioblastoma, endometrial carcinoma, melanoma, and
adenocarcinoma of the prostate (28, 50). PTEN is a lipid phos-
phatase that converts the mitogenically active lipid phospha-
tidylinositol (3,4,5)-triphosphate (PIP3) to PIP2(32). The im-
portance of the lipid phosphatase activity of PTEN for
tumorigenesis was recently highlighted by the discovery that
activating mutations in PIK3CA, encoding the phosphatidyl-
inositol 3-kinase alpha (PI3K?) subunit, are also commonly
present in human cancer (22, 45).
PIP3mitogenic signaling is well known to proceed via acti-
vation of the PIP3-dependent serine threonine kinases Akt1 to
-3, which phosphorylate downstream effectors, including TSC2,
BAD, FKHR1, and FKHLR1 (6, 12, 33, 42, 53). The identities
of these Akt substrates and their relevance to cancer patho-
genesis are rapidly emerging. Importantly, a subset of these
Akt substrates are transcription factors (most prominently,
FKHR1 and FKHRL1), which converge on the nucleus to
modulate the expression of PIP3effector genes. The identity of
the “PIP3transcriptome” remains largely unknown, but it is an
intensely active area of investigation.
The p53 tumor suppressor gene is perhaps the best-known
and best-studied transcription factor whose function is critical
to human cancer pathogenesis. The best-established function
of p53 is as a transcriptional activator that induces the expres-
sion of genes that can induce apoptosis and/or senescence-like
cell cycle arrest. In most untransformed cells, p53 is quiescent.
However, p53 is induced during the process of cancer patho-
genesis to provide its tumor-suppressing activity. Though the
identity of the “natural inducer” of p53 during human tumor-
igenesis has been long debated, numerous stimuli have been
identified that clearly lead to potent p53 induction in vitro.
These include both extracellular insults—radiation, DNA-
damaging chemotherapeutics, spindle poisons, antimetabo-
lites, and oxygen deprivation—and intracellular stimuli—on-
cogene activation, cellular aging, and oxygen radical formation
(1, 7, 10, 16, 17, 23, 52).
Oncogene activation in particular has been appealing as a
potential inducer of p53, since it is considered likely that on-
cogene activation precedes p53 inactivation during the patho-
genesis of most, if not all, human tumors. The expression of
activated oncogenes induces the expression of p14ARF, which
sequesters Hdm2 and inhibits its E3 ubiquitin ligase activity
(13, 19, 30, 35, 51, 55). This leads to an increase in the half-life
of p53 and its functional activation. However, despite the focus
on oncogenes as potential inducers of p53, several important
caveats have remained. First, most studies have been per-
formed in murine, not human, cells (47). Second, most human
studies have employed ectopic overexpression of oncogenes,
leading to a general concern that oncogene-induced activation
of p53 could be an artifact of overexpression. This concern has
been compounded by the fact that it has not yet been demon-
strated that deletion of an endogenous activated oncogene can
reduce p53 levels and activity in any human cell line.
* Corresponding author. Mailing address: Lombardi Comprehen-
sive Cancer Center, Georgetown University School of Medicine, 3970
Reservoir Road, NW, NRB E304, Washington, DC 20057. Phone:
(202) 687-1340. Fax: (202) 687-7505. E-mail: waldmant@georgetown
?Published ahead of print on 23 October 2006.
Recent studies have uncovered important intersections be-
tween the PI3K and p53 signaling pathways. Several studies
have suggested that activation of PI3K signaling via mutations
in PTEN could lead to inactivation of p53 via alteration of
Hdm2 expression and/or nuclear localization or via direct bind-
ing of PTEN to p53 (9, 15, 34). These studies helped to explain
the observation that mutations of PTEN and p53 are mutually
exclusive in stromal cells during the early stages of human
breast cancer pathogenesis (26). However, other studies have
suggested that activation of PI3K signaling can lead to activa-
tion of p53. Most prominently, a recent study by Chen et al.
demonstrated that tissue-specific inactivation of PTEN in the
mouse prostate led to activation of p53 and subsequent cellular
senescence (11). Consistent with this, prostate-specific deletion
of both PTEN and p53 led to the formation of prostate adeno-
We have recently pursued a loss-of-function genetic ap-
proach to identify the functions of PTEN in human cancer cells
by using human somatic cell gene targeting. In our initial
studies using PTEN gene-targeted human cancer cells, we
demonstrated that PTEN controls a DNA damage-inducible
cell size checkpoint in human cells (27). Here, we present
further studies that employ PTEN gene-targeted human can-
cer cells to identify PTEN functions.
Microarray analysis revealed that a host of well-known p53-
regulated genes were upregulated following PTEN deletion in
human cells. Subsequent experiments employing a variety of
complementary genetic approaches, including additional gene-
targeted human cancer cell lines, stable short hairpin RNA
(shRNA), and ectopic transgene overexpression confirmed this
observation, extended it to oncogenic PIK3CA, and demon-
strated that it was partially dependent on Akt1 and likely
independent of p14ARF. Therefore, we demonstrate here that
PTEN inactivation leads to activation of p53 responses in un-
transformed and transformed human cells, provide several in-
sights into the mechanism of p53 activation by PI3K signaling,
and demonstrate that like PTEN loss, oncogenic activation of
PIK3CA can also lead to induction of p53 responses.
