Transcriptional regulation of fibronectin by p21-activated kinase-1 modulates pancreatic tumorigenesis

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is the eighth largest cause of cancer-related mortality across the world, with a median 5-year survival rate of less than 3.5%. This is partly because the molecules and the molecular mechanisms that contribute to PDAC are not well understood. Our goal is to understand the role of p21-activated kinase 1 (Pak1) signaling axis in the progression of PDAC. Pak1, a serine/threonine kinase, is a well-known regulator of cytoskeletal remodeling, cell motility, cell proliferation and cell survival. Recent reports suggest that Pak1 by itself can have an oncogenic role in a wide variety of cancers. In this study, we analyzed the expression of Pak1 in human pancreatic cancer tissues and found that Pak1 levels are significantly upregulated in PDAC samples as compared with adjacent normals. Further, to study the functional role of Pak1 in pancreatic cancer model systems, we developed stable overexpression and lentiviral short hairpin RNA-mediated knockdown (KD) clones of Pak1 and studied the changes in transforming properties of the cells. We also observed that Pak1 KD clones failed to form tumors in nude mice. By adopting a quantitative PCR array-based approach, we identified fibronectin, a component of the extracellular matrix and a mesenchymal marker, as a transcriptional target of Pak1 signaling. The underlying molecular mechanism of Pak1-mediated transformation includes its nuclear import and recruitment to the fibronectin promoter via interaction with nuclear factor-κB (NF-κB)-p65 complex. To our knowledge, this is the first study illustrating Pak1-NF-κB-p65-mediated fibronectin regulation as a potent tumor-promoting mechanism in KRAS intact model.Oncogene advance online publication, 24 February 2014; doi:10.1038/onc.2013.576.

Full text

ORIGINAL ARTICLE
Transcriptional regulation of fibronectin by p21-activated kinase-1
modulates pancreatic tumorigenesis
S Jagadeeshan
1,6
, YR Krishnamoorthy
1,6
, M Singhal
1,6
, A Subramanian
1,6
, J Mavuluri
1
, A Lakshmi
1
, A Roshini
1
, G Baskar
2
,
M Ravi
2
, LD Joseph
3
, K Sadasivan
4
, A Krishnan
5
, AS Nair
5
, G Venkatraman
2
and SK Rayala
1
Pancreatic ductal adenocarcinoma (PDAC) is the eighth largest cause of cancer-related mortality across the world, with a median
5-year survival rate of less than 3.5%. This is partly because the molecules and the molecular mechanisms that contribute to PDAC
are not well understood. Our goal is to understand the role of p21-activated kinase 1 (Pak1) signaling axis in the progression of
PDAC. Pak1, a serine/threonine kinase, is a well-known regulator of cytoskeletal remodeling, cell motility, cell proliferation and cell
survival. Recent reports suggest that Pak1 by itself can have an oncogenic role in a wide variety of cancers. In this study, we
analyzed the expression of Pak1 in human pancreatic cancer tissues and found that Pak1 levels are significantly upregulated in
PDAC samples as compared with adjacent normals. Further, to study the functional role of Pak1 in pancreatic cancer model
systems, we developed stable overexpression and lentiviral short hairpin RNA-mediated knockdown (KD) clones of Pak1 and
studied the changes in transforming properties of the cells. We also observed that Pak1 KD clones failed to form tumors in nude
mice. By adopting a quantitative PCR array-based approach, we identified fibronectin, a component of the extracellular matrix and a
mesenchymal marker, as a transcriptional target of Pak1 signaling. The underlying molecular mechanism of Pak1-mediated
transformation includes its nuclear import and recruitment to the fibronectin promoter via interaction with nuclear factor-kB
(NF-kB)–p65 complex. To our knowledge, this is the first study illustrating Pak1–NF-kB–p65-mediated fibronectin regulation as a
potent tumor-promoting mechanism in KRAS intact model.
Oncogene advance online publication, 24 February 2014; doi:10.1038/onc.2013.576
Keywords: Pak1; fibronectin; PDAC; NF-kB–p65; KRAS intact model
INTRODUCTION
Pancreatic cancer is the thirteenth most common type of cancer
worldwide. More than 95% of pancreatic cancers are ductal
adenocarcinomas and remain as one of the most devastating
malignancies.
1,2
Owing to the lack of symptoms during early
stages, most patients present with the disease at an advanced
stage during diagnosis, which is difficult to treat and manage.
Therefore, the 5-year survival rates are very low and are estimated
to be less than 3.5%.
3
In terms of clinically effective therapeutic
options, nothing much has changed significantly over the past
few years.
4,5
Our limited understanding of the pathobiology of
pancreatic ductal adenocarcinoma (PDAC) has been a barrier to
progress in developing suitable therapeutic strategies. Research
till date on PDAC has concentrated on understanding the basic
causative factors inducing tumorigenesis. Hence, there is an
increasing need to understand the novel signaling pathways that
contribute to the progression of PDAC, which might eventually
translate into potential therapeutic models of intervention.
It is accepted that key genes involved in pancreatic cancer
progression encode proteins that function as critical regulators of
signal–transduction networks that regulate cell proliferation,
differentiation and survival.
6
Studies have demonstrated that
several signaling cascades important for normal pancreatic
development are also dysregulated during the proliferation,
survival and metastasis of pancreatic ductal adenocarcinoma
cells.
7–9
Despite an in-depth research on various signaling
pathways involved in transforming properties that have been
implicated in the development of PDACs, treatments targeting
these pathways have proven to be ineffective. More than 90% of
pancreatic cancers carry oncogenic KRAS mutations at codon
12,
10,11
which cause a constitutive activation of the G protein RAS,
eventually leading to abnormal activation of various downstream
signaling pathways involving p21-activated kinase 1 (Pak1). Pak1
is a serine/threonine kinase that functions as a downstream
activator for various oncogenic signaling pathways. Pak1 is a well-
known regulator of cytoskeletal remodeling, cell proliferation, cell
survival and cell motility, all of which result in tumor formation
and cell invasiveness.
