SOCS2 correlates with malignancy and exerts growth-promoting effects in prostate cancer

Deregulation of cytokine and growth factor signaling due to an altered expression of endogenous regulators is well recognized in prostate cancer (PCa) and other cancers. Suppressor of cytokine signaling 2 (SOCS2) is a key regulator of the GH, IGF, and prolactin signaling pathways that have been implicated in carcinogenesis. In this study, we evaluated the expression patterns and functional significance of SOCS2 in PCa. Protein expression analysis employing tissue microarrays from two independent patient cohorts revealed a significantly enhanced expression in tumor tissue compared with benign tissue as well as association with Gleason score and disease progression. In vitro and in vivo assays uncovered the involvement of SOCS2 in the regulation of cell growth and apoptosis. Functionally, SOCS2 knockdown inhibited PCa cell proliferation and xenograft growth in a CAM assay. Decreased cell growth after SOCS2 downregulation was associated with cell-cycle arrest and apoptosis. In addition, we proved that SOCS2 expression is significantly elevated upon androgenic stimulation in androgen receptor (AR)-positive cell lines, providing a possible mechanistic explanation for high SOCS2 levels in PCa tissue. Consequently, SOCS2 expression correlated with AR expression in the malignant tissue of patients. On the whole, our study linked increased SOCS2 expression in PCa with a pro-proliferative role in vitro and in vivo.


Introduction
Prostate cancer (PCa) represents the most common malignancy in men in the Western world (Jemal et al. 2011).The development and progression of PCa are strongly associated with the dysfunction of various signaling pathways.Preserved androgen signaling is one of the main characteristics of the disease, especially in advanced and castration-resistant PCa (Locke et al. 2008, Lonergan & Tindall 2011).Furthermore, cytokine and growth factor signaling cascades are frequently deregulated due to an altered expression of endogenous key regulators such as protein inhibitors of activated STAT (PIAS), Src-homology 2 (SH2)-containing protein tyrosine phosphatases, and suppressors of cytokine signaling (SOCS; Larsen & Ropke 2002, Heinrich et al. 2003, Tan & Nevalainen 2008, Dutt & Gao 2009, Puhr et al. 2009, Hoefer et al. 2012, Singh et al. 2012).
SOCS proteins have originally been described as the feedback inhibitors of cytokine-induced Janus kinase (JAK/STAT) signaling (Endo et al. 1997, Naka et al. 1997, Starr et al. 1997).However, they have also been reported to be associated with other pathways such as nuclear factor kappa B signaling (Ryo et al. 2003) and insulin signaling (Rui et al. 2002).The SOCS family consists of eight members: SOCS1-7 and cytokine-inducible SH2containing protein (CIS).They contain an N-terminal region of variable length, a central SH2 domain, and a conserved C-terminal region called SOCS box.SOCS members can act as E3 ubiquitin ligases toward associated proteins (Kamura et al. 1998(Kamura et al. , 2004) ) and are rapidly induced upon the release of multiple growth factors and cytokines, with STAT molecules being the main transducers of these signals (Croker et al. 2008).
The involvement of SOCS1 and SOCS3 in PCa has been extensively investigated and both tumor suppressive and oncogenic roles have been reported (Bellezza et al. 2006, Neuwirt et al. 2007, 2009, Puhr et al. 2009, 2010, Pierconti et al. 2011).However, to date, only a limited number of studies have investigated the expression and functional significance of SOCS2 in PCa.In other malignancies, the upregulation or downregulation of SOCS2 has already been demonstrated (Arany et al. 2001, Schultheis et al. 2002, Wikman et al. 2002, Sutherland et al. 2004).Furthermore, Socs2 knockout as well as transgenic mice have been reported to display gigantism (Metcalf et al. 2000, Greenhalgh et al. 2002), suggesting a dual role for SOCS2 in growth regulation.SOCS2 is preferentially upregulated by growth hormone (GH; Greenhalgh et al. 2002), erythropoietin (Starr et al. 1997, Wang et al. 2004), interleukins (ILs; Starr et al. 1997, Rico-Bautista et al. 2006), and prolactin (Pezet et al. 1999).Furthermore, SOCS2 is implicated in insulin-like growth factor (IGF) 1 and insulin signaling cascades (Dey et al. 1998, Sadowski et al. 2001).Deregulation of either of these signaling cascades has already been reported to be associated with PCa (Roberts 2004, Monti et al. 2007, Cox et al. 2009, Culig 2011, Goffin et al. 2011, Culig & Puhr 2012, Nakonechnaya et al. 2013).Therefore, we hypothesize that SOCS2 is a crucial regulator in PCa.To address this question, in this study, we investigated SOCS2 expression in tissue samples and carried out in vitro and in vivo assays to uncover its functional relevance in PCa.Our data clearly demonstrate a growth-promoting role for SOCS2 and provide an explanation for a high SOCS2 expression in malignant tissue via androgenic regulation.