MATERIALS AND METHODS
Cell lines. HCT116 PTEN?/?and PTEN?/?cells were created using human
somatic cell gene targeting and were described in a previous study (27). BJ-
hTERT and RPE-hTERT cells were obtained from Clontech (Mountain
View, CA). Normal human astrocytes (NHA) were obtained from AllCells
LLC (Emeryville, CA) and cultured as recommended. HEK293, MCF10A,
and A172 were obtained from the ATCC (Manassas, VA) and cultured as
recommended. HEK293T cells were obtained from Richard Pestell (Thomas
Microarray analysis. Global gene expression profiles were measured using
Affymetrix U133A and U133B microarrays (with 22,283 and 22,645 probe sets,
respectively) in two PTEN?/?cell lines (HCT116 parental cells and HCT116
Neor, with random integration of the PTEN targeting vector) and three inde-
pendently derived PTEN?/?cell lines. To do this, total RNA was prepared using
standard TRIZOL-based techniques. Fragmented cRNA was prepared using the
method described in the Affymetrix Genechip Expression Analysis technical
manual. Hybridization, washing, and imaging steps were performed in the Lom-
bardi Comprehensive Cancer Center Macromolecular Shared Resource. Raw
images were preprocessed using Microarray Suite 5.0 software (Affymetrix, Santa
Clara, CA). Data analysis was performed with the BRB Array software package,
a publicly available suite of analysis tools created by the NCI Biometric Research
Branch. A class comparison to identify differentially expressed genes was per-
formed using univariate analysis. In brief, the randomized-variance t test, a
variant of the standard t test, was used to compare the two classes (PTEN?/?and
PTEN?/?cells). A multivariate permutation test was employed to control the
number and proportion of false positives. For more detailed statistical informa-
tion, see http://linus.nci.nih.gov/BRB-ArrayTools.html.
qRT-PCR. Total RNA was prepared by standard TRIZOL-based methods.
Quantitative reverse transcription-PCR (qRT-PCR) was performed in an iCycler
(Bio-Rad, Hercules, CA) using TaqMan gene expression assays (Applied Bio-
systems, Foster City, CA) and the Superscript III Platinum One Step qRT-PCR
System (Invitrogen, Carlsbad, CA), according to the manufacturers’ specifica-
tions. Relative gene expression levels were calculated using the 2??(?CT)
method, normalizing to the expression of the ?2-microglobulin housekeeping
gene. All assays were performed at least in triplicate.
Immunoblotting and immunoprecipitation. For immunoprecipitation, 200 ?g
of whole-cell lysate was incubated with mouse ?-FLAG preconjugated M2 aga-
rose beads (SIGMA, St. Louis, MO) at 4°C for 24 h. The beads were washed
three times with lysis buffer, boiled in 2? gel loading buffer, and analyzed by
immunoblotting. For immunoblotting, protein lysates were separated by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene
difluoride membranes, probed with primary and horseradish peroxidase-coupled
secondary antibodies, and visualized by enhanced chemiluminescence (Pierce,
Rockford, IL). Bands were quantified using Scion Image densitometry software
(Scion Corporation, Frederick, MD). Primary antibodies were obtained from
Abcam (Cambridge, MA; p14ARF), Cascade Bioscience (Winchester, MA;
PTEN clone 6H2.1), Calbiochem (San Diego, CA; p53 clone DO-1), Cell Sig-
naling (Danvers, MA; p-Akt S473 clone 193H12; pan-Akt; Akt1 clone 2H10;
p14ARFclone 4C6/4), Upstate (Temecula, CA; ?-H2AX clone JBW301), Santa
Cruz Biotechnology (Santa Cruz, CA; p14ARFclone C-18; Hdm2 clone SMP14),
Zymed (South San Francisco, CA; p21 clone EA10), Neomarkers (Freemont,
CA; tubulin ? clone DM1A), and SIGMA (St. Louis, MO; FLAG polyclonal
Lentiviral shRNA. Constructs for stable depletion of PTEN, p53, and Akt1
were obtained from The RNAi Consortium (37) via SIGMA and Open Biosys-
tems (Huntsville, AL). For each gene, four or five premade constructs were
obtained and tested to identify those able to achieve efficient knockdown at the
protein level. Negative control constructs in the same vector system (vector alone
and scrambled shRNA) were created by Robert Weinberg (Massachusetts
Institute of Technology) and David Sabatini (Massachusetts Institute of
Technology) and obtained from Addgene (Cambridge, MA). The lentiviral
helper plasmids pHR?8.2?R and pCMV-VSV-G were also obtained from
Robert Weinberg via Addgene. All plasmids were prepped, and their integrity
was confirmed by restriction analysis. The integrity of all shRNA inserts was
confirmed by sequencing.
To prepare transient virus stocks, 1.5 ? 106293T cells were plated in 10-cm
dishes. The next day, the cells were cotransfected with shRNA constructs (3 ?g),
together with pHR?8.2?R and pCMV-VSV-G helper constructs (3 ?g and 0.3
?g, respectively), using FuGENE 6 (Roche, Indianapolis, IN). The media were
changed the next day, and the following day, virus-containing media were har-
vested. The viral stocks were centrifuged and filtered to remove any nonadherent
Next, HCT116, BJ-hTERT, RPE-hTERT, or MCF10A cells were infected
with shRNA lentiviruses. To do this, the cells were plated at subconfluent
densities. The next day, the cells were infected with a cocktail of 1 ml virus-
containing medium, 3 ml regular medium, and 8 ?g/ml Polybrene. The medium
was changed 1 day postinfection, and selective medium was added 2 days postin-
fection (2 ?g/ml puromycin for HCT116, BJ-hTERT, and MCF10A; 10 ?g/ml for
RPE-hTERT). After 3 days of puromycin selection, the mock-infected cells had
all died. Stably infected pooled clones were studied.
Creation of wild-type and mutant human PIK3CA retroviruses. A wild-type
human PIK3CA cDNA was obtained from the Harvard Institute of Proteomics.
This cDNA was PCR amplified with high-fidelity VENT Polymerase (New
England Biolabs, Ipswich, MA) with an amino-terminal FLAG tag and cloned
into pDNR-CMV (Clontech, Mountain View, CA). Of note, Peter Vogt’s labo-
ratory has demonstrated that amino-terminally epitope-tagged chicken PI3K
p110? retains its biological and biochemical properties (21). The insert was
sequenced in its entirety. Next, site-directed mutagenesis was employed to create
two mutant derivatives—early stop (W11STOP) and a common oncogenic mu-
tation in the catalytic domain (H1047R). These inserts were then shuttled into
the pLP-LNCX retroviral expression vector (Clontech) using an in vitro Cre/Lox
recombination. The integrity of these clones was confirmed by restriction analysis
and sequencing of critical junctions.
