Ubiquitin-specific protease 10 (USP10) is known to deubiquitylate its target proteins, mainly to enhance their stabilities. USP10 maintains p53 protein levels and controls epigenetic changes induced by the androgen receptor (AR). GTPase-activating protein-binding protein 2 (G3BP2), an androgen-responsive gene, is known as the main component of stress granules (SG) that interacts with USP10 in SGs. This study explores the roles of USP10 in prostate cancer progression in p53, G3BP2, and AR signaling. Using chromatin immunoprecipitation (ChIP) and sequence analysis, it was found that USP10 is transcriptionally induced with AR recruitment to an intronic region. Furthermore, USP10 regulates androgen-mediated signaling and cell growth. USP10 maintained G3BP2 protein stability by reducing polyubiquitylation. G3BP2-dependent growth activation and p53 nuclear export that reduced p53 signaling were repressed by USP10 knockdown. Clinically, USP10 was expressed primarily in the cytoplasm of prostate cancer tissues. High levels of USP10 expression were strongly correlated with high levels of AR, G3BP2, and p53 in the cytoplasm. High expression of USP10 was significantly associated with poor prognosis of patients with prostate cancer. Taken together, USP10 has a repressive effect on p53 signaling for cell growth by regulating G3BP2 expression. These findings highlight an important oncogenic aspect of USP10 through its modulation of the p53–G3BP2 complex and AR signaling in prostate cancer.

Implications: These findings elucidate the oncogenic role of USP10 in prostate cancer through an increase in G3BP2 protein that inhibits p53 activity, in addition to the promotion of AR signaling. Mol Cancer Res; 16(5); 846–56. ©2018 AACR.

The protein p53 is known to function as a tumor-suppressive gene and regulator of the cell cycle, DNA repair, apoptosis, and senescence (1). Past reports indicate that p53 plays an important role in cancer progression because the p53 pathway is frequently inactivated by mutations or genomic deletions in many human cancers (2–4). Therefore, p53 is recognized as a desirable target for cancer prevention and therapy. Moreover, posttranslational modifications of p53 protein are important to regulating transcriptional activity, protein stability, and cellular localization (5–8).

Prostate cancer is the most frequently diagnosed cancer in men. The actions of androgen and its cognate nuclear receptor, androgen receptor (AR), are essential for the development and proliferation of prostate cancer (9–11). When bound to androgens, ARs translocate to nuclei and mainly activate target gene transcription. The epigenetic status of cells is modulated by AR binding and the subsequent recruitment of coactivators or corepressors to its binding sites. AR overexpression is frequently observed in tissues with advanced prostate cancer (12–16). Therefore, androgen deprivation therapy is the first-line treatment for advanced prostate 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 genes are needed to increase our understanding of the mechanism underlying the progression to advanced prostate cancer.

We recently revealed that androgen regulates p53 localization by inducing GTPase-activating protein-binding protein 2 (G3BP2), which is an AR target gene (17). G3BP2 associates with p53 and SUMO E3 ligase RAN-binding protein 2 (RanBP2), promoting p53 nuclear export via increased p53 sumoylation (17). Elevated G3BP2 expression has been shown to repress docetaxel-mediated apoptosis and promote CRPC tumor growth (17). Moreover, we found, through a clinicopathologic analysis, that G3BP2 overexpression is associated with poor outcomes in patients with prostate cancer (17).

Ubiquitylation is a reversible reaction that posttranslationally modulates the stability of target proteins. Removal of ubiquitin is mediated by a specialized class of enzymes called deubiquitylating enzymes (DUB) that cleave the isopeptide bond that links ubiquitin to its substrate. Ubiquitin-specific protease 10 (USP10) is a DUB that deubiquitylates its target proteins to enhance their stability (18–20). An important target protein of USP10 is p53, and p53 protein levels are regulated by USP10 in human cancers (20). USP10 has been also implicated in controlling epigenetic conditions by deubiquitylating histone proteins in the nucleus. H2A.Z is a variant of the core histone H2A (21). H2A.Z has been shown to regulate gene transcription, functioning in either a positive or negative manner. Modification of H2A.Z is a key event that defines the epigenetic role of H2A.Z. Monoubiquitylation of H2A.Z is associated with transcriptional silencing. USP10 was found to deubiquitylate monoubiquitylated H2A.Z and positively regulate AR-mediated transcription of PSA (22). Therefore, these previous reports have indicated that USP10 functions as a modulator of the p53 pathway by increasing p53 protein levels and activating transcription through histone modification.

Recent studies have shown that G3BP2 interacts with USP10 (23–25). Both proteins have been observed to colocalize in SGs, which are stress-inducible cytoplasmic structures containing mRNAs (26–28). The formation of SGs is essential for the recovery of cells from stress, and SGs inhibit apoptosis of cancer cells in response to various types of stress, such as exposure to arsenite, heat shock, hypoxia, and viral infections. Mammalian cells activate protective mechanisms to prevent the accumulation of altered DNA and proteins. Because polysome-released mRNAs are transferred to SGs in an inactive state, SGs function as a transient storage site for mRNAs during periods of stress. The biological significance of SGs includes contributing to cell survival and inhibition of apoptosis following exposure to stress inducers (24, 29).

Because AR and G3BP2, both USP10-binding partners (21, 23–25), mediate important signaling pathways in prostate cancer, we hypothesized that USP10 has a role in prostate cancer progression. In the current study, we revealed a new oncogenic role of USP10 as an inhibitor of p53 signaling through modulation of G3BP2 protein levels. We found that USP10 regulates a G3BP2-mediated repressive effect on p53 activity. Furthermore, we found that USP10 is a target of AR and is upregulated by androgen treatment, leading to a positive feedback loop of AR signaling in prostate cancer. Moreover, our clinicopathologic analysis of prostate cancer specimens indicated the importance of USP10 in prostate cancer tissues in which G3BP2 was highly expressed. Taken together, our findings support the novel oncogenic role of USP10 in regulating the p53–G3BP2 complex, AR signaling, and prostate cancer progression.