12,13
Alterations in Pak1 expression have
been detected in a wide variety of human tumors.
14
Pak1 was
shown to regulate cyclin D1 levels through the activation of
nuclear factor (NF)-kB and thus contributing to increased tumor
cell proliferation.
15
The hematopoietic-specific guanine nucleotide
exchange factor VAV1 reportedly contributes to tumorigenic
properties of pancreatic cancer cells through the regulation of
signaling axis involving Pak1.
16
The upstream regulators of Pak
family members, Rac1 and Cdc42, have been previously
1
Department of Biotechnology, Indian Institute of Technology Madras (IITM), Chennai, India;
2
Department of Human Genetics, Sri Ramachandra University, Chennai, India;
3
Department of Pathology, Sri Ramachandra University, Chennai, India;
4
Department of Plastic and Reconstructive Surgery, Government Medical College, Thiruvananthapuram,
India and
5
Rajiv Gandhi Centre for Biotechnology (RGCB), Thiruvananth apuram, India. Correspondence: Dr SK Rayala, Department of Biotechnology, IIT Madras, Chennai 600036,
India or Dr G Venkatraman, Department of Human Genetics, Sri Ramachandra University, Porur, Chennai 600116, India or Dr AS Nair, Rajiv Gandhi Centre for Biotechnology
(RGCB), Thycaud Post, Poojappura, Thiruvananthapuram 695014, Kerala, India.
E-mail: rayala@iitm.ac.in or ganeshv@sriramachandra .edu.in or sasha@rgcb.res.in
6
These authors contributed equally to this work.
Received 10 June 2013; revised 3 December 2013; accepted 7 December 2013
Oncogene (2014), 1–10
&
2014 Macmillan Publishers Limited All rights reserved 0950-9232/14
www.nature.com/onc

implicated in pancreatic cancer initiation and progression.
17,18
Rac1 has been shown as a mediator of transforming growth
factor-b-1-induced effects on cell migration and Smad signaling in
pancreatic cancer cell lines.
19
Among the several important
therapeutic targets discovered so far for PDAC, all the receptor
tyrosine kinases, growth factor receptors, gastrin receptors and Src
kinases are known to activate Pak1 kinase.
20
Cox-2 and b-catenin
have been identified as downstream targets of Pak1 signaling.
21,22
Thus, Pak1 seems to be significantly involved in the multiple
signaling pathways dysregulated or hyperactivated in PDACs. This
study focuses on understanding the biology of Pak1 and its
contribution to the etiology of pancreatic cancer.
RESULTS
Clinical significance of Pak1
Pak1 expression is dysregulated in PDAC tissues and cell lines.In
order to study the role of Pak1 in PDAC, we initially analyzed the
expression of Pak1 protein in 14 paired samples of human PDAC
tumors and adjacent normal pancreatic tissue by western blotting,
and found that Pak1 levels are significantly upregulated in 9 out of
14 tumors as compared with that in normal tissue (Figure 1a). We
further analyzed the Pak1 mRNA levels in human PDAC tissue
samples (n ¼ 16 for PDAC) and normal pancreatic tissue (n ¼ 17 for
normal) by quantitative PCR (qPCR) and observed approximately
fivefold upregulation in the expression of Pak1 in PDAC tumors as
compared with the normal pancreatic tissue (Figure 1b). Further-
more, to evaluate the significance of our above findings, we
examined the expression levels of Pak1 in human tissue
microarray containing 60 cases of PDAC tissue samples with the
corresponding adjacent normal tissue by immunohistochemistry.
Although 70% of the PDAC tumors stained positive for Pak1, more
than 40% of the tumors exhibited intense staining for Pak1
(Table 1). The representative images of the stained tissue array are
shown in Figure 1c and Supplementary Figure S1. Next, in order to
choose the appropriate model system for our in vitro study, we
evaluated the expression of Pak1 protein in seven PDAC cell lines
and in the immortalized, nontumorigenic HPDE6 (E6/E7) cell line
by western blot and qPCR analysis. Results showed elevated
expression of Pak1 in all of the malignant cell lines compared with
the nontumorigenic control (Figures 1d and e). Together, these
results suggested that elevated Pak1 levels in PDAC tumors and
cell lines might contribute to the transforming properties of
pancreatic cells.
Transforming and tumorigenic properties of Pak1
The dysregulated expression of Pak1 in PDAC tissue samples
prompted us to examine its significance in the tumorigenic
properties of pancreatic tumor cells.
Generation of stable Pak1 overexpressing and Pak1 shRNA Knock-
down model cells. To explore the role of Pak1 as an upstream
determinant of oncogenic transformation in pancreatic cancers,
we generated stable clones of Pak1 overexpression in Mia Pa Ca 2
cell line. Stable overexpression of ectopic Pak1 expression was
confirmed by immunoprecipitation of T7-tagged Pak1. The
overexpression was also confirmed by running whole-cell lysates
and probing for Pak1 as well as immunofluorescence for Pak1
using confocal microscopy (Figure 2a and Supplementary Figure
S2). Similarly, we also developed stable lentiviral Pak1 short
hairpin RNA (shRNA) Knockdown clones in MDA Panc 28 cell line.
The downregulation of Pak1 was confirmed by western blot, qPCR
and immunofluorescence (Figure 3a and Supplementary Figure
S2). More than 90% knockdown (KD) in the levels of Pak1 was
observed in the Pak1 KD clones compared with the control
nontarget (NT) shRNA-expressing clone.
Figure 1. Pak1 is upregulated in PDAC. (a) Lysates from paired samples of human PDAC tumors and adjacent normal pancreatic tissue were
analyzed for Pak1 expression by western blotting. (b) mRNA levels of Pak1 in human PDAC and normal pancreatic tissue samples.