Tissue microarray and immunohistochemistry
A tissue microarray (TMA) (Innsbruck-TMA) containing tissue cores obtained from 90 PCa patients who underwent radical prostatectomy at the University Hospital Innsbruck was constructed and immunohistochemically stained as described elsewhere (Hoefer et al. 2012).In addition, SOCS2 expression in metastases was assessed using two TMAs constructed from tissue cores obtained from ten patients with lymph node and bone metastases respectively.Evaluation was done by an uropathologist (G S) using the semi-quantitative scoring system 'quickscore' (Detre et al. 1995).A second TMA (Bonn-TMA) comprising tissue specimens collected from 25 PCa patients who underwent radical prostatectomy at the University Hospital Bonn, Germany, was constructed and stained as described above.The study was approved by the institutional review boards.Staining intensity was evaluated by an independent pathologist (V S), defining a score between 0 (negative staining) and 3 (intense staining).The following antibodies were used: anti-SOCS2 (H-74) (1:100; Santa Cruz Biotechnology) and anti-androgen receptor (AR) (1:400; Epitomics, Burlingame, CA, USA).

Methylation analysis
Bisulfite-converted DNA from FFPE tissue samples was prepared as described previously (Dietrich et al. 2012) (see Supplementary Materials and methods).Relative DNA methylation levels of the SOCS2 gene locus referred to as total DNA as determined with a methylation-unspecific b-actin (ACTB) assay were assessed using a quantitative duplex PCR.PCR composition and thermal cycling were used as described previously (Dietrich et al. 2012).
Cell culture and reagents PC3, DU145, LNCaP, BPH-1, 22RV1, DUCaP, LAPC4, RWPE-1, and VCaP cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured as indicated.The DUCaP-R subline was established by cultivation in the presence of 1 nM R1881 (Pfeiffer et al. 2010).The identity of the cell lines was confirmed by short tandem repeat analysis.Benign stromal PM151T and epithelial EP156T cells were cultured as described elsewhere (Kogan et al. 2006).For androgen stimulation, the cells were starved in RPMI containing 10% (v/v) charcoal-stripped FCS for 24 h and then treated with R1881 (DuPont NEN Products, Boston, MA, USA).To antagonize androgen-induced effects, LNCaP cells were incubated with 5 mM of bicalutamide for 24 h.LAPC4 cells, which are usually maintained in a medium containing dihydrotestosterone (DHT), were grown in RPMI containing 10% (v/v) charcoal-stripped FCS with or without 100 nM DHT (Sigma-Aldrich).

RNA isolation and qRT-PCR
RNA isolation, RT, and qRT-PCR were carried out as described elsewhere (Puhr et al. 2009) (see Supplementary Materials and methods).

Proliferation measurement
Proliferation was assessed using [ 3 H]thymidine incorporation assay as described in Hoefer et al. (2012).The medium was supplemented with 10% FCS.Viability was measured by WST assay.The cells were seeded in 96-well plates and grown in the medium.To the plates, 10 ml of WST reagent (Roche) were added for 60 min.Absorbance was assessed using Chameleon 5025 (HVD Life Sciences, Vienna, Austria).

Apoptosis measurement and cell-cycle distribution
Apoptotic cells and cell-cycle distribution were assessed as described previously (Hoefer et al. 2012) and analyzed using the ModFit LT 3.2 software.

Microarray dataset generation and analysis
The microarray dataset was generated at the Expression Profiling Unit of the Medical University Innsbruck following the manufacturer's protocols as described in Martowicz et al. (2013).The analysis of the in total 30 Affymetrix HuGene-1.0-ST-v1GeneChips was carried out as described in Rainer et al. (2012) using functions from Bioconductor packages (Gentleman et al. 2004).The raw and pre-processed data were been submitted to GEO (accession number: GSE 50936).