Cell culture and reagents

VCaP and 293T cells were obtained from Saitama Medical University (Saitama, Japan) and grown in DMEM medium supplemented with 10% FBS, 50 U/mL penicillin, and 50 μg/mL streptomycin. 22Rv1 and LNCaP cells were obtained from ATCC and grown in RPMI medium supplemented with 10% FBS, 50 U/mL penicillin, and 50 μg/mL streptomycin. All cell authentications were validated as the expected cell type by short tandem repeat analyses in 2015 and routinely checked for mycoplasma contamination using a Mycoplasma Detection Kit (Jena Bioscience). We maintained stocks of low-passage cells and restarted our cell culture with a fresh vial at least once a month. The antibodies used in this study were USP10 #5553 from Cell Signaling Technology; G3BP2 (86135) and USP10 (109219) from Abcam, Ub (FL76), BAX (N-20), and p53 (Do1) from Santa Cruz Biotechnology; and β-actin from Sigma. Other antibodies and reagents used have been described previously (30, 31). The following reagents were purchased from the indicated companies: MG132 (Abcam), sodium arsenite (Sigma-Aldrich), hydrogen peroxide (Wako), and cycloheximide (Roche). The cells were treated with 1 mmol/L sodium arsenite or 1 mmol/L hydrogen peroxide for the indicated times.

Immunoblot analysis and immunoprecipitation

For the immunoprecipitation assay, we incubated lysates with anti-G3BP2 or normal rabbit IgG overnight at 4°C. The mixture was then incubated with protein G-Sepharose beads (Amersham) and rotated for 2 hours at 4°C. After four washes with NP-40 lysis buffer [(150 mmol/L NaCl, 1% NP40, and 50 mmol/L Tris-HCl (pH 8.0)], beads were mixed in SDS sample buffer [50 mmol/L Tris-HCl (pH 6.8), 1% SDS, 10% glycerol]. We boiled the samples for 5 minutes and separated their proteins by SDS-PAGE. To detect ubiquitin-conjugated proteins, we immunoprecipitated G3BP2 under denatured conditions. Cells were lysed in 100 μL SDS lysis buffer [50 mmol/L Tris-HCl (pH 7.5), 0.5 mmol/L EDTA, 1% SDS, 1 mmol/L DTT, and protease inhibitor cocktail] and boiled the lysates for 10 minutes. The lysates were then diluted in 1 mL 0.5% P40 buffer, and protein complexes were immunoprecipitated with anti-G3BP2 rabbit polyclonal antibody. For the immunoblotting assay, we detected ubiquitylated proteins using anti-ubiquitin mouse mAb and anti-G3BP2 rabbit polyclonal antibody. Immunoblotting was performed as described previously (32).

Cell proliferation assay

Cells were cultured in 24-well plates at 5 × 103 cells per well. Cells were trypsinized and counted using the trypan blue exclusion method. For the MTS assay, cells were cultured in 96-well plates at 3 × 103 cells per well. We used CellTiter 96 Aqueous MTS reagent (Promega) according to the manufacturer's instructions to determine cell viability. The experiments were performed in quintuplicate.

Immunofluorescence

Cells grown on 12-mm circular coverslips (Matsunami) in 24-well plates were fixed with 4% paraformaldehyde in PBS for 10 minutes at room temperature and permeabilized with 0.5% Triton X-100/PBS for 2 minutes. Cells were washed in PBS, blocked with 5% normal goat serum/PBS for 30 minutes, and then incubated with primary antibodies in 5% normal goat serum/PBS overnight at 4°C. Next, cells were washed three times with PBS and incubated with anti-mouse IgG conjugated to Alexa Fluor 546 and anti-rabbit IgG conjugated to Alexa Fluor 488 (Life Technologies) in goat serum/PBS for 1 hour. Nuclei were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI). Cells were washed 3 times with PBS, coverslips were mounted in glycerol, and cells were visualized using an Olympus confocal laser scanning microscope (FV10).

IHC

We obtained 104 prostate cancer samples from surgeries performed at the University of Tokyo Hospital (Tokyo, Japan). Our study (G10044-2) was approved by the University of Tokyo ethics committee and conducted in accordance with Declaration of Helsinki. Informed consent was obtained from each patient before surgery. The ages of the patients ranged from 52 to 78 years (mean, 67 years), and pretreatment serum PSA levels ranged from 1.2 to 136 ng/mL (mean, 16.9 ng/mL). Formalin-fixed tissues were embedded in paraffin and sectioned. A Histofine Kit (Nichirei), based on the streptavidin-biotin amplification method, was used for the IHC analysis of USP10 (Cell Signaling Technology) and G3BP2 (Abcam). The antigen–antibody complex was visualized using a 3,3′-diaminobenzidine solution [1 mmol/L 3,3′-diaminobenzidine, 50 mmol/L Tris-HCl buffer (pH 7.6), and 0.006% H2O2]. To stain p53, the streptavidin–biotin amplification/peroxidase-catalyzed signal amplification (CSA) system (Dako) was used as described in an earlier report (17). In the IHC analysis, the immunoreactivity of more than 1,000 carcinoma cells was evaluated in each case, and the percentage immunoreactivity [labeling index (LI)] was determined by specialized pathologists. Expression of AR was analyzed previously in this cohort (31). Cases with an LI >10% for G3BP2 and USP10 staining were considered “high expression.” This 10% cut-off value is frequently used for reproducible reports of various immunostaining assays (33).

