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ORIGINAL ARTICLE
Src kinase potentiates androgen receptor transactivation function and
invasion of androgen-independent prostate cancer C4-2 cells
M Asim
1,2,4
, IA Siddiqui
1,4
, BB Hafeez
1
, A Baniahmad
2,3
and H Mukhtar
1
1
Department of Dermatology, University of Wisconsin, Madison, WI, USA;
2
Medical Faculty, Institute of Human Genetics and
Anthropology, University of Jena, Jena, Germany and
3
Department of Biosciences, University of Kuopio, Kuopio, Finland
Prostate cancer is one of the most prominent malignancies
of elderly men in many Western countries including
Europe and the United States with increasing trend
worldwide. The growth of normal prostate as well as of
prostate carcinoma cells depends on functional androgen
receptor (AR) signaling. AR manifests the biological
actions of androgens and its transcriptional activity is
known to be influenced by signal transduction pathways.
Here we show that Src, a nonreceptor tyrosine kinase, is
overexpressed in androgen-independent prostate carcino-
ma C4-2 cells. Interestingly, the expression of Src was
found to progressively increase (up to threefold) in
transgenic adenocarcinoma of mouse prostate mice as a
function of age and cancer progression. Blocking Src
kinase function by a specific inhibitor, PP2, resulted in
decreased AR transactivation function on two different
reporters, mouse mammary tumor virus (MMTV) and
prostate-specific antigen (PSA). Consistent with this,
overexpression of a functional Src mutant also led to a
dramatic decrease in AR transactivation potential in a
hormone-dependent manner. Interference with Src func-
tion in C4-2 cells led to decreased recruitment of AR on
the target gene PSA enhancer and also resulted in the
abrogation of hormone-dependent PSA transcript induc-
tion. Src inhibition also led to a dramatic decrease in the
cell invasion in addition to decreasing the cellular growth.
We suggest that targeting Src kinase could be an effective
strategy to inhibit prostate cancer growth and metastasis.
Oncogene (2008) 27, 3596–3604; doi:10.1038/sj.onc.1211016;
published online 28 January 2008
Keywords: androgen receptor; casodex; invasion; pros-
tate cancer; PP2; Src kinase
Introduction
Prostate cancer (PCa) is an endocrine malignancy,
which initially depends on male sex hormones, testos-
terone and dihydrotestosterone for growth. Androgen
receptor (AR), which is a member of nuclear hormone
receptor superfamily, manifests the biological action of
male hormones. AR has a modular structure containing
the N terminus harboring transcriptional activation
domain(s) (or transactivation domain), a central DNA-
binding domain (DBD) and a C-terminal ligand-binding
domain (LBD). Binding of androgens to the LBD
induces conformational changes in the AR and subse-
quently results in the shuttling of the receptor to the
target cell nucleus where it forms homodimer that is
recruited on the androgen response element (ARE)
present in the regulatory element on the target genes
such as prostate-specific antigen (PSA) and regulates
growth of prostate gland by modulating the expression
of target genes implicated in growth and proliferation
(Young et al., 1992; Perry et al., 1996).
To block AR function, androgen ablation using
antiandrogens, in particular by Casodex (bicalutamide),
remains the hormone therapy for PCa. This treatment
unfortunately proves ineffective as the androgen-inde-
pendent tumor eventually emerges in a short span of 3–4
semesters for which there is no curative therapy (Isaacs,
2000). It is noteworthy that hormone-refractory PCa
cells nevertheless depend on functional AR signaling for
growth as their growth is severely compromised if AR is
depleted in cells using RNAi or ribozyme (Chen et al.,
1998, 2004; Haag et al., 2005; Liao et al., 2005). This
makes AR a prime drug target and AR-regulated genes
as potential biomarkers in the treatment and diagnosis
of advanced PCa, respectively.
