Association of USP10 with G3BP2 Inhibits P53 Signaling And

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. 2018 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 2018 American Association for Cancer Research. is mediated by a specialized class of enzymes called

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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 that USP10 has a role in prostate ously (32). cancer progression. In the current study, we revealed a new oncogenic role of USP10 as an inhibitor of p53 signaling Cell proliferation assay through modulation of G3BP2 protein levels. We found that Cells were cultured in 24-well plates at 5 103 cells per well. USP10 regulates a G3BP2-mediated repressive effect on p53 Cells were trypsinized and counted using the trypan blue exclu- activity. Furthermore, we found that USP10 is a target of AR and sion method. For the MTS assay, cells were cultured in 96-well is upregulated by androgen treatment, leading to a positive plates at 3 103 cells per well. We used CellTiter 96 Aqueous MTS feedback loop of AR signaling in prostate cancer. Moreover, our reagent (Promega) according to the manufacturer's instructions to clinicopathologic analysis of prostate cancer specimens indi- determine cell viability. The experiments were performed in cated the importance of USP10 in prostate cancer tissues in quintuplicate. which G3BP2 was highly expressed. Taken together, our findings support the novel oncogenic role of USP10 in regu- Immunofluorescence lating the p53–G3BP2 complex, AR signaling, and prostate Cells grown on 12-mm circular coverslips (Matsunami) in cancer progression. 24-well plates were fixed with 4% paraformaldehyde in PBS for 10 minutes at room temperature and permeabilized with Materials and Methods 0.5% Triton X-100/PBS for 2 minutes. Cells were washed in PBS, blocked with 5% normal goat serum/PBS for 30 minutes, Cell culture and reagents and then incubated with primary antibodies in 5% normal VCaP and 293T cells were obtained from Saitama Medical goat serum/PBS overnight at 4C. Next, cells were washed University (Saitama, Japan) and grown in DMEM medium three times with PBS and incubated with anti-mouse IgG supplemented with 10% FBS, 50 U/mL penicillin, and 50 mg/ conjugated to Alexa Fluor 546 and anti-rabbit IgG conjugated mL streptomycin. 22Rv1 and LNCaP cells were obtained from to Alexa Fluor 488 (Life Technologies) in goat serum/PBS ATCC and grown in RPMI medium supplemented with 10% for 1 hour. Nuclei were counterstained with 40,6-diamidino- FBS, 50 U/mL penicillin, and 50 mg/mL streptomycin. All cell 2-phenylindole (DAPI). Cells were washed 3 times with PBS, authentications were validated as the expected cell type by coverslips were mounted in glycerol, and cells were visualized shorttandemrepeatanalysesin2015androutinelychecked using an Olympus confocal laser scanning microscope for mycoplasma contamination using a Mycoplasma Detection (FV10).

