Published OnlineFirst August 12, 2013; DOI: 10.1158/1535-7163.MCT-12-0920
Molecular
Cancer
Therapeutics
Cancer Therapeutics Insights
PI3K Stimulates DNA Synthesis and Cell-Cycle Progression
via Its p55PIK Regulatory Subunit Interaction with PCNA
Guihua Wang1,2, Xiaonian Cao1, Senyan Lai1, Xuelai Luo1, Yongdong Feng2, Xianmin Xia1,
Paul M. Yen3, Jianping Gong2, and Junbo Hu1
Abstract
Previously, we have shown that p55PIK, an isoform of class IA phosphoinositide 3-kinase (PI3K),
specifically interacts with important cell-cycle regulators, such as retinoblastoma (Rb), to promote cellcycle progression. Here, we used the glutathione S-transferase pull-down assay to identify other p55PIKinteracting proteins besides Rb in a Rb-deficient cell line and found that p55PIK interacted with
proliferation cell nuclear antigen (PCNA), which plays a key role in coordinating both initiation of the
leading strand DNA replication and discontinuous lagging strand synthesis. Overexpression of p55PIK
increased, and knockdown decreased, DNA synthesis and DNA replication by modulating the binding of
DNA polymerase d (Pold) to PCNA. Moreover, a cell-permeable peptide containing the N-terminal–
binding domain of p55PIK (TAT–N24) disrupted the p55PIK–PCNA interaction in cancer cells, and also
inhibited the DNA synthesis and tumor growth in cell culture and in vivo. Altogether, our results show that
the p55PIK–PCNA interaction is important in regulating DNA synthesis and contributes to tumorigenesis.
Furthermore, the p55PIK–PCNA interaction provides a potential new target for anticancer drug development. Mol Cancer Ther; 12(10); 2100–9. 2013 AACR.
Introduction
Class IA phosphoinositide 3-kinase (PI3K) signaling
critically regulates many cellular processes and is involved in the initiation and progression of tumors (1, 2).
PI3K consists of a p110 catalytic subunit (a, b, or d) that
dimerizes with one of the several regulatory subunits
(p85a, p85b, p55a, p55g, or p50a). There are three genes
encoding these regulatory subunits: PIK3R1, PIK3R2, and
PIK3R3. p85a, p55a, and p50a are products of PIK3R1,
whereas p85b and p55g (also named p55PIK) are products
of PIK3R2 and PIK3R3, respectively (2, 3). Regulatory
subunits have no enzymatic activity, but are needed to
recruit p110 subunits to specific cellular sites and thus
regulate the catalytic activity of PI3K (4, 5). All regulatory
subunits contain two SH2 domains as well as an intermeAuthors' Affiliations: 1Cancer Research Institute; 2Molecular Medical
Center, Tongji Hospital, Huazhong University of Science and Technology,
Wuhan, China; and 3Laboratory of Hormonal Regulation, Cardiovascular
and Metabolic Disease Program, Duke-National University of Singapore
Graduate Medical School, Singapore, Singapore
Corresponding Authors: Junbo Hu, Cancer Research Institute, Tongji
Hospital, Huazhong University of Science and Technology, 1095 Jiefang
Road, Wuhan 430030, China. Phone: 86-27-83665295; Fax: 86-2783662696; E-mail: [email protected]; Jianping Gong, Molecular Medical Center, Tongji Hospital, Huazhong University of Science and Technology, Wuhan 430030 China. E-mail: [email protected]; and Paul
M. Yen, Laboratory of Hormonal Regulation, Cardiovascular and Metabolic
Disease Program, Duke-National University of Singapore Graduate Medical School, 1095 Jiefang Road, Singapore, 540000 Singapore. E-mail:
[email protected]
doi: 10.1158/1535-7163.MCT-12-0920
2013 American Association for Cancer Research.
2100
diate iSH2 domain that binds to the p110 catalytic subunit.
Regulatory subunits have distinct N-terminal sequences
that enable the specific regulation of different PI3K signaling pathways (6, 7).
The association of PI3K catalytic subunits (p110) with
cancer development and progression is well documented (8). The PIK3CA gene is mutated in many cancers,
including colon, thyroid, and breast cancers (8, 9).
PIK3CB is the principal catalytic isoform involved in
mediating PTEN-deficient tumorigenesis (10). PIK3CD
has been used as a key therapeutic target for hematologic malignancies (11). In contrast, our current understanding of the role(s) of specific PI3K regulatory subunits in cancer is limited. So far, there have been only a
few reports describing the possible roles of PIK3R1
(encoding p85a, p55a, and p50a) and PIK3R3 (encoded
p55PIK) in tumorigenesis (12, 13).
Previously, we showed that the p55PIK regulatory
subunit was important for cell proliferation and tumor
growth, and was overexpressed in some cancers (14, 15).
