Endocrine-Related Cancer (2007) 14 1007–1019
Suppressor of cytokine signalling-3 isup-regulated by androgen in prostatecancer cell lines and inhibits androgen-mediated proliferation and secretion
Hannes Neuwirt, Martin Puhr, Ilaria T Cavarretta, Michael Mitterberger,Alfred Hobisch1 and Zoran Culig
Department of Urology, Innsbruck Medical University, Anichtrasse 35, A-6020 Innsbruck, Austria1General Hospital Feldkirch, Carinagasse 45, A-6800 Feldkirch, Austria
(Correspondence should be addressed to Z Culig; Email: [email protected])
Abstract
Suppressors of cytokine signalling (SOCS) are induced by interleukins (ILs) and various peptidehormones and may prevent sustained activation of signalling pathways. We have previously shownthat SOCS-3 antagonizes regulation of cellular events by cAMP and is expressed in human prostatecancer. To investigate possible effects of androgen on SOCS-3 protein expression, two prostatecancer cell lines (PC3-AR and LAPC4) were treated with different concentrations of R1881. Westernblot analyses revealed induction of SOCS-3 protein expression in both cell lines by androgen, aneffect which can be blocked by the anti-androgen bicalutamide. To further characterize the effects ofR1881 on the SOCS-3 gene, promoter–reporter assay and real-time PCR were performed. We foundno influence of androgen on promoter activity or SOCS-3 mRNA levels, thus suggesting a post-transcriptional effect of androgen. Concordant with our previous findings, we show a significantincrease of SOCS-3 protein after androgen treatment in cells in which transcription was blocked, butnot in those with impaired translation. In order to understand implications of SOCS-3 regulation byandrogen, we used SOCS-3-negative LNCaP–IL-6 cells and stably transfected them with atetracycline-responsive SOCS-3 Tet-On plasmid. We report that androgenic effects on cellproliferation and prostate-specific antigen secretion are significantly diminished following up-regula-tion of SOCS-3. In conclusion, androgen up-regulates SOCS-3 protein via post-transcriptionaleffects. SOCS-3 inhibits androgen-stimulated proliferation by influencing cell cycle regulation. Takentogether with previous findings showing androgen receptor activation by IL-6, our results imply thatandrogen and cytokine signalling pathways interact at multiple levels in prostate cancer.
Endocrine-Related Cancer (2007) 14 1007–1019
Introduction
Prostate cancer is the most common malignant disease
in men, except for non-melanoma skin cancer (Jemal
et al. 2006). Its development and progression are
intimately connected with androgenic stimulation
because of expression of the functional androgen
receptor (AR) protein in androgen-dependent and
independent tumour stages (Hobisch et al. 1996, Linja
et al. 2001). AR down-regulation in cell lines that
represent androgen-independent prostate tumours
slows down cancer cell growth (Zegarra-Moro et al.
2002). Moreover, AR is active as levels of its target
protein, prostate-specific antigen (PSA), aremeasurable
Endocrine-Related Cancer (2007) 14 1007–1019
1351–0088/07/014–001007 q 2007 Society for Endocrinology Printed in Gr
in sera of patientswith prostate cancer (Grossmann et al.
2001). In general, AR is activated in a ligand-dependent
manner. Increased activation because of receptor
mutations, AR amplification and the presence of growth
factors or cytokines that act in a ligand-independent or
synergistic manner occurs in prostate cancer (Culig
et al. 2003). AR coactivators that potentiate ligand-
dependent activation may be increasingly expressed in
prostate cancer. Ligand-independent activation of the
AR by ErbB-2 contributes to the in vivo progression of
the LAPC4 prostate tumour (Craft et al. 1999). Several
groups showed that interleukin-6 (IL-6) activates
AR-mediated transcription in a ligand-independent
eat Britain
DOI:10.1677/ERC-07-0172
Online version via http://www.endocrinology-journals.org
H Neuwirt et al.: Androgenic regulation of SOCS-3
manner (Hobisch et al. 1998, Chen et al. 2000, Lin et al.
2001). Additionally, IL-6 potentiates the effects of low
concentrations of androgen. Implications of AR
activation by IL-6 on regulation of cellular events are
not fully understood. Although IL-6 inhibits LNCaP
prostate cancer cell proliferation, a subline selected in
its presence acquires a growth advantage (Hobisch et al.
2001). Higher tissue levels of IL-6 measured in prostate
cancer patients may reflect decreased androgenic
suppression of the promoter or loss of the tumour
suppressor retinoblastoma that is a negative regulator of
its expression (Bellido et al. 1995, Steiner et al. 2003).
Pleiotropic effects of IL-6 in target tissues occur
because of differential activation of signalling pathways
of Janus kinase (JAK)/signal transducer and activators
of transcription (STAT), MAP kinases and phospha-
tidylinositol 3-kinase. STAT3, which is phosphorylated
and translocated into the nucleus upon IL-6 treatment, is
known to act as an oncogene in most malignancies. It
may prevent tumour cell death, stimulate expression
of angiogenic factors and induce expression of
extracellular matrix degradation enzymes thus resulting
in enhancedmetastatic potential (Turkson& Jove 2000,
Buettner et al. 2002). Its role in prostate cancer is still
controversially discussed.