Next, amphotropic retroviruses were created and used to infect MCF10A cells.
To do this, 293T cells were cotransfected using FuGENE 6 with the retroviral
expression vectors described above, together with the appropriate human helper
plasmid (38). The medium was changed 1 day postinfection, and retrovirus-
containing medium was harvested on day 3 postinfection. MCF10A cells were
VOL. 27, 2007 ACTIVATION OF p53 BY MUTATIONS IN PTEN AND PIK3CA663
infected with retrovirus in the presence of 8 ?g/ml Polybrene. G418-resistant
clones were selected for 2 weeks in the presence of 1.0 mg/ml G418, pooled, and
Flow cytometry. Cells were fixed in 70% ethanol and stained in phosphate-
buffered saline containing 0.1% Triton X-100, 50 ?g/ml RNase, and 50 ?g/ml
propidium iodide. The DNA content was measured on a FACSort flow cytom-
eter (Becton Dickinson, Franklin Lakes, NJ), and data were analyzed using
ModFit software (Verity Software House, Topsham, ME). At least 20,000 cells
were analyzed per sample.
Measurement of cell size. Cells were trypsinized in 0.5 ml, added to 0.5 ml of
serum-containing medium, and further diluted in 10 ml of Isoton II. Cell diam-
eters were determined using a Multisizer 3 Coulter Counter (Beckman Coulter,
Fullerton, CA). At least 10,000 cells were counted per measurement.
PIK3CA human somatic cell gene targeting. The PIK3CA targeting vector was
constructed in an adeno-associated virus (AAV) backbone for delivery via in-
fection and was designed to delete exon 2. Exon 2 was chosen because its flanking
exons (which compose the homology arms) contain few repeat elements and
because any rare exon 2-skipping events would drive the message out of frame.
To create this vector, homology arms were PCR amplified from a human
genomic DNA template using VENT high-fidelity polymerase (New England
Biolabs) and sequentially cloned into the AAV-MCS vector (Stratagene, La
Jolla, CA). The cloned homology arms were sequenced to eliminate those with
PCR-generated mutations. Next, a PCR-generated pTK-Neorgene was cloned
between the homology arms. After the junctions were sequenced, the completed
targeting vector was prepped. Transient viral stocks were created by cotransfec-
tion of targeting vector into 293-AAV cells, together with pAAV-RC and
pHELPER, two plasmids that provide proteins needed for viral replication and
packaging (both from Stratagene). The virus was harvested and used to infect
Soft-agar growth assay. Five thousand cells were plated in 0.3% agar layered
on top of 0.6% agar in 35-mm2plates. After 2 weeks, the colonies were stained
with 0.005% crystal violet and counted in an Omnicon 3600 automated colony
counter (BioLogics, Inc., Manassas, VA).
Microarray data accession numbers. The complete microarray data sets have
been submitted to the NCBI Gene Expression Omnibus database under acces-
sion number GSE6263.
Upregulation of p53-regulated genes in PTEN?/?cells. In
an effort to identify genes whose expression is modulated by
the presence or absence of PTEN, microarray analysis was
performed. To do this, we measured the gene expression pro-
files in a previously described isogenic set of HCT116 cells in
which the endogenous wild-type PTEN genes had been deleted
by gene targeting (27). In an effort to eliminate false positives,
we studied two HCT116 PTEN?/?cell lines (parental cells and
a clone with random integration of the targeting vector) and
three independently derived HCT116 PTEN?/?cell lines using
the Affymetrix U133A/B chip set, as described in Materials and
Methods. Data analysis was performed using the BRB Array
Depicted in Tables 1 and 2 are lists of the differentially
expressed genes on the U133A microarray that met the strin-
gent P ? 0.001 statistical-significance cutoff. The fact that three
of the probe sets in Table 2 correspond to PTEN (representing
all three PTEN probe sets on the U133A chip) provides a
valuable internal control, confirming the quality of the data
Two of the genes most highly upregulated in the PTEN?/?
cells were p21WAF1/CIP1and GDF15/MIC-1, two well known
p53-regulated genes (14, 29). To further examine the hypoth-
esis that p53-regulated genes in general were upregulated in
PTEN?/?cells, we examined a more comprehensive list of
differentially expressed genes (which met a slightly less strin-
gent P value cutoff of ?0.005) to identify additional p53-reg-
ulated genes that were upregulated in PTEN?/?cells.
A number of additional p53-regulated genes were identified
(listed in Fig. 1A), including NOXA (increased 2.4-fold; a BH3
motif-containing member of the BCL2 family) (39), PIG3 (in-
creased 2.1-fold; a proapoptotic protein with homology to re-
dox-controlling proteins) (41), and PLK2 (increased 2.1-fold;
confers resistance to spindle damage) (8). These expression
differences were confirmed by qRT-PCR (Fig. 1A). The results
raised the intriguing possibility that deletion of PTEN led to
activation of p53. Of note, it is not surprising that microarray
analysis did not detect an increase in p53 itself in PTEN?/?
cells, since p53 is thought to be regulated primarily at the
Though these experiments strongly suggested that p21,
GDF15, NOXA, PIG3, and PLK2 were upregulated in
PTEN?/?cells through a p53-dependent mechanism, they did
not prove it. To formally demonstrate this, we infected
HCT116 PTEN?/?and PTEN?/?cells with p53 shRNA or
control lentiviruses and established stable pooled clones.
