p21-activated kinase 4 regulates ovarian cancer cell
proliferation, migration, and invasion and contributes
to poor prognosis in patients
Michelle K. Y. Siua, Hoi Yan Chana, Daniel S. H. Konga, Esther S. Y. Wonga, Oscar G. W. Wonga, Hextan Y. S. Nganb,
Kar Fai Tamb, Hongquan Zhangc, Zhilun Lic, Queeny K. Y. Chana, Sai Wah Tsaod, Staffan Strömbladc,
and Annie N. Y. Cheunga,1
Departments ofaPathology,bObstetrics and Gynecology, anddAnatomy, University of Hong Kong, Pokfulam, Hong Kong; andcCenter for Biosciences,
Department of Biosciences and Nutrition, Karolinska Institutet, SE-141 83, Stockholm, Sweden
Edited* by Tak Wah Mak, Campbell Family Institute for Breast Cancer Research, Ontario Cancer Institute at Princess Margaret Hospital, University Health
Network, Toronto, ON, Canada, and approved September 7, 2010 (received for review July 9, 2009)
Ovarian cancer is a lethal gynecological malignancy, and to improve
survival, it is important to identify novel prognostic and therapeutic
targets. In this study, we present a role for p21-activated kinase 4
(Pak4) in ovarian cancer progression. We show a significant associa-
tion between increased expression of Pak4 and its activated form,
phosphorylated (p)-Pak4 Ser474, with metastasis of ovarian cancers,
shorter overall and disease-free survival, advanced stage and high-
grade cancers, serous/clear cell histological subtypes, and reduced
chemosensitivity. Pak4 overexpression was also observed in ovarian
cancer cell lines. Pak4 and p-Pak4 expression were detected both in
vivo. Stable knockdown of Pak4 in ovarian cancer cell lines led to re-
duced cell migration, invasion, and proliferation, along with reduced
and decreased matrix metalloproteinase 2 (MMP2) expression. Con-
versely, Pak4 overexpression promoted ovarian cancer cell migration
and induced cell proliferation through the Pak4/c-Src/EGFR pathway
that controls cyclin D1 andCDC25A expression. Stable knockdown of
Pak4 also impeded tumor growth and dissemination in nude mice.
This report reveals the association between Pak4 and important clin-
icopathologic parameters, suggesting Pak4 to be a significant prog-
nostic marker and potential therapeutic molecular target in ovarian
cancer. The implied possible cross-talk between Pak4 and EGFR sug-
gests the potential of dual targeting of EGFR and Pak4 as a unique
therapeutic approach for cancer therapy.
prognostic marker|therapeutic target
attributed to its commonly subtle symptoms until widespread me-
patients with advanced disease (3). To improve the survival of
patients with ovarian cancer, it is important to investigate genes
governing metastasis and to identify novel prognostic markers and
p21-activated serine/threonine kinases (Paks) are major effec-
tors of the small Rho GTPases Rac1 and Cdc42, which play im-
portant roles in cell morphology, cytoskeletal reorganization,
apoptosis, survival, and angiogenesis—all prerequisite steps for
metastasis (4). Six mammalian Paks have been identified and
classified into group I (Paks1–3) and group II (Paks4–6) (4). We
ovarian cancer, affecting cell migration and invasion via the p38
pathway (5). Pak4 was first identified as an effector of Cdc42 es-
sential for regulating cytoskeleton reorganization (6). Subsequent
studies indicated that Pak4 could protect cells from apoptosis
anchorage-independent growth (8, 12). Many of these functions
varian cancer is a gynecological cancer associated with high
mortality worldwide (1), and its incidence in Asian countries,
are dependent on Pak4 kinase activity. Moreover, Pak4 is up-
regulated in most cancer cell lines (12). In athymic mice, over-
in ovarian cancer (14). Indeed, amplification of Pak4 gene was
expression may relate to cancer progression and clinical outcome.