(c) Representative immuno-stained (against Pak1) paraffin-embedded tissue sections of paired normal and PDAC tumor tissue array.
(d) Western blot and (e) qPCR analysis of Pak1 expression in indicated PDAC cell lines and an immortalized normal pancreatic epithelial cell
line (HPDE6/E6E7). Vinculin/b-actin was used as loading control.
Pak1 signaling regulates fibronectin transcription
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Pak1 regulates the transforming properties of pancreatic cancer
cells. Further, in order to establish the possible role of Pak1 in the
transforming properties of pancr eatic cells, we per formed
proliferation, colony formation, soft agar, migration and invasion
assays with the Pak1 overexpression and KD models of Mia Pa Ca
2 and MDA Panc 28, respectively. Proliferation a ssay results
showed that upon Wt-Pak1 overexpression in Mia Pa Ca 2 cell
line, the cell proliferation rate goes up significantly as compared
with the empty vector carrying cell line (Figure 2b). On the other
hand, MDA Panc 28 KD clones expressing lesser Pak1 than the NT
control clone exhibited decreased proliferation (Figure 3b). In the
col ony formation assay, we observed larger number of colonies
for med in Wt-Pak1 overexpression clone compared with the
empty vector control in Mia Pa Ca 2 cells (Figure 2c). On the other
hand, we observed fewer number of colo nies formed in Pak1
downregulated clones in MDA Panc 28 co mpared with the NT
shRNA-expressing clone (Figure 3 c). Further, t o assess the role of
Pak1 in promoting anchorage-independent growth, w e carried
out soft agar assay with both overexpression and KD models of
Mia Pa Ca 2 and MDA Panc 28, respectively. Results showed that
ectopic expressio n of Wt-Pak1 increased Mia Pa Ca 2 cell line’s
ability to form colonies in an anchorage-independent manner
compared with the empty vector clones (Fi gure 2d). Similarly,
stable KD of Pak1 in MDA Panc 28 reduced the number of
col onies formed in the soft agar as say (Figure 3d). Ne xt, to assess
the role of Pak1 o n the invasive and migratory behavior of
pancreatic cancer cells, we examined the clones using Boyden
chambers coated with or without matrigel, respectively. We
observed that stabl e overexpress ion of Pak1 in Mia Pa Ca 2
res ulted in a multiple-fold increase in the number of c ells
migrating as well as inv ading (Figures 2e and f). Likewise, there is
a multiple-fold decreas e in the number of cells migra ting and
invading towards the bo ttom well in Pak1 downregulated clones
in MDA Panc 28 compared with the NT shRNA -expressing clone
(Figures 3e and f). Similar results were obs erved with NT and
Pak1 KD clones made in Mia Pa Ca 2 (S upplementary Figure S3).
Consistent with these results, in vitro wound healing assay
showed that Pak1 overexpres sion lead s to improved wound
Table 1. Immunohistochemistry data of Pak1 expression in tissue samples
Negative Weak positive Intense staining
Adjacent normal 58.3% (35 of 60) 35% (21 of 60) 6.7% (4 of 60)
Pancreatic ductal adenocarcinoma 31.7% (19 of 60) 25% (15 of 60) 43.3% (26 of 60)
Figure 2. Pak1 regulates the transforming property of pancreatic cells. (a) Western blot analysis showing the stable overexpression of Pak1 in
Mia Pa Ca 2 cell line. Top panel shows immunoprecipitated T7-Pak1 from the clones, bottom two panels are direct lysates. Pak1 overexpression
accelerates (b) cell proliferation; (c) colony formation; (d) anchorage-independent soft agar growth; (e) migration; (f ) invasion and (g) wound
healing properties in stable Mia Pa Ca 2 clones. Each value represents the mean
±
s.e.m. *Po0.05, **Po0.005 compared with vector clones.
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& 2014 Macmillan Publishers Limited Oncogene (2014) 1 – 10

healing and migration rates as compared with the control vector
carrying Mia Pa Ca 2 cell line (Figure 2g ). Correspondingly, stable
KD o f Pak1 in MDA Panc 28 resulted in a significant reduction in
the wound healing capacity (Figure 3g). All these results suggest
that Pak1 is the key effector node in controlling the downstream
signaling axis in PDAC.
Transforming potential of Pak1 in wild-type KRAS model.As
mutations in the KRAS oncogene account for more than 90% of
human pancreatic cancers, we intended to check whether Pak1
can transform pancreatic cells independent of mutant KRAS. To
this end, we transiently overexpressed Wt-Pak1 in BxPC 3 cell line
(having Wt-KRAS) and performed colony formation and ancho-
rage-independent soft agar assays with these cells. Results
showed that Pak1-transfected BxPC 3 cells showed significantly
increased ability to form colonies in both anchorage-dependent
and -independent manner compared with the empty vector
transfect (Figures 4a and b). Simultaneously, to prove the other
way, we developed stable lentiviral Pak1 shRNA-mediated KD
clones in BxPC 3 cell line. The downregulation of Pak1 was
confirmed by qPCR, western blot and immunofluorescence
(Figure 4c and Supplementary Figure S2). More than 95% KD in
the levels of Pak1 was observed in the Pak1 knockdown (KD1)
clone as compared with the control NT shRNA-expressing clone.
We performed colony formation, soft agar, migration and invasion
assays with these clones and results showed that stable KD of
Pak1 in BxPC 3 reduced the number of colonies formed in the
clonogenic and in soft agar assay (Figures 4d and e). In addition, a
significant reduction in the number of cells migrated and invaded
as compared with the BxPC 3 NT clone (Figures 4f and g). Taken
together, our results illustrate the potential role of Pak1 in
transforming pancreatic cells with intact KRAS (Wt).