Suppressor of cytokine signalling (SOCS)-3 belongs
to the SOCS family, which consists of eight members,
named SOCS 1–7 and CIS. These proteins contain a
central SH2 domain and the SOCS box, a conserved
40-residue C-terminal motif (Yoshimura et al. 1995,
Endo et al. 1997, Naka et al. 1997, Starr et al. 1997,
Hilton et al. 1998). In addition, SOCS-1 and SOCS-3
have a kinase inhibitory region constituted of 12 amino
acids (Sasaki et al. 1999). Cytokines like IL-6
stimulate a rapid induction of SOCS proteins, which
inhibit various steps of the pathway, in particular in the
case of IL-6 the JAK/STAT3 signalling. They exert
their action through different mechanisms such as
binding to the phosphorylated cytokine receptor or
JAK (Tan & Rabkin 2005). SOCS can reduce
phosphorylation of JAKs or STATs, STAT dimeri-
zation or import to the nucleus and target the complex
of SOCS-3 with JAK/STAT for proteasomal
degradation (Naka et al. 1997, Starr et al. 1997, Song
& Shuai 1998, Larsen & Ropke 2002). Thus, SOCS
proteins are believed to be negative feedback loop
regulators of cytokine-mediated signalling pathways.
Expression and function of SOCS proteins have
been investigated in several malignant diseases. Those
studies revealed that SOCS are implicated in regulation
of cellular proliferation and apoptosis. In squamous
cell carcinoma (SCC;Weber et al. 2005) or lung cancer
(He et al. 2003b), SOCS-3 is lost because of gene
1008
promoter methylation. Hence, silencing of the SOCS-3
gene causes a sustained activation of STAT3, resulting
in enhanced proliferation and a more malignant
phenotype. Introducing SOCS-3 in SCC yielded
inhibition of cell proliferation and enhanced apoptotic
rate accompanied by decreased protein expression
levels of Bcl-2.
However, in breast and prostate cancers, the two
most common hormone-dependent malignancies in
female and male SOCS-3 respectively is not lost. In
prostate cancer cells, SOCS-3 antagonizes the effects
of cAMP resulting in inhibition of apoptosis and
promotion of cellular proliferation (Bellezza et al.
2006). In breast cancer, it was shown that oestradiol
up-regulates SOCS-3 mRNA levels (Leong et al. 2004,
Matthews et al. 2005). In this context, it is of interest
that SOCS-3 causes enhanced phosphorylation of ERK
kinases and therefore may exert proliferative effects on
breast cancer cell lines (Raccurt et al. 2003). On
the basis of those results, we hypothesized that
androgens are implicated in regulation of SOCS-3
expression in prostate cancer. In this study, we show
that SOCS-3 is up-regulated by androgen via a post-
transcriptional effect on mRNA translation. We report
that up-regulation of SOCS-3 using a tetracycline-
responsive construct inhibits androgen-stimulated
proliferation and secretion. Moreover, SOCS-3 signi-
ficantly diminishes stimulation of cell cycle regulatory
proteins cdk2, cdk4, cyclins E and D1 by androgen.
Materials and methods
Cell culture and chemicals
Prostate cancer PC3-AR cells were donated by Dr
Andrew Cato (Research Center Karlsruhe, Germany).
LAPC4 cells were kindly provided by Dr Charles
Sawyers (University of California, Los Angeles). CV1
cells were purchased from ATCC (Rockville, MD,
USA). LAPC4 cells were maintained in IMDM
containing 15% foetal calf serum (FCS) supplemented
with 10 nM R1881 (Perkin–Elmer, Montreal, Canada).
All other cell lines were cultured in RPMI 1640
(HyClone, Logan, UT, USA). Media were supple-
mented with 10% FCS and penicillin/streptomycin
(PAA Laboratories, Pasching, Austria). Medium for
LNCaP–IL-6/B and D cells was additionally supple-
mented with 70 mg/ml hygromycin B (Merck). These
clones derived from LNCaP–IL-6 cells which were
described previously (Hobisch et al. 2001). DMSO,
cycloheximide and actinomycin D were purchased
from Sigma. The synthetic androgen methyltrienolone
(R1881) is a product of DuPont NEN Products
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Endocrine-Related Cancer (2007) 14 1007–1019
(Boston, MA, USA), and IL-6 was purchased from
R&D (Minneapolis, MN, USA). The SOCS-3 promoter
construct was a kind gift of Dr David Jablons
(University of California, San Francisco). All experi-
ments were performed in media containing either 3%
or in case of clones 0.2% steroid-free FCS (HyClone).
Controls were treated with vehicle only, which was
either DMSO or ethanol. None of these solvents
exceeded a dilution of 0.1% either in controls or in
treated samples.