Herein, ovarian cancer was adopted as the model for the study
of Pak4. The expression and subcellular localization of Pak4 and
phosphorylated(p)-Pak4 Ser474(the activated form) (12) in ovar-
ian cancer cell lines and tumors was assessed and correlated with
clinicopathological parameters. We also investigated the potential
role and downstream pathways of Pak4 in ovarian cancer cell
migration, invasion, and proliferation, and the induction effect of
follicle stimulating hormone (FSH) and hepatocyte growth factor
(HGF) on Pak4. Our findings suggest that Pak4 may play a role in
the progression of ovarian tumors, and is a potentially useful
molecular prognostic marker and therapeutic target.
Overexpression of Pak4 and Activated Pak4 Correlates with Pro-
gression of Ovarian Cancer and Prognosis of Patients. By immuno-
histochemistry, we observed mild nuclear and strong cytoplasmic
Pak4 (Fig. S1A) and strong nuclear and moderate cytoplasmic
p-Pak4 (Fig. 1A) immunoreactivity in ovarian cancer tissue and
ascitic fluid samples (Fig. S1B). In contrast, there was only mod-
erate staining in borderline tumors and weak or no staining in be-
higher nuclear and cytoplasmic Pak4 and cytoplasmic p-Pak4 im-
munoreactivity was detected in ovarian cancers and borderline
tumors than in benign cystadenomas/inclusion cysts (all P < 0.05;
Table S1). At mRNA level, significantly higher Pak4 was also
found in ovarian cancers and borderline tumors than in benign
cystadenomas as evaluated by qPCR (all P < 0.05; Fig. 1B). Up-
cancer cell lines compared with normal ovarian epithelium cell
lines (Fig.1C).Moreover, increasednuclear and cytoplasmicPak4
and p-Pak4 expression were significantly associated with serous/
survival (all P < 0.05; Table S1, Fig. S1D). Multivariate analysis
Author contributions: M.K.Y.S. and A.N.Y.C. designed research; M.K.Y.S., H.Y.C., D.S.H.K.,
and E.S.Y.W. performed research; O.G.W.W., H.Y.S.N., K.F.T., H.Z., Z.L., Q.K.Y.C., S.W.T.,
and S.S. contributed new reagents/analytic tools; M.K.Y.S. and A.N.Y.C. analyzed data;
and M.K.Y.S. and A.N.Y.C. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| October 26, 2010
| vol. 107
| no. 43www.pnas.org/cgi/doi/10.1073/pnas.0907481107
revealed that cytoplasmic Pak4, nuclear and cytoplasmic p-Pak4,
for overall survival (all P < 0.05; Table S2), whereas cytoplasmic
Pak4 and p-Pak4, disease stage, and chemosensitivity continued to
be significant predictors for disease-free survival (all P < 0.05;
Table S2). Significantly higher cytoplasmic Pak4 and p-Pak4 ex-
IV) and poor histological differentiation (grade 3) and at meta-
static foci (all P < 0.05; Table S1). Furthermore, high nuclear and
cytoplasmic Pak4 expression was significantly associated with re-
sistance to chemotherapy (all P < 0.05; Table S1).
Pak4 and p-Pak4 Localization in the Nucleus and Cytoplasm of Ovarian
Cancer Cells. Immunofluorescent staining displayed strong nuclear
and moderate cytoplsmic Pak4 and p-Pak4 immunoreactivity in
biogenesis (Fig. 2A). Four possible nuclear localization signals
(NLSs), responsible for nuclear accumulation (16), and a leucine
were identified when the Pak4 amino acid sequence was analyzed
using the NLS-recognizing program PSORTII (16) (Fig. 2B).
Subcellular expression of Pak4 and p-Pak4 in cytoplasmic and
nuclear fractions of OVCAR-3 was confirmed by immunoblot
analysis (Fig. 2C). Moreover, ectopic expression of Flag-tagged wt
Pak4 in SKOV-3 revealed that exogenous Pak4 was localized to
boththecytoplasm andthenucleus (Fig.2D, ii andvi). Incontrast,
no immunofluoresecence was detected in the negative control,
untransfected cells immunostained with anti-Flag antibody. Fur-
thermore, cells with ectopically expressed Flag-tagged wt Pak4
were double stained with anti-Flag and anti-Pak4/anti–p-Pak4
the specificity of the anti-Pak4 and anti–p-Pak4 antibodies.