Tumorigenic potential of Pak1 in nude mouse model. Consistent
with in vitro results, MDA Panc 28 shRNA Pak1 KD clones failed to
form tumors in nude mice, whereas the NT shRNA-expressing
clone formed palpable tumors (Figure 5a). These results showed
that Pak1 drives the tumorigenic property in pancreatic cancer
cells in vivo.
Molecular mechanism of Pak1-induced tumorigenesis
Fibronectin—a novel molecular target of Pak1. The evidence from
the above results suggested that Pak1 might have an important
role in the regulation of pancreatic cell’s invasive and metastatic
behavior. To identify the downstream molecular targets that might
be regulated through Pak1 signaling and also associated with the
tumorigenesis and metastatic potential of cells, we performed qPCR
expression profiling of 84 genes associated with tumorigenesis and
metastasis utilizing MDA Panc 28 NT shRNA-expressing clone NT
and Pak1 shRNA-expressing KD clone KD1 (Figure 5b). A complete
list of genes analyzed through the gene array and the genes that
are modulated by Pak1 activity are given as Supplementary Table 2.
The shortlisted genes that are significantly modulated by Pak1 are
given as Supplementary Table 3. As determined by this method, we
identified fibronectin as one of the potential targets that was
drastically downregulated in the Pak1 KD clone as compared with
NT clone. We further validated fibronectin in both overexpression
and KD models of Mia Pa Ca 2, MDA Panc 28 and BxPC 3,
respectively using individual qPCR assays and immunoblotting for
fibronectin. Results showed that fibronectin mRNA and protein
levels were significantly increased in Wt-Pak1 overexpressing Mia
Pa Ca 2 cell line as compared with the empty vector transfect
(Figure 5c). Similarly, there is a significant decrease in the
fibronectin mRNA and protein levels in the stable KD of Pak1 in
MDA Panc 28 and BxPC 3 clones as compared with the NT clones
Figure 3. Pak1 silencing decreases the transforming property of pancreatic cells. shRNA-mediated downregulation of Pak1 in stable MDA Panc
28 clone as confirmed by (a) Western blot and qPCR. Pak1 downregulation reduces (b) cell proliferation; (c) colony formation;
(d) anchorage-independent soft agar growth; (e) migration; (f ) invasion and (g) wound healing properties. Each value represents the
mean
±
s.e.m. *Po0.05, **Po0.005 compared with non-target clones.
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(Figure 5c). In addition, we analyzed for the expression of
extracellular fibronectin (fibronectin EDA domain) by performing
western blotting using fibronectin EDA domain specific antibody
and results showed that there is a significant change in its
expression with the modulation of Pak1 (Supplementary Figure
S4A). We also assayed for the secretory fibronectin in the cell
culture conditioned media using ELISA kit and results showed a
significant change in its levels with the modulation of Pak1
(Supplementary Figure S4B).
Consistent with this, overexpression of Wt-Pak1 significantly
augmented fibronectin promoter activity in the Mia Pa Ca 2 cell line
(Figure 5d), whereas silencing of Pak1 significantly downregulated
fibronectin promoter activity in MDA Panc 28 cell line (Figure 5e).
Further, to determine whether kinase activity of Pak1 is required for
the regulation of fibronectin promoter, we used Wt-Pak1, Pak1-
T423E (kinase active) and Pak1-K299R (kinase dead) constructs and
performed fibronectin promoter luciferase assay in parental Mia Pa
Ca 2 cell line. Results showed that expression of Pak1-T423E led to
an increase in fibronectin promoter activity, whereas expression of
Pak1-K299R significantly decreased basal promoter activity
(Figure 6a). Interestingly, depletion of fibronectin using siRNA
significantly abated Pak1’s ability to form colonies in colony
formation assay and soft agar assay; it showed significant reduction
in the number of cells migrated and invaded as compared to the
control siRNA transfect in Wt-Pak1 stable overexpressing Mia Pa Ca
2 clone (Figures 6b–e). On the contrary, transient overexpression of
fibronectin cDNA in Pak1 KD clones showed increase in the
tumorigenic phenotype of Pak1 KD clones as evidenced by increase
in migration potential, soft agar and colony forming ability
(Supplementary Figure S5). Taken together, these results indicate
that Pak1 can promote tumorigenic properties in part through the
regulation of fibronectin promoter.
Pak1 regulates fibronectin via NF-kB–p65 pathway. The observa-
tion that active Pak1 (T423E) induced and kinase dead Pak1
(K299R) diminished fibronectin promoter activity prompted
us to investigate the mechanism of regulation of fibronectin
transcription by Pak1 in cancer cells. It was previously reported
that activated Pak1 gets localized to the nucleus.
23
In addition,
Pak1 has been shown in various cellular contexts to modulate
gene expression of nuclear factor of activated T cell,
phosphofructokinase-muscle isoform, tissue factor and tissue
Figure 4. Pak1 modulates the transforming property of KRAS wild-type pancreatic cells. Western blot analysis showing the transient
overexpression of Pak1 in BxPC 3 cell line. Pak1 overexpression accelerates (a) colony formation and (b) anchorage-independent soft agar
growth in transiently Pak1-overexpressed BxPC 3 cell line. Each value represents the mean
±
s.e.m. *Po0.05 compared with vector clones.
(c) shRNA-mediated downregulation of Pak1 in stable BxPC 3 cell line as confirmed by western blot and qPCR. Pak1 downregulation abrogates
(d) colony formation and (e) anchorage-independent soft agar growth, (f) invasion and (g) migration. Each value represents the mean
±
s.e.m.
*Po0.05, **Po0.005 compared with non-target clones.
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factor pathway inhibitor through the regulation of several
transcription factors, as it lacks transcriptional activity of its
own.
24
It was also shown that Pak1 has a role in the activation of
NF-kB.
25
Recently, it was reported that p65 subunit of NF-kB
activates fibronectin transcription.