siRNA

Silencer select siRNAs targeting USP10 (s17368, s17365), AR (s1538), and p53 (s607) were purchased from Thermo Fisher Scientific. All siRNA experiments were performed at siRNA concentrations of 10 nmol/L. Cells were transfected with siRNAs using Lipofectamine RNAiMAX (Thermo Fisher Scientific) 48 to 72 hours before each experiment.

qRT-PCR

Total RNA was isolated using ISOGEN reagent (Nippon Gene). First-strand cDNA was generated using a PrimeScript RT Reagent Kit (Takara). The resulting cDNA was then analyzed by qRT-PCR using KAPA SYBR Green Mix (KAPA Biosystems) on a Stepone Real-Time PCR system (Thermo Fisher Scientific). The primer sequences used are shown below or were described previously (30, 31, 34, 35). USP10 forward: 5′-TTTTAAATGCCACCGAACCTATC-3′; reverse: 5′-CCAGCCATTCAGACCGATCT-3′.

Chromatin immunoprecipitation-qPCR

Chromatin immunoprecipitation (ChIP) was performed as described previously (17). In short, cells were crosslinked with 1% formaldehyde. After 10 minutes of incubation, we stopped fixation by adding 0.2 mol/L glycine. Cells were lysed, and the lysates were incubated on ice for 15 minutes. We sonicated the lysates to shear the chromatin DNA. After centrifugation, supernatants were incubated with specific antibodies overnight at 4°C with rotation. Protein G agarose beads were added, and the mixture was incubated for an additional 2 hours. The beads were washed and incubated at 65°C overnight to reverse the crosslinking in ChIP elution buffer. We purified the DNA by ethanol precipitation. Fold enrichment relative to the input was measured by qPCR using an ABI StepOne System and KAPA SYBR Green PCR mix for qPCR. The sequences of primers that targeted the negative control locus (N.C: GAPDH locus) were described previously (17). The sequences of primers targeting USP10 AR-binding site (ARBS) used were as follows: forward: 5′-GCAGGACTCGACAAGTTTGG-3′, reverse: 5′-AACCCCCAAGATCCTTTCTC-3′.

Statistical analysis

We performed all experiments at least twice and confirmed the results were similar. In most cell-based experiments, we used two-sided Student t tests to determine statistical significances and calculate P values of differences between groups. Cancer-specific survival curves were determined using the Kaplan–Meier method. The statistical significances of differences were calculated using the log-rank test. The association between immunoreactivity and clinicopathologic factors was evaluated using Student t test, a cross-table by χ2 testing, or correlation coefficient (r) and regression equation. One-way ANOVA was used to analyze the association between p53 and USP10 expression levels. We used GraphPad Prism 5 software or MS Excel for the statistical analyses.

USP10 is an androgen-regulated gene with an AR-binding site in an intronic region

USP10 has been reported to enhance androgen-dependent epigenetic regulation in the nucleus of AR-positive LNCaP prostate cancer cells (21). In the current study, we examined the effect of androgen on USP10 expression. First, based on the results of our high-throughput analysis of ARBSs determined by ChIP and sequencing (ChIP-seq) in two AR-positive LNCaP and VCaP cell lines (32, 36), we found marked AR recruitment to an intronic region of USP10 (Fig. 1A; Supplementary Fig. S1A). These results were validated by a ChIP-qPCR analysis (Fig. 1B; Supplementary Fig. S1B). Positive regulation of USP10 by androgen was confirmed by a qRT-PCR analysis of LNCaP and VCaP cells (Fig. 1C; Supplementary Fig. S1C). Moreover, this androgen-dependent increase in USP10 was inhibited by treatment with the AR antagonist bicalutamide (Fig. 1D) or AR knockdown using siRNA (Fig. 1E). Androgen-dependent induction of USP10 was also observed at the protein level by Western blot analysis (Fig. 1F; Supplementary Fig. S1D). Thus, our analyses indicate positive regulation of USP10 expression by androgen and AR in prostate cancer cells.

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 = 3). Data, average + 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 = 3) was performed to measure the expression level of USP10. Data, average + SD. D, Androgen-dependent induction of USP10 was inhibited by AR blockade. We added bicalutamide 5 μmol/L or vehicle to LNCaP cells 3 hours before androgen treatment for 24 hours. qRT-PCR analysis (n = 3) was performed to measure the expression level of USP10. Data, average + SD. **, P < 0.01. E, Androgen-dependent induction of USP10 was inhibited by AR knockdown. We treated LNCaP cells with siControl (−) or siAR (+) for 48 hours before androgen treatment for 24 hours. Left, Western blot analysis was performed to detect AR protein. β-Actin is a loading control. Right, qRT-PCR analysis (n = 3) was performed to measure the expression level of USP10. Data, average + 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. β-Actin is a loading control. IB, immunoblot.

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 = 3). Data, average + 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 = 3) was performed to measure the expression level of USP10. Data, average + SD. D, Androgen-dependent induction of USP10 was inhibited by AR blockade. We added bicalutamide 5 μmol/L or vehicle to LNCaP cells 3 hours before androgen treatment for 24 hours. qRT-PCR analysis (n = 3) was performed to measure the expression level of USP10. Data, average + SD. **, P < 0.01. E, Androgen-dependent induction of USP10 was inhibited by AR knockdown. We treated LNCaP cells with siControl (−) or siAR (+) for 48 hours before androgen treatment for 24 hours. Left, Western blot analysis was performed to detect AR protein. β-Actin is a loading control. Right, qRT-PCR analysis (n = 3) was performed to measure the expression level of USP10. Data, average + 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. β-Actin is a loading control. IB, immunoblot.