Pak4 Regulation of Gene Transcription. Having found the nuclear
localization of Pak4, we attempted to determine the effect of Pak4
expressing wt Pak4 as a fusion protein with a GAL4-DNA binding
domain significantly increased the GAL4-Luc activity by 1.5- to 2-
fold in SKOV-3. The expression of the fusion protein (∼93 kDa)
was detected by immunoblot analyses (Fig. S2A). Moreover,
a dose-dependent activation wasobservedwhen the concentration
of construct used for transfection was increased, suggesting that
Pak4 may influence gene transcription (Fig. 2E). To further in-
vestigate the effect of the NLSs for Pak4 nuclear accumulation,
four Pak4 NLS mutants were generated. The GAL4-Luc activities
were significantly reduced in Pak4 NLS1 and NLS3 mutants when
compared with wt Pak4 (Fig. 2F). Mutation of NLS1 and NLS3
also inhibited exogenous Pak4 nuclear accumulation as detected
by immunoblotting using nuclear fractions, suggesting that NLS1
Pak4 Regulation of Cell Migration and Invasion Is Dependent on PAK4
Kinase Activity, c-Src, MEK-1/ERK1/2, and MMP2. The specific tran-
Pak4 in OVCAR-3 and OVCA420, ovarian cancer cell lines with
relatively high Pak4 expression, was detected. In a wound-healing
assay, a slower migration rate was found in siPak4 OVCA420 as
compared with control cells (Fig. 3B). In Transwell migration and
invasion assays, significantly reduced migration and invasion were
observed in OVCAR-3 andOVCA420 upon siPak4 (Fig. S3 B and
C) or shPak4 (Fig. 3 C and D) knockdown. We then aimed to un-
ravel Pak4 downstream signaling pathways. Focal adhesion kinase
(FAK), c-Src, and extracellular signal-regulated kinase (ERK1/2)
together with other signaling and adaptor proteins form large cell-
matrix adhesion complexes that constitute a key machinery in
cancer cell migration, invasion, and metastasis, in part through the
Interestingly, knockdown of Pak4 in OVCA420 reduced c-Src and
benign ovarian cystadenomas, serous (ii) and mucinous (vi) borderline ovarian tumors and serous (iii), mucinous (vii), clear-cell (iv), and endometrioid (viii)
ovarian carcinomas. (Insets) Regions with higher magnification. (B) qPCR analysis of Pak4 mRNA in ovarian tumors. The fold change of Pak4 mRNA was
calculated with respect to the lowest expression of Pak4 in cystadenomas. (C) mRNA (Left) and protein (Right) expression of Pak4 in immortalized ovarian
epithelial cell lines and ovarian cancer cell lines as determined by qPCR (*P < 0.05 compared with HOSE 11-12) and immunoblot analysis, respectively.
Overexpression of Pak4 and p-Pak4 (the activated form) in ovarian cancer. (A) Immunohistochemical staining of p-Pak4 in serous (i) and mucinous (v)
Siu et al.PNAS
| October 26, 2010
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expression was also reduced in shPak4 OVCA420 both in protein
lysates (Fig. 3E) and in conditioned media (Fig. 3F).
To further investigate the effect and signaling of Pak4 in ovarian
celllinewithrelativelylow Pak4 expression,werestably transfected
dead Pak4. Wt Pak4 and ca Pak4 significantly increased cell mi-
gration and invasion when compared with the FLAG control with
inhibited cell migration and invasion (Fig. S4C), indicating that
Pak4 kinase activity promotes cell migration and invasion. In addi-
tion, c-Src and ERK1/2 activity were induced together with MMP2
expression, and this induction was further enhanced by ca Pak4 ki-
nase activity (Fig. 4B). To test the effect of Pak4-induced c-Src and
ERK1/2 activities on cell migration and invasion, and to elucidate
Pak4 overexpressing SKOV-3 cells were treated with a c-Src in-
hibitor (PP1), two MEK-1 inhibitors (U0126 and PD98059), or
a MMP2 inhibitor (OA-Hy). PP1 inhibited both basal and Pak4-
mediated cell migration and invasion (Fig. 4A). By immunoblot
analyses, PP1 abolished c-Src activation, but did not alter ERK1/2
activation or MMP2 expression in SKOV-3 cells with or without
2 activation and MMP2 expression is independent of c-Src. How-
ever, U0126 attenuated Pak4-induced migration and invasion (Fig.