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IHC the negative control locus (N.C: GAPDH locus) were We obtained 104 prostate cancer samples from surgeries described previously (17). The sequences of primers targeting performed at the University of Tokyo Hospital (Tokyo, Japan). USP10 AR-binding site (ARBS) used were as follows: forward: 50- Our study (G10044-2) was approved by the University of GCAGGACTCGACAAGTTTGG-30, reverse: 50-AACCCCCAA- Tokyo ethics committee and conducted in accordance with GATCCTTTCTC-30. Declaration of Helsinki. Informed consent was obtained from each patient before surgery. The ages of the patients ranged Statistical analysis from 52 to 78 years (mean, 67 years), and pretreatment serum We performed all experiments at least twice and confirmed the PSA levels ranged from 1.2 to 136 ng/mL (mean, 16.9 ng/mL). results were similar. In most cell-based experiments, we used two- Formalin-fixed tissues were embedded in paraffinandsec- sided Student t tests to determine statistical significances and tioned. A Histofine Kit (Nichirei), based on the streptavidin- calculate P values of differences between groups. Cancer-specific biotin amplification method, was used for the IHC analysis of survival curves were determined using the Kaplan–Meier method. USP10 (Cell Signaling Technology) and G3BP2 (Abcam). The The statistical significances of differences were calculated using the 0 antigen–antibody complex was visualized using a 3,3 -diami- log-rank test. The association between immunoreactivity and 0 nobenzidine solution [1 mmol/L 3,3 -diaminobenzidine, 50 clinicopathologic factors was evaluated using Student t test, a 2 mmol/L Tris-HCl buffer (pH 7.6), and 0.006% H2O2]. To stain cross-table by c testing, or correlation coefficient (r) and regres- p53, the streptavidin–biotin amplification/peroxidase-cata- sion equation. One-way ANOVA was used to analyze the asso- lyzed signal amplification(CSA)system(Dako)wasusedas ciation between p53 and USP10 expression levels. We used described in an earlier report (17). In the IHC analysis, the GraphPad Prism 5 software or MS Excel for the statistical analyses. immunoreactivity of more than 1,000 carcinoma cells was evaluated in each case, and the percentage immunoreactivity Results [labeling index (LI)] was determined by specialized patholo- gists. Expression of AR was analyzed previously in this cohort USP10 is an androgen-regulated gene with an AR-binding site in (31). Cases with an LI >10% for G3BP2 and USP10 staining an intronic region were considered "high expression." This 10% cut-off value is USP10 has been reported to enhance androgen-dependent frequently used for reproducible reports of various immunos- epigenetic regulation in the nucleus of AR-positive LNCaP pros- taining assays (33). tate cancer cells (21). In the current study, we examined the effect of androgen on USP10 expression. First, based on the results of siRNA our high-throughput analysis of ARBSs determined by ChIP and Silencer select siRNAs targeting USP10 (s17368, s17365), AR sequencing (ChIP-seq) in two AR-positive LNCaP and VCaP cell (s1538), and p53 (s607) were purchased from Thermo Fisher lines (32, 36), we found marked AR recruitment to an intronic Scientific. All siRNA experiments were performed at siRNA con- region of USP10 (Fig. 1A; Supplementary Fig. S1A). These results centrations of 10 nmol/L. Cells were transfected with siRNAs were validated by a ChIP-qPCR analysis (Fig. 1B; Supplementary using Lipofectamine RNAiMAX (Thermo Fisher Scientific) 48 to Fig. S1B). Positive regulation of USP10 by androgen was con- 72 hours before each experiment. firmed by a qRT-PCR analysis of LNCaP and VCaP cells (Fig. 1C; Supplementary Fig. S1C). Moreover, this androgen-dependent qRT-PCR increase in USP10 was inhibited by treatment with the AR antag- Total RNA was isolated using ISOGEN reagent (Nippon Gene). onist bicalutamide (Fig. 1D) or AR knockdown using siRNA First-strand cDNA was generated using a PrimeScript RT Reagent (Fig. 1E). Androgen-dependent induction of USP10 was also Kit (Takara). The resulting cDNA was then analyzed by qRT-PCR observed at the protein level by Western blot analysis (Fig. 1F; using KAPA SYBR Green Mix (KAPA Biosystems) on a Stepone Supplementary Fig. S1D). Thus, our analyses indicate positive Real-Time PCR system (Thermo Fisher Scientific). The primer regulation of USP10 expression by androgen and AR in prostate sequences used are shown below or were described previously cancer cells. (30, 31, 34, 35). USP10 forward: 50-TTTTAAATGCCACCGAACC- TATC-30; reverse: 50-CCAGCCATTCAGACCGATCT-30. Positive feedback loop in AR signaling formed by USP10 Previously, PSA induction by androgen was shown to be Chromatin immunoprecipitation-qPCR regulated by USP10 in LNCaP cells (21). Here, we investigated Chromatin immunoprecipitation (ChIP) was performed as the effect of USP10 knockdown on other androgen signals in described previously (17). In short, cells were crosslinked with prostate cancer cells by knocking down USP10 in LNCaP and 1% formaldehyde. After 10 minutes of incubation, we stopped VCaP cells and treating cells with DHT or vehicle. We observed fixation by adding 0.2 mol/L glycine. Cells were lysed, and the significant repression of androgen-dependent induction of several lysates were incubated on ice for 15 minutes. We sonicated the AR target genes, suggesting a positive role of USP10 in androgen- lysates to shear the chromatin DNA. After centrifugation, super- mediated signaling in these cells (Fig. 2A; Supplementary Fig. natants were incubated with specific antibodies overnight at 4C S2A). In contrast, overexpression of USP10 enhanced androgen- with rotation. Protein G agarose beads were added, and the mediated gene induction (Fig. 2B; Supplementary Fig. S2B). mixture was incubated for an additional 2 hours. The beads Consistent with these results, androgen-dependent growth of were washed and incubated at 65C overnight to reverse the LNCaP and VCaP cells was repressed by USP10 knockdown (Fig. crosslinking in ChIP elution buffer. We purified the DNA by 2C; Supplementary Fig. S2C and S2D). Thus, USP10 appears to ethanol precipitation. Fold enrichment relative to the input was form a positive feedback loop in androgen signaling in prostate measured by qPCR using an ABI StepOne System and KAPA SYBR cancer cells and in androgen-mediated cell growth. However, Green PCR mix for qPCR. The sequences of primers that targeted knockdown of endogenous USP10 had no significant effects on