The unique 24-amino acid N-terminal domain of
p55PIK (N24) specifically binds to important cell-cycle
regulators such as retinoblastoma (Rb) protein. N24
peptide expression inhibits the cell-cycle progression
and induces cell differentiation by blocking the p55PIK
interaction with Rb (16–18). Recently, we showed that
non-Rb proteins can also interact with p55PIK and
regulate the cell-cycle progression, particularly during
the S-phase, when DNA synthesis occurs in the cell
cycle. In this article, we report that p55PIK specifically
interacts with proliferation cell nuclear antigen (PCNA)
Mol Cancer Ther; 12(10) October 2013
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Published OnlineFirst August 12, 2013; DOI: 10.1158/1535-7163.MCT-12-0920
PI3K–PCNA Interaction Stimulates DNA Synthesis
through the N-terminal end of p55PIK (N24) and this
interaction regulates the DNA synthesis in cancer cells
by modulating the PCNA interaction with DNA polymerase d (Pold). We also show that the p55PIK–PCNA
interaction is important in tumorigenesis and its inhibition with a novel peptide inhibitor decreases tumor
growth.
Materials and Methods
Cell culture, plasmid and adenoviral constructs, and
transfection
The culture of thyroid carcinomas cell line FTC236 and
colon cancer cell line HT29 was described previously (17)
and all cell lines were authenticated. Adenovirus GFP and
p55PIK–GFP were constructed as following. The pTrackcytomegalovirus (CMV), Ad-Easy1, and Escherichia coli
bacteria BJ5183 were purchased from Stratagene. The
cDNA-encoding p55PIK was first amplified from pFLAG-CMV 4 (Sigma) using primers CCGGTACCACCATGTACAATACGGTGTGG and CCCTCGAGTTATCTGCAAAGCGAGGGCAT and then cloned into the EcoR V
site of the pTrack–CMV vector. The PmeI-linearized
pTrack–CMV plasmid was mixed with adenoviral backbone AdEasy1 to cotransform into E. coli BJ5183. The
recombinant adenovirus vector expressing p55PIK was
purified to transfect HEK293A cells, and the production of
adenovirus particles was confirmed by visualizing the
GFP signals in the cells. The amplification and purification
of recombinant virus was conducted as described previously (17).
The vector expressing p55PIK was constructed in
pcDNATM6/myc-His by cloning cDNA encoding p55PIK
using primers (forward: CGGCTAGCATGTACAATACGGTGTGGAGTAT and reverse: CCCTCGAGTTATCTGCAAAGCGAGGGCAT) and the vector expressing PCNA
in bacteria was constructed in pGEX-4T-1 using primers
for PCNA (forward: CGGGATCCATGTTCGAGGCGCGCCTGGT and reverse: CCCTCGAGCTAAGATCCTTCTTCATCC). For the purification of p55PIK and PCNA
proteins, bacteria were lysed in 8 mol/L urea and the
affinity purification of His-p55PIK and glutathione Stransferase (GST)–PCNA was carried out according to
the standard protocols. The purified fusion proteins were
dialyzed against PBS (pH 9.5), filtered, and stored in
aliquots at 4 C.
Flow cytometry for cell-cycle analysis and BrdUrd
incorporation
For the flow cytometry analysis, cells were detached by
trypsinization, washed twice with cold PBS, and resuspended in 80% ethanol for at least 30 minutes at 20 C.
Then, cells were washed once with PBS and incubated on
ice for 10 minutes with propidium iodide (PI; 50 mg/mL).
Finally, the stained cells were analyzed using FACSCalibur from BD Biosciences. Aggregated cells revealed by
forward scattering were filtered out of the dataset before
the analysis. To determine G0–G1, S, and G2–M populations, the settings for 2N and 4N peaks were defined
www.aacrjournals.org
within each experiment and applied to all samples within
a given experiment.
Bromodeoxyuridine (BrdUrd) incorporation was
done as described previously (17). Briefly, cells were
cultured in a 6-well plate overnight, then infected with
Ad-GFP or Ad-p55PIK–GFP adenovirus for 48 hours,
followed by 30-minute incubation of BrdUrd at 10mg/
mL. After aspirating the medium, the cells were immediately fixed for more than 8 hours at 20 C. After
immunostaining using the BrdUrd antibody, the DNA
synthesis rate was assessed by calculating the percentage of BrdUrdþ cells out of the total cell count. Three
biologic repeats were carried out for the final statistical
analysis.
Real-time PCR, Western blot analysis,
immunostaining, and immunoprecipitation
Procedures involving human subjects were approved
by the Huazhong University of Science and Technology
Ethics Committee and the formal form was explained to
each subject for full understanding and consent. All animal experiments were carried out by following the Animal Study Guidelines of Huazhong University of Science
and Technology (Wuhan, China). Human tissue samples
were pathologically separated as normal or tumor tissues.
Total RNA was isolated using the TRIzol method by
following the manufacture’s protocols. Reverse transcription and real-time PCR were conducted as described
previously (17).
The Western blot analysis was conducted as described
previously (17). Antibodies to Akt1/2/3, p-Akt(Thr308),
p55PIK, p85a, p85b, p110a, p110b, a-tublin, lamin B,
BrdUrd, anti-mouse immunoglobulin G (IgG), and PCNA
were purchased from Santa Cruz Biotechnology. Antibodies to Ki-67 and to DNA Pold were from BD and Sigma,
respectively.