There was an 80% decrease in p53 mRNA in cells infected
with p53 shRNA lentivirus, as measured by qRT-PCR; the
corresponding reduction in p53 protein is depicted in the inset
in Fig. 1B. Next, we employed qRT-PCR to measure the ex-
pression of the five signature p53-regulated genes. As depicted
in Fig. 1B, p53 depletion led to a statistically significant pref-
TABLE 1. Genes increased in PTEN?/?cells (P ? 0.001)
Signal intensity in:
Interferon-induced protein 44
G-protein-coupled receptor 2
G-protein-coupled receptor 2
G-protein-coupled receptor 2
Solute carrier family 7
Ring and zinc finger domain
Proteolipid protein 1
Amyloid beta binding protein
SH3/SH2 adaptor protein
Growth and transformation
Chromosome 12 open
reading frame 5
Heat shock protein
1.9 2,232 4,267
TABLE 2. Genes decreased in PTEN?/?cells (P ? 0.001)
Signal intensity in:
Ras-like small GTPase
RNA binding motif protein 4
Zinc finger protein 574
Similar to tubulin beta 2
664 KIM ET AL.MOL. CELL. BIOL.
erential reduction in expression of each of the five genes in
HCT116 PTEN?/?cells. Of note, p53 depletion virtually
equalized expression of the p21 gene (considered to be a par-
ticularly robust and important p53-regulated gene) in HCT116
PTEN?/?and PTEN?/?cells. We hypothesize that the varia-
tions in the extents of reduction of the other genes are due to
complexities in their transcriptional regulation and/or varia-
tions in their sensitivities to the remaining p53 protein present
in the p53 knockdown cells. When taken together, our exper-
iments provide strong support for the contention that PTEN
deletion leads to the activation of a p53-dependent gene ex-
pression program in human cells.
Upregulation of p53 levels and activity in proliferating
PTEN?/?cells. To more directly test the hypothesis that p53
was activated in PTEN?/?cells, we measured the levels of p53,
p21, pAkt (S473), pan-Akt, and ?-tubulin in proliferating
HCT116 PTEN?/?, PTEN?/?, and PTEN?/?cell lines. To
rule out the possibility of clone-specific artifacts, we studied
two independently derived clones of each genotype. As shown
in Fig. 2A, deletion of PTEN led to upregulation of p53 and
p21 protein levels.
We considered two possible models to begin to explain the
upregulation of p53 levels and activity in PTEN?/?cells. First,
it was possible that deletion of PTEN simply led to upregula-
tion of the basal level and activity of p53. Second, it was equally
possible that deletion of PTEN changed the p53 “set point,”
resulting in an enhancement not only of its baseline level, but
also in the extent to which it could be induced by DNA dam-
age. To distinguish between these two models, we next mea-
sured p53 levels in etoposide-treated HCT116 PTEN?/?,
PTEN?/?, and PTEN?/?cells. As shown in Fig. 2B, the levels
of p53 after induction with etoposide were identical in HCT116
PTEN?/?, PTEN?/?, and PTEN?/?cells. When taken to-
gether with the data depicted in Fig. 2A, this experiment dem-
FIG. 1. Functional activation of p53 responses in PTEN?/?cells. (A) p53-regulated genes whose expression is upregulated in PTEN?/?cells,
as determined by both microarray analysis and qRT-PCR. (B) Formal demonstration of p53 dependence. HCT116 PTEN?/?and PTEN?/?cells
were infected with control (pLKO.1) and p53 shRNA lentiviruses, and the expression levels of the listed p53-regulated genes were measured by
qRT-PCR. The y axis represents the difference (n-fold) in gene expression between HCT116 PTEN?/?and PTEN?/?cells. The extent of p53
depletion as measured by Western blotting is depicted in the inset. As indicated, p53 depletion led to a reduction in the expression of each of the
five genes preferentially in HCT116 PTEN?/?cells. The error bars indicate standard deviations.
VOL. 27, 2007 ACTIVATION OF p53 BY MUTATIONS IN PTEN AND PIK3CA 665
onstrated that PTEN deletion led to an elevation in the basal
levels of p53 during normal growth but did not similarly in-
crease the extent to which p53 could be induced by DNA
Downregulation of p53 in PTEN?/?cells after treatment
with LY294002. To formally implicate the lipid phosphatase
activity of PTEN in its ability to regulate p53, we measured
levels of p53 in HCT116 PTEN?/?cells after treatment with
the PI3K inhibitor LY294002. As expected, treatment of
HCT116 PTEN?/?cells with LY294002 rescued the effect of
PTEN deletion, substantially reducing p53 protein levels (Fig.
3A). This experiment directly implicated the lipid phosphatase
activity of PTEN in its ability to regulate p53.
PI3K inhibition on p53 levels in two related cell lines, A172, a
human glioma cell line with naturally occurring biallelic muta-
tional inactivation of PTEN, and NHA, which represent the
PTEN-proficient precursor cell type for glioma. As shown in Fig.
3B and C, pharmacological inhibition of PI3K led to a reduction
in p53 levels in PTEN-deficient A172 cells, but not in their pre-
cursor PTEN-proficient NHA. These results confirmed the initial
results obtained in HCT116 cells and extended them to a genet-
FIG. 2. Increased expression of p53 and p21 proteins in PTEN?/?cells. (A) p53 and p21 levels in proliferating HCT116 PTEN?/?, PTEN?/?,
and PTEN?/?cells. Immunoblotting was performed with the antibodies indicated. (B) p53 levels in the same cell lines after DNA damage. HCT116
cells with the PTEN genotypes indicated were treated with 5 ?g/ml etoposide for 24 h. Immunoblotting was performed with the antibodies
666KIM ET AL.MOL. CELL. BIOL.
ically unmodified human cell system that differs in the presence
and absence of mutant PTEN genes.
Enhanced p53 protein stability in PTEN?/?cells. p53 levels
are thought to be regulated primarily at the posttranscriptional
level, generally at the level of protein stability. Thus, we tested
if the half-life of p53 was prolonged in PTEN?/?cells. To do
this, we measured the levels of p53 protein in isogenic sets of
proliferating HCT116 PTEN?/?and PTEN?/?cells at various
time points after treatment with cycloheximide to block addi-
tional protein synthesis. As shown in Fig. 4A and B, there was
an approximately fourfold increase in the half-life of p53 fol-
lowing PTEN deletion.
p53 stability is regulated by Hdm2, a ubiquitin ligase that
interacts with and ubiquitinates p53, targeting it for degrada-
tion. Mitogenic stimuli that activate p53 increase the expres-
sion of the Hdm2-binding partner p14ARF, which sequesters
Hdm2 and consequently increases the half-life of p53. To de-
termine if the p53 activation in PTEN?/?cells was dependent
on p14ARF, we measured levels of p14ARFprotein by Western
blotting in HCT116 PTEN?/?and PTEN?/?cells (Fig. 4C).