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Figure 1. USP10 is regulated by AR binding to the intronic region. A, Identiﬁcation of ARBSs in the intronic region of USP10. Our AR ChIP-seq analysis data (32) in LNCaP cells were used to detect the ARBS in USP10 locus. LNCaP cells were treated with vehicle or DHT 10 nmol/L for 24 hours. B, Validation of AR binding by ChIP-qPCR. LNCaP cells were treated with vehicle or DHT 10 nmol/L for 24 hours. ChIP was performed using anti-AR antibody or nonspeciﬁc IgG. Enrichment of ARBS in USP10 and negative control locus (N.C.) was measured by real-time PCR (n ¼ 3). Data, average þ SD. , P < 0.01. C, Androgen-dependent induction of USP10 in prostate cancer cells. LNCaP cells were treated with DHT 10 nmol/L for the indicated times. qRT-PCR analysis (n ¼ 3) was performed to measure the expression level of USP10. Data, average þ SD. D, Androgen-dependent induction of USP10 was inhibited by AR blockade. We added bicalutamide 5 mmol/L or vehicle to LNCaP cells 3 hours before androgen treatment for 24 hours. qRT-PCR analysis (n ¼ 3) was performed to measure the expression level of USP10. Data, average þ SD. , P < 0.01. E, Androgen-dependent induction of USP10 was inhibited by AR knockdown. We treated LNCaP cells with siControl () or siAR (þ) for 48 hours before androgen treatment for 24 hours. Left, Western blot analysis was performed to detect AR protein. b-Actin is a loading control. Right, qRT-PCR analysis (n ¼ 3) was performed to measure the expression level of USP10. Data, average þ SD. , P < 0.05; , P < 0.01. F, Induction of USP10 at protein level by androgen in prostate cancer cells. LNCaP cells were treated with DHT 10 nmol/L or vehicle for 24 and 48 hours. b-Actin is a loading control. IB, immunoblot.

growth of LNCaP and VCaP cells in the absence of androgen hydrogen peroxide (H2O2; ref. 19). Therefore, we hypothesized (Fig. 2C; Supplementary Fig. S2C) or on that of the 22Rv1 that USP10 modulates G3BP2 expression for SG formation or androgen-independent prostate cancer cells (Supplementary Fig. negatively regulates p53 activity. S3A). We analyzed the expression levels of USP10 and its binding To test this hypothesis, we first examined the interaction partner, G3BP2, by Western blot analysis (Supplementary Fig. of G3BP2 with USP10 in prostate cancer cells and in SGs. We S3B). The expression of both USP10 and G3BP2 was enhanced by immunoprecipitated G3BP2 and performed a Western blot androgen treatment. Furthermore, we observed increased G3BP2 analysis of LNCaP cell lysates (Fig. 3A). We revealed the mRNA levels in the presence of androgen (Supplementary Fig. endogenous interaction of G3BP2 with USP10 in LNCaP cells. S3C). However, G3BP2 is expressed at a relatively low level in Furthermore, we also observed the interaction of G3BP2 22Rv1 cells, suggesting that transcriptional activation of G3BP2 with USP10 in cells under stress conditions (sodium might be important for USP10-mediated growth promotion. arsenite and H2O2 treatment) by immunoprecipitation analysis (Fig. 3B). USP10 colocalizes with G3BP2 in the cytoplasm and SGs in We then analyzed the subcellular localization of USP10 in prostate cancer cells prostate cancer cells using LNCaP cells overexpressing Flag- We previously reported negative regulation of the p53 pathway G3BP2 and both anti-Flag mouse monoclonal and anti-USP10 with the export of p53 from the nucleus to the cytoplasm by rabbit polyclonal antibodies (Fig. 3C). Although USP10 has been androgen-regulated G3BP2, based on results of SUMO-mediated reported to function as an epigenetic modulator in the nucleus modification of p53 (17). In our previous report, overexpression (21), we determined, by immunofluorescence analysis, that of G3BP2 reduced p53 activity, and knockdown of G3BP2 USP10 was mainly expressed in the cytoplasm, suggesting that induced p53-mediated apoptosis of prostate cancer cells. Nota- USP10 may play a major role in the cytoplasm. Interestingly, bly, G3BP2 has been reported to interact with USP10 in the G3BP2 was enriched in SGs in response to stress-inducing treat- cytoplasm as well as in SGs, which are formed in response to ments, whereas USP10 expression was observed in both SGs and stress-inducing treatments with hypoxia, sodium arsenite, or the cytoplasm (Fig. 3C). We also observed the colocalization of

