Published OnlineFirst January 29, 2018; DOI: 10.1158/1541-7786.MCR-17-0471 Oncogenes and Tumor Suppressors Molecular Cancer Research Association of USP10 with G3BP2 Inhibits p53 Signaling and Contributes to Poor Outcome in Prostate Cancer Ken-ichi Takayama1, Takashi Suzuki2, Tetsuya Fujimura3, Satoru Takahashi4, and Satoshi Inoue1,5 Abstract Ubiquitin-specific protease 10 (USP10) is known to deubiqui- repressed by USP10 knockdown. Clinically, USP10 was expressed tylate its target proteins, mainly to enhance their stabilities. primarily in the cytoplasm of prostate cancer tissues. High levels USP10 maintains p53 protein levels and controls epigenetic of USP10 expression were strongly correlated with high levels of changes induced by the androgen receptor (AR). GTPase- AR, G3BP2, and p53 in the cytoplasm. High expression of USP10 activating protein-binding protein 2 (G3BP2), an androgen- was significantly associated with poor prognosis of patients with responsive gene, is known as the main component of stress prostate cancer. Taken together, USP10 has a repressive effect on granules (SG) that interacts with USP10 in SGs. This study p53 signaling for cell growth by regulating G3BP2 expression. explores the roles of USP10 in prostate cancer progression in These findings highlight an important oncogenic aspect of USP10 p53, G3BP2, and AR signaling. Using chromatin immunoprecip- through its modulation of the p53–G3BP2 complex and AR itation (ChIP) and sequence analysis, it was found that USP10 is signaling in prostate cancer. transcriptionally induced with AR recruitment to an intronic region. Furthermore, USP10 regulates androgen-mediated signal- Implications: These findings elucidate the oncogenic role of ing and cell growth. USP10 maintained G3BP2 protein stability USP10 in prostate cancer through an increase in G3BP2 protein by reducing polyubiquitylation. G3BP2-dependent growth acti- that inhibits p53 activity, in addition to the promotion of AR vation and p53 nuclear export that reduced p53 signaling were signaling. Mol Cancer Res; 1–11. Ó2018 AACR. Introduction Prostate cancer is the most frequently diagnosed cancer in men. The actions of androgen and its cognate nuclear receptor, andro- The protein p53 is known to function as a tumor-suppressive gen receptor (AR), are essential for the development and prolif- gene and regulator of the cell cycle, DNA repair, apoptosis, and eration of prostate cancer (9–11). When bound to androgens, ARs senescence (1). Past reports indicate that p53 plays an important translocate to nuclei and mainly activate target gene transcription. role in cancer progression because the p53 pathway is frequently The epigenetic status of cells is modulated by AR binding and the inactivated by mutations or genomic deletions in many human subsequent recruitment of coactivators or corepressors to its cancers (2–4). Therefore, p53 is recognized as a desirable target for binding sites. AR overexpression is frequently observed in tissues cancer prevention and therapy. Moreover, posttranslational mod- with advanced prostate cancer (12–16). Therefore, androgen ifications of p53 protein are important to regulating transcrip- deprivation therapy is the first-line treatment for advanced pros- tional activity, protein stability, and cellular localization (5–8). tate cancer. Although androgen deprivation therapy is initially effective for hormone-sensitive cancers, long-term treatment often results in castration-resistant prostate cancer (CRPC) with enhanced AR signaling (13, 15). Thus, investigations of AR target 1 Department of Functional Biogerontology, Tokyo Metropolitan Institute of genes are needed to increase our understanding of the mechanism Gerontology, Itabashi-ku, Tokyo, Japan. 2Department of Pathology, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan. 3Department of underlying the progression to advanced prostate cancer. Urology, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, We recently revealed that androgen regulates p53 localization Tokyo, Japan. 4Department of Urology, Nihon University School of Medicine, by inducing GTPase-activating protein-binding protein 2 Itabashi-ku, Tokyo, Japan. 