ZBTB7A Mediates the Transcriptional Repression Activity of the Androgen Receptor in Prostate Cancer Dong Han1, Sujun Chen2,3, Wanting Han1, Shuai Gao1, Jude N

Published OnlineFirst August 23, 2019; DOI: 10.1158/0008-5472.CAN-19-0815

Cancer Molecular Cell Biology Research

ZBTB7A Mediates the Transcriptional Repression Activity of the Androgen Receptor in Prostate Cancer Dong Han1, Sujun Chen2,3, Wanting Han1, Shuai Gao1, Jude N. Owiredu1, Muqing Li1, Steven P. Balk4, Housheng Hansen He2,3, and Changmeng Cai1

Abstract

Loss of expression of context-specific tumor suppressors is a binding sites by AR and negatively regulated the transcrip- critical event that facilitates the development of prostate tional activity of E2F1 on DNA replication genes. Finally, cancer. Zinc finger and BTB domain containing transcriptional ZBTB7A suppressed the growth of castration-resistant repressors, such as ZBTB7A and ZBTB16, have been recently prostate cancer (CRPC) in vitro and in vivo, and overexpres- identified as tumor suppressors that play important roles in sion of ZBTB7A acted in synergy with high-dose testosterone preventing prostate cancer progression. In this study, we used treatment to effectively prevent the recurrence of CRPC. combined ChIP-seq and RNA-seq analyses of prostate cancer Overall, this study provides novel molecular insights of the cells to identify direct ZBTB7A-repressed genes, which are role of ZBTB7A in CRPC cells and demonstrates globally its enriched for transcriptional targets of E2F, and identified critical role in mediating the transcriptional repression that the androgen receptor (AR) played a critical role in the activity of AR. transcriptional suppression of these E2F targets. AR recruitment of the retinoblastoma protein (Rb) was required to Significance: ZBTB7A is recruited to the E2F–Rb binding strengthen the E2F–Rb transcriptional repression complex. sites by AR and negatively regulates the transcriptional activ- In addition, ZBTB7A was rapidly recruited to the E2F–Rb ity of E2F1 on DNA replication genes.

Introduction portion of these relapsed tumors still express AR and AR-regulated genes. Although AR is known for its transcriptional activator Prostate cancer is one of the most common cancers in men. The function, it can also act as a transcriptional repressor to suppress development of primary prostate cancer depends on the activity of the expression of a subset of genes, including AR itself, androgen androgen receptor (AR), a ligand-dependent nuclear receptor synthetic genes, and genes mediating DNA replication and transcription factor. The standard treatment of prostate cancer is repair (4, 5). Mechanistically, we have shown that AR globally surgical or medical castration (known as androgen deprivation recruits hypophosphorylated retinoblastoma protein (Rb) to the therapy, ADT) to reduce circulating androgens. However, patients promoters/enhancers of DNA replication gene loci and strength- invariably relapse into more aggressive castration-resistant pros- ens the activity of E2F–Rb suppressor complex (6). Therefore, loss tate cancer (CRPC) with increased expression and restored activity of this tumor suppressor activity of AR after ADT is likely to be one of AR (1). Although CRPC can be further treated with more mechanism contributing to the progression to CRPC (1). This aggressive ADT using agents such as abiraterone and enzaluta- transcriptional repressor activity of AR also provides one of the mide (2, 3), the tumors generally relapse within 1 year and a large mechanisms for the high-dose testosterone therapy in patients with CRPC (7–10). In addition to AR, the recently reported zinc finger and BTB 1Center for Personalized Cancer Therapy, University of Massachusetts Boston, domain containing transcription repressors, such as ZBTB7A and Boston, Massachusetts. 2Princess Margaret Cancer Center/University Health ZBTB16 3 , can also function as tumor suppressors in preventing the Network, Toronto, Ontario, Canada. Department of Medical Biophysics, Uni- – ZBTB7A versity of Toronto, Toronto, Ontario, Canada. 4Beth Israel Deaconess Medical progression of prostate cancer (11 14). , also known as LRF POKEMON – Center, Boston, Massachusetts. / , consists of a protein interacting BTB domain at the N-terminus and DNA binding zinc fingers at the C- Note: Supplementary data for this article are available at Cancer Research ZBTB7A fi Online (http://cancerres.aacrjournals.org/). terminus (13). Even though has been identi ed as a proto-oncogene in other cancer types, a recent study using a D. Han, S. Chen, and W. Han contributed equally to this article. transgenic mouse model indicates that it functions as a tumor Corresponding Authors: Changmeng Cai, University of Massachusetts Boston, suppressor in prostate cancer and that loss of its expression can 100 Morrissey Blvd, ISC/4/4720, Boston, MA 02125; Phone: 617-287-3537; drive the development of aggressive invasive tumor in Pten-null Fax: 617-287-3537; E-mail: [email protected], or Housheng Hansen He, prostate epithelial cells by bypassing the Pten-loss induced cel- Princess Margaret Cancer Center/University Health Network, Toronto, Ont. M5G1L7, Canada; E-mail: [email protected] lular senescence (15). Mechanistically, ZBTB7A was shown to repress the activity of SOX9, a proto-oncogene in prostate can- Cancer Res 2019;79:5260–71 cer (16), and to impair the SOX9 regulation of an RB-targeting doi: 10.1158/0008-5472.CAN-19-0815 miRNA, thus allowing cells to bypass the Pten-loss induced 2019 American Association for Cancer Research. senescence (15). Although its tumor suppressor activity has been