change c-Src activation, suggesting that MEK-1/ERK1/2 mediates
Pak4-induced MMP2 expression (Fig. 4B). PD98059 (Fig. 4C) and
OA-Hy (Fig. 4D) also showed inhibition on Pak4-mediated migra-
tion and invasion. In addition, specific siRNAs of c-Src, MEK-1, or
MMP2 were used to treat wt, ca, or kinase-dead Pak4 over-
expressing cells, and all of these siRNAs inhibited Pak4-mediated
migration and invasion (Fig. S4 A–C). These findings show that
Pak4 acts through at least two downstream pathways to promote
ovarian cancer cell migration and invasion—one including c-Src
and the other including MEK-1/ERK1/2 signaling to MMP2. We
also tested the effect of NLS mutants on cell migration and inva-
sion, but no significant difference was found between cells tran-
siently transfected with NLSs mutants and with wt Pak4 (Fig. S2B).
Pak4 Regulation of Cell Proliferation Involves c-Src and EGFR. We
found that both wt and ca Pak4 significantly induced SKOV-3 cell
proliferation after 12 d. More obvious effect was observed in ca
Pak4 transfected cells (Fig. 5A). Conversely, significantly reduced
proliferation was observed in OVCA420 12 d after shPak4
knockdown (Fig. 5E). c-Src has been reported to mediate phos-
phorylation of epidermal growth factor receptor (EGFR) on
Tyr845 leading to modulation of receptor function, such as in-
crease in DNA content (20). We sought to investigate if Pak4-
mediated c-Src activation may lead to phosphorylation of EGFR
on Tyr845 and affect ovarian cancer cell proliferation. We ob-
served that wt Pak4, and to a higher degree ca Pak4, induced p-
EGFR Tyr845in SKOV-3 cells (Fig. 5B), whereas knockdown of
Pak4 in OVCA420 reduced p-EGFR Tyr845(Fig. 3E). The Pak4-
induced EGFR phosphorylation was blocked by PP1, suggesting
that c-Src signaling mediates Pak4-induced EGFR phosphoryla-
tion. Intriguingly, PP1 (Fig. 5A) and two distinct EGFR inhib-
itors (CL387, 785 and PD153035; Fig. 5C) all blocked the Pak4-
mediated increase in SKOV-3 cell proliferation. PP1 and CL also
inhibited basal cell proliferation. To further elucidate downstream
targets of Pak4/c-Src/EGFR-regulated SKOV-3 cell proliferation,
the mRNA expression of G1-phase-associated cyclin D1, cyclin
D3, and CDC25A, and G2/M-associated cyclin B1 was determined
that ca Pak4 induced cyclin D1 and CDC25A mRNA expression
(Fig. 5F) but had no effect on cyclin D3 and cyclin B1 expression
(Fig. S4D). Further, CL blocked ca Pak4-induced cyclin D1 and
abolished ca Pak4 induced cyclin D1 protein expression (Fig. 5G).
These findings suggest that Pak4 regulation of cell proliferation
involves the c-Src and EGFR pathway leading to the regulation of
Pak4 on cell survival. TUNEL assay showed lack of significant dif-
ference in the proportion of apoptotic cells in OVCAR-3 and
implying that Pak4 may not be critical for ovarian cancer cell sur-
vival, at least in the absence of apoptotic stimulus.
ovarian cancer cells and activation of gene transcription by Pak4. (A) Sub-
cellular localization of Pak4 and p-Pak4 in OVCAR-3 determined by confocal
p-Pak4 antibody (v) along with mouse anti C23-nucleolin antibody (ii and vi).