Real-time PCR
Cells were grown in six-well plates. Upon treatment
with R1881, RNA isolation, cDNA synthesis and
quantitative real-time PCR were performed as pre-
viously published (Bellezza et al. 2006). Each
experiment was carried out in triplicates. TATA box-
binding protein (TBP) was chosen as an endogenous
expression standard (Savinainen et al. 2002). PCR
products were measured using the ABI Prism 7500 Fast
RT-PCR System (Applied Biosystems, Rotkreuz,
Switzerland). Ct values of SOCS-3 and TBP as
assessed by (Version 1.3) were used to calculate the
dCt using Microsoft Excel 2002. Values obtained in
control cells were defined as 100% and those from
treated cells were expressed as percent of control.
Western blot
Western blots, including harvesting and cell lysis, were
performed as previously reported (Bellezza et al. 2006).
In the same experiments, protein levels of SOCS-3 and
AR were analysed in cytosolic and nuclear fractions.
The following antibodies were used: anti-SOCS-3
(1:1000; Acris Antibodies, Hiddenhausen, Germany),
anti-AR (1:1000; Biogenex, San Ramon, CA, USA),
anti-GAPDH (1:100 000; Chemicon International,
Temecula, CA, USA), anti-b-actin (1:1 000 000;
Chemicon), anti-c-Myc (1:1000; Santa Cruz
Biotechnology, Santa Cruz, CA, USA), anti-cdk2
and anti-cdk4 (both 1:200; Biosource, Solingen,
Germany), anti-cyclin D1 (1:750; LabVision, Fremont,
CA, USA) and anti-cyclin E (1:500; Santa Cruz). The
membranes were scanned and quantified using Odyssey
Infrared Imaging System (LiCor Biosciences, Lincoln,
NE, USA).
Immunofluorescence
PC3-AR cells were grown on microscope slides for
48 h and stimulated with 1 nM R1881 for 48 h. Cells
were fixed with buffered 5% paraformaldehyde for
20 min. After incubations with primary antibodies,
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slides were washed four times with PBS followed by a
secondary antibody step (Alexa Fluor 555, Invitrogen)
for 30 min. Primary antibodies used were: anti-
SOCS-3 (1:200; Acris Antibodies), anti-AR (1:200;
Biogenex) and mouse negative control (Dako, Vienna,
Austria). After rinsing the slides twice with PBS,
fluorescence was analysed using a Zeiss Axio Imager
M1 microscope (Carl Zeiss, Oberkochen, Germany).
Luciferase activity assay
PC3-AR, CV1 and PC3 cells were seeded at a density of
1!104 cells per well in black-walled 96-well plates
(Corning, Corning, NY, USA). Cells were transfected
using Lipofectamin2000 (Invitrogen) according to the
manufacturer’s protocol with the following plasmids:
SOCS-3 promoter construct as published by He et al.
(2003a), pHRL-null plasmid as a control for transfection
efficiency and in case of AR-negative cells pSG5-AR
expressionplasmid (Hobisch et al. 1998).Cellswere then
stimulated with either IL-6 (25 ng/ml) as a positive
control or with 1 nM R1881. Luciferase activity was
assessed using Dual-Glo luciferase activity assay
(Promega) according to the manufacturer’s protocol
with a luminometer (Chameleon Microplate Reader,
Hidex, Turku, Finland).
Generation of stably transfected cell lines
For inducible expression of N-terminal myc-tagged
SOCS-3 in LNCaP–IL-6 cells, a tetracycline-respon-
sive expression vector pBIG2i (Strathdee et al. 1999)
containing the coding region of SOCS-3 and the myc
tag was used (Tonko-Geymayer et al. 2002). The
plasmid was a kind gift of Prof. Wolfgang Doppler
(Division of Medical Biochemistry, Innsbruck Medical
University). In order to obtain stably transfected cells,
1!106 LNCaP–IL-6 cells were transfected with 3 mgplasmid using Amaxa electroporation (Amaxa,
Cologne, Germany) according to the manufacturer’s
protocol. Transfected cells were selected in RPMI
1640 medium containing 70 mg/ml hygromycin B,
10% FCS, 1% penicillin/streptomycin and 1%
glutamine (Invitrogen). Single cell clones were
obtained using clone cylinders (Sigma–Aldrich).
Selected clones were cultivated in RPMI 1640
medium containing 10% tetracycline-free FCS
(HyClone) followed by treatment with 2 mg/ml
doxycycline (Sigma–Aldrich) for 2 days. SOCS-3
levels were measured by western blot. Two clones,
LNCaP–IL-6/B and D (from now referred to as clone B
and D) were chosen for further experiments because of
similar expression levels of the AR and phenotype
when compared with parental LNCaP–IL-6 cells.
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H Neuwirt et al.: Androgenic regulation of SOCS-3
[3H]Thymidine incorporation assay
Thymidine incorporation assay was carried out as
reported previously (Bellezza et al. 2006). The
treatment times were as follows: after 24 h of
stimulation with doxycycline (2 mg/ml), cells were
treated with different concentrations of R1881 (1, 10,
100, 500 and 1000 pM) for 48 h.
PSA measurement
For measurement of PSA secretion, clones B and D
were grown in 24-well plates (Corning) for 24 h with
or without doxycycline and subsequently stimulated
with different concentrations of R1881 (10, 100 and
1000 pM) for 48 h. PSA content was measured using
Advia CentaurXP Immunoassay System (Siemens,
Munich, Germany), and cell number was assessed by
Casy Cell Counter and Analyser (Schaerfe Systems,
Reutlingen, Germany). PSA levels were finally
adjusted to cell number.