5Division of Gene Regulation and Signal Transduction, (G3BP2), which is an AR target gene (17). G3BP2 associates with Research Center for Genomic Medicine, Saitama Medical University, Hidaka, p53 and SUMO E3 ligase RAN-binding protein 2 (RanBP2), Saitama, Japan. promoting p53 nuclear export via increased p53 sumoylation Note: Supplementary data for this article are available at Molecular Cancer (17). Elevated G3BP2 expression has been shown to repress Research Online (http://mcr.aacrjournals.org/). docetaxel-mediated apoptosis and promote CRPC tumor growth Corresponding Author: Satoshi Inoue, Department of Functional Biogerontol- (17). Moreover, we found, through a clinicopathologic analysis, ogy, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, that G3BP2 overexpression is associated with poor outcomes in Tokyo 173-0015, Japan. Phone: 813-5800-8834; Fax: 814-2984-4541; E-mail: patients with prostate cancer (17). [email protected] Ubiquitylation is a reversible reaction that posttranslationally doi: 10.1158/1541-7786.MCR-17-0471 modulates the stability of target proteins. Removal of ubiquitin Ó2018 American Association for Cancer Research. is mediated by a specialized class of enzymes called www.aacrjournals.org OF1 Downloaded from mcr.aacrjournals.org on September 25, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst January 29, 2018; DOI: 10.1158/1541-7786.MCR-17-0471 Takayama et al. deubiquitylating enzymes (DUB) that cleave the isopeptide bond Kit (Jena Bioscience). We maintained stocks of low-passage that links ubiquitin to its substrate. Ubiquitin-specific protease 10 cells and restarted our cell culture with a fresh vial at least once (USP10) is a DUB that deubiquitylates its target proteins to a month. The antibodies used in this study were USP10 #5553 enhance their stability (18–20). An important target protein of from Cell Signaling Technology; G3BP2 (86135) and USP10 USP10 is p53, and p53 protein levels are regulated by USP10 in (109219) from Abcam, Ub (FL76), BAX (N-20), and p53 human cancers (20). USP10 has been also implicated in control- (Do1) from Santa Cruz Biotechnology; and b-actin from ling epigenetic conditions by deubiquitylating histone proteins in Sigma. Other antibodies and reagents used have been the nucleus. H2A.Z is a variant of the core histone H2A (21). H2A. described previously (30, 31). The following reagents were Z has been shown to regulate gene transcription, functioning in purchased from the indicated companies: MG132 (Abcam), either a positive or negative manner. Modification of H2A.Z is a sodium arsenite (Sigma-Aldrich), hydrogen peroxide (Wako), key event that defines the epigenetic role of H2A.Z. Monoubi- and cycloheximide (Roche). The cells were treated with quitylation of H2A.Z is associated with transcriptional silencing. 1 mmol/L sodium arsenite or 1 mmol/L hydrogen peroxide USP10 was found to deubiquitylate monoubiquitylated H2A.Z for the indicated times. and positively regulate AR-mediated transcription of PSA (22). Therefore, these previous reports have indicated that USP10 Immunoblot analysis and immunoprecipitation functions as a modulator of the p53 pathway by increasing For the immunoprecipitation assay, we incubated lysates p53 protein levels and activating transcription through histone with anti-G3BP2 or normal rabbit IgG overnight at 4C. The modification. mixture was then incubated with protein G-Sepharose beads Recent studies have shown that G3BP2 interacts with USP10 (Amersham) and rotated for 2 hours at 4C.Afterfourwashes (23–25). Both proteins have been observed to colocalize in SGs, with NP-40 lysis buffer [(150 mmol/L NaCl, 1% NP40, and which are stress-inducible cytoplasmic structures containing 50 mmol/L Tris-HCl (pH 8.0)], beads were mixed in SDS mRNAs (26–28). The formation of SGs is essential for the recovery sample buffer [50 mmol/L Tris-HCl (pH 6.8), 1% SDS, 10% of cells from stress, and SGs inhibit apoptosis of cancer cells in glycerol]. We boiled the samples for 5 minutes and separated response to various types of stress, such as exposure to arsenite, their proteins by SDS-PAGE. To detect ubiquitin-conjugated heat shock, hypoxia, and viral infections. Mammalian cells acti- proteins, we immunoprecipitated G3BP2 under denatured con- vate protective mechanisms to prevent the accumulation of ditions. Cells were lysed in 100 mL SDS lysis buffer [50 mmol/L altered DNA and proteins. Because polysome-released mRNAs Tris-HCl (pH 7.5), 0.5 mmol/L EDTA, 1% SDS, 1 mmol/L DTT, are transferred to SGs in an inactive state, SGs function as a and protease inhibitor cocktail] and boiled the lysates transient storage site for mRNAs during periods of stress. The for 10 minutes. The lysates were then diluted in 1 mL 0.5% biological significance of SGs includes contributing to cell survival P40 buffer, and protein complexes were immunoprecipitated and inhibition of apoptosis following exposure to stress inducers with anti-G3BP2 rabbit polyclonal antibody. For the immu- (24, 29). noblotting assay, we detected ubiquitylated proteins using Because AR and G3BP2, both USP10-binding partners anti-ubiquitin mouse mAb and anti-G3BP2 rabbit polyclonal (21, 23–25), mediate important signaling pathways in prostate antibody. Immunoblotting was performed as described previ- cancer, we hypothesized