Merge images displays Pak4 colocalization with C23-nucleolin (iii). p-Pak4 was
not colocalized with C23-nucleolin in merge image (vii). White arrowheads
indicated the cytoplasmic localization of Pak4 and p-Pak4. (iv and viii) DAPI
staining of nuclei. (B) Schematic drawing illustrating four possible nuclear lo-
calizationsignals(NLSs1–4)and aleucinezipperpatternandtheir positionson
Pak4 protein. (C) Pak4 and p-Pak4 in subcellular protein fractions (T, total cell
lysate; C, cytoplasmic fraction; N, nuclear fraction) of OVCAR-3. (D) Immuno-
fluorescent staining of Flag-tagged wt Pak4 overexpressing SKOV-3 cells by
rabbit anti-Pak4 antibody (i) or rabbit anti–p-Pak4 antibody (v) along with
mouse anti-Flag tag antibody (ii and vi). Exogenous wt Pak4 was detected in
the nucleus and cytoplasm of SKOV-3 cells and colocalized with fluorescent
staining as detected by anti-Pak4 antibody (iii) or rabbit anti–p-Pak4 antibody
(vii). (iv and viii) DAPI staining of nuclei. (E Upper) Schematic illustration of
GAL4-Luc reporter plasmid, GAL4-BD vector (control), and GAL4-BD-wt Pak4
construct. (Lower) The GAL4-Luc activity of the reporter gene as fold of con-
trol; n = 3; *P < 0.05. (F) The GAL4-Luc activity in SKOV-3 cells expressing wt
Pak4 or four NLS Pak4 mutants as fold of control; n = 3. (G) Immunoblot
analyses of exogenous Pak4 in nuclear fractions extracted from SKOV-3 cells
expressing wt Pak4 or Pak4 NLS mutants using anti-GAL4 DNA-BD antibody.
Localization of Pak4 and p-Pak4 in the nucleus and cytoplasm of
| www.pnas.org/cgi/doi/10.1073/pnas.0907481107Siu et al.
Pak4 Regulation of Tumor Growth and Dissemination in Nude Mice.
cells were prepared (Fig. S3E) and inoculated s.c. or i.p. to nude
shPak4 ES-2 cells was significantlylower than in tumors formed by
17 d after i.p. inoculation with control cells showed extensive dis-
semination, particularly throughout the mesentery with ascites
formation in three of the seven mice (Fig. 6B). In contrast, mice
injected with shPak4 ES-2 cells showed much less dissemination,
with only focal deposits at the mesentery, and no ascites formation
the control mice (0.184 ± 0.079 g; P < 0.05).
HGF and FSH Regulate Pak4 in Ovarian Cancer Cells. Given that most
of Pak4-mediated functions are kinase dependent, and activated
Pak4 was found in ovarian cancer cells, we investigated HGF as
a potential activator of Pak4. HGF has been reported to exert
potent effects on ovarian cancer cell migration and invasion
Pak4 as described above. We found that HGF induced OVCA420
cell migration and invasion along with Pak4, c-Src, and ERK1/2
phosphorylation. However, the HGF-induced migration, invasion,
and phosphorylations were all reversed by shPAK4 (Fig. S5). We
also investigated the effect of FSH on Pak4 expression in ovarian
to play important roles in ovarian cancer development (22, 23). In
dependent manner in OVCA420 cells (Fig. S5).
In this study, we demonstrated significant up-regulation of total
and phosphorylated Pak4 in ovarian cancer tissue and ascitic fluid
samples and cell lines when compared with benign ovarian epi-
thelium. More importantly, we found a significant correlation
between high Pak4 expression with extent of ovarian cancers,
shorter overall and disease-free survival. These findings suggest
Pak4 to be a significant prognostic marker in ovarian cancer. Pak4
immunocytochemistry may also be explored to enhance the sen-
sitivity of detecting metastatic cancer cells in ascitic fluid.