Statistical analyses
SPSS (V15.0) and GraphPad Prism 4 were used for statistical analyses.The Mann-Whitney U test or t-test was carried out to calculate significances between two groups.Correlation analysis was carried out using Pearson's method.Differences in relapse-free survival, defined as biochemical recurrence, were assessed using the Kaplan-Meier plots and log-rank test.Differences were considered statistically significant when P was !0.05.

SOCS2 expression increases with malignancy and inversely correlates with time to disease recurrence
To evaluate SOCS2 expression patterns in benign and malignant prostate tissue, we used a radical prostatectomy specimen TMA of two independent PCa cohorts.Ninety malignant and 79 benign cores of PCa specimens obtained in Innsbruck (Innsbruck-TMA) were evaluable after immunohistochemical staining.A representative benign gland (Fig. 1A) revealed a low SOCS2 expression in the surrounding stromal compartment (especially in smooth muscle fibers), whereas epithelial cells expressed higher levels of SOCS2.Within the epithelial compartment, the basal cell layer exhibited a much more intense SOCS2 staining than luminal cells.A similar phenomenon was observed in benign epithelial and stromal prostate cell lines.We detected the highest SOCS2 expression in EP156T cells, which predominantly represent a basal epithelial phenotype (Kogan et al. 2006), lower SOCS2 expression in the phenotypically luminal epithelial RWPE-1 cells, and the weakest SOCS2 expression in the stromal cell line PM151T (Fig. 1B).Quantitative analysis of the TMA demonstrated a significantly elevated SOCS2 expression in malignant areas compared with benign tissue, as well as in high-vs low-Gleason score samples (Fig. 1C and D).Furthermore, SOCS2 expression was significantly higher in bone and lymph node metastases compared with benign tissue (Fig. 1D).In primary tumor tissue, SOCS2 expression was positively correlated with the Gleason score (Fig. 1E), and relapse-free survival was significantly shorter in patients with a high SOCS2 expression (Fig. 1F).IHC staining of a second cohort of patients (Bonn-TMA) confirmed an elevated SOCS2 expression in low-as well as high-Gleason score tumors (Supplementary Figure 1, see section on supplementary data given at the end of this article).Testing for SOCS2 gene hypermethylation, which is frequently reported for other tumor entities (Sutherland et al. 2004, Culig 2013), revealed no alteration in malignant tissue (Fig. 1G), consistent with the high expression of the protein in PCa.

SOCS2 expression regulates PCa cell growth in vitro and in vivo
All the tested prostate cell lines expressed both SOCS2 mRNA and protein, as measured by qRT-PCR and western blotting respectively (Fig. 2A and B).The observed discrepancy between mRNA and protein levels indicates a tight regulation of SOCS2 protein stability via proteasomal degradation.Cellular fractionation and subsequent western blotting revealed SOCS2 expression predominantly in the cytoplasmic fraction (Fig. 2C).To uncover a possible influence of SOCS2 on cell growth, we carried out functional assays following SOCS2 depletion.For this purpose, PC3, DU145, and LNCaP cells were stably transfected with doxycycline-inducible SOCS2 shRNA constructs and treated with doxycycline for 6 days to achieve prolonged SOCS2 downregulation (Fig. 3A).Subsequent [ 3 H]thymidine incorporation and clonogenic assays revealed a significant decrease in both proliferation (Fig. 3B) and colony formation ability (Fig. 3C) in cells in which SOCS2 was silenced.We obtained similar results with both specific shRNA sequences; however, growth inhibition with shSOCS2-1 was more prominent.The WST assay confirmed decreased cell viability after SOCS2 downregulation (Fig. 3D).In line with these findings, SOCS2 overexpression increased the proliferation and clonogenic potential in PC3 cells (Supplementary Figure 2, see section on supplementary data given at the end of this article).
To confirm a possible anti-proliferative effect of SOCS2 knockdown on tumor growth in vivo, we applied a CAM assay using PC3 shSOCS2-1 or shLuc cells.In the past three decades, the CAM assay has become an accepted and reliable in vivo model to replace animal experiments for testing different treatments (Armstrong et al. 1982, Kunzi-Rapp et al. 2001, Taizi et al. 2006, Tavaszi & Budai 2006, Saw et al. 2008).In xenografts derived from SOCS2 knockdown cells, we observed a significantly reduced tumor area after 5 days on the CAM (Fig. 4A and D).Immunohistochemical staining revealed a reduced SOCS2 expression in the tumor cells as well as a significant decrease in the proportion of Ki67-positive cells (Fig. 4B and C).