It has been proposed that in the absence of a ligand,
the AR activation could take place by cross talk with
various growth factors known to be components of
protein kinase pathways. For example, it has been
demonstrated that epidermal growth factor and its
receptor, keratinocyte growth factor, insulin-like growth
factor-1, protein kinase A (PKA), mitogen-activated
protein kinase (MAPK), as well as interleukin 6 (IL-6)
could activate the AR signaling (Culig et al., 1994;
Abreu-Martin et al., 1999; Craft et al., 1999; Sadar,
1999; Jenster, 2000). An additional mechanism under-
lying ligand-dependent activation of the AR by these
Received 3 October 2007; revised 29 November 2007; accepted 3
December 2007; published online 28 January 2008
Correspondence: Dr H Mukhtar, Department of Dermatology,
University of Wisconsin, 1300 University Avenue, Medical Science
Center, 25B, Madison, WI 53706, USA.
E-mail: hmukhtar@wisc.edu or Dr A Baniahmad, Medical Faculty,
Institute of Human Genetics and Anthropology, University of Jena,
Jena 07743, Germany.
E-mail: aban@mti.uni-jena.de
4
These authors contributed equally to this work.
Oncogene (2008) 27, 3596–3604
&
2008 Nature Publishing Group
All rights reserved 0950-9232/08 $30.00
www.nature.com/onc
alternative pathways may involve phosphorylation of
either the AR or its associated cofactor proteins (Sadar,
1999; Dotzlaw et al., 2002; Ueda et al., 2002).
In this study we demonstrate that Src, a nonreceptor
tyrosine kinase, is overexpressed in PCa and interference
with Src function results in inhibition of PCa growth via
regulation of AR function and decreased cell invasion.
We also demonstrate that Src inhibition results in the
interference of AR functioning. We suggest that Src
inhibition, either by relatively safer chemical inhibitors
or by natural diet-based compounds may be an effective
strategy for inhibition of PCa growth and invasion.
Results
Src kinase is overexpressed in androgen-independent
prostate carcinoma cells
Western blot analysis was performed to screen for
endogenous Src expression in a panel of normal as well
as PCa cells. Here, in addition to normal prostate
epithelial cells (PrEC), androgen-dependent LNCaP
prostate carcinoma, androgen independently growing
PC3, 22Rn1 and C4-2 cells were included. Interestingly,
C4-2 cells are derivatives of LNCaP cells and express
functional endogenous AR; they can grow in an
androgen-independent manner making them an excel-
lent model representing transition of the initial andro-
gen-dependent disease to an androgen-independent state
(Thalmann et al., 1994). The data indicate that the
expression of Src kinase was modest in PrEC normal
human PCa cells (Figure 1a). The expression of Src was
significantly higher in androgen-independent PCa cell
lines PC3 (two fold), C4-2 (three fold) and slightly
higher in 22Rn1 cells when compared with normal PrEC
(Figure 1a).
Interestingly, we also found that Src expression is
progressively increased during the progression of PCa in
transgenic adenocarcinoma of mouse prostate
(TRAMP) model that spontaneously develops prostate
tumor with increasing age (Greenberg et al., 1995). A
progressive increase in the Src protein expression and its
phosphorylation at Tyr
418
in the activation loop of
kinase domain that upregulates its activity was observed
in TRAMP mice as cancer progressed from not
detectable cancer at 8 weeks to well-differentiated
carcinoma at 16 weeks and finally to moderately
differentiated carcinoma at 24 weeks (Figure 2a). The
expression of Src protein in nontransgenic littermates
was found to be constant (data not shown). Similar
22Rν
ν
1
PrEC
LNCaP
C4-2
PC3
Src
β-actin
1 2.1 2.9 1.7 1.5
Control Casodex
PP2 PP2+ Casodex
200
400
600
800
1000
1200
Apoptosis induction
(% Control)
PP2
Casodex +
++
+
20
40
60
80
100
120
Relative cell number
PP2
PP2
Control
Figure 1 Src kinase is overexpressed in androgen independently growing C4-2 prostate carcinoma cells and its blockade induces
apoptosis in C4-2 cells. (a) Western blot analysis of endogenous Src expression was carried out as described in ‘Materials and methods’.
Equal loading of protein was confirmed by stripping the membrane and reprobing it with b-actin. Values on top of bands represent
relative densities normalized to b-actin. (b) Immunofluorescence analysis to detect apoptotic cells based on the detection of annexin V
was carried out as described in ‘Materials and methods’. (c) Quantification of apoptosis is presented. (d) A total of 200 000 C4-2 cells
were cultured in 10 cm tissue culture dishes in T-media containing 10% normal serum for a period of 6 weeks. Cells were then provided
media twice a week along with 1 mMPP2. After 6 weeks cells were counted and photographed. The bar graph shows relative cell
number. Experiments were repeated three times with similar observations and representative figures are shown.