26
Based on these, we
hypothesized that Pak1 might be regulating fibronectin via NF-
kB–p65. To check this possibility, we next performed a sequence
analysis of the fibronectin promoter using the ConSite prediction
software to identify the putative binding sites for transcriptional
factors that might be responsible for Pak1-mediated regulation of
fibronectin transcription. We found that NF-kB is one of the
potential transcriptional factors that bind to fibronectin promoter.
As Pak1’s role in activation of NF-kB is already established, we
hypothesized that this might be an appropriate candidate to study
Pak1-mediated regulation of fibronectin. To examine this
possibility, we initially checked for the levels of phospho-p65 in
the overexpression and KD models of Mia Pa Ca 2, MDA Panc 28
and BxPC 3, respectively by western blotting. Consistent with the
role of Pak1 in the activation of NF-kB as reported earlier, results
showed that there is a significant increase in the level of phospho-
p65 in the Wt-Pak1 overexpressing Mia Pa Ca 2 cell line as
compared with the empty vector transfect. Similarly, there is a
significant decrease in the phospho-p65 levels in the stable KD of
Pak1 in MDA Panc 28 and BxPC 3 clones as compared with the NT
clones (Figure 6f). There is no change in the levels of total p65.
Further, we observed a synergistic increase in the fibronectin
promoter activity upon co-transfection of p65 along with Wt-Pak1
(Figure 6g), whereas shRNA-mediated p65 depletion resulted in a
significant decrease in the Pak1-induced fibronectin promoter
activity (Figure 6h). These results clearly indicate that Pak1 could
regulate fibronectin via NF-kB–p65 pathway.
Pak1 recruitment onto the fibronectin promoter. Further, to gain a
deeper insight into the molecular mechanism involved in NF-kB-
mediated Pak1 regulation of fibronectin promoter, we focused on
the nuclear role of Pak1. To start with, we examined for the levels
of nuclear Pak1 in Pak1-overexpression clones of Mia Pa Ca 2 by
subcellular fractionation, followed by western blotting. Consistent
with the previous reports that active Pak1 translocates to the
nucleus, we observed that there is a significant increase in the
amount of Pak1 in the nuclear compartment in the Wt-Pak1-
overexpressing Mia Pa Ca 2 clone. We also observed an increase in
the nuclear levels of p65 in the Pak1 overexpression clones of Mia
Pa Ca 2 (Figure 7a). Consistent with this, confocal analysis for Pak1
in Pak1-overexpressing Mia Pa Ca 2 showed increased nuclear
localization of Pak1, whereas the KD clones of Pak1 in MDA Panc
28 and BxPC 3 showed decreased nuclear localization
(Supplementary Figure S2). Further, to establish that nuclear
Pak1 is essential for the activation of fibronectin promoter, we
used a Pak1 NLS mutant construct
23
and showed that this mutant
is not able to induce fibronectin promoter activity (Figure 7b).
From the above results, it was clear that kinase activity of Pak1 and
its nuclear localization are required for the activation of
fibronectin promoter via NF-kB–p65. Therefore, to establish the
molecular cooperation between these molecules in the regulation
of fibronectin promoter, we examined the recruitment of Pak1
onto the predicted 700 bp fibronectin core promoter region using
a chromatin immuno precipitation (ChIP)-based promoter walk
Figure 5. Fibronectin is a novel molecular target of Pak1. (a) Tumor xenograft images of MDA Panc 28/NT and MDA Panc 28/KD1 shRNA clones
implanted subcutaneously in nude mice. Western blot confirming Pak1 downregulation in MDA Panc 28 clones before injecting into mice.
Graph showing tumor growth in mouse xenografts. (b) Heat map of the human metastatic PCR array performed on MDA Panc 28 Pak1
knockdown clones. (c ) qPCR and western blot analysis for fibronectin levels in Pak1 overexpressing and knockdown clones. (d) Mia Pa Ca 2
cells were co-transfected with 0.5 mg of 1.2 k b pFN1-luciferase reporter and 0.5 mg of Pak1 or plasmid pcDNA 3. 1. After 24 h, the cells were
lysed, luciferase activity was measured (n ¼ 3) and normalized with b-galactosidase activity. Each value represents the mean
±
s.e.m.
**Po0.005 compared with vector control. (e) MDA Panc 28 cell lines were transfected with Pak1 siRNA/control siRNA. After 48 h of
transfection, the cells were transfected with 1.0 mg of 1.2 kb pFN1-luciferase reporter and with a pGL3-basic luciferase reporter. After 24 h, the
cells were lysed, luciferase activity was measured (n ¼ 3) and was normalized with b-galactosidase activity. Each value represents the
mean
±
s.e.m. **Po0.005, ***Po0.001, compared with control siRNA. Western blot analysis confirming Pak1 downregulation after siRNA
transfection of MDA Panc 28 cells.
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with three different sets of primers covering the 700 bp region.
Results showed that Pak1 was recruited to the fibronectin
promoter at a region between  13 to  220 bp, which is about
200 bp region (Set 1; Figure 7ci). This was confirmed by
sequencing the PCR product of ChIP. We also observed that NF-
kB–p65 is also recruited to the same region as Pak1 (Figure 7cii).