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demonstrated in mouse prostate cancer cells, the activities of the chromatin into 500 to 800 bp fragments, followed by immu- ZBTB7A at the chromatin level in human prostate cancer cells noprecipitation. The qPCR analysis was carried out using the remain to be characterized. Using a combined analysis of the SYBR Green method on the QuantStudio 3 real-time PCR system ZBTB7A cistrome and transcriptome, we have mapped the bind- (Thermo Fisher Scientific). The primers for MCM7-pro, BLM-pro, ing sites of ZBTB7A and identified direct ZBTB7A-regulated genes. FANCI-pro, and TK1-pro were previously listed (6). Significantly, the direct ZBTB7A-repressed genes were enriched for the activation function of E2Fs, suggesting that ZBTB7A may RT-PCR and immunoblotting function to repress their oncogenic activities in prostate cancer The expression of genes was measured using real-time RT-PCR cells. analyses with Taqman one-step RT-PCR reagents (Thermo Fisher Because our previous studies indicated AR can function as a Scientific) and results were normalized to coamplified GAPDH. transcriptional repressor to suppress DNA replication genes The primer and probe sets for the following genes: MCM2, MCM7, through enhancing the chromatin binding of Rb that reinforces FANCI, BLM, TK1, PCAT1, and GAPDH were purchased as inven- the suppressor activity of E2F–Rb complex (6), we next deter- toried mix from Applied Biosystems at Thermo Fisher Scientific. mined how ZBTB7A chromatin binding in prostate cancer cells For immunoblotting, cells were lysed with RIPA buffer with globally impacts the transcriptional activity of AR. By co- protease inhibitors (Thermo Fisher Scientific) and anti-ZBTB7A analyzing the previous reported AR cistrome database in prostate (Bethyl), anti-AR PG21 (Millipore), anti-Rb, anti-E2F1 (Cell cancer cells (4, 16), we show that a significant portion of ZBTB7A Signaling), anti-V5, anti-HA (Sigma), anti-HDAC1, anti-GAPDH, binding sites overlap with AR binding sites and that these ZBTB7A or anti-b-actin (Abcam) antibodies were used. Immunoblotting and AR overlapping sites are significantly associated with the results shown are representative of at least 3 independent repression activity of AR on gene transcription. More importantly, experiments. we also show that ZBTB7A binding at those AR repression sites is rapidly increased upon androgen stimulation and the increased RNAi and transfection binding is highly associated with E2F–Rb binding, indicating that siRNAs against ZBTB7A and nontarget control (NTC) were AR-recruited ZBTB7A may cooperate with Rb in regulating the purchased from Dharmacon and transfected into cells using transcriptional activity of E2Fs. By co-immunoprecipitation lipofectamine 2000 (Thermo Fisher Scientific). C4-2-shZBTB7A assays, we demonstrated that ZBTB7A can physically interact with cells were generated using lentiviral shRNA against ZBTB7A or AR, Rb, and E2F1, further indicating that ZBTB7A may be an NTC (Dharmacon). VCaP-tet-shZBTB7A cells were generated additional component of the AR–Rb repressor complex. Further- using tetracycline-inducible lentiviral shRNA against ZBTB7A or more, we carried out in vitro and in vivo studies to examine the NTC (Dharmacon). C4-2-tet-ZBTB7A cells were generated by effects of overexpression of ZBTB7A on CRPC tumor growth and stable infection with lenti-virus containing tetracycline- results show that overexpression of ZBTB7A in CRPC cells sig- inducible V5-tagged ZBTB7A cDNA in C4-2 cells. COS-7 cells nificantly reduced the cancer development, and that overexpres- cultured in DMEM with 10% FBS were transiently transfected with sion of ZBTB7A can synergize with high-dose testosterone therapy plasmids expressing HA-tagged AR 1-539aa, AR 1-628aa, AR 538- in treating CRPC. Overall, this study has provided novel insights 919aa, or AR 662-919aa for overnight, followed by immunopre- into the tumor suppressor activity of ZBTB7A in prostate cancer cipitation assay. cells and identified ZBTB7A as a critical mediator required for AR- dependent transcriptional repression activity. ChIP-seq and RNA-seq For ChIP-seq, VCaP cells were fixed and then lysed, and the chromatin was sheared to 300 to 500 bp in size using the Materials and Methods Bioruptor sonicator (Diagenode). The chromatin was incubat- Cell lines and cell culture ed with ZBTB7A antibody bound to protein G beads (Thermo The VCaP and C4-2 cell lines were purchased from ATCC. All Fisher Scientific) for overnight. After immunoprecipitation, the cell lines were recently authenticated using short tandem samples were reversely crosslinked in 65Cwaterbath, repeat (STR) profiling by DDC Medical and tested for Mycoplasma and DNA was extracted with QIAquick PCR Purification Kit contamination (negative result) by using MycoAlert Mycoplasma (Qiagen). The ChIP-seq libraries were prepared using ThruPlex Detection Kit (Lonza). VCaP cells were cultured in DMEM medi- DNA-seq 48D Kit (Rubicon Genomics) and then sequenced um with 10% FBS (Gibco). VCaP-tet-shZBTB7A (tetracycline- using Illumina HiSeq 2500. For RNA-seq, VCaP cells trans- inducible ZBTB7A silencing) cells were maintained in DMEM fected with siZBTB7A or siNTC were harvested for RNA medium with 10% tetracycline-free FBS. C4-2 and C4-2- extraction using RNeasy mini kits (Qiagen), followed by shZBTB7A (stable ZBTB7A silencing) cells were cultured in RNA-SeqlibrarypreparationwithTruSeqStrandedRNALTKit RPMI1640 medium supplemented with 2% FBS plus 8% CSS (Illumina). (charcoal-dextran stripped FBS; Gibco). C4-2-tet-ZBTB7A (tetra- For data analysis, ChIP-seq raw reads were aligned to hg19 cycline-inducible ZBTB7A overexpressing) cells were maintained using bwa (version 0.7.2-r351). The resulted sam files are con- in RPMI1640 medium with 2% tetracycline-free FBS plus 8% CSS. verted to bam with samtools [version 0.1.18 (r982:295)]. MACS2 For androgen stimulation assays, cells were generally grown to (version 2.0.10.20131216) was used to call peak on the bam files. 50% to 60% confluence in medium containing 5% CSS for 2 to bedGraph files containing signal per million reads produced from 3 days and then treated with DHT or inhibitors for indicated time. MACS2 was converted to bigwig files with ucsctool kit (315). The R package ChIPpeakAnno (version 3.10.1) was used for analyzing Chromatin immunoprecipitation peak intervals. deepTools (version 2.4.1) was used to extract and For preparation of chromatin immunoprecipitation (ChIP), visualize signal from bigwig files. The RNA-seq differential gene dispensed cells were formalin fixed, lysed, and sonicated to break expression analysis was performed using TopHat pipeline on