Src kinase and prostate cancer
M Asim et al
3597
Oncogene
results were observed by immunohistochemical analysis
of the prostate tissue of TRAMP mice (Figure 2b).
Mice with moderately differentiated (24 weeks) exhib-
ited strong staining for Src, with negligible staining in
their nontransgenic counterparts. This staining was
especially seen in the epithelia of prostatic acini and
also in the stroma. These results clearly indicate
the progressive increase in Src expression during the
progression of PCa.
Inhibition of Src kinase induces apoptosis and in
combination with Casodex further enhances apoptotic
response in androgen-independent prostate cancer
C4-2 cells
To address the role of Src overexpression in PCa cells,
C4-2 cells, which showed a robust expression of Src,
were chosen for further studies. Cells were treated with
PP2, a specific chemical inhibitor of Src kinase.
Treatment of C4-2 cells with potent antiandrogens, in
particular by Casodex, had marginal effect on cellular
growth similar to untreated control cells indicating their
androgen-refractory/independent behavior (Figures 1b
and c). However, PP2 treatment led to a dramatic
increase (seven fold) in the apoptosis induction identi-
fied based on the increased expression of annexin V, an
early marker of apoptosis. C4-2 cells co-treated with
PP2 and Casodex exhibited a further heightened
apoptosis induction (ten fold; Figures 1b and c). To
address the role of Src kinase in growth promotion, a
cell growth assay was performed. Incubation of C4-2
cells with Src inhibitor led to a 50% decrease in cell
growth (Figure 1d). These findings indicate that Src
overexpression in androgen-independent PCa can serve
to defy response to antiandrogen therapy.
Src specifically potentiates AR transactivation function in
C4-2 cells
AR transactivation is critical for PCa progression and
the antiandrogen therapy is known to inactivate AR
function by decreasing its transactivation. We therefore
determined the possible role of Src in modulating AR
transactivation function using luciferase reporter assays.
C4-2 cells were transfected with MMTV-Luc and
PSA-Luc androgen-responsive reporters. We found that
AR-mediated transactivation repressed to almost 50%
on both mouse mammary tumor virus (MMTV) and
PSA reporter elements by treatment of cells with Src
inhibitor PP2 (Figure 3a). This suggests that in androgen
independently growing PCa cells, AR is still functional
on its target genes and that its transactivation function is
potentiated by endogenously overexpressed Src kinase.
To test, whether PP2-mediated decrease in target gene
expression was specific to androgen-responsive promo-
ters and was not due to decrease in global transcription,
another control reporter plasmid pCMV-LacZ, which is
not responsive to androgens was used. As expected, the
reporter activity remained unaffected by Src inhibition
(data not shown) suggesting that PP2 treatment specifi-
cally decreases AR transactivation on its target genes.
To further confirm these findings reporter assays were
performed with ectopically expressed Src-wt (wild-type)
or Src dominant-negative mutant in C4-2 cells. As
compared to control, where a robust hormone induction
was observed (Figure 3b), PP2 co-treatment led to a
decrease in AR transactivation. However, ectopic
expression of Src-wt did not further increase hormone-
dependent AR transactivation suggesting that both
ectopic wt-Src and endogenous kinases act through the
similar pathway to enhance AR transactivation. Con-
sistent with the inhibitor data, interference with wt-Src
function by ectopic overexpression of dominant-nega-
tive Src mutant led to a potent decrease in AR
transactivation in a hormone-dependent manner, in line
with the inhibitor data (Figure 3a). This indicates that
enhanced transactivation by AR observed in C4-2
cells is in part boosted by overexpression of Src
kinase as evident from PP2 chemical as well as Src
8 Wks 16 Wks 24 Wks
Src
β-actin
Non-Transgenic
Trans
g
enic
1.0 1.0 1.7 1.9 2.7 2.9
1.0 1.3 5.7 14.8 12.7 14.9
p-Src
Figure 2 Expression of Src in the dorsolateral prostate during
progressive stages of prostate cancer development in transgenic
adenocarcinoma of mouse prostate (TRAMP) mice. (a) Protein
levels of Src and phospho-Src (Tyr 416) by immunoblot analysis.