Statistical analysis
For each treatment group, statistical distribution was
determined using Kolmogorov–Smirnov test. In case of
non-Gaussian distribution, non-parametric tests were
applied as follows: to assess the overall significance for
experiments with more than one treatment group, we
used Kruskal–Wallis test; additionally, Mann–Whitney
U test was applied when appropriate. In case of Gaussian
distribution two-sided Student’s t-test was used. For
comparing datasets in PSA and proliferation experi-
ments, two-way ANOVA for non-repeated measure-
mentswas applied.Bonferroni’s post hoc testwas used to
correct for multiple testing. Corrected P values !0.05
were defined as statistically significant and encoded in
figures (*P!0.05, **P!0.01, ***P!0.001). All
statistical analyses were performed using SPSS 12.0
software (SPSS, Chicago, IL, USA). For detailed
description of statistics, please refer to the figure legends.
Results
SOCS-3 is up-regulated by androgen in two
prostate cancer cell lines
In order to investigate the influence of androgenic
stimulation on protein expression of SOCS-3, two
prostate cancer cell lines, in particular PC3-AR and
LAPC4 were used. PC3-AR cells were treated with
increasing concentrations of the synthetic androgen
R1881, whereas in LAPC4, which are maintained in
medium supplemented with 10 nM R1881, levels
of androgen were decreased to 1 and 0.1 nM. In the
1010
PC3-AR cell line, we report that with increasing
concentrations of androgen SOCS-3 expression levels
increase significantly by threefold in the cytosol
(Fig. 1A) without affecting SOCS-3 levels inside the
nucleus (data not shown). In the case of LAPC4 cells,
we evince a decrease of SOCS-3 (Fig. 1B) protein
levels with decreasing concentrations of R1881 (10, 1
and 0.1 nM; PZ0.011).
To further confirm these results, we stimulated both
cell lines with the same concentrations of R1881 in the
presence of 1 and 5 mM anti-androgen bicalutamide.
Corroborating our findings, under those conditions
changing amounts of androgen did not yield an
alteration of SOCS-3 protein in cytosol (Fig. 1A) and
nucleus (data not shown). In LAPC4 cells, treatment
with bicalutamide decreased SOCS-3 levels to about
60% of control. Additionally, we treated AR-negative
PC3 cells with 0.1 and 1 nM R1881 and found no
change in SOCS-3 levels (Fig. 1C). Taken together,
these results strongly suggest an androgenic effect on
SOCS-3 expression.
To analyse the time course of androgen-mediated
SOCS-3 up-regulation, we performed experiments by
stimulating PC3-AR cells for 24, 48 and 72 h with
different concentrations of R1881 (0.1 and 1 nM). As
shown in Fig. 2, we found the strongest induction at
48 h (PZ0.0064). AR levels were assessed in all
experiments mentioned above. As expected, we found
higher AR expression after androgenic stimulation in a
dose-dependent manner with increasing concentrations
of R1881 (data not shown). In LAPC4 cells, this
regulation was also found, meaning a dose-dependent
decrease tow60% of control with decreasing androgen
concentrations. In nuclear extracts, this alteration was
more prominent, for example, 13-fold AR increase in
PC3-AR cells (data not shown). To corroborate our
findings with western blot, we performed immuno-
fluorescence experiments using PC3-AR cells. Both
SOCS-3 and AR levels were enhanced in cells
stimulated with 1 nM R1881 for 48 h (Fig. 1D).
Up-regulation of SOCS-3 by androgen
is dependent on translation
To further investigate androgen-mediated SOCS-3
up-regulation, we questioned whether SOCS-3
promoter coupled with a firefly luciferase reporter
can be directly stimulated by R1881 in PC3-AR and
CV1 cells. For this purpose, the cells were transfected
with a plasmid containing the 5 0 region of the human
SOCS-3 promoter (He et al. 2003a). CV1 cells, which
lack AR, were co-transfected with the pSG5-AR
plasmid (Hobisch et al. 1998). IL-6 stimulation was
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Figure 2 Time course of SOCS-3 expression after androgenicstimulation. PC3-AR cells were treated with R1881 for 24, 48 and72 h. Results show meanGS.D. (nZ5). Statistics: Mann–WhitneyU: 48 h Co versus 48 h 0.1, 1 nM R1881: PcZ0.0106*, 0.0064**;72 h Co versus 72 h 0.1, 1 nM R1881: PcZ0.0208*, 0.0106*.
Figure 1 SOCS-3 expression levels assessed by western blot. (A) PC3-AR cells were treated for 48 h with the indicatedconcentrations of R1881 with or without pre-treatment with bicalutamide (1 and 5 mM). Results represent meanGS.D. (nZ8; numberof independent experiments carried out in duplicates). (B) LAPC4 cells, maintained in medium supplemented with 10 nM R1881,were incubated for 48 h with decreasing concentrations of androgen with or without bicalutamide. Results shown are meanGS.D.(nZ8). (C) PC3 cells were treated with different concentrations of R1881 for 48 h. Results represent meanGS.D. (nZ4). (D)Immunofluorescence of PC3-AR cells grown on microscope slides and treated with 1 nM R1881 for 48 h. Statistics: (A)Mann–Whitney U: Co versus 0.1 nM R1881: PcZ0.000095***; Co versus 1 nM R1881: PcZ0.00266**; (B) Mann–Whitney U:Co versus 1, 0.1 nM R1881, 10 nM R1881C5 mM Bic: PcZ0.0117*, 0.0117*, 0.0049**.