Using three different commercially available p14ARFantibod-
ies and an effective positive control, it was clear that neither
HCT116 PTEN?/?nor HCT116 PTEN?/?cells express de-
tectable levels of p14ARFprotein, regardless of the length of
the exposure. These results are consistent with a published
report stating that HCT116 cells do not express p14ARFpro-
tein as measured with a single different commercially available
p14ARFantibody (54). Next, we tested whether the expression
of Hdm2 itself might be downregulated in HCT116 PTEN?/?
cells. As depicted in Fig. 4D, there was no reduction in Hdm2
levels in HCT116 PTEN?/?and PTEN?/?cells (in fact, there
was a slight increase, consistent with the fact that Hdm2 ex-
pression is known to be activated by p53). When taken to-
gether, our data suggest that the mechanism of p53 activation
in PTEN?/?cells is independent of p14ARFand is not caused
by a reduction in the levels of Hdm2 protein.
Akt1 dependence on p53 upregulation in PTEN?/?cells.
The mitogenic activity of activated PI3K signaling requires
activation of Akt. We wondered if the ability of activated PI3K
signaling to activate p53 similarly required activation of Akt.
We focused our studies on Akt1, as this is the Akt isoform
most highly expressed in HCT116 cells (44). To test this, we
infected HCT116 PTEN?/?cells with control or Akt1 shRNA
lentiviruses, established pooled clones, and measured levels of
Akt1, p53, and ?-tubulin proteins by Western blotting and
levels of p21 and GDF15 mRNAs by qRT-PCR. Depletion of
Akt1 led to an approximately 50% reduction in p53 protein
levels (Fig. 5A) and a corresponding reduction in the mRNA
FIG. 3. Reduction in p53 levels by LY294002 in PTEN?/?cells. (A) HCT116 PTEN?/?cells were treated with 0, 30, 60, or 90 ?M LY294002
for 24 h. Protein lysates were prepared, and immunoblots were performed with the primary antibodies indicated. (B) A172 cells and (C) NHA were
treated with 0, 2, 10, 30, or 60 ?M LY294002 for 24 h. Protein lysates were prepared, and immunoblots were performed with the primary antibodies
VOL. 27, 2007 ACTIVATION OF p53 BY MUTATIONS IN PTEN AND PIK3CA667
levels of two p53-regulated genes, p21 and GDF15 (Fig. 5B).
Next, we transiently transfected HCT116 PTEN?/?cells with a
myr-Akt expression vector and measured levels of p53 by
Western blotting. There was no increase in p53 levels (not
shown). When taken together, our data indicate that Akt ac-
tivation is necessary but not sufficient for PIP3-induced p53
activation in HCT116 cells.
Upregulation of p53 levels by PTEN depletion in untrans-
formed human cells. To confirm and extend the results ob-
tained in HCT116 cells, we measured the effects of stable
shRNA-mediated depletion of PTEN on p53 levels in telo-
merase-immortalized retinal pigment epithelial cells (RPE-
hTERT) and telomerase-immortalized BJ fibroblasts (BJ-
hTERT). As shown in Fig. 6A, depletion of PTEN led to a
twofold increase in p53 levels in each of these cell lines. There
was no increase in levels of p16INK4a(Fig. 6B).
PTEN depletion causes p53-dependent senescence-like growth
arrest in untransformed human cells. Depletion of PTEN in
FIG. 4. Enhanced p53 stability in PTEN?/?cells. (A) HCT116 PTEN?/?and PTEN?/?cells were treated with 100 ?g/ml cycloheximide (CHX)
for the times indicated. Protein lysates were prepared, and immunoblots were performed with the antibodies indicated. (B) Bands were quantified
using Scion Image densitometry software (Scion Corporation, Frederick, MD), and half-lives were determined using linear-regression analysis.
(C) Protein lysates were prepared from HeLa, HCT116 PTEN?/?, and PTEN?/?cells, and immunoblots were performed with three different
p14ARFprimary antibodies. (D) Protein lysates from two different HCT116 PTEN?/?and HCT116 PTEN?/?cell lines were studied by
immunoblotting with an Hdm2 antibody.
668 KIM ET AL.MOL. CELL. BIOL.
untransformed human cells not only led to p53 activation but
also led to a dramatic morphological change. PTEN-depleted
RPE-hTERT cells became flattened and enlarged, as though
they had undergone senescence (Fig. 6C). Immunofluores-
cence revealed the accumulation of ?-H2AX foci, an important
marker of senescence (Fig. 6C) (46). Attempts to passage the
RPE-hTERT cells revealed that they remained healthy but had
stopped dividing. Flow cytometry confirmed that the cells had
arrested, with a virtual absence of S phase (Fig. 6D). Addi-
tionally, PTEN depletion led to cellular enlargement, a classic
feature of senescence (Fig. 6E). Similar cell cycle and cell size
results were also obtained in PTEN-depleted BJ-hTERT cells
(Fig. 6D and E), though these cells were able to maintain a
viable senescence-like growth arrest for only 1 week. Of note,
attempts to demonstrate elevated levels of senescence-associ-
ated ?-galactosidase activity in these apparently senescent cells
were unsuccessful, perhaps due to the high levels of basal
?-galactosidase activity (data not shown). Taken together,
these results indicate that depletion of PTEN in untrans-
formed human cells can lead to p53 induction and a senes-
cence-like cell cycle arrest.