Immunostaining was carried out by Guge Biotech.
Briefly, cells were seeded onto a coverslip overnight. The
coverslips were then washed twice with PBS and fixed in
4% paraformaldehyde for 30 minutes at room temperature. Immunostained images were acquired and merged
by confocal laser scanning microscopy (LSM-410; Carl
Zeiss).
For immunoprecipitation (IP), total protein extracted
from 1 107 cultured cells was incubated for 1 hour at 4 C
with 10 mL Protein A/G beads. Then, beads were removed
and anti-p55PIK or anti-PCNA antibodies (Santa Cruz
Biotechnology; 1:100 mouse) and mouse anti-IgG antibodies (Santa Cruz Biotechnology; 1:100) were added to
incubate overnight at 4 C. Next, 50 mL protein A/G beads
were added to incubate for additional 5 hours. Finally,
beads were collected, washed five times with PBS, and
mixed with 2 SDS-PAGE sample buffer, and the collected supernatant was boiled for 5 minutes for the Western
blot analysis to detect p85a, p85b, p110a, p110b, and DNA
Pold. For quantitative immunoprecipitation, different
amounts of total protein were loaded for the Western blot
analysis.
Mol Cancer Ther; 12(10) October 2013
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Wang et al.
Animal study
Four- to six-weeks athymic female nude mice weighing
15 to 20 g were obtained from Shanghai Laboratory
Animal Center. Mice were fed under specific pathogenfree conditions in a temperature- and humidity-controlled
environment. All animal experiments were in accordance
with the Institutional Animal Research Guidelines
approved by the Ethics Committee. For tumor-growth
studies, HT29 cells with minimal expression of Rb
(HT29Rb) were established by stably transfecting HT29
cells with pcDNA–p55PIK vectors; control HT29 cells
were stably transfected with pcDNA vectors. Cells were
collected, washed, and resuspended in the culture medium and then injected subcutaneously into each mouse.
Tumor size was measured every other day from the day
tumors were visible. Mice were sacrificed at indicated
times after inoculation and the tumors were removed for
analysis.
The expression and purification of TAT–N24 and control TAT–N24M fusion proteins used in the animal study
have been described previously (18).
Lipid emulsion injection was formulated from equal
volumes of TAT–N24 or TAT–N24M proteins and
injectable lipid emulsion (30% Intralipid; Sino-Swed Pharmaceutical Corp, Ltd.). For experiments investigating the
TAT–N24 effect on the xenograft tumor growth, mice
were injected with 200 mL TAT–N24 or TAT–N24M lipid
emulsion via tail vein on the same day shortly after tumor
inoculation. The mice in each group, consisting of 4 to 6
animals, received injections of 200 mL protein–lipid emulsion. Injections were repeated every 2 days for 10 to 20
days.
Statistical analysis
The data are presented as mean SD. P values were
calculated using an unpaired Student t test.
Results
PI3K interacts with PCNA through the N-terminal
ends of p55PIK (N24 domain)
We used the GST pull-down assay to identify proteins
other than Rb that interacted with the N24 domain of
p55PIK. For this purpose, we incubated the cell lysates
from FTC236 cells that do not express detectable Rb (17)
with His-tagged TAT–N24 fusion proteins (18), and
pulled down bound proteins with Niþþ-agarose bead
columns. One of the identified proteins was PCNA, which
is known to play important roles in the DNA synthesis (19,
20). The interaction of PCNA with N24 was specific as the
mutant N24 did not bind PCNA (Fig. 1A). In addition, the
PCNA interaction with N24 was direct as the purified
GST–PCNA protein could bind to N24 in vitro (Fig. 1B).
We next examined whether the full-length p55PIK
binds to PCNA in the cell culture and in vivo. First, we
incubated purified GST–PCNA proteins with His-p55PIK
and determined that indeed, GST–PCNA interacts with
p55PIK in an in vitro binding assay (Fig. 1C). Because there
are several isoforms of PI3K regulatory subunits, we
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Mol Cancer Ther; 12(10) October 2013
examined whether the p55PIK–PCNA interaction was
specific for p55PIK. Only p55PIK interacted with PCNA
as other members of PI3K regulatory subunit family, such
as p85a and p85b, did not bind with PCNA in FTC236
cells, consistent with the notion that p55PIK interaction
with PCNA was mediated by its unique N24 domain in
p55PIK (Fig. 1D). Moreover, the presence of p110b catalytic subunit of PI3K among the PCNA-bound proteins
complexes strongly suggests that dimerized p110/P55
PI3K interacts with PCNA in cells. Furthermore, immunofluorescent staining using specific antibodies confirmed the colocalization of PCNA and p55PIK in the
nuclei of cultured cancer cells (Fig. 1E).