The above Pak4 expression profiling and our in vitro and in vivo
studiesoncell migration andinvasionhighlight thecontribution of
Pak4 to ovarian cancer progression and metastasis, concurring
with the role of Pak4 in cytoskeleton reorganization and cell mi-
gration at least in part executed in the cytoplasm (6) via phos-
were able toshow a linkbetweenPak4 andc-Src,MEK-1/ERK1/2,
our experiments on the mechanisms regarding Pak4 regulation of
downstream pathways, including Pak4 to c-Src and Pak4 to MEK-
migration and invasion and reduced c-Src and ERK1/2
activation as well as MMP2 expression. (A) Stable
knockdown of Pak4 mRNA and protein in OVCAR-3 and
OVCA420 as detected by qPCR (Upper) and immunoblot
analysis (Lower), respectively. (B) Wound-healing assay
OVCA420. In vitro migration (C) and invasion assays (D)
using Transwell membrane without or with Matrigel
coating, respectively. (Upper) Representative images of
migrating or invading OVCA420 cells. (Lower) Cell mi-
gration or invasion from OVCAR-3 and OVCA420 pre-
sented as percentage of control; n = 3; **P < 0.005. (E)
Immunoblot analysis on FAK, c-Src, ERK1/2, p38, MMP2,
and EGFR expression and/or activation in control and
shPak4 OVCA420. (F) Immunoblot analysis of MMP2
expression using conditioned media prepared from
control and shPak4 OVCA420.
Pak4 abrogation inhibited ovarian cancer cell
and invasion in ovarian cancer cells in-
volved c-Src and MEK-1/ERK1/2/MMP2
pathways. (A, C, and D) In vitro migra-
SKOV-3 cells stably transfected with
Flag-tagged wt Pak4, ca Pak4, or con-
trol vector in the presence or absence
of PP1 (c-Src inhibitor), U0126 (MEK-1
inhibitor), PD98059 (MEK-1 inhibitor),
OA-Hy (MMP2 inhibitor), or DMSO
(vehicle). Cell migration or invasion
presented as percentage of control;
noblot analyses of exogenous Flag-
tagged Pak4 and MMP2 levels; and
c-Src and ERK1/2 activities in SKOV-3
cells expressing wt or ca Pak4 in the
presence or absence of PP1 or U0126.
(C, Lower) Immunoblot analysis of
expressing SKOV-3 cells in the pres-
ence or absence of PD98059.
Siu et al. PNAS
| October 26, 2010
| vol. 107
| no. 43
1/ERK1/2 to MMP2. These pathways are intimately linked with
migration and invasion is kinase dependent.
In addition to effects on cell migration and invasion, we also
demonstrated the cell proliferation enhancement effect of Pak4
in ovarian cancer cells, in line with the tumor-promoting effect of
ca Pak4 overexpression in NIH 3T3 cells in athymic mice (13). We
are also excited by the identification of a downstream pathway of
Pak4, the Pak4 to c-Src to EGFR pathway that controls cyclin D
expression and ovarian cancer cell proliferation. EGFR is known
to be important in the etiology of several common human cancers,
including that of the lung, colon, stomach, head, and neck.
EGFR-targeting monoclonal antibodies and small molecule ty-
rosine kinase inhibitors are currently in clinical use or trial for
cancer therapy. However, the frequent occurrence of primary and
acquired resistance to these agents limits the clinical efficacy of
monospecific EGFR-targeted therapy (25, 26). For instance, tar-
geting EGFR alone does not appear optimistic in ovarian cancer
patients (27). Even in cancers with a good response, such as lung
cancers, resistance often develops and subsequent salvage treat-
ment is difficult. In this study, a cross-talk between Pak4, c-Src,
and EGFR in ovarian cancer is contemplated. Exploration for
dual targeting of EGFR and Pak4 may be examined in future
studies on ovarian or other cancers as possible promising novel
therapeutic approaches. Cyclin D1, a D-type cyclin regulating G1-
phase cell-cycle progression, has been identified as a critical
downstream effector of mutant EGFR signaling in non-small cell
lung cancer and as an alternative target of therapy (28). In ovarian
cancer, cyclin D1 and CDC25A expression were associated with
patient survival (29, 30). The link we identified between Pak4, c-
Src, EGFR, cyclin D1, and CDC25A not only suggests that Pak4
may regulate cell proliferation through the alteration of G1-phase
cell cycle progression, but also provides insights into the use of
alternative multitargeted therapy.