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Figure 2. USP10 regulates AR signaling for androgen-mediated cell growth. A, Androgen-mediated gene inductions were inhibited by USP10 knockdown. LNCaP cells were treated with siControl or siUSP10 #1 and #2 for 48 hours. Cells were treated with DHT 10 nmol/L or vehicle for 24 hours. qRT-PCR analysis (n ¼ 3) was performed to measure the expression level of AR-regulated genes. Data, average þ SD. , P < 0.01. B, Overexpression of USP10 enhances androgen-dependent signals in VCaP cells. Cells were transfected with pcDNA3.0-USP10 or control vector. After 48-hour incubation, cells were treated with vehicle or DHT 10 nmol/L for 24 hours. qRT-PCR analysis (n ¼ 3) was performed to measure the expression level of AR-regulated genes. Data represent the average þ SD. , P < 0.01. C, Androgen-dependent cell growth of LNCaP cells was modulated by USP10 knockdown. Cells were treated with siControl or siUSP10 #1 and #2. After 3-day incubation, the number of cells were counted (n ¼ 4). Data, average þ SD. , P < 0.01.

USP10 with Flag-G3BP2 in SGs (Fig. 3C). In addition to the stability of G3BP2 by inhibiting polyubiquitylation, probably by interaction of endogenous G3BP2 with USP10, we observed deubiquitylating G3BP2. interactions between exogenous Flag-G3BP2 and USP10 transfected into 293T cells (Fig. 3D). The middle region (140–252 aa) Cell proliferation and p53 nuclear export induced by G3BP2 are of G3BP2 was found to be responsible for its interactions with inhibited by USP10 knockdown in G3BP2-overexpressing cells USP10 in the current analysis (Fig. 3E). Taken together, these We then investigated the alternative p53 regulatory pathway by observations demonstrate the interaction of USP10 with G3BP2 modulating G3BP2 and USP10 expression. We previously in both the cytoplasm and SGs of prostate cancer cells. reported that overexpression of G3BP2 in LNCaP cells induced cytoplasmic export of p53 and cell proliferation (17). By Western USP10-mediated regulation of G3BP2 protein levels by blot analysis, we determined that exogenous G3BP2 protein levels reducing polyubiquitylation were repressed by USP10 inhibition in LNCaP cells overexpres- Next, we investigated whether USP10 maintains the G3BP2 sing Flag-G3BP2 (Fig. 5A). In line with G3BP2 inhibition, G3BP2- protein levels. Our Western blot analysis showed USP10 knock- dependent p53 nuclear export was reversed, and nuclear enrich- down–mediated repression of G3BP2 protein (Fig. 4A; Supple- ment of p53 protein was observed in LNCaP cells overexpressing mentary Fig. S2D). We also observed that MG132 treatment Flag-G3BP2 (Fig. 5B). We next investigated the effect of USP10 reversed siUSP10-mediated inhibition of G3BP2, suggesting the knockdown on G3BP2-mediated growth induction. Interestingly, ubiquitin–proteasome pathway is involved in repression of this the MTS assay indicated that G3BP2-dependent induction of cell protein. We then treated LNCaP cells that overexpressed Flag- growth was impaired by USP10 knockdown. In contrast, growth G3BP2 with cycloheximide to block protein synthesis and ana- inhibition was not observed in vector control cells, suggesting that lyzed protein stability by Western blot analysis (Fig. 4B). G3BP2 USP10 plays a positive role in prostate cancer cell proliferation in protein levels were severely decreased with USP10 inactivation, G3BP2-overexpressing cells (Fig. 5C). indicating that protein stability of G3BP2 is positively regulated Next, we analyzed how p53 target genes are affected by USP10 by USP10. Because USP10 is known to deubiquitylate its target knockdown in G3BP2-overexpressing cells. We demonstrated proteins and enhance protein stability, we investigated whether signiﬁcant upregulation of NOXA and p21 at the mRNA level USP10 regulates polyubiquitylation of G3BP2. As expected, we and BAX at the protein level in response to USP10 knockdown, found that USP10 knockdown enhances the polyubiquitylation suggesting that inhibited p53 activity by G3BP2 was reversed of G3BP2 (Fig. 4C). These results suggest that USP10 increases the (Fig. 5D). Moreover, we observed that cell growth inhibition in