Sequence analysis of this 200 bp (region 1) of the fibronectin
promoter using the ConSite prediction software revealed that NF-
kB binding consensus sequence GGGACAGCCC lies in the same
region. These results suggest that both Pak1 and NF-kB–p65
might be recruited to the same region onto the fibronectin
promoter. To confirm this, we performed a sequential double ChIP
analysis with Pak1 followed by p65 antibody and found that
indeed Pak1 and NF-kB–p65 are corecruited onto the region 1
(Figure 7ciii). To further demonstrate the importance of NF-kB–
p65 in Pak1-mediated fibronectin upregulation, we silenced NF-
kB–p65 expression using shRNA and examined for the recruitment
of Pak1 onto the region 1. Results showed that there is a
significant decrease in the amount of Pak1 recruited to the region
1 upon silencing NF-kB–p65 (Figure 7civ). Further, in order to
investigate the functional significance of this 200 bp region in the
transcriptional regulation of fibronectin by Pak1, we made a
fibronectin promoter construct deleting this 200 bp region where
Pak1–NF-kB–p65 are recruited, and performed reporter luciferase
assay. Results showed that Pak1 was not able to induce the
fibronectin promoter activity without the 200 bp region
(Figure 7d). Consistently, Pak1 did not show activation of
fibronectin promoter luciferase, when point mutations were made
in the NF-kB–p65 binding consensus site on fibronectin promoter
(from GGGACAGCCC to T GGACAGCCC) (Figure 7d). Next, to
examine the direct recruitment of Pak1 and NF-kB-p65 to the
fibronectin promoter, we performed electrophoretic mobility shift
Figure 6. Pak1 modulates fibronectin via NF-kB pathway. (a) Mia Pa Ca 2 cells were co-transfected with 0.5 mg of 1.2 kb pFN1 -luciferase
reporter and 0.5 mg of Pak1, or Pak1-T423E or Pak1-K299R or pCMV control vector. After 24 h, the cells were lysed, luciferase activity was
measured (n ¼ 3) and was normalized with b-galactosidase activity. Each value represents the mean
±
s.e.m. **Po0.005, compared with vector
control. (b) Western blot analysis confirming fibronectin downregulation after siRNA transfection of Mia Pa Ca 2 cells. Vector and Pak1
overexpressed Mia Pa Ca 2 cells transfected with FN1 siRNA/control siRNA were subjected to (b) colony formation, (c) anchorage-independent
soft agar, (d) migration and (e) invasion. Each value represents the mean
±
s.e.m. **Po0.005, ***Po0.001, compared with control siRNA (f ).
western blot showing the phosphorylation status of p65 in Pak1 modulated clones. (g) BxPC 3 cells were co-transfected with 0.5 mg of 1.2 kb
pFN1-luciferase reporter and 0.5 mg of Pak1 or p65 plasmid or both. After 24 h, the cells were lysed, luciferase activity was measured ( n ¼ 3)
and was normalized with b-galactosidase activity. Each value represents the mean
±
s.e.m. *Po0.05, **Po0.005, compared with vector control
(h). BxPC 3 cells were co-transfected with 0.5 mg of 1.2 kb pFN1-luciferase reporter and 0.5 mg of Pak1 and/or p65 shRNA plasmid/scramble
shRNA. After 24 h, the cells were lysed, luciferase activity was measured (n ¼ 3) and was normalized with b-galactosidase activity. Each value
represents the mean
±
s.e.m. ***Po0.001 compared with the scrambled shRNA treated.
Pak1 signaling regulates fibronectin transcription
S Jagadeeshan et al
7
& 2014 Macmillan Publishers Limited Oncogene (2014) 1 – 10

assay using the oligonucleotides containing the NF-kB consensus
sequence GGGAGAGCCC, and binding of the Pak1 and NF-kB–p65
was analyzed. Results showed the formation of DNA–protein
complexes in the Wt consensus sequence, whereas there is no
binding in the mutant probe. In addition, the observed complex
was highly inhibited in the presence of Pak1 and p65 antibody
alone or together. Furthermore, electrophoretic mobility shift
assay using nuclear extracts from NF-kB–p65 shRNA showed a
significant decrease in the DNA–protein complex (Figure 7e).
Pak1 physically interacts with NF-kB–p65. It has been previously
reported that Pak1 activates NF-kB–p65, resulting in the
p65 phosphorylation followed by nuclear localization of NF-kb–
p65 complex. However, our above finding that Pak1 and
NF-kB–p65 are corecruited onto the fibronectin promoter raises
the possibility of physical interaction between these two proteins.
Results from in vitro pulldown and in vivo coimmunoprecipitation
experiments showed that indeed these two proteins are physically
associated with each other (Supplementary Figure S6).
Figure 7. Pak1 recruitment onto the fibronectin promoter. (a) Western blot showing increased p65 and Pak1 in the nuclear lysate of Pak1
clones indicating the nuclear import of p65 induced by Pak1 overexpression. PARP and a-tubulin were used as nuclear and cytoplasmic
loading controls. (b) BxPC 3 cells were co-transfected with 0.5 mg of 1.2 kb pFN1-luciferase reporter and 0.5 mg of Pak1 or NLS Pak1 mutant
plasmid or vector control. After 24 h, the cells were lysed, luciferase activity was measured (n ¼ 3) and activity was normalized with b-
galactosidase activity. Each value represents the mean
±
s.e.m. **Po0.005, compared with vector. (c) Schematic representation of the FN1
promoter, showing the regions analyzed. ChIP analysis showing the recruitment of (ci) PAK1 and (cii)p65 on the human FN1 promoter in Mia
Pa Ca 2 cells. (ciii) Double ChIP analysis of PAK1–p65 complex recruitment onto the FN1 chromatin region 1 (set 1) in Mia Pa Ca 2 cells. The first
ChIP was carried out with an anti-PAK1 antibody followed by second ChIP with anti-p65 antibody. (civ) ChIP analysis showing the reduction in
the recruitment of Pak1 to FN1 promoter after knockdown p65 using shRNA plasmid. p65 shRNA-mediated knockdown confirmed by western
blotting. Input represents around 1–10% of the total immunoprecipitated DNA. (d) Bx PC 3 cells were co-transfected with 0.5 mg of 1.2 kb
pFN1-luciferase reporter or 200 bp deletion of pFN1-luciferase reporter (pDel) or SDM mutant of pFN1-luciferase reporter (pSDM) and/or
0.5 mg of Pak1 plasmid. After 24 h, the cells were lysed, luciferase activity was measured (n ¼ 3) and was normalized with b-galactosidase
activity. Each value represents the mean
±
s.e.m. *Po0.05, **Po0.005, compared with vector. (e) Electrophoretic mobility shift assay (EMSA)
analysis of p65 and PAK1 binding to the human fibronectin (FN1) promoter using the wild-type biotin-labeled probe (*Probe), cold probe and
biotin-labeled mutant (Mut) oligonucleotides encompassing p65 consensus sites using Mia Pa Ca 2 nuclear lysates. # Indicates the specific
band of interest. EMSA using nuclear extracts from NF-kB–p65 shRNA showing a significant decrease in the DNA–protein complex.