Anticancer therapies often induce apoptosis. Although we ob-
served that Pak4 had no significant effect on apoptosis under
regular culture conditions, Pak4 has been found to protect cells
fromapoptosis inducedby variousapoptoticstimuli,including UV
irradiation, serum deprivation, and TNFα (4, 7). We also revealed
a significant association between high Pak4 expression and che-
mortality in ovarian cancer patients. As such, molecular manipu-
lation of Pak4 to enhance chemosensitivity of ovarian cancer may
provide a useful direction to putative targeted therapy. However,
induced by chemotherapy needs to be elucidated in more detail.
We have also demonstrated, at subcellular level, Pak4 and p-
Pak4 immunoreactivity in both the nucleus and the cytoplasm of
ovarian cancer cells. A dose-dependent increase of GAL4-Luc
activity was also observed when the concentration of Pak4 con-
struct was increased, suggesting that Pak4 may influence gene
transcription. We further found that two PAK4 NLS motifs, NLS1
ovarian cancer cells implicate a function for Pak4 in the nucleus.
Pak1, another Pak isoform, was also shown to be associated with
nuclear chromatin and modulate gene transcription (31). The
same study also revealed nuclear localization of Pak1 upon stim-
ulation by EGF. Moreover, nucleocytoplasmic shuttling of Pak5
has also been found to be vital for cell survival under stress (32).
Studies on Pak4 nucleocytoplasmic shuttling will be worthwhile to
further elucidate the nuclear functions of Pak4, as well as whether
Pak4 kinase activity may affect gene transcription. We further
observed nuclear Pak4 immunoreactivity in the nucleolus, a sub-
nuclear organelle that forms the ribosomal DNA repeats and is
responsible for the synthesis, process, and assembly of ribosomal
subunits (33). Apart from cell growth and cell proliferation, the
nucleolus may also play a role in aging, cell cycle control, and
sensing cellular stress (33). The localization of Pak4 in the nucle-
olus suggests that Pak4 could potentially exert effects on such
functions, including cell proliferation and cell cycle control, as
shown in the present study.
We also identified HGF as a potential activator of Pak4 in
ovarian cancer cells, consistent with the effect of HGF observed
in other epithelial cells (34). The blockage of induced cell mi-
gration, invasion, and activation of Pak4, c-Src, and ERK1/2 in
HGF-treated ovarian cancer cells after stable knockdown of
Pak4 further suggests that Pak4, c-Src, and ERK1/2 are putative
downstream effectors of HGF to mediate cell migration and
invasion functions in ovarian cancer cells. This also concurs with
Pak4overexpressingSKOV-3cellsinthepresenceorabsenceofPP1,CL387,785orPD153035.(E)Cell proliferation rateofOVCA420incontrol andshPak4after 12
d displayed as fold change compared with control; n = 3; **P < 0.005. (B and D) Immunoblot analysis of p-EGFR Tyr845and EGFR expression in SKOV-3 cells
overexpressing wt or ca Pak4 in the presence or absence of PP1, CL or PD153035. (F) mRNA expression of cyclin D1 and CDC25A in ca Pak4 overexpressing cells
displayed as percentage of control (Flag vector-transfected SKOV-3 cells with DMSO) in the presence or absence of CL by qPCR; n = 3; *P < 0.05. (G) Immunoblot
analysis of cyclin D1 expression in ca Pak4 overexpressing cells in the presence or absence of PP1 or CL.
Pak4-induced proliferation involved the c-Src/EGFR pathway that controls cyclin D1 and CDC25A expression. (A and C) Cell proliferation rate of wt or ca
of mice inoculated i.p. with shPak4 ES-2 cells or control cells. Arrows, tumors.
Pak4 depletion impeded tumor growth and dissemination in nude
| www.pnas.org/cgi/doi/10.1073/pnas.0907481107Siu et al.
the effects of HGF on ovarian cancer cell migration and invasion
through the activation of ERK1/2, as found in previous studies
(21). Besides HGF, the up-regulation of Pak4 by FSH in ovarian
cancer cells suggests that FSH is a putative upstream mediator
accounting for Pak4 overexpression in ovarian cancer cells.