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Positive feedback loop in AR signaling formed by USP10

Previously, PSA induction by androgen was shown to be regulated by USP10 in LNCaP cells (21). Here, we investigated the effect of USP10 knockdown on other androgen signals in prostate cancer cells by knocking down USP10 in LNCaP and VCaP cells and treating cells with DHT or vehicle. We observed significant repression of androgen-dependent induction of several AR target genes, suggesting a positive role of USP10 in androgen-mediated signaling in these cells (Fig. 2A; Supplementary Fig. S2A). In contrast, overexpression of USP10 enhanced androgen-mediated gene induction (Fig. 2B; Supplementary Fig. S2B). Consistent with these results, androgen-dependent growth of LNCaP and VCaP cells was repressed by USP10 knockdown (Fig. 2C; Supplementary Fig. S2C and S2D). Thus, USP10 appears to form a positive feedback loop in androgen signaling in prostate cancer cells and in androgen-mediated cell growth. However, knockdown of endogenous USP10 had no significant effects on growth of LNCaP and VCaP cells in the absence of androgen (Fig. 2C; Supplementary Fig. S2C) or on that of the 22Rv1 androgen-independent prostate cancer cells (Supplementary Fig. S3A). We analyzed the expression levels of USP10 and its binding partner, G3BP2, by Western blot analysis (Supplementary Fig. S3B). The expression of both USP10 and G3BP2 was enhanced by androgen treatment. Furthermore, we observed increased G3BP2 mRNA levels in the presence of androgen (Supplementary Fig. S3C). However, G3BP2 is expressed at a relatively low level in 22Rv1 cells, suggesting that transcriptional activation of G3BP2 might be important for USP10-mediated growth promotion.

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 = 3) was performed to measure the expression level of AR-regulated genes. Data, average + 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 = 3) was performed to measure the expression level of AR-regulated genes. Data represent the average + 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 = 4). Data, average + SD. **, P < 0.01.

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 = 3) was performed to measure the expression level of AR-regulated genes. Data, average + 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 = 3) was performed to measure the expression level of AR-regulated genes. Data represent the average + 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 = 4). Data, average + SD. **, P < 0.01.

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USP10 colocalizes with G3BP2 in the cytoplasm and SGs in prostate cancer cells

We previously reported negative regulation of the p53 pathway with the export of p53 from the nucleus to the cytoplasm by androgen-regulated G3BP2, based on results of SUMO-mediated modification of p53 (17). In our previous report, overexpression of G3BP2 reduced p53 activity, and knockdown of G3BP2 induced p53-mediated apoptosis of prostate cancer cells. Notably, G3BP2 has been reported to interact with USP10 in the cytoplasm as well as in SGs, which are formed in response to stress-inducing treatments with hypoxia, sodium arsenite, or hydrogen peroxide (H2O2; ref. 19). Therefore, we hypothesized that USP10 modulates G3BP2 expression for SG formation or negatively regulates p53 activity.

To test this hypothesis, we first examined the interaction of G3BP2 with USP10 in prostate cancer cells and in SGs. We immunoprecipitated G3BP2 and performed a Western blot analysis of LNCaP cell lysates (Fig. 3A). We revealed the endogenous interaction of G3BP2 with USP10 in LNCaP cells. Furthermore, we also observed the interaction of G3BP2 with USP10 in cells under stress conditions (sodium arsenite and H2O2 treatment) by immunoprecipitation analysis (Fig. 3B).

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 μm. 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. ΔC:1-326, ΔC2:1-224, ΔN:140-483, ΔN2:252-483 a.a. of G3BP2 protein.

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 μm. 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. ΔC:1-326, ΔC2:1-224, ΔN:140-483, ΔN2:252-483 a.a. of G3BP2 protein.

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We then analyzed the subcellular localization of USP10 in prostate cancer cells using LNCaP cells overexpressing Flag-G3BP2 and both anti-Flag mouse monoclonal and anti-USP10 rabbit polyclonal antibodies (Fig. 3C). Although USP10 has been reported to function as an epigenetic modulator in the nucleus (21), we determined, by immunofluorescence analysis, that USP10 was mainly expressed in the cytoplasm, suggesting that USP10 may play a major role in the cytoplasm. Interestingly, G3BP2 was enriched in SGs in response to stress-inducing treatments, whereas USP10 expression was observed in both SGs and the cytoplasm (Fig. 3C). We also observed the colocalization of USP10 with Flag-G3BP2 in SGs (Fig. 3C). In addition to the interaction of endogenous G3BP2 with USP10, we observed interactions between exogenous Flag-G3BP2 and USP10 transfected into 293T cells (Fig. 3D). The middle region (140–252 aa) of G3BP2 was found to be responsible for its interactions with USP10 in the current analysis (Fig. 3E). Taken together, these observations demonstrate the interaction of USP10 with G3BP2 in both the cytoplasm and SGs of prostate cancer cells.

USP10-mediated regulation of G3BP2 protein levels by reducing polyubiquitylation

Next, we investigated whether USP10 maintains the G3BP2 protein levels. Our Western blot analysis showed USP10 knockdown–mediated repression of G3BP2 protein (Fig. 4A; Supplementary Fig. S2D). We also observed that MG132 treatment reversed siUSP10-mediated inhibition of G3BP2, suggesting the ubiquitin–proteasome pathway is involved in repression of this protein. We then treated LNCaP cells that overexpressed Flag-G3BP2 with cycloheximide to block protein synthesis and analyzed protein stability by Western blot analysis (Fig. 4B). G3BP2 protein levels were severely decreased with USP10 inactivation, indicating that protein stability of G3BP2 is positively regulated by USP10. Because USP10 is known to deubiquitylate its target proteins and enhance protein stability, we investigated whether USP10 regulates polyubiquitylation of G3BP2. As expected, we found that USP10 knockdown enhances the polyubiquitylation of G3BP2 (Fig. 4C). These results suggest that USP10 increases the stability of G3BP2 by inhibiting polyubiquitylation, probably by deubiquitylating G3BP2.