Equal loading of protein was confirmed by stripping the blot and
reprobing with b-actin antibody. Western blot analysis was
conducted in five animals in each group, and only representative
blots are shown. Values on top of bands represent relative densities
normalized to b-actin. (b) Immunohistochemical analysis as
detailed in ‘Materials and methods’, the protein levels were
determined in the dorsolateral prostate of 8-, 16-, and 24-week-
old TRAMP mice. Immunostaining data were confirmed in two
slides from five animals. Photomicrographs (magnification, 20
and 40) represent immunohistochemical staining for Src in
TRAMP mice with moderately differentiated carcinoma (24
weeks).
Src kinase and prostate cancer
M Asim et al
3598
Oncogene
mutant-mediated inhibition of wt-Src function, in both
cases it led to a decrease in AR transactivation function.
Src kinase inhibition decreases AR recruitment on PSA
enhancer and modulates PSA expression
To determine the effect of Src inhibition on AR
recruitment to its target genes, chromatin immunopre-
cipitation (ChIP) assay was performed with C4-2 cells.
A substantial recruitment of AR was observed on
androgen-responsive PSA enhancer in the absence of
androgen agonist R1881 (Figure 4a). Treatment of C4-2
cells with R1881 led to a further increase in AR
recruitment indicating hormone responsiveness of these
cells. Intriguingly, C4-2 cells pretreated with Src
inhibitor exhibited drastic loss of AR recruitment on
PSA enhancer in the absence of R1881. However, AR
was strongly re-recruited upon treatment of PP2-
pretreated cells with R1881.
0.2
0.4
0.6
0.8
1.0
1.2
Luciferase units
(LacZ corrected)
PP2
MMTV
+
0.2
0.4
0.6
0.8
1.0
1.2
1.4
PSA
+
PP2
Luciferase units
(LacZ corrected)
Fold activation (RLU)
without R1881
with R1881
control PP2 Src-wt Src-mut
50
100
150
200
250
300
350
1
Figure 3 Src kinase inhibition attenuates androgen receptor (AR)
transactivation on reporter genes in C4-2 cells. (a) C4-2 cells were
seeded out in 10% serum containing T-media and transfected with
mouse mammary tumor virus (MMTV)-Luc or prostate-specific
antigen (PSA)-Luc reporter plasmids as detailed in ‘Materials and
methods’ section. Luciferase values obtained without the inhibitor
in each case were set as 1. The graph represents the activation of
reporter expression as LacZ normalized relative luciferase units. (b)
C4-2 grown in T media supplemented with 10% charcoal-stripped
fetal bovine serum (FBS) and transfected with MMTV-Luc
reporter along with expression plasmid Src-wt or dominant-
negative Src. Cells were treated as described in the ‘Materials and
methods’ section. Luciferase values were normalized with renilla
luciferase internal control. Values obtained without the inhibitor
and ligand were set as 1. The graph represents the activation of
reporter as fold hormone induction. Experiment was repeated three
times with similar observations, a representative figure is shown.
PP2
R1881
IgG Control
α
α
-AR
Input
++
++
PP2
R1881 ++
++
PP2
R1881 ++
++
q real time RT-PCR PSA
transcript level (normalized)
0.001
0.002
0.003
0.004
0.005
0.006
0.007
20
40
60
80
PSA (in ng)
Figure 4 Src inhibition modulates the recruitment of androgen
receptor (AR) on prostate-specific antigen (PSA) enhancer and
attenuates PSA transcript induction in C4-2 cells. (a) A total of
750 000 C4-2 cells were grown for 4 days in 10% fetal bovine serum
(FBS) containing media and were further grown for 48h in
hormone-depleted FBS containing T-media and treated with 1mM
PP2. Before lysis, cells were treated with ligands R1881 (10
8
M)for
1 h after which chromatin immunoprecipitation (ChIP) procedure
was followed as described in the ‘Materials and methods’. Purified
DNA was PCR amplified by specific primers spanning the enhancer
region of PSA containing ARE III and pictured. (b)Intotal200000
C4-2 cells per well were seeded out in hormone-depleted FBS
containing T-media in six-well tissue culture dishes. After 24 h cells
were treated with R1881 (10
10
M) for 48 h. Afterwards total cellular
RNA was isolated, reverse transcribed to cDNA and amplified by
light cycler using specific primers and control primers for actin. The
graph represents the actin-normalized values of the PSA transcript.