Overexpression of p55PIK increased DNA Pold
bound with PCNA and stimulated DNA synthesis
To identify the roles of p55PIK–PCNA interaction
in cell-cycle progression, we overexpressed p55PIK in
Rb-deficient FTC236 cells to prevent any concomitant
p55PIK–Rb interaction. We transformed FTC236 cells using
an adenovirus construct (Ad-p55PIK–GFP) that separately
expressed GFP and full-length p55PIK under the control of
two CMV promoters. The adenovirus construct expressing
only GFP (Ad-GFP) was used as a vector control. We
analyzed cells by fluorescence-activated cell sorting 48
hours after the infection, and found little change in the
cell-cycle distribution of cells infected with Ad-GFP compared with noninfected control cells. In contrast, treatment
of cells with Ad-p55PIK–GFP caused a significant increase
in the cell number at S-phase (33.8% S-phase cells in vector
control group vs. 40% in cells overexpressing p55PIK).
More importantly, the expression of p55PIK increased
the number of cells that incorporated BrdUrd (Fig. 2A),
showing that p55PIK increases the DNA synthesis.
PCNA plays a central role in coordinating both initiation of the leading strand DNA replication and the discontinuous lagging strand synthesis during the DNA
synthesis. During the DNA synthesis process, the amount
of DNA Pold binding to PCNA determines the rate of the
DNA synthesis (19). Accordingly, we examined the association of Pold to PCNA in FTC236 cells that overexpressed p55PIK. Overexpression of p55PIK had no effects
on the protein expression of PCNA and Pold (Fig. 2B).
However, p55PIK significantly increased the Pold binding
to PCNA (Fig. 2C), indicating that the p55PIK–PCNA
interaction may enhance the Pold association with PCNA,
leading to increased DNA synthesis in cells overexpressing p55PIK. Consistent with these observations, p21
protein, which inhibits the cell proliferation during the
cell cycle, also was decreased in FTC236 cells overexpressing p55PIK (Fig. 2B).
Knockdown of p55PIK inhibited cell-cycle
progression by blocking DNA synthesis
Next, we examined the effects of p55PIK on cell-cycle
distribution in Rb-deficient FTC236 cells after the knockdown by siRNA against p55PIK. Data in Fig. 3A showed
an increased number of cells in the S-phase. Results from
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Published OnlineFirst August 12, 2013; DOI: 10.1158/1535-7163.MCT-12-0920
PI3K–PCNA Interaction Stimulates DNA Synthesis
A
Ni ++ agarose
B
Ni ++ agarose
Pull down
Pull down
24
N
T–
TA
te
sa
Ly
M
24
N
–
T
TA
WB: αPCNA
M
24
24
N
N
–
–
T
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Ly
GST–PCNA
WB: αPCNA
C
IP
D
te
sa
Ly
Ni ++ agarose pull down
WB: αPCNA
A
CN
αP
G
Ig
WB:
αp55PIK
WB: αp55PIK
GST–PCNA
–
+
+
His-p55PIK
+
+
–
E
αp110β
αp85α
αp85β
αPCNA
αp55PIK
Merge
Figure 1. PI3K interacts with PCNA directly through the N-terminal ends of p55PIK (N24 domain). A, association of PCNA with N24. Lysates from FTC236 cells
þþ
were incubated with TAT–N24 or TAT–N24M. fusion proteins. Ni agarose-bound proteins were analyzed by Western blotting (WB) using a monoclonal
anti-PCNA antibody. B, interaction of GST-PCNA with TAT–N24. Purified GST-PCNA was incubated with TAT–N24 or TAT–N24M for 2 hours, Niþþ
agarose was added and pull-down proteins were analyzed by Western blotting using a monoclonal anti-PCNA antibody. C, interaction of recombinant
His-p55PIK fusion protein with GST-PCNA in vitro. E. coli–expressed and purified fusion proteins His-p55PIK and GST-PCNA were mixed and incubated
for 2 hours; Niþþ agarose pull-down proteins were analyzed by Western blotting using monoclonal anti-PCNA antibody and anti-p55PIK antibody.
D, specific interaction of p55PIK and PCNA in cultured cells. FTC236 cell lysates were immunoprecipitated (IP) with an anti-PCNA antibody or nonspecific
mouse IgG. Immunoprecipitated proteins were analyzed by Western blotting using anti-p55PIK antibody, anti-p85a antibody, anti-p85b antibody or antip110b antibody as indicated. E, colocalization of p55PIK and PCNA in cultured cells. FTC236 cells on coverslips were stained with a monoclonal anti-PCNA
antibody and fluorescein-labeled rabbit anti-mouse IgG (green) and with an anti-p55PIK antibody and phycoerythrin-labeled sheep anti-goat IgG (red). The
images were taken on a laser-scanning confocal microscope as described. Areas of colocalization appear yellow.
BrdUrd incorporation assay also showed that the DNA
synthesis in p55PIK knockdown cells was inhibited. Data
from the cell-cycle distribution analysis showed that the
knockdown of either p55PIK or PCNA in FTC236 cells led
to cell-cycle arrest at S and G2–M phases (Fig. 3A and B).
Of note, the knockdown of PCNA increased the cell death
and apoptosis, whereas p55PIK did not significantly affect
these processes (Fig. 3A and B). These results are consistent with our previous observations that p55PIK signaling
did not play any significant role in the apoptosis (17).
