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.
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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FIG. 12. Model of p53 activation by PIP3signaling. Inactivation of
PTEN or activation of PIK3CA leads to an increase in cellular levels
of PIP3and subsequent activation of Akt. This leads to simultaneous
activation of mitogenesis and activation of p53-dependent cellular
senescence. Oncogenes are depicted in red and tumor suppressor
genes in blue.
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