(c) Media supernatant from the above experiment was used to test
for PSA secretion using kit as described in ‘Materials and methods’.
Experiment was repeated three times with similar observations, a
representative figure is shown.
Src kinase and prostate cancer
M Asim et al
3599
Oncogene
To test the implication of the modulation of AR
recruitment by Src blockade on target gene expression,
real-time RT-PCR and enzyme-linked immunosorbent
assay (ELISA) for PSA were performed. Treatment with
R1881 led to further three fold induction of PSA gene
transcripts, in agreement of enhanced AR recruitment
under that condition (Figure 4b). R1881-mediated
induction of PSA mRNA expression was lost in C4-2
cells pretreated with PP2. Consistent with this, the
induction of PSA secretion in response to hormone was
also abolished in C4-2 cells pretreated with PP2
(Figure 4c). Taken together, results suggest that a basal
level of PSA transcripts are expressed in C4-2 cells in the
absence of agonist. Agonist R1881 leads to enhanced
AR transactivation function on target genes reflected in
the induction of PSA gene transcription. PP2 treatment
(the extent blockade was not tested, therefore a milder
expression) suggests that induction of PSA transcription
by AR in response to agonist is significantly contributed
by Src signal transduction pathway, inhibiting Src
activity therefore decreased AR target gene expression
and hormone-responsive induction. Treatment of C4-2
cells growing in androgen-depleted T-media with Src
kinase inhibitor PP2 alone however, leads to loss of AR
recruitment, surprisingly no further decrease in PSA
gene transcript was observed. This suggests that the C4-
2 cells express basal level of PSA mRNA independent of
AR transactivation function. However, induction of
PSA expression by AR requires not only enhanced AR
occupancy but also Src-mediated signaling plays an
important role in AR transactivation leading to
enhanced PSA expression.
Src kinase inhibition suppresses colony formation in C4-2
cells
As shown above (Figure 1d) incubation of C4-2 cells
with Src inhibitor led to a 50% decrease in cell growth.
The long-term functional consequence of PP2-mediated
decrease in AR transactivation and reduced target gene
expression in response to agonist was tested in relation
to cellular growth. C4-2 cells stably expressing either
wt-Src or Src dominant-negative mutants were therefore
generated. C4-2 cells stably overexpressing Src-wt
formed large-sized colonies indicating their accelerated
growth (Figure 5), which was comparable to colonies
derived from C4-2 stably transfected with empty vector.
However, stable clones of C4-2 overexpressing the
dominant-negative mutant form of Src showed a
marked reduction in colony size indicating the growth-
promoting effect of Src kinase. Taken together these
results indicate that androgen independently growing
cells overexpress Src kinase that is associated with
enhanced growth.
Src inhibition decreases C4-2 cell invasion
As the tumor metastasis by invading cells remains a
major cause of therapy failure in PCa patients, an
invasion assay was performed to test whether inhibition
of Src may modulate invasive potential of C4-2 cells,
which are known to have high metastatic potential
(Thalmann et al., 2000). To rule out the fact that a
decrease in cell number may result in decreased
invasion, a time frame of 20 h at which no apoptosis
was detectable was selected. While untreated and
Casodex-treated C4-2 cells showed a vigorous migration
through extracellular matrix, cells treated with PP2
showed a potent inhibition of their migratory capability
(Figure 6a). The combination of PP2 and antiandrogen
Casodex did not further decrease the invasion by these
cells suggesting that Src-assisted invasion of C4-2 cells
may occur independent of genomic action of AR
function. Nevertheless, it may involve nongenomic AR
actions whereby MAPK–Ras–Raf axis stimulation by
AR signaling (Peterziel et al., 1999) may play a role in
invasion and metastasis. To further strengthen these
findings, stable clones overexpressing wt or mut Src were
generated and the impact of Src kinase was tested on cell
migration through extra cellular matrix. Here, no
change in cell migration was observed between cells
overexpressing either empty vector or Src-wt
(Figure 6b), interestingly however, C4-2 cells over-
expressing mutant form of Src showed drastic reduction
in their migratory capacity (Figure 6b, last panel).