In summary, Pak4 is overexpressed and activated in ovarian
cancers, suggesting that Pak4 expression and activation play im-
and p-Pak4 in ovarian cancer cells may imply unique nuclear Pak4
increased Pak4 and/or activated Pak4 expression were associated
with cancer metastasis, reduced patient survival, late stages, and
increased resistance to chemotherapy. The mechanisms by which
Pak4 affects ovarian cancer cell progression include the control of
c-Src, MEK-1/ERK1/2, MMP2, and c-Src/EGFR. Given that ki-
nase inhibitors for the Pak family are being developed as thera-
peutics for cancer therapy (35–37), it is of great interest to explore
Pak4 as a therapeutic molecular target either alone or in combi-
such as EGFR. The latter combined approach may be particularly
promising for salvage of patients with various human cancers
manifesting primary or secondary resistance to EGFR antagonist,
a targeted therapy of increasing use in oncology patients.
Materials and Methods
Clinical Samples, Cell Culture, in Vivo Studies, Treatments, and General
Methods. Clinical samples, cell culture, subcellular proteins extraction, in
vivo studies, HGF, and FSH treatments and other pertinent methods, including
various functional assays, are described in SI Materials and Methods (5, 38).
Plasmid, Transfection, Treatments with Inhibitors or siRNAs, and Luciferase
Assay. To stably express Pak4 in SKOV-3, cells were transfected with Flag-tagged
wt Pak4, ca Pak4 (445N/474E), kinase-dead Pak4 (M350), or the control vector
p3XFLAG-CMV-10 (11) using Lipofectamine 2000 (Invitrogen) and then se-
lected with G418 (800 μg/mL) (5). For drug or siRNAs treatment, Pak4 over-
expressing cells were plated 6 or 24 h before treating with the c-Src inhibitor
PP1 (20 μM), the two MEK-1 inhibitors U0126 (20 μM) and PD 98059 (50 μM),
the two EGFR inhibitors CL387, 785 (1 μM) and PD153035 (2 μM), vehicle
(DMSO), or siRNAs (100 nM; Ambion) of c-Src, MEK-1, MMP2, or control. All
inhibitors were purchased from Calbiochem except PP1 (Biomol). After 48 h
(for PP1, U0126, PD98059, and siRNAs) or 12 d (for CL and PD153035 with
change of medium and drugs in every 3 d), cells were harvested for real-time
PCR and/or immunoblot analyses. To generate the Pak4 fusion protein with
GAL4-DNA binding domain construct, wt Pak4 was amplified and subcloned
in-frame into the vector pCMV-BD (Stratagene). To generate Pak4 NLS
mutants, a QuikChange Kit (Stratagene) was used, and the lysine residues in
the four NLSs were mutated to alanines using pCMV-BD wt Pak4 as template.
wt and NLS mutants of Pak4 were determined by immunoblotting. The con-
structs or the control vector were transiently transfected into SKOV-3 cells
along with pFR-Luc reporter plasmid (Stratagene). After 24 h, luciferase assay
was performed using Promega luciferase assay system. The transcription ac-
tivation domain of NF-κB fused with GAL4 was used as positive control. To
transiently silencePak4inOVCAR-3 andOVCA420,100nMeachofsiGENOME
Smart-pool for Pak4 and siControl nontargeting siRNA pool (Dharmacon) was
used. Cells were plated for migration and invasion assays 48 h after trans-
fection. To stably silence Pak4, cells were transfected with a set of shRNA
constructs against human Pak4, pRS-shPak4 (Origene),andthen selected with
puromycin (1.5 μg/mL) (5, 38). The pRS vector was used as controls.
ACKNOWLEDGMENTS. We thank the Faculty Core Facility, Dr. Chi Keung Lau
for providing valuable advice and technical help for in vivo studies and Dr.
Kelvin Chan for his valuable comments. This work was supported by the
Hong Kong Anti-Cancer Society Grant (to M.K.Y.S.), Hong Kong Research
Grants Council Grant (HKU 750306M) (to A.N.Y.C.), the University of Hong
Kong Seed Funding and Small Project Funding (to A.N.Y.C. and M.K.Y.S.),
and the Center for Biosciences, the Swedish Cancer Society, and the Swedish
Research Council Grants (to S.S.).
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Siu et al.PNAS
| October 26, 2010
| vol. 107
| no. 43