SOCS2 knockdown leads to cell-cycle arrest and increased apoptosis
To elucidate the mechanism underlying the decreased cell growth after SOCS2 downregulation, we measured apoptosis and cell-cycle distribution.The percentage of apoptotic cells was slightly increased in PC3 and DU145 cells after SOCS2 silencing compared with the control cells.However, LNCaP cells were more sensitive and displayed a 40% apoptosis rate after SOCS2 knockdown (Fig. 5A).These findings were confirmed by the measurement of cleaved PARP (cPARP) levels by western blotting.As expected, LNCaP shSOCS2 cells exhibited a massive increase in cPARP levels compared with the respective controls, while this effect was less distinct in PC3 and DU145 cells (Fig. 5B).To further uncover the molecular basis for altered cell proliferation after SOCS2 knockdown, we analyzed cell-cycle distribution in the control as well as shSOCS2 cells.As has been mentioned above, LNCaP cell line responded with a massive increase in the subG1 proportion, which represents the apoptotic population (Supplementary Figure 3, see section on supplementary data given at the end of this article).However, we observed an altered cell-cycle distribution pattern in PC3 and DU145 cells after SOCS2 downregulation compared with the control cells (Fig. 5C).Statistical analysis revealed an S-phase growth arrest in SOCS2-depleted PC3 and DU145 cells and in addition an increase in the percentage of G2/M-phase cells in PC3 shSOCS2 cell line.The G0/G1phase proportion was significantly decreased in both cell lines after SOCS2 knockdown (Fig. 5D).

SOCS2 expression is upregulated by androgens
Finally, we aimed to provide a possible explanation for the significantly increased SOCS2 expression in the malignant tissue of PCa patients.AR is able to activate STAT5 (Tan et al. 2008), which in turn has been shown to upregulate SOCS2 in head-and-neck squamous cell carcinoma (HNSCC; Sen et al. 2011).Therefore, we hypothesized that SOCS2 expression might be influenced upon androgenic stimulation in PCa.To address this issue, we used Affymetrix GeneChip expression data of LNCaP, DUCaP, and VCaP cells cultured in the absence or presence of R1881.SOCS2 was identified as an androgen-regulated gene (Fig. 6A).We confirmed this finding also at the mRNA and protein levels, demonstrating androgenic SOCS2 upregulation in a dose-dependent manner in LNCaP cells.Furthermore, treatment with the antiandrogen bicalutamide was sufficient to reverse this effect (Fig. 6B).Androgen-induced SOCS2 expression increase was in addition time dependent and peaked between 8 and 24 h in LNCaP, DUCaP, and VCaP cells (Fig. 6C).To further confirm androgen responsiveness of SOCS2, we expected that androgen withdrawal would result in a diminished SOCS2 expression.Hence, we depleted LAPC4 cells, which are usually grown in a medium containing DHT.As expected, we observed a significantly decreased SOCS2 expression in cells grown in the steroid-free medium, compared with cells cultured in the presence of DHT (Fig. 6D).Finally, we were able to translate these findings to the patients, demonstrating a significant correlation between SOCS2 and AR expression in patient samples (Fig. 6E).