However, because the knockdown of PCNA significantly
increased the apoptosis and cell death, PCNA is likely to
be involved in other pathways, in addition to the pathway
(s) mediated by the p55PIK–PCNA interaction, to regulate
the cell-cycle progression and/or prevent cell death.
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p55PIK stimulates tumorigenesis in vivo
We initially created a cell line that had minimal expression of Rb protein in HT29 colon cancer cells using short
hairpin RNA (shRNA) against Rb (HT29Rb cells). To further examine the role of p55PIK-mediated Rb-independent
interactions in tumorigenesis, we established a cell line
stably overexpressing p55PIK by transforming HT29Rb
cells with Ad-p55PIK in HT29Rb cells (HT29Rb-p55PIK; Fig.
4A).
We then injected HT29Rb-p55PIK cells subcutaneously
into athymic nude mice and tumors were harvested after
12 days. Mice injected with HT29Rb-p55PIK cells had larger
tumors than mice injected with control HT29Rb cells (Fig.
4B). The immunohistochemical analysis of tumors confirmed the increased expression of p55PIK and showed
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Ad
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S: 40.0 %
P
S: 33.8 %
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Wang et al.
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S: 34.0 %
Cell-cycle analysis
p55PIK
R2: 33.2%
R2: 29.3%
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R2: 46.4%
50
BrdUrd+ cells (%)
GFP
GAPDH
DNA synthesis
30
20
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B
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WB: DNA Polδ
WB: PCNA
WB: PCNA
WB: DNA Polδδ
IP: αPCNA
WB: GAPDH
Figure 2. Overexpression of p55PIK increased Pold bound with PCNA and stimulated DNA synthesis. A, left, increased p55PIK expression. Cultured FTC236
cells were infected with Ad-p55PIK-GFP or control Ad-GFP adenovirus. Lysates from parental cells (control), Ad-GFP–infected cells, and Ad-p55PIK-GFP–
infected cells were loaded on SDS-PAGE gels and p55PIK protein expression was assessed by Western blotting (WB) using anti-p55PIK antibody.
Top, right, overexpressing p55PIK increased the cell-cycle progression. Cell-cycle analyses were conducted on uninfected FTC236 cells, cells infected with
Ad-GFP or Ad-p55PIK-GFP as described in Materials and Methods. Representative histograms from an individual experiment and similar results were
obtained in three independent experiments. Overexpression of p55PIK stimulated the DNA synthesis. FTC236 cells were infected with the Ad-p55PIK-GFP or
Ad-GFP. BrdUrd was added 48 hours after infection, and cells were cultured for an additional 30 minutes. Cells then were collected and immunostained for
BrdUrd and counterstained for nucleic acid with PI. BrdUrd incorporation into DNA and DNA content in nuclei were determined by flow cytometry
analysis. Representative data from an individual experiment; similar results were obtained in three independent experiments; the data are presented
þ
quantitatively with error bars and appropriate statistical analysis ( , P < 0.01). Note that the percentage of cells in active DNA synthesis is defined as BrdUrd
cells/total cells (the events in R2 area/total events). B, effects of overexpressing p55PIK on p21, PCNA, and Pold proteins in FTC236 cells.
Cellular lysates were prepared and proteins were analyzed by Western blotting using antibodies against p21, PCNA, Pold, and p55PIK as described in
Materials and Methods. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression also was analyzed with an appropriate antibody to show the equal
loading of proteins in every well. C, overexpressing p55PIK increased the association of PCNA with Pold in FTC236 cells. Cellular lysates from cells
overexpressing p55PIK or control cells were prepared and immnunoprecipitated with anti-PCNA antibodies; Pold proteins associated with PCNA were
analyzed by Western blotting using antibodies against PCNA and Pold.
that p55PIK overexpression increased Ki-67, a marker of
cell proliferation (Fig. 4C).
Cell-permeable TAT–N24 blocked the binding of
p55PIK with PCNA and inhibited DNA synthesis and
tumor growth in vivo
The unique N-terminal end (N24) of p55PIK is the
interaction site for both Rb and PCNA. Previously, we
showed that the cell-permeable TAT–N24 fusion protein inhibited cell proliferation and induced cell differ-
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Mol Cancer Ther; 12(10) October 2013
entiation in cancer and leukemia cells expressing Rb by
blocking p55PIK-mediated signaling pathways (18). We
thus examined whether TAT–N24 could affect the binding of p55PIK and PCNA, and modulate the signaling
pathways mediated by p55PIK and PCNA interaction.