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Figure 3. Interaction of USP10 with G3BP2 in cytoplasm and stress granules in prostate cancer cells. A, USP10 interacts with G3BP2 in prostate cancer cells. We treated LNCaP cells for 24 hours. Cell lysates were obtained for immunoprecipitation (IP) by anti-USP10 antibody. IB, immunoblot. B, USP10 interaction with G3BP2 is enhanced in stress-induced condition. Both LNCaP and 22Rv1 cells were treated with 1 mmol/L sodium arsenite (AS),

1 mmol/L hydrogen peroxide (H2O2), or vehicle for 1 hour. Cell lysates were obtained for immunoprecipitation by anti-G3BP2 antibody. C, Colocalization of USP10 with G3BP2 in SGs and cytoplasm of prostate cancer cells. Immunoﬂuorescence analysis was performed in LNCaP cells overexpressing Flag-tagged G3BP2. Cells were treated with 1 mmol/L AS,

1 mmol/L H2O2, or vehicle for 1 hour. We stained cells with anti-Flag mouse monoclonal (red) and anti-USP10 rabbit polyclonal antibodies (green). Colocalization of both signals in SGs was magniﬁed. Scale bar, 10 mm. D, Interaction of exogenous USP10 with G3BP2. 293T cells were transfected with Flag-G3BP2 and USP10. After 48-hour incubation, cell lysates were obtained. Immunoprecipitation using anti-Flag antibody was performed. We performed Western blot analysis to detect USP10 and Flag-tagged G3BP2. E, Identiﬁcation of interaction domain in G3BP protein. We transfected 293T cells with Flag-tagged G3BP2 or its deletion mutants. We performed immunoprecipitation to detect the interaction of exogenous G3BP2 with endogenous USP10. DC:1-326, DC2:1-224, DN:140-483, DN2:252-483 a. a. of G3BP2 protein.

response to USP10 knockdown in G3BP2-overexpressing cells activity. Taken together, USP10 appears to have a positive effect on was reversed by p53 knockdown (Fig. 5E and F). These results cell proliferation and survival through its interactions with G3BP2 indicate that USP10 knockdown suppressed the expression of that results in increased protein stability and inhibition of p53 G3BP2 and cell growth and that this suppression was at least activity (Fig. 5G). partially dependent on p53. In addition, we overexpressed USP10 in 22Rv1 and LNCaP High levels of USP10 expression correlate with high levels of cells. USP10 overexpression increased both p53 and G3BP2 G3BP2 and a poor prognosis for patients with prostate cancer expression (Supplementary Fig, S4A). We found, by immunoﬂu- We previously revealed that G3BP2 was upregulated in a subset orescence analysis, that both nuclear and cytoplasmic p53 protein of prostate cancer tissues and could be an independent prognostic levels were enhanced by USP10 overexpression (Supplementary marker in patients with prostate cancer (17). In the current study, Fig. S4B) because USP10 deubiquitylates and stabilizes p53. By we analyzed the clinical signiﬁcance of USP10 expression in qRT-PCR analysis, we also observed repressive effects of USP10 prostate cancer by IHC (Fig. 6A). We used a cohort of patients overexpression on several p53 target genes (Supplementary Fig. with prostate cancer other than that analyzed in the previous study S4C). This result suggests that the USP10-mediated increase in (17) to investigate expression of USP10 and G3BP2. We obtained G3BP2 abrogates the increase in p53 activity because of nuclear prostate cancer specimens (n ¼ 104) by performing total prosta- export. We also observed increased cell growth with USP10 over- tectomies in our hospital. IHC results indicated that USP10 expression (Supplementary Fig. S4D), consistent with activated expression was upregulated in a subset of prostate cancer tissues AR (Fig. 2C), increased G3BP2 expression, and repressed p53 compared with that in benign prostate tissues (Fig. 6A, P < 0.0001,