Discussion
Deregulation of endogenous feedback inhibitors of JAK/ STAT signaling is well recognized in PCa and other cancers.We have previously demonstrated that SOCS1, SOCS3, and PIAS1 are upregulated in prostate tumors compared with benign tissue and have fundamental roles in proliferation and apoptosis (Neuwirt et al. 2009, Puhr et al. 2009, Hoefer et al. 2012).SOCS2 is hypermethylated in ovarian and breast cancers, in which its reduced expression is associated with the activation of STAT3, indicating an increased cytokine responsiveness in these tumors (Sutherland et al. 2004).SOCS2 mRNA has been found to be downregulated in pulmonary cancer, hepatocellular cancer and PCa (Wikman et al. 2002, Hendriksen et al. 2006, Qiu et al. 2013).On the other hand, SOCS2 acts as an oncogene in advanced stages of chronic myeloid leukemia as well as in precursors of anal cancer, where it is significantly upregulated (Arany et al. 2001, Schultheis et al. 2002).However, other studies that have investigated SOCS2 in PCa have addressed different aspects of SOCS2 function and have been carried out under different experimental conditions (Hendriksen et al. 2006, Iglesias-Gato et al. 2013, Zhu et al. 2013).
In the present study, we investigated SOCS2 expression patterns in benign and low-and high-Gleason score tumor samples as well as in prostate cell lines.Differences between SOCS2 mRNA and protein levels observed in the cell lines are not surprising, because SOCS2 protein is highly unstable as a consequence of active degradation (Siewert et al. 1999).In tissue samples, we detected significantly increased SOCS2 levels in malignant areas and were furthermore able to positively correlate SOCS2 expression with increasing Gleason scores.In addition, we could demonstrate that patients with a high SOCS2 expression are more likely to experience biochemical tumor relapse.These findings are partially in line with those of a recent study (Zhu et al. 2013), which identified an increased SOCS2 expression in malignant vs benign tissue.However, in contrast to our findings, that study observed a significant upregulation only in low-Gleason score tumors, whereas in  high-Gleason score tumors only a trend toward a high SOCS2 expression was observed.SOCS2 expression was found to be associated with longer relapse-free survival.Besides ethnic differences, a diverse designation of tumors into low-and high-Gleason score groups, as well as different IHC scoring methods, might explain these discrepancies.As SOCS2 is an androgen-regulated gene, ethnic differences between the study populations may be important for the analysis of data.Although more studies comparing AR target gene expression in different ethnic groups have to be carried out, one can expect that differences occur in certain tumor subgroups as a consequence of dissimilar AR transcriptional activities.
Nevertheless, the generally elevated SOCS levels found in PCa were consistently observed in two independent patient cohorts.Furthermore, it should be mentioned that Iglesias-Gato et al. have very recently reported a higher SOCS2 immunoreactivity in parallel with a Gleason score increase in a Swedish population (2013).
In concordance with the tissue expression profile, our functional assays point to a potential growth-promoting activity of SOCS2 in PCa.First, SOCS2 downregulation leads to a significantly diminished tumor growth in vivo.Secondly, SOCS2 knockdown results in a substantial decrease in cell proliferation and clonogenicity in ARnegative and -positive cell lines.We could link this growth inhibition to S-phase and G2/M-phase cell-cycle arrests with a slight increase in apoptosis in AR-negative PC3 and DU145 cells.Interestingly, SOCS2-depleted LNCaP cells exhibit a dramatic increase in apoptosis, suggesting an additional role for SOCS2 in AR-positive cells, which renders them more sensitive to SOCS2 knockdown.
The concept of SOCS2 as a growth promoter rather than as an inhibitor is supported by data obtained from other studies.Both Socs2 knockout and transgenic mice display gigantism (Metcalf et al. 2000, Greenhalgh et al. 2002).This observation can be explained by the circumstance that SOCS2 plays a dual role in growth regulation, depending on its concentration.At low levels, SOCS2 inhibits several cascades such as GH, prolactin, and IL signaling, whereas at high levels, SOCS2 restores or potentiates responsiveness to these growth factors (Favre et al. 1999, Pezet et al. 1999, Tannahill et al. 2005, Piessevaux et al. 2006).In this context, SOCS2 has been demonstrated to enhance GH, IL2, and IL3 signals and subsequent proliferation via proteasomal degradation of other SOCS members, thereby overcoming their inhibitory effects (Favre et al. 1999, Tannahill et al. 2005).These findings suggest SOCS2 to be a positive regulator of proliferation when expressed at high levels.PCa is characterized by the deregulation of several signaling pathways including GH, prolactin, IL6, and the IGF/insulin axis (Roberts 2004, Monti et al. 2007, Cox et al. 2009, Culig 2011, Goffin et al. 2011, Culig & Puhr 2012, Nakonechnaya et al. 2013), which can induce SOCS2 expression (Trengove & Ward 2013).We therefore hypothesize that in PCa SOCS2 is generally highly expressed as a consequence of these oncogenic events and thus acts as an accelerator of proliferative signals in this disease.Hence, it is not surprising that SOCS2 knockdown in PCa cells resulted in growth inhibition.