Figure 4.

USP10 positively regulates G3BP2 protein stability by blocking polyubiquitylation. A, USP10 regulates p53 and G3BP2 protein expression by proteasome-dependent pathway. 22Rv1 and LNCaP cells were treated with siControl or siUSP10 #1 and #2 for 72 hours. Cells were treated with MG132 (50 μg/mL) for 5 hours before cell lysis. Western blot analysis was performed to detect indicated proteins. IB, immunoblot. B, G3BP2 protein level decreased by USP10 knockdown. LNCaP cells overexpressing Flag-G3BP2 were treated with 20 μg/mL cycloheximide (CHX). After indicated times, cell lysates were obtained for Western blot analysis. C, USP10 blocks polyubiquitylation of G3BP2 in prostate cancer cells. 22Rv1 cells were treated with siControl or siUSP10 #1 and #2 for 72 hours. Cells were treated with MG132 (50 μg/mL) for 5 hours before cell lysis. Cell lysates were immunoprecipitated under denature condition using anti-G3BP2 antibody. Western blot analysis was performed using anti-ubiquitin antibody.

Figure 4.

USP10 positively regulates G3BP2 protein stability by blocking polyubiquitylation. A, USP10 regulates p53 and G3BP2 protein expression by proteasome-dependent pathway. 22Rv1 and LNCaP cells were treated with siControl or siUSP10 #1 and #2 for 72 hours. Cells were treated with MG132 (50 μg/mL) for 5 hours before cell lysis. Western blot analysis was performed to detect indicated proteins. IB, immunoblot. B, G3BP2 protein level decreased by USP10 knockdown. LNCaP cells overexpressing Flag-G3BP2 were treated with 20 μg/mL cycloheximide (CHX). After indicated times, cell lysates were obtained for Western blot analysis. C, USP10 blocks polyubiquitylation of G3BP2 in prostate cancer cells. 22Rv1 cells were treated with siControl or siUSP10 #1 and #2 for 72 hours. Cells were treated with MG132 (50 μg/mL) for 5 hours before cell lysis. Cell lysates were immunoprecipitated under denature condition using anti-G3BP2 antibody. Western blot analysis was performed using anti-ubiquitin antibody.

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Cell proliferation and p53 nuclear export induced by G3BP2 are inhibited by USP10 knockdown in G3BP2-overexpressing cells

We then investigated the alternative p53 regulatory pathway by modulating G3BP2 and USP10 expression. We previously reported that overexpression of G3BP2 in LNCaP cells induced cytoplasmic export of p53 and cell proliferation (17). By Western blot analysis, we determined that exogenous G3BP2 protein levels were repressed by USP10 inhibition in LNCaP cells overexpressing Flag-G3BP2 (Fig. 5A). In line with G3BP2 inhibition, G3BP2-dependent p53 nuclear export was reversed, and nuclear enrichment of p53 protein was observed in LNCaP cells overexpressing Flag-G3BP2 (Fig. 5B). We next investigated the effect of USP10 knockdown on G3BP2-mediated growth induction. Interestingly, the MTS assay indicated that G3BP2-dependent induction of cell growth was impaired by USP10 knockdown. In contrast, growth inhibition was not observed in vector control cells, suggesting that USP10 plays a positive role in prostate cancer cell proliferation in G3BP2-overexpressing cells (Fig. 5C).

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 = 4). Data, average + 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 = 3). Data, average + 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. β-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 = 4). Data, average + 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.

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 = 4). Data, average + 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 = 3). Data, average + 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. β-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 = 4). Data, average + 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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Next, we analyzed how p53 target genes are affected by USP10 knockdown in G3BP2-overexpressing cells. We demonstrated significant upregulation of NOXA and p21 at the mRNA level and BAX at the protein level in response to USP10 knockdown, suggesting that inhibited p53 activity by G3BP2 was reversed (Fig. 5D). Moreover, we observed that cell growth inhibition in response to USP10 knockdown in G3BP2-overexpressing cells was reversed by p53 knockdown (Fig. 5E and F). These results indicate that USP10 knockdown suppressed the expression of G3BP2 and cell growth and that this suppression was at least partially dependent on p53.

In addition, we overexpressed USP10 in 22Rv1 and LNCaP cells. USP10 overexpression increased both p53 and G3BP2 expression (Supplementary Fig, S4A). We found, by immunofluorescence analysis, that both nuclear and cytoplasmic p53 protein levels were enhanced by USP10 overexpression (Supplementary Fig. S4B) because USP10 deubiquitylates and stabilizes p53. By qRT-PCR analysis, we also observed repressive effects of USP10 overexpression on several p53 target genes (Supplementary Fig. S4C). This result suggests that the USP10-mediated increase in G3BP2 abrogates the increase in p53 activity because of nuclear export. We also observed increased cell growth with USP10 overexpression (Supplementary Fig. S4D), consistent with activated AR (Fig. 2C), increased G3BP2 expression, and repressed p53 activity. Taken together, USP10 appears to have a positive effect on cell proliferation and survival through its interactions with G3BP2 that results in increased protein stability and inhibition of p53 activity (Fig. 5G).