(f) Schematic representation of Pak1 regulation of fibronectin via Pak1–NF-kB–p65 complex.
Pak1 signaling regulates fibronectin transcription
S Jagadeeshan et al
8
Oncogene (2014) 1 – 10 & 2014 Macmillan Publishers Limited

DISCUSSION
Previously, few of the upstream regulators of Pak1 pathway, such
as EGFR
27
and VAV1 protein,
16
have been shown to be involved
and dysregulated in PDAC. However, to our knowledge, this is the
first study to directly implicate the role of Pak1 in pancreatic
tumorigenesis. Recently, Pak1 has emerged as a potential
therapeutic target owing to its oncogenic role in several cancer
types.
28–31
Results from this study show that a majority of PDACs
and PDAC cell lines express high levels of Pak1, clearly indicating
that Pak1 has an important role in PDAC tumorigenesis. This was
supported and validated in vitro by Pak1 overexpression and Pak1
downregulated cell line models and in vivo by an animal model.
Importantly, our data from BxPC 3 cell line clearly demonstrated
that Pak1 modulates transforming properties in Wt-KRAS
pancreatic model also implying Pak1 as a potential oncogene in
KRAS intact tumorigenesis (which need to be explored further).
Further, for investigating the possible mechanism by which Pak1
could promote PDAC tumorigenesis, we utilized a qPCR array that
encoded several classes of protein factors including extracellular
matrix components, cell adhesion, cell cycle, cell growth and
proliferation, apoptosis, transcription factors and regulators and
other genes related to tumor metastasis. Among these, three
genes relevant to PDAC were analyzed, revealing that fibronectin
showed consistent and significant change, whereas Syk did not
show significant change and insulin like growth factor-1 (IGF-1)
was not detectable in the cell lysates (Supplementary Figure S7).
Hence, we established fibronectin as the potential transcriptional
target of Pak1. Fibronectin is an important component of the
extracellular matrix and has been shown to have a vital role in cell
migration, invasion and metastasis of tumors.
32,33
As Pak1 lacks
transcriptional activity of its own, we looked for intermediate
transcriptional factor that could mediate fibronectin transcription.
Previous studies have reported that p65 subunit of NF-kB has a
role in activating fibronectin transcription.
26
It is already reported
that Pak1 regulates cyclin D1 via NF-kB pathway.
14
In addition,
Pak1 signaling has been reported to play a crucial role in the
activation and nuclear translocation of p65 subunit of NF-kB.
25
Owing to the fact that the connection between Pak1 and NF-kB–
p65 is previously established and that there is a potent NF-kB
binding site on fibronectin promoter, we hypothesized that Pak1
might be regulating fibronectin via NF-kB. This was supported and
confirmed by promoter luciferase studies, Pak1–p65 interaction
studies, ChIP experiments and electrophoretic mobility shift assay
analysis. Further, our results from active-, kinase dead- and NLS
Pak1 mutants also showed that Pak1 kinase activity and its nuclear
localization are essential for induction of fibronectin transcription.
This clearly supports the previous observation that nuclear
localization of Pak1 has an important role in promoting
tumorigenic properties of cancer cells.
34
In summary, the study
illustrated that Pak1 interacts with and modulates the activity of
NF-kB–p65 subunit and together as a complex might move into
the nucleus where they bind with the fibronectin promoter and
enhance its transcription, thus promoting the invasion and
metastatic potential of the pancreatic cancer cells (Figure 7f).
Given the fact that Pak1 is a central player in growth factor
signaling and morphogenetic processes that control cell prolifera-
tion, migration, invasion and cytoskeleton organization and now
being recognized as a transcriptional regulator for several genes, it
would be worthwhile to explore Pak1 inhibitors that interfere with
Pak1 activity to deliver therapeutic benefits in several cancers.
MATERIALS AND METHODS
Antibodies and reagents
Pak1, phospho-p65 and PARP antibody were obtained from Cell Signaling
Technology (Beverly, MA, USA), Vinculin and b-actin from Sigma-Aldrich
(St Louis, MO, USA), T7 from Bethyl Laboratories (Montgomery, TX, USA),
p65 and a-tubulin from Santa Cruz Biotechnology (Dallas, TX, USA), and
fibronectin—total and EDA (IST-9) from BD Biosciences (Bedford, MA, USA)
and Abcam (Cambridge, MA, USA) respectively. Fibronectin human ELISA
kit was purchased from Abcam. siRNA: Pak1 was obtained from Cell
Signaling Technology, FN1 from SA Biosciences (Valencia, CA, USA) and
control siRNA from Santa Cruz Biotechnology. Plasmids: pCMV6-Myc-Pak1,
pCMV6-Myc-Pak1-T423E and pCMV6-Myc-Pak1-K299R were obtained from
Addgene, Cambridge, MA, USA – deposited by Dr Jonathan Chernoff, Fox
Chase Cancer Centre, Philadelphia. p65 plasmid, anti-p65 shRNA and
scramble shRNA plasmid were obtained from Dr K Boume
´
diene, University
of Caen Lower Normandy, France, pFN1 (1.2 Kb) Luc Promoter (pGL3–FN1
Luc) from Dr Eric S White, University of Michigan Medical School, USA. All
the vectors (pcDNA3.1-A,B,C and pcDNA6-V5-HisA,B,C) were purchased
from Invitrogen (Carlsbad, CA, USA). pcDNA3.1-C-T7-Pak1, pcDNA6-V5-
Pak1, and pGEX-GST-Pak1 were cloned from pCMV6-myc-Pak1. Pak1 NLS
mutant was made by site-directed mutagenesis using conditions and
primers as described earlier.