Discussion
The transcriptional activation function of AR is not
only essential for the normal sexual development in men
but is also implicated in the progression of PCa (Chen
Src-wt Src-mutEmpty vector
Figure 5 Src inhibition decreases growth of C4-2 cells. The stable clones of C4-2 overexpressing Src-wt or Src-mut were generated
as described in ‘Materials and methods’. Experiment was repeated three times with similar observations, a representative
photomicrograph is shown.
Src kinase and prostate cancer
M Asim et al
3600
Oncogene
et al., 2004; Notini et al., 2005). Src kinase is activated in
breast carcinoma where it has been shown to promote
agonistic action of tamoxifen (Shah and Rowan, 2005).
However, its role remains poorly understood in PCa.
Our observation that Src expression proportionates with
enhanced androgenic independence suggests that Src
kinase may play a nonredundant function in potentiat-
ing AR function in PCa cells and thereby may serve as
an important therapeutic target. Also, in TRAMP
model expression and activating phosphorylation of
Src is enhanced indicating its association with PCa
growth that is regulated by AR. It is evident from the
experiment that C4-2 cells show a basal leaky expression
of PSA gene transcripts in the absence of agonist
R1881 (Figure 3b), which may be explained largely by
the agonist-independent recruitment of AR on PSA
enhancer or may due to the androgen-independent leaky
expression of PSA.
The AR is synthesized as a single 110 kDa protein,
which becomes rapidly phosphorylated to a 112 kDa
protein (Brinkmann et al., 1992). A recent report has
suggested that AR tyrosine phosphorylation is in fact
induced by growth factors and elevated in hormone-
refractory prostate tumors and that mutation in these
tyrosine residues significantly inhibits the growth of PCa
cells under androgen-depleted conditions (Guo et al.,
2006). The study further showed a positive correlation
of AR tyrosine phosphorylation with Src activity in
human PCa. Src kinase is known to be overexpressed in
PCa and enhances the AR function at very low
concentration of agonist R1881 (Castoria et al., 2003).
In the androgen-depleted environment, the recruitment
of AR on PSA enhancer was completely abolished
without altering basal expression. It is possible that
phosphorylation of various domains may have different
functional consequences on receptor activation, suggest-
ing that Src enhances the transactivation function of AR
and may also cooperate in its ability to bind to target
genes.
Another set of co-regulatory molecules such as AR
co-repressors repress AR transactivation function and
therefore inhibit PCa cell growth and their expression is
decreased in some PCa cells (Baniahmad, 2005; Wang
et al., 2005; Dehm and Tindall, 2007). We have recently
shown that stable overexpression of co-repressors alien
in LNCaP cells leads to an antihormone-dependent
decrease in cell growth (Moehren et al., 2007). Also,
PKA pathway leads to reduced co-repressor SMRT
binding to AR and thus could be a pathway by which
PKA activates AR (Dotzlaw et al., 2002) and a similar
Control Casodex
PP2 + CasodexPP2
Empty Src-wt Src-mut
Figure 6 Src inhibition decreases invasive potential of C4-2 cells. (a,b) A total of 70 000 wt-C4-2 cells or Src stable clones were
suspended in culture media and the experiment was performed as described in ‘Materials and methods’. Wt C4-2 cells were treated with
PP2 and allowed to invade for 20 h followed by staining as per manufacturer’s protocol. Experiment was repeated three times with
similar observations and representative photomicrographs of invading cells are shown.
Src kinase and prostate cancer
M Asim et al
3601
Oncogene
possibility might apply also for Src pathway. The
decrease in the AR transactivation observed may
therefore be due to the enhanced interaction of co-
repressors to AR and thereby a decrease in AR
transactivation and local chromatin condensation asso-
ciated with lower accessibility of transcription factors.
In addition a decrease in the phosphorylation of
critical AR residues in the amino terminus by Src
inhibition may also explain the observed decrease in AR
transactivation. C4-2 cells also express PSA at a basal
level, which could be attributed to the androgen-
independent marginal recruitment of AR allowing the
expression of PSA, in line with a report suggesting PSA
basal expression in androgen independently growing
cells do not require AR binding (Jia and Coetzee, 2005).