High levels of USP10 expression correlate with high levels of G3BP2 and a poor prognosis for patients with prostate cancer

We previously revealed that G3BP2 was upregulated in a subset of prostate cancer tissues and could be an independent prognostic marker in patients with prostate cancer (17). In the current study, we analyzed the clinical significance of USP10 expression in prostate cancer by IHC (Fig. 6A). We used a cohort of patients with prostate cancer other than that analyzed in the previous study (17) to investigate expression of USP10 and G3BP2. We obtained prostate cancer specimens (n = 104) by performing total prostatectomies in our hospital. IHC results indicated that USP10 expression was upregulated in a subset of prostate cancer tissues compared with that in benign prostate tissues (Fig. 6A, P < 0.0001, McNemar test). USP10 protein was markedly enriched in the cytoplasm of cancer cells. Consistent with results of the previous study, we found that high levels of G3BP2 expression were also associated with poor prognosis in this cohort (Supplementary Fig. S5A and S5B). High levels of USP10 expression were 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 (Supplementary Table S1). These findings are in line with our experimental 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.

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 = 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 = 39) is significantly associated with high expression of G3BP2 (n = 12). C, High expression of USP10 is a prognostic factor for prostate cancer patients (P = 0.0083). Kaplan–Meier analysis using the log-rank test was performed. D, Plots of TCGA RNA expression profiles in prostate adenocarcinoma (n = 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.

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 = 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 = 39) is significantly associated with high expression of G3BP2 (n = 12). C, High expression of USP10 is a prognostic factor for prostate cancer patients (P = 0.0083). Kaplan–Meier analysis using the log-rank test was performed. D, Plots of TCGA RNA expression profiles in prostate adenocarcinoma (n = 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.

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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 + SD.

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 + SD.

Close modal

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 prostate cancer. We revealed that G3BP2-dependent growth induction was impaired by USP10 knockdown through downregulation of G3BP2 at the protein level. This effect was cancelled by p53 knockdown, suggesting that p53 signaling is important in this growth inhibition. More importantly, we found that cytoplasmic expression of p53 is significantly associated with high levels of USP10 in prostate cancer tissues. These observations indicate that USP10 enhances G3BP2 expression to promote the translocation of stabilized p53 protein to the cytoplasm to reduce p53 signaling. Meanwhile, it is also possible that USP10 deubiquitylates other unknown oncogenic proteins in the cytoplasm. Screening to identify USP10-binding proteins would be helpful in clarifying substrates of this enzyme.

Other reports have presented a different mechanism of action by which G3BP1, another member of the G3BP family (40), inhibits the p53 pathway (41). G3BP1 inhibits the deubiquitylation of p53 by interacting with USP10, which negatively regulates p53 protein expression. It is tempting to speculate that upregulation of G3BP2 by USP10 also represses activation of the p53 pathway by blocking USP10-mediated upregulation of p53 at the protein level and that two androgen-induced proteins, USP10 and G3BP2, cooperate to inhibit p53 activity through both nuclear export and protein degradation.

Other important features of USP10 are its regulation by androgen in prostate cancer cells and AR binding to intronic regions. We also observed the upregulation of USP10 by androgen in LNCaP and VCaP cells. A past analysis revealed that USP10 regulates histone modification by AR in AR-binding sites to induce PSA (21). We found that USP10 knockdown reduced the expression of other AR target genes in prostate cancer cells. In contrast, overexpression of USP10 enhanced AR-mediated transcription. Thus, USP10 plays a role in a positive feedback loop to enhance the activity of AR. Activation of this pathway could be another indication of USP10 as an oncogene in prostate cancer, because AR signals are central to prostate cancer progression. We also showed that androgen-dependent cell proliferation was repressed in LNCaP and VCaP cells. These results may indicate the importance of USP10 in AR signaling and androgen-dependent cell growth in prostate cancer.

Overall, USP10 may modulate apoptosis and cell growth in several ways: (i) p53 protein stabilization by deubiquitylation; (ii) G3BP2 induction that represses p53 activity by nuclear export; (iii) SG formation through the maintenance of G3BP2 protein levels. Several interesting reports regarding USP10 have described the localization of USP10 in SGs interacting with G3BPs (23–25), although the biological significance of USP10 localization in SGs has been largely unknown. We observed USP10 colocalization with G3BP2 in prostate cancer cells; however, the cytoplasmic distribution of USP10 was not fundamentally different than that of G3BP2 following treatment with stress-inducing reagents. Previous reports have noted the antiapoptotic effects of SGs in cancer cells (29). These multiple functions of USP10 in apoptosis might be dependent on the condition of the tumor cells. For example, in other tissues such as renal or gastric cancer tissues, USP10 has been reported to be a tumor suppressor gene (20, 42, 43).

In summary, our findings suggest that USP10 is a potent regulator of G3BP2 protein expression in prostate cancer cells that represses p53 signaling. USP10 can promote cell survival and growth, dependent on G3BP2-mediated signaling. In addition, USP10 forms a positive feedback loop in AR signaling. Although the tumor-suppressive function of USP10 through the deubiquitylation of p53 protein has been reported as important in several tissues, this alternative mechanism of p53 regulation by USP10 confers oncogenic characteristics in some phases of cancer progression, such as CRPC development.

No potential conflicts of interest were disclosed.