TAT–N24 was able to specifically block the binding of
p55PIK with PCNA and decreased the association of
Pold with PCNA in a dose-dependent manner (Fig. 5A
and B). These data were consistent with the observations that TAT–N24 induced the cell-cycle arrest at
Molecular Cancer Therapeutics
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Published OnlineFirst August 12, 2013; DOI: 10.1158/1535-7163.MCT-12-0920
55
PI
K
si
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si
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S: 28.8 %
l
55
PI
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S: 29.1 %
tro
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S: 45.8%
Cell-cycle analysis
si
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PI3K–PCNA Interaction Stimulates DNA Synthesis
p55PIK
R2: 32.2%
R2: 35.8%
R2: 23.3%
GAPDH
A
CN
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tro
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S=31.4%
S: 64.5%
A
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Cell-cycle analysis
si
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tro
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S: 33.5%
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si
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DNA synthesis
GAPDH
R2: 32.3%
R2: 31.3%
40
35
30
25
20
15
10
5
0
**
NA
PC
sil
ro
nt
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DNA synthesis
R2: 22.1%
Percentage of BrdUrdpositive cells (%)
PCNA
Figure 3. Knockdown of p55PIK and PCNA led to cell-cycle arrest at S-phases and inhibited DNA synthesis. A, left, downregulated p55PIK expression in
FTC236 cells. FTC236 cells were transfected with siRNA against p55PIK (si-p55PIK) and control RNA (si-control). Lysates from parental cells (control), sip55PIK–transfected cells, and si-control–transfected cells were loaded on a gel and p55PIK protein expression was assessed by Western blotting using
anti-p55PIK antibody. Top, right, downregulated p55PIK induced the cell-cycle arrest at S and G2–M phases. Cell-cycle analyses were conducted on control
FTC236 cells, cells transfected with si-p55PIK or si-control. Representative histograms from an individual experiment and similar results were obtained in
three independent experiments. Bottom right, downregulated p55PIK decreased the DNA synthesis in FTC236 cells. FTC236 cells were transfected with the
si-p55PIK or si-control. BrdUrd was added 48 hours after infection, and cells were cultured for an additional 30 minutes. Cells then were collected and
immunostained for BrdUrd and counterstained for nucleic acid with PI. BrdUrd incorporation into DNA and DNA content in nuclei were determined by flow
cytometry analysis. Representative data from an individual experiment; similar results were obtained in three independent experiments. Note that the
percentage of cells in active DNA synthesis is defined as BrdUrdþ cells/total cells (the events in R2 area/total events). B, left, downregulation of PCNA in
Rb-deficient cells. FTC236 cells were transfected with siRNA against PCNA (si-PCNA) or control small RNA (si-control). Lysates from parental cells (control),
si-PCNA–transfected cells, and si-control-transfected cells were loaded on a gel and PCNA protein expression was assessed by Western blotting using
anti-PCNA antibody. Top, right, downregulated PCNA induced cell-cycle arrest at S-phase and increased cell death in Rb-deficient cells. Cell-cycle analyses
were conducted on FTC236 cells transfected with si-PCNA or si-control as described in Materials and Methods. Representative histograms from an individual
experiment and similar results were obtained in three independent experiments; the data are presented quantitatively with error bars and appropriate statistical
analysis ( , P < 0.01; , P < 0.05). Bottom right, downregulated PCNA inhibited DNA synthesis. FTC236 cells were transfected with the si-p55PIK or si-control.
BrdUrd was added 48 hours after infection, and cells were cultured for an additional 30 minutes. Cells then were collected and immunostained for BrdUrd and
counterstained for nucleic acid with PI. BrdUrd incorporation into DNA and DNA content in nuclei were determined by flow cytometry analysis. Representative
data from an individual experiment; similar results were obtained in three independent experiments; the data are presented quantitatively with error bars and
appropriate statistical analysis ( , P < 0.01; , P < 0.05). Note that the percentage of cells in active DNA synthesis is defined as BrdUrdþ cells/total cells (the
events in R2 area/total events).
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5P
IK
Wang et al.
bR
H
T2
9
H
T2
9
H
T2
9
R
b-
p5
A
p55PIK
Rb
GAPDH
**
0.6
HT29Rb- (1/6)
HT29Rb-p55PIK (6/6)
Tumor weight (g)
B
0.5
0.4
0.3
0.2
0.1
0.0
HT29Rb-
C
Ki-67
p55PIK
S-phase and inhibited the DNA synthesis in FTC236
and HT29Rb-p55PIK cells (data not shown), and suggests
that TAT–N24 can inhibit p55PIK effects on non-Rb
signaling pathways, including those mediated by the
p55PIK–PCNA interaction.
We next examined the effects of TAT–N24 on the tumor
growth of HT29Rb-p55PIK cells in athymic nude mice.
HT29Rb-p55PIK cells were injected into mice subcutaneously, and 2 mg TAT–N24 (0.1 g/kg body weight) or control
TAT–N24M were injected intravenously into tail veins of
mice every 2 days for 20 days. The volumes of tumors
were measured twice a week before mice were sacrificed
and their tumors were weighed (Fig. 6A). The mean
xenograft tumor weight decreased by 54% in mice receiving TAT–N24 compared with mice receiving TAT–N24M
(Fig. 6A and B).