23
pRC-FN1-Wt and pRC-FN1-EDA domain was
kindly gifted by Dr Andre
´
s F Muro, ICGEB, Trieste, Italy. All the recombinant
plasmids and lentiviral particles used in this manuscript have the clearance
and approval of DBT—Institutional Biosafety Committee (IBSC) of IIT
Madras. Additional primer details are provided in Supplementary Table 1.
Cell lines and tissues
The PDAC cell lines Mia Pa Ca 2 and Panc 1 were procured from NCCS,
India. Capan 1 was kindly gifted by Dr Subhash C. Chauhan (Cancer Biology
Research Center, Sanford Research, USA); Capan 2 by Prof Ilona Silins
(Institute of Environmental Medicine, Karolinska Institutet, Sweden); MDA
Panc 28 by Dr Marsha Fraizer (University of Texas, M. D. Anderson Cancer
Center, USA); MDA Panc 48 by Dr Paul J Chiao (University of Texas, M. D.
Anderson Cancer Center, USA); and BxPC 3 by Dr Uddalak Bharadwaj
(Baylor College of Medicine, USA). Human immortalized ductal epithelial
cells HPV16 E6/E7 (HPDE6/E6E7) was purchased from Applied Biological
Materials (Richmond, BC, Canada). Human pancreatic tissue samples were
collected and the study was approved by the Institutional Human Ethics
Committee.
Generation of stable clones
Stable Pak1 overexpression clones were generated upon transfection of
pcDNA3.1-T7-Pak1 plasmid and corresponding empty vector in Mia Pa Ca
2, followed by selection with G418 (MP Biomedicals, Santa Ana, CA, USA).
Pak1 KD clones were generated by transducing MDA Panc 28, Mia Pa Ca 2
and BxPC 3 with MISSION lentiviral particles coding for shRNA sequence
against PAK1 and NT shRNA as the negative control (Sigma-Aldrich) and
were selected for stable integration using puromycin (MP Biomedicals).
Immunohistochemistry
Tissue microarrays containing 60 tumors and adjacent normal tissue were
purchased from US Biomax (Rockville, MD, USA) and ISU ABXIS AccuMax
(San Diego, CA, USA). Pak1 expression was evaluated using an indirect
immunoperoxidase procedure (ABC-Elite, Vector Laboratories, Burlingame,
CA, USA). Staining results were scored by the pathologist. Severity of
cytoplasmic staining of tumor cells and rate of staining were graded semi-
quantitatively as follows: 0 ¼ o1%, 1 þ¼1–10%, 2 þ¼11–50%,
3 þ¼51–80%, 4 þ¼480% of tumor cells stained. Based on the score,
Pak1 expression was categorized as negative (no cytoplasmic staining in
tumor cells, 0,1 þ ), weak (low staining, 2 þ ), and high (diffuse moderate to
strong staining in tumor cells, 3 þ and 4 þ ).
Cell migration and invasion assays
Cells (5  10
4
) were suspended in serum-free media and added to the
upper chamber of an insert (Biocoat-BD Biosciences), and the insert was
placed in a 24-well plate containing media supplemented with 10% fetal
bovine serum. The migrated/invaded cells were stained and scored.
Anchorage-independent colony formation assay
Cells were suspended in complete media containing 0.5% agarose. Cells
were overlaid onto a bottom layer of solidified 0.8% agarose in media at
cell concentrations of 5  10
3
cells per well and incubated for 2 weeks,
stained and counted.
Pak1 signaling regulates fibronectin transcription
S Jagadeeshan et al
9
& 2014 Macmillan Publishers Limited Oncogene (2014) 1 – 10

Generation of SDM and deletion construct of fibronectin promoter
Using pGL3-FN (1.2 Kb)-luc plasmid as template, mutations were made in
the NF-kB consensus site within the region  318 to  13 by mutating
G-T in the position  97 using QuikChange kit (Stratagene, La Jolla, CA,
USA). The deletion construct excluding the Pak1 binding region (  318 to
 78) was made with NEB Q5 polymerase, using specific primers, followed
by ligation of the digested fragments. Additional details are provided in
Supplementary methods.
Animal studies
Mice (nu/nu) that were 6 weeks old were obtained from the Animal
Breeding Facility, Reliance Life Sciences, India, after obtaining ethical
clearance. Mice were divided into two groups and injected subcutaneously
on both the sides using a 25-gauge needle with MDA Panc 28 stable Pak1
KD clones versus the NT clones. The growth of xenografts was monitored.
PCR array studies
Human tumor metastasis PCR array for 84 genes involved in tumorigenesis
and metastasis was purchased from SA Biosciences. Target genes whose
expression was differentially regulated (at least fivefold difference) in the
clones were selected. Additional details are provided in Supplementary
methods and Supplementary Tables 2 and 3.
Statistical analysis
Data are expressed as the mean
±
s.e.m. and analyzed by Student’s t-test
using SigmaPlot (SYSTAT software Inc., Chicago, IL, USA). The P-value,
Po0.05 is considered significant.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
ACKNOWLEDGEMENTS
We thank Silpa for help in mutagenesis and cloning and Swarnalatha for help with
Pak1 lentiviral clones. Thanks to Sekar Sathiya, Rohan Prasad, Hemadev and the
entire team of the Centre for Toxicology and Developmental Research (CEFT), Sri
Ramachandra University, Chennai, for help with animal experiments. We thank the
Department of Biotechnology (DBT), Government of India for the financial support to
SKR (grant no.: BT/PR13559/Med/30/283/2010) and Indian Institute of Technology
Madras (IITM) for all other facilities.
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Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)
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Oncogene (2014) 1 – 10 & 2014 Macmillan Publishers Limited