Suppressor of cytokine signalling-3 is up-regulated by androgen in prostate cancer cell lines and...

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.


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


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

www.endocrinology-journals.org


(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,


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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[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

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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


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**.


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


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

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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*.


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.

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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,


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**.


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


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.

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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**.


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


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*.


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


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


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.



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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