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Oncogenic Role of USP10 in Prostate Cancer

significantly associated with high levels of G3BP2 (Fig. 6B) and with poor outcomes in patients with prostate cancer (Fig. 6C). In addition, The Cancer Gene Atlas (TCGA) RNA-seq data revealed that USP10 mRNA levels were significantly associated with G3BP2 in prostate cancer tissues, suggesting that USP10 is highly expressed in tumors with high levels of G3BP2 because both genes are regulated by AR (Fig. 6D). Using public microarray data (Oncomine), we also found that USP10 expression was upregulated in prostate cancer tissues compared with that in benign tissues from patients in other cohorts (Fig. 6E). We next investigated the association between USP10 expression and p53 localization in cancer cells (Fig. 7A and B). Interestingly, high levels of USP10 expression were significantly associated with cytoplasmic expression of p53. In contrast, nuclear p53 expression was not associated with USP10 expression, suggesting the importance of USP10 in regulating cytoplasmic p53 expression. Moreover, USP10 expression was correlated with AR expression (Supple- mentary Table S1). These findings are in line with our experi- mental results, which showed that USP10 forms a positive feedback loop in AR signaling and positively regulates G3BP2 protein to repress p53 activity. These clinicopathologic findings and results of the cell-based analyses demonstrate the oncogenic roles of USP10 in the progression of prostate cancer.

Discussion One of the important roles of USP10 in cancer cells is the deubiquitylation of p53, a major tumor suppressor (19, 20). Here, we present the ﬁrst evidence that USP10 levels correlate with a poor prognosis for patients with prostate cancer, suggesting other oncogenic roles for USP10 in prostate cancer progression. An AR target gene, G3BP2, exports p53 to the cytoplasm, reducing p53 activity (17, 37). G3BP2 is known to play an oncogenic role in prostate cancer progression, promoting tumor growth and inhibiting apoptosis (17). In addition, G3BP2 has been associated with a poor prognosis for breast cancer patients, because it is involved in breast cancer tumor initiation (38, 39). In the current study, we determined an alternative pathway by which USP10 inhibits p53 activity through increasing G3BP2 expression and the export of the p53 protein to cytoplasm, illustrating the oncogenic potential of USP10. The results of the USP10 IHC analysis suggest that USP10's association with G3BP2 could be important in Figure 4. prostate cancer. We revealed that G3BP2-dependent growth USP10 positively regulates G3BP2 protein stability by blocking polyubiquitylation. A, USP10 regulates p53 and G3BP2 protein expression induction was impaired by USP10 knockdown through down- by proteasome-dependent pathway. 22Rv1 and LNCaP cells were treated regulation of G3BP2 at the protein level. This effect was cancelled with siControl or siUSP10 #1 and #2 for 72 hours. Cells were treated with MG132 by p53 knockdown, suggesting that p53 signaling is important in (50 mg/mL) for 5 hours before cell lysis. Western blot analysis was performed to this growth inhibition. More importantly, we found that cyto- detect indicated proteins. IB, immunoblot. B, G3BP2 protein level decreased plasmic expression of p53 is signiﬁcantly associated with high by USP10 knockdown. LNCaP cells overexpressing Flag-G3BP2 were treated levels of USP10 in prostate cancer tissues. These observations with 20 mg/mL cycloheximide (CHX). After indicated times, cell lysates were obtained for Western blot analysis. C, USP10 blocks polyubiquitylation indicate that USP10 enhances G3BP2 expression to promote the of G3BP2 in prostate cancer cells. 22Rv1 cells were treated with siControl or translocation of stabilized p53 protein to the cytoplasm to reduce siUSP10 #1 and #2 for 72 hours. Cells were treated with MG132 (50 mg/mL) for p53 signaling. Meanwhile, it is also possible that USP10 deubi- 5 hours before cell lysis. Cell lysates were immunoprecipitated under denature quitylates other unknown oncogenic proteins in the cytoplasm. condition using anti-G3BP2 antibody. Western blot analysis was performed Screening to identify USP10-binding proteins would be helpful in using anti-ubiquitin antibody. clarifying substrates of this enzyme. Other reports have presented a different mechanism of action McNemar test). USP10 protein was markedly enriched in the by which G3BP1, another member of the G3BP family (40), cytoplasm of cancer cells. Consistent with results of the previous inhibits the p53 pathway (41). G3BP1 inhibits the deubiquityla- study, we found that high levels of G3BP2 expression were also tion of p53 by interacting with USP10, which negatively regulates associated with poor prognosis in this cohort (Supplementary p53 protein expression. It is tempting to speculate that upregula- Fig. S5A and S5B). High levels of USP10 expression were tion of G3BP2 by USP10 also represses activation of the p53