Discussion
In the current study, we used two Rb-deficient cell lines
to show that the p55PIK regulatory subunit of PI3K can
bind to PCNA in addition to Rb. This interaction enhanced
the binding of Pold to PCNA and was associated with the
increased DNA synthesis. Furthermore, the unique N24
2106
Mol Cancer Ther; 12(10) October 2013
HT29Rb-p55PIK
Figure 4. p55PIK stimulates
tumorigenesis in vivo. A, increased
p55PIK expression in HT29 cells
with minimal level of Rb proteins
Rb-)
Rb
cells were
(HT29 . HT29
transfected with pcDNA-p55PIK or
control vector pcDNA and clones
with increased expression of
p55PIK were selected in the
presence of G418 (HT29Rb-p55PIK)
and further studied. Cell lysates
from HT29Rb (control), pcDNAp55PIK-transfected, and pcDNAtransfected HT29Rb cells were
loaded on a gel and p55PIK or Rb
protein expression was assessed
by Western blotting using antip55PIK antibody or anti-Rb
antibody. B, tumor xenograft
growth from cells HT29Rb or
HT29Rb-p55PIK. Cells were
subcutaneously injected into nude
mice (1 105 per site, six sites).
Two weeks later, mice were
sacrificed and tumors were
collected and weighed.
(mean þ SD; n ¼ 6; , P < 0.01;
HT29Rb-p55PIK group compared
with HT29Rb group). C, bottom,
increased expression of p55PIK
and Ki-67 in HT29Rb-p55PIK tumor
xenografts. Immunohistochemical
analyses were conducted on tumor
xenografts to examine the
expression of p55PIK as well as
Ki-67 proteins.
domain in p55PIK was sufficient for the binding of p55PIK
to PCNA as cell-permeable TAT–N24 abolished the
p55PIK–PCNA interaction. This, in turn, led to DNA
synthesis inhibition and cell-cycle arrest at the S-phase.
It is well known that multiple PI3K signaling pathways
are involved in cell-cycle progression; however, the precise mechanism(s) used by PI3K to regulate the DNA
synthesis is not well understood (21, 22). Our findings
provide insight into a novel PI3K signaling mechanism
that directly modulates the DNA synthesis and cell-cycle
progression.
PCNA is an essential component of the chromosomal
DNA replisome (19). In eukaryotes, both DNA leading
strand replication and completion of Okazaki fragments
synthesis require PCNA and DNA polymerases such as
Pola, Pold, and Pole (19, 23, 24). Our results showed that
the overexpression of p55PIK increased the association of
Pold with PCNA and led to increased DNA synthesis. We
currently are working on determining the precise mechanism for PI3K regulation of DNA synthesis. However,
one major potential mechanism may involve p21Waf/Cip,
the major protein that competes with Pold for binding
to PCNA, and inhibits DNA synthesis (25). It is possible
Molecular Cancer Therapeutics
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Published OnlineFirst August 12, 2013; DOI: 10.1158/1535-7163.MCT-12-0920
PI3K–PCNA Interaction Stimulates DNA Synthesis
A
C
on
tr
ol
TA
T–
N
M
24
TA
24
N
T– WB:
αHA (TAT–N24)
αp55PIK
αPolδ
αPCNA
isolated by the ability of its SH2 domains to bind phosphorylated tyrosine in signaling proteins (26, 27). However, its unique N24 domain specifically interacts with Rb
and the p55PIK–Rb interaction is important for cell-cycle
progression at G0–G1 phases (16). In our current study, we
identified a new N24 interacting partner, PCNA, that is
involved in cell-cycle progression at the S-phase. The fact
that other PI3K regulatory subunits do not associate with
Rb or PCNA suggests that each PI3K regulatory subunit
may specify distinct functions and may help explain how
PI3K can be involved in diverse, and sometimes opposing,
IP: αPCNA
B
M )
24 L
-N g/m TAT–N24 (µg/mL)
T
μ
tr A
on T 200 50 100 150 200
WB:
C
(
A
αPolδ
αPCNA
IP: αPCNA
that p55PIK overexpression decreases the expression of
p21Waf/Cip during cell-cycle progression, leading to an
increase of Pold binding to PCNA. This hypothesis is
supported by our findings that the overexpression of
p55PIK decreases the expression of p21Waf/Cip, whereas
the inhibition of p55PIK signaling increases the expression of p21Waf/Cip (Fig. 2B).
In the N-terminal region of p55PIK (N24), there is no
apparent PIP box or other motifs found in PCNA-binding
proteins, suggesting that it may contain a new PCNAbinding domain (19). The finding that N24 was both
necessary and sufficient for PCNA binding excludes the
possibility that SH2, SH3, bcr, and proline-rich domains in
regulatory subunits (p85, p55, and p50) are involved in its
interaction to PCNA. The p55PIK–PCNA and p55PIK–Rb
interactions through binding to N24 suggest that p55PIK
has important roles in the determination of PI3K activities.
It is now becoming apparent that different isoforms of
both p110 catalytic subunits and regulatory subunits of
PI3K may perform unique functions. p55PIK was first
www.aacrjournals.org
Control
TAT-N24M
TAT-N24
0.3
0.2
*
*
*
0.1
0
Day 4
Day 12
Day 20
B
Control
TAT–N24M
TAT–N24
0.5
Tumor weight (g)
Figure 5. TAT–N24 blocked the binding of p55PIK with PCNA and
decreased association of PCNA with Pold. A, TAT–N24 blocked the
binding or p55PIK with PCNA in cultured cells. FTC236 cells were
incubated with TAT–N24 (200 mg/mL) or control TAT–N24M for 24 hours.