Next, we tested whether p53 activation was required for the
senescence-like growth arrest caused by PTEN depletion. In
particular, we determined whether codepletion of PTEN and
p53 could rescue the deleterious effects of PTEN depletion
and enable the cells to continue proliferation. To do this, we
coinfected RPE-hTERT cells with various combinations of
lentiviruses encoding PTEN shRNAs, p53 shRNAs, and sev-
eral different negative control shRNAs. After selection in pu-
romycin, pooled clones were studied by Western blotting to
confirm PTEN and/or p53 knockdown (Fig. 7A) and then
studied in several different phenotypic assays to assess the
effects on cellular proliferation and cell cycle control. As pre-
viously demonstrated, stable depletion of PTEN led to perma-
nent growth arrest characterized by a complete absence of
S-phase cells (Fig. 7B and C). However, coinfection with p53
shRNAs, but not control shRNAs, rescued this effect, enabling
the cells to enter S phase, continue proliferation, and maintain
FIG. 5. Effects of Akt1 depletion on p53 activation in PTEN?/?cells. (A) HCT116 PTEN?/?cells were infected with the lentiviruses indicated,
and pooled clones were established and studied by immunoblotting them to document levels of Akt1, p53, and ?-tubulin proteins. (B) HCT116
PTEN?/?and HCT116 PTEN?/?cells were infected with control and Akt1 shRNA lentiviruses, pooled clones were established, and total RNA
was prepared and studied by qRT-PCR to document the relative expression levels of p21 and GDF15. The y axis represents the difference in gene
expression (n-fold) between HCT116 PTEN?/?and PTEN?/?cells. As indicated, Akt1 depletion led to a reduction in p21 and GDF15
preferentially in PTEN?/?cells. The error bars represent standard deviations.
VOL. 27, 2007 ACTIVATION OF p53 BY MUTATIONS IN PTEN AND PIK3CA669
their normal size, despite the absence of PTEN (Fig. 7B to D).
These results were obtained with multiple independent PTEN
and p53 shRNA constructs, and similar cell cycle and cell size
results were obtained in BJ-hTERT fibroblasts (not shown).
These data demonstrate that p53 is necessary for the senes-
cence-like growth arrest seen in untransformed human cells
and caused by PTEN inactivation.
Activation of p53 by ectopic expression of oncogenic
PIK3CA in human cells. Direct mutational activation of PI3K
signaling can occur by either inactivation of PTEN or onco-
genic activation of PIK3CA. Therefore, we tested whether
activation of PIK3CA, like inactivation of PTEN, could lead
to activation of p53 in MCF10A premalignant breast epi-
thelial cells. MCF10A cells were a particularly relevant
model system for these experiments, since oncogenic muta-
tions of PIK3CA are found in a substantial fraction of hu-
man breast cancers (2, 43).
To do this, we created retroviral expression vectors express-
FIG. 6. Effects of PTEN depletion in untransformed human cells. BJ-hTERT fibroblasts and RPE-hTERT epithelial cells were infected with
the lentiviruses indicated, and pooled clones were prepared as described in Materials and Methods. (A and B) Protein lysates were prepared, and
immunoblots were performed with the primary antibodies indicated. p16INK4alevels were undetectable in BJ-hTERT cells. (C) (a and b) pLKO.1-
and PTEN-shRNA-infected RPE-hTERT cells were imaged via phase-contrast light microscopy. The scale bars represent 200 ?m. (c and d)
pLKO.1- and PTEN-shRNA-infected RPE-hTERT cells were double stained for ?-H2AX (green) and DAPI (blue) and imaged by fluorescence
microscopy. The scale bar represents 10 ?m. (D) RPE-hTERT and BJ-hTERT cells were infected with control or PTEN shRNA lentiviruses, flow
cytometry was performed as described in Materials and Methods, and the S-phase fraction was quantified using ModFit software (Verity Software
House, Topsham, ME). (E) The sizes of the cells described in panel C were measured using a Multisizer 3 Coulter Counter (Beckman Coulter,
FIG. 7. p53 is required for the cell cycle arrest caused by PTEN depletion. RPE-hTERT cells were infected in combination with lentiviruses
encoding PTEN shRNAs, p53 shRNAs, or control shRNAs as indicated. (A) Protein lysates were prepared, and immunoblotting was performed
using the primary antibodies indicated. (B) S phase was quantified using ModFit software (Verity Software House, Topsham, ME) after flow
cytometry analysis, as described in Materials and Methods. (C) Cells were harvested and counted every 1 or 2 days to document proliferation.
(D) Cell size was measured using a Multisizer 3 Coulter Counter (Beckman Coulter, Fullerton, CA).
VOL. 27, 2007 ACTIVATION OF p53 BY MUTATIONS IN PTEN AND PIK3CA671
ing various wild-type and mutant forms of human PIK3CA, as
described in Materials and Methods. Next, we created ampho-
tropic human PIK3CA retroviruses, infected MCF10A cells,
established pooled G418-resistant clones, and measured levels
of p53 by Western blotting. As shown in Fig. 8A, there was a
1.7-fold increase in p53 levels in MCF10A cells expressing
oncogenic human PIK3CA. Next, we created total RNA from
the various pooled clones and employed qRT-PCR to measure
the levels of two p53-regulated genes, p21 and GDF15. As
shown in Fig. 8B, there was a substantial increase in the ex-
pression of both genes in cells expressing oncogenic, but not
wild-type, PIK3CA. Importantly, this increase was reversed by
coexpression of a p53 shRNA, formally demonstrating its p53
dependence. Taken together, these studies demonstrate that
ectopic expression of oncogenic PIK3CA can induce p53 levels
and activity in human breast epithelial cells.
Modulation of p53 levels and activity by endogenous onco-
genic PIK3CA in human cancer cells. We were intrigued by
our results demonstrating that ectopic overexpression of on-
cogenic PIK3CA could lead to p53 activation but were con-
cerned that it could be an artifact of overexpression. To rule
out this possibility, we pursued an approach based on PIK3CA
human somatic cell gene targeting to measure the effects of the
endogenous, naturally occurring allele of oncogenic PIK3CA
present in HCT116 cells on p53 levels and activity (44).