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Figure 5. USP10 knockdown inhibits G3BP2-dependent growth induction in prostate cancer cells. A, G3BP2 protein expression is repressed by USP10 knockdown in LNCaP cells overexpressing Flag-G3BP2. LNCaP cells overexpressing G3BP2 were treated with siControl (siCt) or siUSP10 #1 and #2 for 72 hours. Western blot analysis was performed to detect the indicated proteins. IB, immunoblot; siCt, siControl. B, Distribution of p53 protein in cytoplasm was reduced by USP10 knockdown. LNCaP cells overexpressing Flag-G3BP2 or control vector were treated with siControl or siUSP10 #1 for 72 hours. Immunoﬂuorescence analysis was performed using anti-Flag (mouse) and anti-p53 (rabbit) antibodies. C, USP10 knockdown inhibits G3BP2-dependent growth induction in prostate cancer cells. LNCaP cells overexpressing Flag-G3BP2 or control vector were treated with siControl or siUSP10 #1 and #2. MTS assay was performed at the indicated time points (n ¼ 4). Data, average þ SD. , P < 0.01. D, USP10 knockdown activates p53 signaling in prostate cancer cells with high G3BP2 expression. LNCaP cells overexpressing Flag-G3BP2 or control vector were treated with siControl or siUSP10 #1. Western blot analysis was performed to detect BAX protein. qRT-PCR analysis was performed to determine p21 and NOXA mRNA level (n ¼ 3). Data, average þ SD. , P < 0.01. IB, immunoblot. E, Knockdown of p53 and USP10 in LNCaP cells overexpressing Flag-G3BP2 or control vector. Western blot analysis was performed to detect G3BP2, p53, and USP10 protein. b-Actin is a loading control. IB, immunoblot; siCt, siControl. F, USP10 knockdown–mediated growth inhibition of prostate cancer cells with high G3BP2 expression was not observed in the absence of p53. Knockdown of p53 and USP10 in LNCaP cells overexpressing Flag-G3BP2 or control vector was performed. After 3-day incubation, cell growth was analyzed by counting cell numbers (n ¼ 4). Data, average þ SD. , P < 0.01. G, Schematic summary of oncogenic roles of USP10 and G3BP2 in prostate cancer. Both USP10 and G3BP2 were upregulated by androgen. USP10 enhances protein level of G3BP2 by blocking polyubiquitylation. G3BP2 has a role in inhibiting apoptosis by promoting p53 nuclear export.

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Oncogenic Role of USP10 in Prostate Cancer

Figure 6. High expression of USP10 is a prognostic factor of prostate cancer patients and associated with high expression of G3BP2. A, USP10 is upregulated in a subset of prostate cancer specimens. IHC of USP10 in prostate cancer and benign prostate tissues (n ¼ 104) was performed. Left, USP10-positive cases in prostate cancer tissues. USP10 was immunolocalized in the cytoplasm of prostate cancer cells. Middle, no or weak USP10 immunoreactivity was observed in most benign tissues. Top right, USP10-negative prostate cancer case. USP10 immunoreactivity was weakly observed. Bottom right, lower magnification in a section including both benign and cancerous regions. Immunoreactivity was detected in the cancerous region in contrast to no staining in benign prostate tissues. B, Higher expression of USP10 (n ¼ 39) is significantly associated with high expression of G3BP2 (n ¼ 12). C, High expression of USP10 is a prognostic factor for prostate cancer patients (P ¼ 0.0083). Kaplan–Meier analysis using the log-rank test was performed. D, Plots of TCGA RNA expression profiles in prostate adenocarcinoma (n ¼ 333). Expression levels of G3BP2 and USP10 were plotted and analyzed. Statistical significance was determined by correlation coefficient (R)and regression analysis. RSEM, RNA-seq by expression expectation maximization. E, USP10 expression levels in prostate cancer and benign tissues were analyzed using Oncomine. Two databases showing high expression of USP10 compared with benign tissues were shown.