However, induction of PSA mRNA by R1881 is
associated with enhanced AR binding (Figure 4a). This
induction of AR-mediated PSA expression may require
Src kinase signaling blocking that leads to decreased
target gene expression analysis (Figures 4b and c). Long-
term PP2 treatment leads to growth inhibition of C4-2
cells in androgen-containing growth factor rich T-
media, suggesting that PP2-mediated Src blockade not
only affects induction of target gene expression, but also
leads to decreased growth of C4-2 cells presumably by
interfering with the expression of target genes that play
important function in cell growth and proliferation.
Another important finding of this work is the promotion
of cell invasion by Src kinase. As a result, interfering
with Src function either chemically or by expressing
dominant-negative Src leads to robust decrease in cell
invasion. Blocking Src function could therefore serve as
an important strategy against metastasis of PCa and
thereby may result in more effective treatment of a
prostate-confined disease either by radiotherapy or
surgery. In addition, Src function may be explored to
understand and better treat other endocrine malignan-
cies. We suggest that employment of Src inhibitors may
therefore offer a great therapeutic advance by prostate-
specific confinement of the tumor, thereby making the
disease manageable by currently available therapies,
both androgen ablation and surgical management.
Materials and methods
Materials
AR and Src antibodies were obtained from Santa Cruz
Biotechnology Inc. (Santa Cruz, CA, USA) and Cell Signaling
(Danvers, MA, USA), respectively. Anti-mouse and anti-
rabbit secondary horseradish peroxidase (HRP) conjugates
were from Amersham Life Science Inc. (Piscataway, NJ,
USA), R1881 from PerkinElmer (Waltham, MA, USA).
Annexin V-Fluos-based immunofluorescent apoptosis staining
kit was from Roche (Berkeley, CA, USA). Guanidium HCl
and hematoxylin were from Sigma Chemicals (St Louis, MO,
USA). RNA purification kit was from Qiagen (Valencia, CA,
USA). BCA protein assay kit was obtained from Pierce
(Rockford, IL, USA). Luciferase assay reagent was purchased
from Promega (Madison, WI, USA). The human PSA ELISA
kit was purchased from Anogen (Ontario, Canada).
Diaminobenzidine was obtained from DakoCytomation
(Carpinteria, CA, USA) and Novex precast Tris-glycine gels
from Invitrogen. PP2 was procured from Calbiochem (San
Diego, CA, USA). Src-wt and mutant plasmids were kind gift
from Dr Sarah Courtneidge.
Protein extraction and western blotting
Protein lysates from cultured cells were prepared, 40 mg protein
was subjected to SDS–polyacrylamide gel electrophoresis
(PAGE) and western blot was performed as previously
described (Siddiqui et al., 2006). The extraction of tumor
proteins from TRAMP mice has been described previously
(Adhami et al., 2004). Densitometric measurements of the
bands in western blot analysis were done using digitalized
scientific software program, UN-SCAN-IT, purchased from
Silk Scientific Corporation (Orem, UT, USA).
Immunohistochemical analysis
Sections (4 mm) were cut from paraffin-embedded prostate
tissues. Immunostaining was performed using specific anti-
bodies with appropriate dilutions and was replaced with either
normal host serum or block for negative controls, followed by
staining with appropriate HRP-conjugated secondary anti-
bodies. The slides were developed in diaminobenzidine and
counter stained with a weak solution of hematoxylin stain as
described previously (Adhami et al., 2004). The stained slides
were dehydrated and mounted in Permount and visualized on
a Zeiss-Axiophot DM HT microscope (Zeiss-Axiophot, Jena,
Germany). Images were captured with an attached camera
linked to a computer.
Apoptosis detection by fluorescence microscopy
The cells were grown on cell culture slides (BD Biosciences,
Rockville, MD, USA) and then treated with 1 mMof PP2 for
48 h period of time. The cells were then incubated for 10 min
with Annexin V-Fluos labeling reagent to detect annexin, an
early marker of apoptosis and analysed using fluorescence
microscopy (excitation at 450–500 nm and detection at 515–
565 nm). Cells were visualized by Zeiss confocal microscope
and green fluorescent cells were scored as apoptotic.