Endocrine-Related Cancer (2007) 14 1007–1019
considered a positive control for activation, whereas
AR-negative PC3 cells were used as a negative control.
Androgenic effect was proved by western blotting,
showing a substantial increase of AR and translocation
to the nucleus (data not shown). As demonstrated in
Fig. 3, we found that neither in PC3-AR nor in CV1
cells androgenic stimulation yields a significant
activation of the SOCS-3 promoter. Then, we asked
whether SOCS-3 mRNA is regulated after androgen
treatment in PC3-AR cells. For this purpose, the cells
were treated with 0.1 and 1 nM R1881 at different time
points (30 min, 1, 2, 8 and 24 h). We found no
up-regulation of SOCS-3 mRNA at any time point
(Fig. 4). In fact, mRNA levels were decreased after
androgen treatment; however, due to the correction for
multiple testing (Bonferroni’s post hoc test), these
results are not statistically significant. Taken together,
these experiments demonstrate that androgen does not
have a direct effect on the SOCS-3 promoter, thus not
enhancing mRNA transcription.
Hence, we asked whether androgen could up-regu-
late SOCS-3 protein levels when transcription is
inhibited. Therefore, PC3-AR cells were pre-treated
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with actinomycin D, followed by a 48-h stimulation
with 0.1 and 1 nM R1881. Actinomycin D, used at a
concentration of 10 nM, did not increase apoptotic
cell death, as measured by flow cytometry using
Nicoletti’s method, over a time period of 72 h (data
not shown). Compared with control conditions, under
which SOCS-3 was up-regulated by twofold with
1011
Figure 3 SOCS-3 promoter activity. PC3-AR, PC3 and CV1 cellswere transfected with a SOCS-3 promoter construct and pHRL-null plasmid. In case of AR-negative CV1 cells an AR-expressionplasmid, pSG5AR, was co-transfected. Cells were subsequentlystimulated with R1881 for 48 h, followed by measurement ofluciferase activity. Data shown are meanGS.D. (nZ4). Statistics:Mann–Whitney U: Co versus IL-6: PcZ0.037*.
Figure 5 SOCS-3 protein levels. PC3-AR cells were pre-treatedfor 1 h with either cycloheximide or actinomycin D in order toblock translation and transcription respectively. Subsequently,cells were treated with R1881 (0.1 and 1 nM) for 48 h. Datarepresent meanGS.D. (nZ5). Statistics: Mann–Whitney U:control group: 0 vs 0.1, 1 nM R1881: PcZ0.0277*, 0.0277*;Actinomycin D group: 0 vs 1 nM R1881: PcZ0.0385*.
H Neuwirt et al.: Androgenic regulation of SOCS-3
1 nM R1881 (PZ0.0277), SOCS-3 protein levels
were similarly increased in cells pre-treated with
actinomycin D (PZ0.0385; Fig. 5). In conclusion,
these data suggest that androgen-stimulated SOCS-3
protein up-regulation is not dependent on
transcription.
To further elucidate these findings, we blocked
translation by pre-treating the cells with cycloheximide
(1 mg/ml) for 1 h followed by R1881 stimulation for
48 h. This concentration of cycloheximide did not alter
the apoptotic rate (data not shown). We report that
blocking translation significantly inhibits androgen-
mediated SOCS-3 up-regulation, thus demonstrating
the need for a functional translation apparatus to
enhance SOCS-3 expression by androgen (Fig. 5).
Up-regulation of SOCS-3 inhibits androgen-
stimulated cell proliferation and PSA secretion
To investigate the implications of SOCS-3 regulation
on cellular function, we intended to use specific siRNA
and knock-down SOCS-3 expression. Although
SOCS-3 siRNA significantly lowered SOCS-3 levels
Figure 4 Quantitative real-time PCR. PC3-AR cells weretreated with R1881 for the indicated time points. Datarepresent meanGS.D. (nZ3). Statistics: Kruskal–Wallis: Pvalues for 30 min, 1, 2, 8 and 24 h: PZ0.055, 0.055, 0.055,0.061 and 0.055.
1012
in PC3 cells (Bellezza et al. 2006), it was not effective
in PC3-AR or LAPC4 cells. To establish an appropriate
model for studies of cellular events, we generated two
sublines, derived from LNCaP–IL-6 cells (Steiner et al.
2003), which were transfected with a tetracycline-
inducible construct encoding for SOCS-3. In two
clones, in particular LNCaP–IL-6/B and D (clone B
and D), doxycycline at a concentration of 2 mg/ml
up-regulated SOCS-3 levels by about four- and sixfold
(B: PZ0.0138; D: PZ0.0243) respectively. After
stimulation for 24 h, the cells were allowed to partially
degrade SOCS-3 under non-stimulating conditions
(without doxycycline) with or without 1 nM R1881.
