Down-regulation of Suppressor of Cytokine Signaling-3 Causes Prostate Cancer Cell Death through Activation of the Extrinsic and Intrinsic Apoptosis Pathways Martin Puhr, 1 Fre ´de ´ric R. Santer, 1 Hannes Neuwirt, 1 Martin Susani, 2 Jeffrey A. Nemeth, 4 Alfred Hobisch, 5 Lukas Kenner, 2,3 and Zoran Culig 1 1 Department of Urology, Innsbruck Medical University, Innsbruck, Austria; 2 Institute of Clinical Pathology, Medical University of Vienna; 3 Ludwig Boltzmann Institute for Cancer Research, Vienna, Austria; 4 Centocor Research and Development, Malvern, Pennsylvania; and 5 Department of Urology, General Hospital Feldkirch, Feldkirch, Austria Abstract Suppressor of cytokine signaling-3 (SOCS-3) acts as a negative feedback regulator of the Janus-activated kinase/ signal transducers and activators of transcription factors signaling pathway and plays an important role in the development and progression of various cancers. To better understand the role of SOCS-3 in prostate cancer, SOCS-3 expression was down-regulated in DU-145, LNCaP-IL-6+, and PC3 cells by consecutive SOCS-3 small interfering RNA transfections. SOCS-3 mRNA and protein expression as measured by quantitative reverse transcription-PCR and Western blot, respectively, were decreased by f70% to 80% compared with controls. We observed a significant decrease in cell proliferation and viability in all SOCS-3–positive cell lines but not in the parental LNCaP cell line, which is SOCS- 3 negative. In this study, we show that down-regulation of SOCS-3 leads to an increased cell death in prostate cancer cell lines. We found a considerable increase in the activation of the proapoptotic caspase-3/caspase-7, caspase-8, and caspase-9. A significant up-regulation of cleaved poly(ADP- ribose) polymerase and inhibition of Bcl-2 expression was observed in all SOCS-3–positive cell lines. Overexpression of Bcl-2 could rescue cells with decreased SOCS-3 levels from going into apoptosis. Tissue microarray data prove that SOCS-3 is highly expressed in castration-refractory tumor samples. In conclusion, we show that SOCS-3 is an important protein in the survival machinery in prostate cancer and is overexpressed in castration-resistant tumors. SOCS-3 knockdown results in an increase of cell death via activation of the extrinsic and intrinsic apoptosis pathways. [Cancer Res 2009;69(18):7375–84] Introduction Dysregulation of the pathway of signal transducer and activator of transcription (STAT) factors has been implicated in various diseases. In many types of carcinomas, the oncogenic role of STAT factors, especially of STAT3, is well documented (1–3). Its overexpression and constitutive activation are correlated with an increased proliferation and metastatic progression. However, in prostate cancer the role of STAT3 is a subject of investigation because its activation is associated with either inhibition or promotion of tumor growth (4, 5). To explain these controversial results, negative feedback regulators of the Janus-activated kinase (JAK)–STAT pathway, especially suppressors of cytokine signaling (SOCS), have become a focus of interest. The SOCS family consists of eight members, SOCS-1 to SOCS-7 and cytokine-inducible SH2 protein. All these proteins contain a NH 2 terminal region of variable length, a central SH2 domain, and a conserved COOH terminal region called SOCS box. This region interacts with Elongin-B, Elongin-C, Cullin-5, and RING-box-2. The complex can recruit E2 ubiquitin transferases (6). Thus, the proteins can act as E3 ubiquitin ligases and mediate the proteasomal degradation of associated proteins. SOCS-1 and SOCS-3 are especially induced by interleukin-6 (IL-6). IL-6 is a multifunctional cytokine that plays an important role in chronic inflammation of the prostate (7, 8) and in prostate carcinogenesis (9). SOCS-1 and SOCS-3 can inhibit IL-6– mediated JAK activation directly through their kinase inhibitory region as evidenced for the first time in mouse BF-ER cells (10). In addition, it was shown in renal cancer cells that SOCS-3 can also bind with high affinity to phosphothyrosine 757 within the IL-6 receptor subunit glycoprotein 130 (11). It is generally accepted that SOCS-3 acts as an important negative feedback regulator of the IL- 6-JAK-STAT signaling pathway (12–14) and plays a crucial role in many biological processes. SOCS-3–deficient mice die as a result of placental defects, whereas enforced expression of SOCS-3 also results in embryonic death (15–17). SOCS-3 is involved in the development and progression of several malignancies. There are indications that SOCS-3 has different functions depending on the tumor origin. In human lung (18), hepatocellular (19), and head and neck cancer (20), SOCS-3 is silenced by hypermethylation, which causes a growth advantage for cancer cells. In contrast, SOCS-3 is detectable in breast cancer (21). In previous studies, we showed that SOCS-3 is expressed in prostate cancer and its expression is inversely correlated with STAT3 phosphorylation, suggesting an inactivation of the JAK-STAT pathway (22). Overexpression of SOCS-3 differentially affects proliferation of prostate cancer cells, depending on androgen sensitivity (22, 23). However, effects of SOCS-3 down-regulation on apoptosis have not been investigated in detail yet. In this study, we show for the first time that sustained down- regulation of SOCS-3 results in reactivation of IL-6–induced STAT3 and in decreased cell proliferation and viability of SOCS-3– positive DU-145, LNCaP-IL-6+, and PC3 prostate cancer cell lines. Increased apoptosis is associated with activation of proapoptotic caspase-3, caspase-8, and caspase-9, increased levels of cleaved Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). L. Kenner and Z. Culig contributed equally to this work. Requests for reprints: Zoran Culig, Department of Urology, Innsbruck Medical University, Anichstrasse 35, A-6020 Innsbruck, Austria. Phone: 43-512-504-24717; Fax: 43-512-504-24817; Email: zoran.culig@i-med.ac.at. I2009 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-09-0806 www.aacrjournals.org 7375 Cancer Res 2009; 69: (18). September 15, 2009 Molecular Biology, Pathobiology, and Genetics Research. on February 23, 2016. © 2009 American Association for Cancer cancerres.aacrjournals.org Downloaded from Published OnlineFirst September 8, 2009; DOI: 10.1158/0008-5472.CAN-09-0806