First, we employed human somatic cell gene targeting to
create clonal derivatives of HCT116 cells in which either the
wild-type or the oncogenic allele of PIK3CA had been deleted.
To do this, we created a human AAV-PIK3CA targeting vector
designed to delete exon 2, as described in Materials and Meth-
ods and depicted in Fig. 9A. Of note, it has recently been
demonstrated that delivering targeting vectors to human cells
via infection with recombinant AAVs can increase the effi-
ciency of targeted integration, obviating the need for a pro-
moterless architecture (18, 25).
Using this gene-targeting virus, we obtained derivatives of
FIG. 8. Activation of p53 by oncogenic PIK3CA. (A) MCF10A cells stably expressing early-stop (W11STOP), wild-type (WT), and oncogenic
FLAG-PIK3CA (H1047R) were harvested. Immunoprecipitation and immunoblotting were performed as described in Materials and Methods with
the antibodies indicated. (B) Total RNA was prepared from the indicated pooled clones, and qRT-PCR was employed to measure levels of p21
and GDF15. The error bars represent standard deviations.
672 KIM ET AL.MOL. CELL. BIOL.
HCT116 cells in which either the oncogenic allele or the wild-
type allele had been deleted (Fig. 9B and C). HCT116 parental
cells and derivatives with deletion of wild-type PIK3CA had
similar morphologies, with mesenchymal features and a scat-
tered, apparently contact-independent growth pattern (Fig.
9D). In contrast, deletion of the oncogenic allele of PIK3CA
led to a dramatic morphology change—the cells assumed a
more epithelium-like morphology and grew in well-organized,
FIG. 9. PIK3CA gene targeting. (A) Homologous recombination between the genomic locus and the AAV targeting vector deletes exon 2 and
replaces it with a thymidine kinase (TK) Neorgene. The PCR primers used for identification of knockouts are indicated, as are the restriction
enzyme cleavage sites and the probe used for Southern blotting-based confirmation of knockouts (KO). (B) Confirmation of PIK3CA targeting
by Southern blot analysis. Fragments corresponding to the untargeted allele and the targeted allele are shown. (C) Sequence-based identification
of the untargeted allele. PCR products from the untargeted allele were sequenced to determine which allele had been deleted in PIK3CA
gene-targeted cells. Asterisks indicate the locations of the PIK3CA mutation A3140G (H1047R), and N denotes heterozygosity. (D) Morphological
features of PIK3CA gene-targeted cells: (a) HCT116 parental cells and derivatives with (b) oncogenic PIK3CA deleted and (c) wild-type PIK3CA
deleted. The scale bar represents 200 ?m.
VOL. 27, 2007 ACTIVATION OF p53 BY MUTATIONS IN PTEN AND PIK3CA673
apparently contact-dependent colonies. A similar morphology
change has been reported by ourselves and others to occur
after targeted deletion of oncogenic K-Ras from several hu-
man cancer cell lines (5, 24, 48).
Next, we measured the levels of p53 and p21 in proliferating
HCT116 cells and PIK3CA gene-targeted derivatives. As de-
picted in Fig. 10A, deletion of the oncogenic, but not the
wild-type, allele of PIK3CA led to an approximately twofold
decrease in p53 and p21 protein levels, as well as the expected
reduction in levels of p-Akt (44). Next, we created total RNA
from the various gene-targeted cell lines and measured levels
of the p53-regulated genes p21 and GDF15 by qRT-PCR (Fig.
10B). Deletion of oncogenic, but not wild-type, PIK3CA led to
substantial reduction in the expression levels of both genes.
Taken together, these studies clearly demonstrate that endog-
enous oncogenic PIK3CA can regulate p53 levels and activity
in a human cancer cell line.
Activation of p53 by oncogenic PIK3CA can have functional
consequences. Finally, we tested whether activation of p53
signaling by oncogenic PIK3CA had functional consequences.
To do this, we returned to our MCF10A model system and
infected MCF10A parental cells with various combinations of
PIK3CA retroviruses, p53 shRNA lentiviruses, and/or appro-
priate control viruses. After double selection in G418 and
puromycin, pooled clones were established and studied. The
extent of p53 depletion after infection with the p53 shRNA
lentivirus was similar to that shown for RPE-hTERT cells in
Fig. 7A. Expression of oncogenic PIK3CA in MCF10A cells
conferred the ability to form colonies in soft agar (Fig. 11A and
B) (20). Depletion of p53 led to a twofold increase in colony
number and an appreciable increase in colony size (Fig. 11A
and B). Of note, p53 depletion alone was unable to confer
anchorage-independent growth. Taken together, these results
demonstrate that mutational activation of PIK3CA can syner-
gize with p53 inactivation in the transformation of human cells.
Here, we have demonstrated that activation of the PI3K
signaling pathway via mutations in PTEN or PIK3CA can lead
to functional activation of p53 signaling in human cells. We
further demonstrated that PI3K-induced p53 activation can
manifest itself as senescence-like growth arrest in untrans-
formed human cells. Our results are consistent with the recent
report by Chen et al. demonstrating that PTEN inactivation
can lead to p53-induced senescence in mouse prostate tumor-
FIG. 10. Effects of PIK3CA deletion on p53 levels and activity. (A) Protein lysates from HCT116 parental cells and PIK3CA gene-targeted cells
were prepared, and immunoblotting was performed using the primary antibodies indicated. (B) Total RNA was prepared from HCT116 parental
cells and PIK3CA gene-targeted derivatives, and qRT-PCR was employed to measure the levels of p21 and GDF15. The error bars represent
674KIM ET AL.MOL. CELL. BIOL.
igenesis (11) and suggest that p53 mutations may be selected
for during human cancer pathogenesis as a way to alleviate
senescence caused by mutational activation of PI3K signaling.