Conception and design: K. Takayama, S. Inoue

Development of methodology: K. Takayama

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): K. Takayama, T. Suzuki

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): K. Takayama, T. Suzuki

Writing, review, and/or revision of the manuscript: K. Takayama, T. Fujimura, S. Takahashi, S. Inoue

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): K. Takayama

Study supervision: S. Inoue

This work was supported by grants of the Cell innovation Program, the P-DIRECT and the P-CREATE from Ministry of Education, Culture, Sports, Science and Technology, Japan (S. Inoue), by the MEXT, Japan, by grants (K. Takayama and S. Inoue) from the JSPS (number 15K15581, 15K15353, 17H04334), Japan, by the Program for Promotion of Fundamental Studies in Health Sciences (S. Inoue), NIBIO, Japan, grants from Takeda Science Foundation, Japan (K. Takayama and S. Inoue), grants from the Terumo foundation for life sciences and arts, Japan (K. Takayama), and grants from the NOVARTIS Foundation for the Promotion of Science, Japan (K. Takayama).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Meek
DW
. 
Tumour suppression by p53: a role for the DNA damage response?
Nat Rev Cancer
2009
;
9
:
714
23
.
2.
Riley
T
,
Sontag
E
,
Chen
P
,
Levine
A
. 
Transcriptional control of human p53-regulated genes
.
Nat Rev Mol Cell Biol
2008
;
9
:
402
12
.
3.
Vogelstein
B
,
Lane
D
,
Levine
AJ
. 
Surfing the p53 network
.
Nature
2000
;
408
:
307
10
.
4.
Chen
Z
,
Trotman
LC
,
Shaffer
D
,
Lin
HK
,
Dotan
ZA
,
Niki
M
, et al
Cruicial role of p53-dependent cellular senescence in suppression of Pten-deficient tumorigenesis
.
Nature
2005
;
436
:
725
30
.
5.
Kruse
JP
,
Gu
W
. 
Modes of p53 regulation
.
Cell
2009
;
137
:
609
22
.
6.
Jenkins
LM
,
Durell
SR
,
Mazur
SJ
,
Appella
E
. 
p53 N-terminal phosphorylation: a defining layer of complex regulation
.
Carcinogenesis
2012
;
33
:
1441
9
.
7.
Brooks
CL
,
Gu
W
. 
The impact of acetylation and deacetylation on the p53 pathway
.
Protein Cell
2011
;
2
:
456
62
.
8.
Li
M
,
Brooks
CL
,
Wu-Baer
F
,
Chen
D
,
Baer
R
,
Gu
W
. 
Mono-versus polyubiquitination: differential control of p53 fate by Mdm2
.
Science
2003
;
302
:
1972
5
.
9.
Cai
C
,
Yuan
X
,
Balk
SP
. 
Androgen receptor epigenetics
.
Transl Androl Urol
2013
;
2
:
148
57
.
10.
Dehm
SM
,
Tindall
DJ
. 
Molecular regulation of androgen action in prostate cancer
.
J Cell Biochem
2006
;
99
:
333
44
.
11.
Debes
JD
,
Tindall
DJ
. 
The role of androgens and the androgen receptor in prostate cancer
.
Cancer Lett
2002
;
187
:
1
7
.
12.
Wang
Q
,
Li
W
,
Liu
XS
,
Carroll
JS
,
Jänne
OA
,
Keeton
EK
, et al
A hierarchical network of transcription factors governs androgen receptor-dependent prostate cancer growth
.
Mol Cell
2007
;
27
:
380
92
.
13.
Chen
CD
,
Welsbie
DS
,
Tran
C
,
Baek
SH
,
Chen
R
,
Vessella
R
, et al
Molecular determinants of resistance to antiandrogen therapy
.
Nat Med
2004
;
10
:
33
9
.
14.
Feldman
BJ
,
Feldman
D
. 
The development of androgen-independent prostate cancer
.
Nat Rev Cancer
2001
;
1
:
34
45
.
15.
Wang
Q
,
Li
W
,
Zhang
Y
,
Yuan
X
,
Xu
K
,
Yu
J
, et al
Androgen receptor regulates a distinct transcription program in androgen-independent prostate cancer
.
Cell
2009
;
138
:
245
56
.
16.
Takayama
K
,
Inoue
S
. 
Transcriptional network of androgen receptor in prostate cancer progression
.
Int J Urol
2013
;
20
:
756
68
.
17.
Ashikari
D
,
Takayama
K
,
Tanaka
T
,
Suzuki
Y
,
Obinata
D
,
Fujimura
T
, et al
Androgen induces G3BP2 and SUMO-mediated p53 nuclear export in prostate cancer
.
Oncogene
2017
;
36
:
6272
81
.
18.
Liu
J
,
Xia
H
,
Kim
M
,
Xu
L
,
Li
Y
,
Zhang
L
, et al
Beclin1 controls the levels of p53 by regulating the deubiquitination activity of USP10 and USP13
.
Cell
2011
;
147
:
223
34
.
19.
Reece
KM
,
Figg
WD
. 
A novel regulator (USP10) of p53: implications for tumor suppression and therapeutic targeting
.
Cancer Biol Ther
2010
;
9
:
583
4
.
20.
Yuan
J
,
Luo
K
,
Zhang
L
,
Cheville
JC
,
Lou
Z
. 
USP10 regulates p53 localization and stability by deubiquitinating p53
.
Cell
2010
;
140
:
384
96
.
21.
Draker
R
,
Sarcinella
E
,
Cheung
P
. 
USP10 deubiquitylates the histone variant H2A.Z and both are required for androgen receptor-mediated gene activation
.
Nucleic Acids Res
2011
;
39
:
3529
42
.
22.
Faus
H
,
Meyer
HA
,
Huber
M
,
Bahr
I
,
Haendler