When compared with cells cultured without R1881,
SOCS-3 levels in cells treated with androgen increased
by w1.5-fold (B: PZ0.0084; D: PZ0.0063; Fig. 6).
Androgens exert a biphasic effect on cellular prolifer-
ation of prostate cancer cells (Sonnenschein et al.
1989, Lee et al. 1995). It was shown that low picomolar
concentrations cause a maximal stimulation of cell
growth. In order to test possible implications of
SOCS-3 regulation for androgen-mediated growth
effects, clones B and D were cultured in the absence
or presence of doxycycline for 24 h, followed by a 48 h
incubation with different concentrations of R1881
(0.001, 0.01, 0.1, 0.5 and 1 nM). We confirm a four-
to sixfold increase of cMyc-SOCS-3 after doxycycline
stimulation as shown above (Figs 6 and 7A, right
panel). Under those conditions, cellular proliferation
was not altered (Fig. 7A, right panel). As shown in
Fig. 7A (left panel), treatment with androgen yielded
stimulation of cell proliferation at concentrations up to
0.1 nM thus confirming previous results (Sonnenschein
et al. 1989, Lee et al. 1995). However, in cells in which
SOCS-3 expression was induced by tetracycline,
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Figure 6 SOCS-3 protein levels in clones B and D. SOCS-3levels were induced by 2 mg/ml doxycycline after 24 htreatment. After induction of SOCS-3, the cells were left withoutdoxycycline for another 24 h with or without androgenicstimulation (1 nM R1881). Data represent meanGS.D. (nZ6).Statistics: Mann–Whitney U: LNCaP–IL-6/B: 3 vs 1, 2 and 4:PcZ0.006**, 0.0264*, 0.0084**; LNCaP–IL-6/D: 3 vs 1, 2 and 4:PcZ0.0024**, 0.0063**, 0.0063**.
Endocrine-Related Cancer (2007) 14 1007–1019
changes in proliferation rate were substantially
abolished. In particular, neither clone BC nor DCcould be stimulated by R1881 (P!0.0001). Inhibitory
concentrations of androgen yielded a more pronounced
repression of cell growth.
Additionally, we tested PSA production as a
parameter for androgen-stimulated transcription
under the same conditions as mentioned above. PSA
levels increased by five- to sevenfold in clones B and D
respectively. Confirming our previous results, PSA
secretion was increased only 2- to 2.5-fold in the
presence of SOCS-3 (PZ0.001; Fig. 7B).
SOCS-3 negatively influences androgen-
mediated stimulation of cell cycle regulators
Molecules that havebeen reported toplaya pivotal role in
androgen-stimulated growth include cdk2, cdk4, cyclins
D1 and E (Lu et al. 1997, Xu et al. 2006). To substantiate
our findings from proliferation assays, we investigated
expression of these cell cycle regulatory proteins after
48 h of androgenic stimulation (1 and 100 pM R1881).
We found that stimulation with R1881 resulted in a
significant increase of cdk2, cdk4, cyclins D1 and E
expression levels by about 1.8-, 1.5-, 1.6- and 2.2-fold
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respectively, in clone B (PZ0.044, 0.0107, 0.044 and
0.011; Fig. 8). In clone D, similar significant regulations
were found; however, the increase was lower than that in
clone B (PZ0.046, 0.013, 0.013 and 0.0233).
In order to elucidate implications of androgenic
regulation of SOCS-3, the same experiments were
performed in the presence of 2 mg/ml doxycycline.
Interestingly, none of the cell cycle regulators was
increased after the treatment with R1881 when
SOCS-3 was up-regulated thus confirming our results
from proliferation assays (Fig. 8). These data show that
SOCS-3 significantly impairs androgen-stimulated
cell proliferation by interfering with regulation of
cell cycle.
Discussion
There is an increasing evidence that SOCS-3, beside its
function as a negative feedback regulator of cytokine
signalling, may exert pleiotropic effects on cell
proliferation and apoptosis. We have previously
shown that SOCS-3 is expressed in prostate cancer
in vitro and in vivo and antagonizes cAMP-mediated
apoptosis in cell lines (Bellezza et al. 2006).
Furthermore, it is known that in breast cancer and
melanoma (Raccurt et al. 2003, Komyod et al. 2007)
levels of SOCS-3 are elevated and contribute to growth
advantage and higher malignant phenotype. Based on
previous data showing regulation of SOCS-3 by
oestrogen (Matthews et al. 2005), we questioned
whether there is an interplay between androgen and
SOCS-3 in prostate cancer cell lines. Furthermore, it
was previously shown that IL-6 acts as a ligand-
independent activator of the AR (Hobisch et al. 1998,
Chen et al. 2000, Lin et al. 2001).