pathway by blocking USP10-mediated upregulation of p53 at the overexpression of USP10 enhanced AR-mediated transcription. protein level and that two androgen-induced proteins, USP10 and Thus, USP10 plays a role in a positive feedback loop to enhance G3BP2, cooperate to inhibit p53 activity through both nuclear the activity of AR. Activation of this pathway could be another export and protein degradation. indication of USP10 as an oncogene in prostate cancer, because Other important features of USP10 are its regulation by andro- AR signals are central to prostate cancer progression. We also gen in prostate cancer cells and AR binding to intronic regions. We showed that androgen-dependent cell proliferation was repressed also observed the upregulation of USP10 by androgen in LNCaP in LNCaP and VCaP cells. These results may indicate the impor- and VCaP cells. A past analysis revealed that USP10 regulates tance of USP10 in AR signaling and androgen-dependent cell histone modiﬁcation by AR in AR-binding sites to induce PSA growth in prostate cancer. (21). We found that USP10 knockdown reduced the expression Overall, USP10 may modulate apoptosis and cell growth in of other AR target genes in prostate cancer cells. In contrast, several ways: (i) p53 protein stabilization by deubiquitylation;

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Figure 7. The association of USP10 expression with cytoplasmic and nuclear p53 immunoreactivity. A, IHC of p53 in prostate cancer tissues. Representative IHC views of cytoplasmic (in a case with G3BP2 and USP10 high expression) and nuclear p53 staining (in a case with G3BP2 and USP10 high expression) are shown. B, High USP10 expression level was signiﬁcantly associated with cytoplasmic p53 localization. Labeling index (LI) of p53 staining in the cytoplasm and nucleus was determined. One-way ANOVA was performed to determine the P values. Data, average þ SD.

(ii) G3BP2 induction that represses p53 activity by nuclear export; Authors' Contributions (iii) SG formation through the maintenance of G3BP2 protein Conception and design: K. Takayama, S. Inoue levels. Several interesting reports regarding USP10 have described Development of methodology: K. Takayama the localization of USP10 in SGs interacting with G3BPs (23–25), Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): K. Takayama, T. Suzuki although the biological signiﬁcance of USP10 localization in SGs Analysis and interpretation of data (e.g., statistical analysis, biostatistics, has been largely unknown. We observed USP10 colocalization computational analysis): K. Takayama, T. Suzuki with G3BP2 in prostate cancer cells; however, the cytoplasmic Writing, review, and/or revision of the manuscript: K. Takayama, T. Fujimura, distribution of USP10 was not fundamentally different than S. Takahashi, S. Inoue that of G3BP2 following treatment with stress-inducing reagents. Administrative, technical, or material support (i.e., reporting or organizing Previous reports have noted the antiapoptotic effects of SGs data, constructing databases): K. Takayama Study supervision: S. Inoue in cancer cells (29). These multiple functions of USP10 in apoptosis might be dependent on the condition of the tumor Acknowledgments cells. For example, in other tissues such as renal or gastric cancer This work was supported by grants of the Cell innovation Program, the tissues, USP10 has been reported to be a tumor suppressor gene P-DIRECT and the P-CREATE from Ministry of Education, Culture, Sports, (20, 42, 43). Science and Technology, Japan (S. Inoue), by the MEXT, Japan, by grants In summary, our ﬁndings suggest that USP10 is a potent (K. Takayama and S. Inoue) from the JSPS (number 15K15581, 15K15353, regulator of G3BP2 protein expression in prostate cancer cells 17H04334), Japan, by the Program for Promotion of Fundamental Studies in that represses p53 signaling. USP10 can promote cell survival and Health Sciences (S. Inoue), NIBIO, Japan, grants from Takeda Science Foun- dation, Japan (K. Takayama and S. Inoue), grants from the Terumo foundation growth, dependent on G3BP2-mediated signaling. In addition, for life sciences and arts, Japan (K. Takayama), and grants from the NOVARTIS USP10 forms a positive feedback loop in AR signaling. Although Foundation for the Promotion of Science, Japan (K. Takayama). the tumor-suppressive function of USP10 through the deubiquitylation of p53 protein has been reported as important in several The costs of publication of this article were defrayed in part by the tissues, this alternative mechanism of p53 regulation by USP10 payment of page charges. This article must therefore be hereby marked confers oncogenic characteristics in some phases of cancer pro- advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate gression, such as CRPC development. this fact.

Disclosure of Potential Conﬂicts of Interest Received August 26, 2017; revised November 28, 2017; accepted January 3, No potential conﬂicts of interest were disclosed. 2018; published OnlineFirst January 26, 2018.

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Association of USP10 with G3BP2 Inhibits p53 Signaling and Contributes to Poor Outcome in Prostate Cancer

Ken-ichi Takayama, Takashi Suzuki, Tetsuya Fujimura, et al.

Mol Cancer Res Published OnlineFirst January 29, 2018.

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