B
. 
The ubiquitin-specific protease USP10 modulates androgen receptor function
.
Mol Cell Endocrinol
2005
;
245
:
138
46
.
23.
Matsuki
H
,
Takahashi
M
,
Higuchi
M
,
Makokha
GN
,
Oie
M
,
Fujii
M
. 
Both G3BP1 and G3BP2 contribute to stress granule formation
.
Genes Cells
2013
;
18
:
135
46
.
24.
Takahashi
M
,
Higuchi
M
,
Matsuki
H
,
Yoshita
M
,
Ohsawa
T
,
Oie
M
, et al
Stress granules inhibit apoptosis by reducing reactive oxygen species production
.
Mol Cell Biol
2013
;
33
:
815
29
.
25.
Jain
S
,
Wheeler
JR
,
Walters
RW
,
Agrawal
A
,
Barsic
A
,
Parker
R
. 
ATPase-modulated stress granules contain a diverse proteome and substructure
.
Cell
2016
;
164
:
487
98
.
26.
Tourrière
H
,
Chebli
K
,
Zekri
L
,
Courselaud
B
,
Blanchard
JM
,
Bertrand
E
, et al
The RasGAP-associated endoribonuclease G3BP assembles stress granules
.
J Cell Biol
2003
;
160
:
823
31
.
27.
Wallace
EW
,
Kear-Scott
JL
,
Pilipenko
EV
,
Schwartz
MH
,
Laskowski
PR
,
Rojek
AE
, et al
Reversible, specific, active aggregates of endogenous proteins assemble upon heat stress
.
Cell
2015
;
162
:
1286
98
.
28.
Bley
N
,
Lederer
M
,
Pfalz
B
,
Reinke
C
,
Fuchs
T
,
Glaß
M
, et al
Stress granules are dispensable for mRNA stabilization during cellular stress
.
Nucleic Acids Res
2015
;
43
:
e26
.
29.
Arimoto
K
,
Fukuda
H
,
Imajoh-Ohmi
S
,
Saito
H
,
Takekawa
M
. 
Formation of stress granules inhibits apoptosis by suppressing stress-responsive MAPK pathways
.
Nat Cell Biol
2008
;
10
:
1324
32
.
30.
Takayama
K
,
Misawa
A
,
Suzuki
T
,
Takagi
K
,
Hayashizaki
Y
,
Fujimura
T
, et al
TET2 repression by androgen hormone regulates global hydroxymethylation status and prostate cancer progression
.
Nat Commun
2015
;
6
:
8219
.
31.
Takayama
K
,
Horie-Inoue
K
,
Katayama
S
,
Suzuki
T
,
Tsutsumi
S
,
Ikeda
K
, et al
Androgen-responsive long noncoding RNA CTBP1-AS promotes prostate cancer
.
EMBO J
2013
;
32
:
1665
80
.
32.
Takayama
K
,
Suzuki
T
,
Fujimura
T
,
Urano
T
,
Takahashi
S
,
Homma
Y
, et al
CtBP2 modulates the androgen receptor to promote prostate cancer progression
.
Cancer Res
2014
;
74
:
6542
53
.
33.
Chishiki
M
,
Takagi
K
,
Sato
A
,
Miki
Y
,
Yamamoto
Y
,
Ebata
A
, et al
Cytochrome c1 in ductal carcinoma in situ of breast associated with proliferation and comedo necrosis
.
Cancer Sci
2017
;
108
:
1510
9
.
34.
Misawa
A
,
Takayama
K
,
Fujimura
T
,
Homma
Y
,
Suzuki
Y
,
Inoue
S
. 
Androgen-induced lncRNA POTEF-AS1 regulates apoptosis-related pathway to facilitate cell survival in prostate cancer cells
.
Cancer Sci
2017
;
108
:
373
9
.
35.
Takayama
K
,
Horie-Inoue
K
,
Suzuki
T
,
Urano
T
,
Ikeda
K
,
Fujimura
T
, et al
TACC2 is an androgen-responsive cell cycle regulator promoting androgen-mediated and castration-resistant growth of prostate cancer
.
Mol Endocrinol
2012
;
26
:
748
61
.
36.
Misawa
A
,
Takayama
K
,
Urano
T
,
Inoue
S
. 
Androgen-induced long noncoding RNA (lncRNA) SOCS2-AS1 promotes cell growth and inhibits apoptosis in prostate cancer cells
.
J Biol Chem
2016
;
291
:
17861
80
.
37.
Kim
MM
,
Wiederschain
D
,
Kennedy
D
,
Hansen
E
,
Yuan
ZM
. 
Modulation of p53 and MDM2 activity by novel interaction with Ras-GAP binding proteins (G3BP)
.
Oncogene
2007
;
26
:
4209
15
.
38.
Gupta
N
,
Badeaux
M
,
Liu
Y
,
Naxerova
K
,
Sgroi
D
,
Munn
LL
, et al
Stress granule-associated protein G3BP2 regulates breast tumor initiation
.
Proc Natl Acad Sci U S A
2017
;
114
:
1033
8
.
39.
French
J
,
Stirling
R
,
Walsh
M
,
Kennedy
HD
. 
The expression of Ras-GTPase activating protein SH3 domain-binding proteins, G3BPs, in human breast cancers
.
Histochem J
2002
;
34
:
223
31
.
40.
Soncini
C
,
Berdo
I
,
Draetta
G
. 
Ras-GAP SH3 domain binding protein (G3BP) is a modulator of USP10, a novel human ubiquitin specific protease
.
Oncogene
2001
;
20
:
3869
79
.
41.
Oi
N
,
Yuan
J
,
Malakhova
M
,
Luo
K
,
Li
Y
,
Ryu
J
, et al
Resveratrol induces apoptosis by directly targeting Ras-GTPase-activating protein SH3 domain-binding protein 1
.
Oncogene
2015
;
34
:
2660
71
.
42.
Zeng
Z
,
Wu
HX
,
Zhan
N
,
Huang
YB
,
Wang
ZS
,
Yang
GF
, et al
Prognostic significance of USP10 as a tumor-associated marker in gastric carcinoma
.
Tumour Biol
2014
;
35
:
3845
53
.
43.
Lin
Z
,
Yang
H
,
Tan
C
,
Li
J
,
Liu
Z
,
Quan
Q
, et al
USP10 antagonizes c-Myc transcriptional activation through SIRT6 stabilization to suppress tumor formation
.
Cell Rep
2013
;
5
:
1639
49
.