In order to test our hypothesis, we used PC3-AR
cells and the AR-positive cell line LAPC4. The reasons
for choosing these two cell lines were on one hand that
both express wild-type AR and on the other hand that
LNCaP cells, which express a mutated AR, are
SOCS-3 negative. In both cell lines, we found a
significant androgenic stimulation of SOCS-3 protein
expression after 48 h. These data strongly corroborate
our hypothesis that SOCS-3 indeed is an androgen-
responsive protein. The lowest concentration of R1881
for treatment of LAPC4 cells in our experiments
that could be used without induction of apoptosis
was 0.1 nM.
To further elucidate these findings, we investigated
androgenic effects on SOCS-3 promoter activity and
mRNA levels using luciferase assay and quantitative
real-time PCR respectively. Neither promoter activity
nor mRNA levels increased after androgen treatment.
1013
Figure 7 (A) Left panel: [3H]thymidine incorporation assay after androgenic stimulation of clone B and D. Cells were seeded at adensity of 1!104 cells per well in 96-well plates. After a 24-h incubation with or without doxycycline, the cells were treated withdifferent concentrations of R1881. MeansGS.E.M. (nZ4). Right panel: Clones B and D were treated with doxycycline for 48 h,followed by quantification of cMyc-SOCS-3 protein levels by western blots and measurement of cell proliferation by [3H]thymidineincorporation. MeanGS.E.M. (nZ4). (B) PSA levels after treatment with or without doxycycline under androgenic stimulation. MeanGS.E.M. (nZ3) Statistics: (A) Two-way ANOVA – overall difference between B versus BC: P!0.0001***; D versus DC: P!0.0001***;corrected Pc values for B versus BC and D versus DC for each concentration: O0.05, !0.01**, !0.05*, !0.05*, O0.05, O0.05and O0.05, !0.05*, !0.05*, !0.05*, O0.05, O0.05. (B) Two-way ANOVA – overall difference between B versus BC:PZ0.0019**; D versus DC: PZ0.001**; corrected Pc values for B versus BC and D versus DC for each concentration: O0.05,O0.05, O0.05, !0.05* and O0.05, O0.05, O0.05, !0.01**.
H Neuwirt et al.: Androgenic regulation of SOCS-3
Interestingly, our results obtained with prostate cells
differ from those published in hepatoma or breast
cancer cell lines in which oestrogen up-regulated
SOCS-3 through transcriptional activation (Leong
et al. 2004, Matthews et al. 2005). It is generally
accepted that AR not only may act as a transcription
factor but also has direct effects on protein translation
or stability. Perry & Tindall (1996) showed that
proliferating cell nuclear antigen, which is required
for DNA replication, is up-regulated by androgen by
increasing protein stability. Our data are comparable
with those of Xu et al. (2006) who reported that D-type
1014
cyclins are up-regulated upon androgen treatment via
post-transcriptional mechanisms. It was demonstrated
that androgens are also capable of activating signalling
pathways, such as that of phosphoinositol
3-kinase/AKT by stimulating AR interaction with the
p85a kinase subunit and Src in the absence of
endogenous receptor transcriptional activity (Castoria
et al. 2003, Sun et al. 2003). Additionally, upon
androgenic stimulation, Src and protein kinase C amediate the phosphorylation of ezrin, a key signalling
molecule that regulates cell survival, adhesion,
migration and invasion of prostate cancer cells (Pang
www.endocrinology-journals.org
Figure 8 Protein levels of cdk2, cdk4, cyclins D1 and E in clones B and D after androgenic stimulation for 48 h. Data representmeanGS.E.M. (nZ5). Statistics: Kruskal–Wallis P values are given for each group above the columns. Mann–Whitney U: cdk2: CloneB Co versus 100 pM R1881: PcZ0.014*; Clone D Co versus 1, 100 pM R1881: PcZ0.014*, 0.037*; cdk4: Clone B Co versus 1,100 pM R1881: PcZ0.014*, 0.037*; Clone D Co versus 1, 100 pM R1881: PcZ0.037*, 0.014*; cyclin D1: Clone B Co versus 100 pMR1881: PcZ0.014*; Clone D Co versus 1, 100 pM R1881: PcZ0.014*, 0.014*; cyclin E: Clone B Co versus 1, 100 pM R1881:PcZ0.014*, 0.014*; Clone D Co versus 100 pM R1881: PcZ0.014*.
Endocrine-Related Cancer (2007) 14 1007–1019
et al. 2004, Chuan et al. 2006). To clarify the
mechanism of regulation of SOCS-3, we blocked
transcription and translation using actinomycin D and
cycloheximide respectively. We found that blockade of
transcription did not significantly inhibit androgen-
mediated up-regulation of SOCS-3, whereas impeding
translation substantially blocked this increase. Taken
together, those data show that SOCS-3 is up-regulated
by androgen in prostate cancer cell lines (PC3-AR and
LAPC4) via post-transcriptional effects. Future studies
may address an issue of androgenic interference with
proteasomal degradation of SOCS-3. In this context, it
is of interest that Zhang et al. (1999) have reported that
such degradation plays a crucial role in regulating
SOCS-3 expression after induction by IL-6.
Up-regulation of SOCS-3 by androgen might have
clinical implications, due to the fact that AR is
expressed at all stages of prostate cancer development
and plays a pivotal role in carcinogenesis. Further-
more, we have reported that SOCS-3 is expressed at a
higher level in malignant than in benign prostate tissue
samples (Bellezza et al. 2006).