Cellular lysates were immunoprecipitated (IP) with an anti-PCNA
antibody. Immunoprecipitated proteins were analyzed by Western
blotting (WB) using anti-HA antibody (for TAT–N24 or TAT–N24M fusion
proteins), anti-p55PIK antibody, anti-Pold antibody as indicated; PCNA
also was analyzed with an appropriate antibody to show the equal
amount of PCNA proteins in every group. B, TAT–N24 fusion proteins
decreased the association of PCNA with Pold in cultured cells. FTC236
cell were incubated with various concentrations of TAT–N24 or TAT–
N24M (200 mg/mL) for 24 hours. Cellular lysates were
immunoprecipitated with an anti-PCNA antibody. Immunoprecipitated
proteins were analyzed by Western blotting using anti-Pold and PCNA
antibodies.
0.4
Tumor volume (cm3)
ol
**
Control
TAT–N24M
TAT–N24
0.25
0
Figure 6. TAT–N24 inhibited tumor growth in vivo. A, intravenous injection
Rb-p55PIK
tumor xenografts
of TAT–N24 fusion proteins inhibited the HT29
growth. Mice inoculated with HT29Rb-p55PIK cells (106/site) were divided
into three groups receiving either 200 mL solvent–lipid emulsion, TAT–
N24 (10 mg/mL emulsion) or TAT–N24M (10 mg/mL emulsion) via tail vein.
The injection was repeated every 2 days. The size of tumors was
measured (mean S.D; n ¼ 5; , P < 0.05; TAT–N24–treated group
compared with TAT–N24M–treated group. B, TAT–N24 inhibitory effects
on the xenograft tumor weight in vivo. Shown are representative tumor
xenografts and mean tumor weights after necropsy (mean S.D; n ¼ 5;
, P < 0.01; TAT–N24–treated group compared with TAT–N24M group).
Mol Cancer Ther; 12(10) October 2013
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Published OnlineFirst August 12, 2013; DOI: 10.1158/1535-7163.MCT-12-0920
Wang et al.
cellular processes. It is interesting to note that the downregulation of p55PIK in Rb-deficient cells led to the cellcycle arrest at the S-phase without any significant effects
on apoptosis or cell death, suggesting that the p55PIK–
PCNA signaling does not directly affect pathways
involved in cell death (Fig. 3A). In contrast, the knockdown of PCNA in Rb-deficient cells not only inhibited
the cell-cycle arrest at the S-phase, but also induced
apoptosis (Fig. 3B). The different effects by p55PIK or
PCNA knockdowns on cell death clearly indicate that
while they may have overlapping functions on DNA
replication, PCNA may also have other roles than p55PIK
on cell survival.
Although PI3K is critical for normal cell function, dysregulated PI3K activities contribute to the development of
cancer and immune-mediated pathologies. Several PI3K
inhibitors are effective in cancer cell lines and animal
models (28). However, their lack of signaling pathway
specificity limits their clinical application, particularly as
only a subset of the many cellular processes regulated by
the PI3K pathway are directly involved in cell proliferation (28, 29). Thus, an important issue in the clinical
application of PI3K inhibitors will be the development of
drugs that can specifically block pathways involved in cell
proliferation with minimal adverse side effects due to the
inhibition of other PI3K-dependent cellular processes.
Our previous and current studies have shown the essential and specific roles of p55PIK–PI3K on Rb- and/or
PCNA-mediated signaling in cell-cycle progression and
DNA synthesis. Furthermore, the blockade of p55PIK
signaling by either siRNA or TAT–N24 did not signifi-
cantly affect other PI3K signaling pathways as it had
little systemic side effects in vivo. Our findings suggest
that targeting specific PI3K regulatory subunits such as
p55PIK could potentially facilitate the development of a
new class of anticancer drugs that have advantages over
present PI3K catalytic subunit inhibitors.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: P.M. Yen, J. Gong, J. Hu
Development of methodology: G. Wang, X. Cao, X. Xia, J. Hu
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): G. Wang, X. Cao, S. Lai, X. Luo, J. Hu
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): G. Wang, X. Cao, X. Xia, J. Hu
Writing, review, and/or revision of the manuscript: G. Wang, Y. Feng, X.
Xia, P.M. Yen, J. Hu
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): G. Wang, X. Luo
Study supervision: Y. Feng, X. Xia, J. Hu
Grant Support
J. Hu was supported by grants of foundation of "973" Program (no.
2009CB521802), National Natural Science foundation (nos. 30872472,
30973496, and 30800569) and PCSIRT1131. P.M. Yen was funded by faculty
funds from the Ministry of Health, Ministry of Education, A StaR, and
Duke-National University of Singapore Graduate Medical School,
Singapore.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
Received September 19, 2012; revised July 1, 2013; accepted July 22, 2013;
published OnlineFirst August 12, 2013.
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