In addition, we proved that SOCS2 is upregulated by androgens in AR-positive cell lines.SOCS2 is induced upon androgenic stimulation in a dose-and timedependent manner at both mRNA and protein levels.Androgen responsiveness of SOCS2 was further confirmed in an experimental setting, where androgens were depleted.Although we cannot exclude alternative mechanisms, a regulation via STAT5 might be a possible explanation.AR increases the transcriptional activity of STAT5 and vice versa (Tan et al. 2008).STAT5 has been shown to stimulate SOCS2 expression in HNSCC (Sen et al. 2011).Furthermore, SOCS2 has been identified as a direct STAT5 target in the liver (Vidal et al. 2007).On the basis of these data and our results, we hypothesize that in AR-positive cells SOCS2 expression is increased due to STAT5 activation following androgen stimulation.This mechanism provides a possible explanation for the elevated SOCS2 expression in prostate tumors, which frequently harbor a highly active androgen signaling (Buchanan et al. 2001, Lonergan & Tindall 2011).
While this manuscript was in preparation, Iglesias-Gato et al. (2013) reported that SOCS2 antagonizes the oncogenic events caused by GH in PCa.It should be mentioned that differences in the results of the two studies may be explained by the presence or absence of GH in the experiments carried out.In the present study, all the experiments were carried out in cells not treated with GH.On the whole, our study is the first to link increased SOCS2 expression in tissue samples of PCa patients with a growth-promoting role for SOCS2 in vitro and in vivo.Furthermore, we demonstrated androgen responsiveness of SOCS2 in AR-positive cell lines, providing a mechanistic explanation for the high SOCS2 expression in PCa via androgenic stimulation.These findings render SOCS2 an interesting candidate for further investigations to clarify specific pathways involved in SOCS2-mediated effects on PCa cell growth.
Figure 1 SOCS2 expression increases with malignancy and inversely correlates with relapse-free survival.(A) SOCS2 immunostaining of a benign prostate gland showing high SOCS2 expression in epithelial cells.Scale bar: 100 mm.(B) qRT-PCR data and representative western blots of three benign prostate cell lines: PM151T (stromal), EP156T (basal epithelial), and RWPE-1 (luminal epithelial).(C) Immunohistochemical SOCS2 staining of representative tissue samples of the Innsbruck-TMAs.Scale bar: 100 mm.(D) Statistical analysis of the Innsbruck-TMAs.Low Gleason: Gleason patterns 3C4 or below.High Gleason: Gleason patterns 4C3 or above.Box-Whiskers plot represents median values and 10-90 percentiles (*P!0.05 and ***P!0.001; Figure 2 SOCS2 is expressed in the cytoplasm of prostate cell lines.(A) SOCS2 mRNA and (B) SOCS2 protein expression in benign and malignant prostate cell lines was assessed by qRT-PCR and western blotting respectively.
Figure 4 SOCS2 expression influences tumor growth in vivo.(A) Bright-field and fluorescence pictures of representative tumors obtained in a CAM assay.Before engraftment, the cells were pretreated with or without doxycycline for 6 days.(B) Immunohistochemical staining of tumor cross-sections for Figure 5 SOCS2 expression influences apoptosis and cell-cycle progression.(A and B) Apoptosis after SOCS2 downregulation was assessed by flow cytometry after PI staining (A), as well as cPARP measurement by western blotting (B) after 6 days of doxycycline treatment.Data represent meansGS.E.M. from three independent experiments (**P!0.01 and ***P!0.001; t-test).
Figure 6 SOCS2 is upregulated upon androgenic stimulation.(A) Affymetrix data of R1881-treated LNCaP, DUCaP, and VCaP cells (***P!0.001; t-test).(B) qRT-PCR data and representative western blot of LNCaP cells treated with increasing concentrations of R1881 and bicalutamide (Bic) or a combination of both for 24 h.Data represent mean values of three independent experiments (*P!0.05; **P!0.01; t-test).(C) qRT-PCR data and representative western blots of LNCaP, DUCaP, and VCaP cells treated with 1 nM of R1881 for 8, 24, 48, and 72 h.Data represent mean values of