To establish a novel cellular model for studies on
implications of SOCS-3 up-regulation, we stably
www.endocrinology-journals.org
transfected LNCaP–IL-6 cells with a tetracycline-
responsive plasmid encoding for SOCS-3. We report
that inductionwith doxycycline increased SOCS-3 levels
in clones B and D. Moreover, SOCS-3 levels were
elevated when compared with controls after subsequent
treatment with R1881 for 24 h. Using previously
published SOCS-3-specific siRNA (Bellezza et al.
2006), we aimed to address the question whether
SOCS-3 has an impact on androgen-stimulated proli-
feration. However, although SOCS-3 levels were
significantly lowered in PC3 cells, we were not able to
reproduce those results in PC3-AR or LAPC4 cells.
Hence, we tested various other SOCS-3 targeting
siRNAs and oligonucleotides from different manufac-
turers (Dharmacon, Ambion) and others that have
previously been published (Leung et al. 2003, Gomez-
Guerrero et al. 2004, Calegari et al. 2005) using four
different transfection reagents/methods. Although trans-
fection efficiency as assessed using siRNA against
GAPDH as a positive control was satisfactory, we were
not able to significantly knock-down SOCS-3 levels.
Thus, we used our stably transfected LNCaP–IL-6/B
and D cells. Androgen treatment was applied with or
without doxycycline-stimulated SOCS-3 expression.
1015
H Neuwirt et al.: Androgenic regulation of SOCS-3
Interestingly, we found that SOCS-3 substantially
diminishes androgen-mediated cell proliferation at
concentrations up to 100 pM R1881, whereas effects of
inhibitory concentrations were even more pronounced
whenSOCS-3 expressionwas elevated. Itwas previously
shown that D-type cyclins and cdks are induced by
androgen thus resulting in enhanced G1–S phase
transition and increased proliferation rate (Xu et al.
2006). To test whether SOCS-3 can impair the
stimulation of protein expression levels of cyclins D1
andE, cdk2 and cdk4,we cultured clonesB andDwith or
without doxycycline followed by androgenic stimu-
lation. It is generally accepted that cell cycle regulatory
genes are direct or post-transcriptional targets of the AR.
Many publications report up-regulation of G1 cyclins,
cdks or reduced expression of retinoblastoma, p16 or cdk
inhibitors in vitro and in vivo (Chen et al. 1996, Lu et al.
1997, Ye et al. 1999, Taneja et al. 2001). Similar to
results published by Xu et al. (2006), we found a
significant up-regulation of all tested cell cycle regulat-
ory molecules by androgen when SOCS-3 was not
induced. However, after induction of SOCS-3 by
doxycycline, these up-regulations were abolished, thus
Figure 9 SOCS-3 is a common negative regulator for androgeprodifferentiation effect as evidenced by PSA increase. Sustained acof SOCS-3 by IL-6. SOCS-3 is also a negative feedback regulator
1016
confirming a negative role of SOCS-3 on androgen-
mediated proliferation.
Taken together with results of our previous
publication (Bellezza et al. 2006), the present study
suggests that SOCS-3 may have cellular context-
dependent effects on tumour growth. Therefore, it is
not surprising that both tumour-suppressive (He et al.
2003b, Niwa et al. 2005, Weber et al. 2005) and
tumour-promoting (Bellezza et al. 2006, Fojtova et al.
2007, Zhou et al. 2007) roles were reported in the
literature.
In conclusion,we have shown that androgen is capable
of up-regulating SOCS-3 protein levels without affecting
transcription. Furthermore, [3H]thymidine incorporation
assays have proven that SOCS-3 can antagonize
proliferative effects of androgen. Confirming these
findings,western blot analyses showed that up-regulation
of cyclins and cdks after androgenic stimulation can be
effectively inhibited by SOCS-3. To our knowledge, this
is the first study reporting a negative feedback loop in
androgenic regulation of cell growth and secretion.
The results of our study, in conjunction with
previous work on androgen and IL-6 signalling in
n and IL-6 pathways in prostate cancer. IL-6 may cause ativation of the JAK/STAT pathway is prevented by up-regulationfor androgen signalling in prostate cancer cells.
www.endocrinology-journals.org
Endocrine-Related Cancer (2007) 14 1007–1019
prostate cancer, may lead to establishment of a novel
concept in growth and differentiation regulation
(Fig. 9). It is based on the fact that androgen and
IL-6 induce the same negative regulator of signalling,
SOCS-3. Thus, the hypothesis that SOCS-3 has an
important role in prostate cancer in vivo warrants
further investigation.
Acknowledgements
The authors gratefully thank Gertraud Sierek, Gerda
Holzl and Elizabeth Tafatsch for their excellent
technical support and Drs Andrew Cato, Charles
Sawyers, Wolfgang Doppler, Michael Haffner and
David Jablons for their providing cell lines and
plasmids. Grant sponsor: Austrian Science Fund FWF
(grants 18193 and W1101 to Z Culig). The authors
declare that there is no conflict of interest that would
prejudice the impartiality of this scientific work.
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