Cell culture and transient transfection
LNCaP cells were grown as previously described (Moehren
et al., 2007). PC3 and 22Rn1 were grown in RPMI-1640
medium containing 10% fetal bovine serum (FBS). Human
PrEC were obtained from Cambrex Bioscience (Walkersville,
MD, USA) and grown in prostate epithelial basal cell medium
(Cambrex Bioscience) according to the manufacturer’s instruc-
tions and the C4-2 cells were in T-media (Invitrogen) contain-
ing 10% FBS (American type culture collection) and 1%
penicillin–streptomycin solution (Invitrogen). Transfections
were performed using a modified calcium-phosphate method
(Dotzlaw et al., 2002). In total, 300 000 C4-2 cells were
seeded out in six-well dishes and transfected with 1 mgof
reporter plasmid MMTV-Luc or PSA-Luc; 0.2 mg either of
pCMV-LacZ or renilla luciferase plasmid served as internal
control; 1 mg either of wt-Src vector or the dominant-negative
Src was used for transfection. Cells were treated with the
agonist R1881 (10
9
M) and harvested 72 h posttransfection
using passive lysis buffer provided with luciferase assay reagent
to measure both luciferase activity and b-galactosidase
or renilla luciferase activity. Independent triplicate experi-
ments were performed each time and were repeated at least
three times. Error bars represent the deviation of the
mean value.
Src kinase and prostate cancer
M Asim et al
3602
Oncogene
Chromatin immunoprecipitation assay
C4-2 ChIP experiment was performed essentially as described
previously (Moehren et al., 2007). ChIP experiments were
repeated at least three times with similar results.
Real-time RT-PCR
Isolation of mRNA and the real-time PCR was performed as
described earlier (Moehren et al., 2007). A total of 200 000 C4-
2 cells per well were seeded out in charcoal-stripped serum
containing T-media in six-well tissue culture dishes. After 24 h
cells were treated with R1881 (10
10
M) for 48 h, total cellular
RNA was isolated, 1 mg RNA was reverse-transcribed to
cDNA and was subjected to amplification by light cycler using
specific primers and control primers against actin.
Enzyme-linked immunosorbent assay
The human PSA ELISA kit was used for the quantitative
analysis of PSA levels in culture medium as per manufacturer’s
protocol. C4-2 cells were treated with Src inhibitor PP2 and/or
Casodex, and 200 ml media was assayed.
Generation of stable clones
A total of 200 000 C4-2 cells were transfected with wt-Src or
mutant Src along with pETE-Hyg plasmid in 5:1 molar ratio
with (total amount being 10 mg) using electroporation kit from
AMAXA (Gaithersburg, MD, USA). The medium was
changed and replaced with fresh T-media 18 h posttransfec-
tion. Stable clones were selected as described previously
(Moehren et al., 2007).
Invasion assay
The assay was performed following the manufacturer’s
protocol. Briefly, 500 ml of complete T-media was added in
lower chamber as chemoattractant and 70 000 cells suspended
in serum free medium were applied to the inner chamber.
Treatment with Src inhibitor and Casodex was given in the
inner chamber either alone or in combination and the cells
were incubated for 20 h in humidified incubator. Invading cells
were stained and photomicrographed as per manufacturer’s
instruction.
Statistical analysis
All assays were repeated in three independent experiments, and
only representative blots are presented. Immunoblots were
scanned by HP PrecisionScan Pro 3.13 (Hewlett-Packard, Palo
Alto, CA, USA). Densitometry measurements of the scanned
bands were done using digitalized scientific software program
UN-SCAN-IT. Data were normalized to b-actin and expressed
as mean±s.e. followed by appropriate statistical analysis.
Acknowledgements
We thank Dr GP Reddy for gift of Casodex, Dr GN
Thalmann for providing C4-2 cells and Dr Sarah Courtneidge
for mammalian expression vectors of wt and mutant Src. This
work was supported by US PHS Grants RO1CA78809;
RO1CA101039, RO1CA120451 and O’Brian center Grant
P50DK065303-01 to HM and Association of International
Cancer research (AICR, UK) and DFG-BA1457/3 grant
to AB.
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