This conclusion is also consistent with at least three other
pieces of data generated in several other laboratories. Mitsuuchi
et al. demonstrated that inhibition of PI3K can lead to dimi-
nution of p21 expression (36). Barbieri et al. demonstrated that
pharmacological inhibition of PI3K signaling can lead to a
reduction in the levels and activity of the p53 homologue p63
(4). Finally, Bar et al. demonstrated that pharmacological in-
hibition of PI3K can lead to a dramatic diminution of the
effects of DNA damage on activation of p53 (3).
Our data have several potentially important implications for
both the PI3K and p53 fields. It has long been known that p53
protein is activated during the process of human tumorigene-
sis. However, the specific nature of the “natural” inducer of
p53 has been controversial. For example, though radiation and
chemotherapeutic drugs can induce p53 activity, it is also true
that p53 mutations routinely occur in untreated tumors. It has
similarly been shown that hypoxia can lead to p53 activation,
but it is also true that p53 mutations routinely occur in “liquid”
tumors, such as leukemias and lymphomas. Finally, activated
oncogenes, such as Ras and Myc, can clearly induce p53, but
this induction has generally been shown in murine systems,
often employing ectopic overexpression of the oncogene.
In contrast, our studies implicating PI3K activation as an
important inducer of p53 have the following comparative
strengths: (i) they implicate a commonly activated pathway as
the upstream inducer; (ii) several of the most potent inducers
of p53 (e.g., DNA damage and oncogenes) are known to also
lead to PI3K pathway activation; (iii) PI3K pathway activation,
though it occurs relatively late during human cancer pathogen-
esis, is still thought to precede p53 mutation; and (iv) our
gene-targeting studies directly implicate endogenous PTEN
and PIK3CA as p53 inducers in both untransformed and trans-
formed human cells. Therefore, we believe that our studies
provide a compelling argument pointing to mutational activa-
tion of PI3K signaling as a particularly important inducer of
p53 in human cells.
Our data also have potentially important clinical implica-
tions. Much work is currently under way to develop PI3K
inhibitors as potential anticancer therapeutics as a means to
treat tumors harboring mutations in PTEN and PIK3CA.
Though this is clearly an appealing strategy, our data predict
that, when used in a p53-proficient setting, pharmacological
inhibitors of PI3K might also have the adverse effect of alle-
viating p53-dependent tumor suppression. As such, it may be
informative to stratify clinical trials of PI3K inhibitors based on
p53 genotype, as our studies predict that PI3K inhibitors could
be more efficacious in treating p53-deficient tumors.
Another prediction of the data presented here is that acqui-
sition of mutations in PTEN or PIK3CA during human cancer
pathogenesis would provide selective pressure for the acquisi-
tion of mutations in p53. Consistent with this prediction, many
of the tumor types in which p53 mutations are most commonly
found (e.g., adenocarcinoma of the colon, adenocarcinoma of
the breast, and glioblastoma) also harbor mutations of PTEN
or PIK3CA. Furthermore, Oki et al. have demonstrated in
human gastric cancer specimens that tumors with inactivating
mutations of PTEN are statistically more likely to also harbor
mutations in p53 than tumors in which PTEN remains intact
Our data also raise a number of intriguing questions. For
example, we have demonstrated that activation of PI3K signal-
ing by PTEN depletion leads to a senescence-like growth arrest
in untransformed human cells, but we also clearly showed that
activation of PI3K signaling can have transforming effects in
other human cell types (e.g., MCF10A cells, HCT116 cells, and
a variety of other human cancer cell lines with PTEN or
PIK3CA mutations used in this and other studies). At first
inspection, these data appear to suggest that PI3K activation
leads to senescence in untransformed human cells but not in
partially or completely transformed cell lines. We believe that
the mechanism(s) for this difference in phenotypic response
will likely be an interesting avenue for further study.
It will also be informative to identify the specific biochemical
mechanism of p53 activation by activated PI3K signaling. In
FIG. 11. Activation of p53 by oncogenic PIK3CA. MCF10A cells
stably expressing early-stop (W11STOP), wild-type (WT), and onco-
genic (H1047R) PIK3CA and p53 shRNAs or control shRNAs as
indicated were plated in soft agar and grown for 2 weeks, and the
colonies were stained with 0.005% crystal violet. The colonies were
photographed (A) and counted (B) as described in Materials and
Methods. The scale bar represents 1.0 mm.
VOL. 27, 2007 ACTIVATION OF p53 BY MUTATIONS IN PTEN AND PIK3CA 675
this study, we have demonstrated that Akt1 is an important
upstream inducer of p53 activation. We also demonstrated that
p14ARFis undetectable in cells in which p53 is activated in
response to activated PI3K signaling, suggesting that p14ARFis
dispensable. Further detailed biochemical and genetic studies
will be required to provide more insight into this important
regulatory relationship. It may also prove informative to de-
termine if the well-known capacity of the PI3K pathway to
undergo autoregulation may impact its ability to regulate p53
In summary, we have demonstrated here that activation of
PI3K signaling by mutations in PTEN or PIK3CA can activate
p53-dependent growth suppression in human cells. When
taken together with other recent studies, our data suggest that
PI3K signaling may be a central node in the regulation of p53
(Fig. 12). Additional work is required to provide the detailed
genetic and biochemical mechanisms for this effect and to
further implicate PI3K signaling as an important endogenous
inducer of p53 in human cancer.
We thank Susette Mueller for assistance with microscopy, Karen
Cresswell and Michelle Lombard for assistance with flow cytometry,
Xiaojun Zou for assistance with microarray processing, Michael John-
son for advice on performing soft-agar assays, and Annabell Oh for
advice on qRT-PCR.
This work was supported by National Institutes of Health grants K01
CA087828 and R01 CA115699 to T.W., American Cancer Society
RPG MGO-112078 to T.W., and the Lombardi Comprehensive Can-
cer Center Support grant P30 CA051008. C.L.B. and C.L. were sup-
ported in part by NIH training grant T32 CA009686.
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