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Galaxy. The GEO accession for ChIP-seq and RNA-seq is ZBTB16 (5%; Fig. 1A), indicating the critical role of this gene GSE123091. family in prostate cancer development. We then compared the expression levels of ZBTB7A and ZBTB16 in clinical cohorts of Cell proliferation assay primary prostate cancer versus metastatic CRPC. As shown C4-2-shNTC and C4-2-shZBTB7A cells were maintained in in Fig. 1B, expression levels of ZBTB7A and ZBTB16 were higher RPMI1640 supplemented with 2% FBS plus 8% CSS. After DHT in the primary prostate cancer cohort (TCGA) than in the CRPC treatments, cells were stained with Muse Count & Viability Assay cohort (SU2C; ref. 18), suggesting that their expression is Kit for 5 minutes and then counted by Muse Cell Analyzer (EMD decreased during CRPC development. Although the transcrip- Millipore). tional repression activity of ZBTB16 in prostate cancer cells has been described (14), the activity of ZBTB7A on chromatin has not Luciferase reporter assay yet been determined. Therefore, we next examined its chromatin HEK293 cells were transfected with a Firefly luciferase reporter binding in prostate cancer cells. ChIP-seq analysis of ZBTB7A in construct containing 800bp promoter of MCM7 gene together VCaP cells (an AR-amplified CRPC cell line) cultured in full serum with a Renilla luciferase reporter construct for 24 hours prior to the condition identified 17,691 high confidence peaks as potential treatments. The activities of Firefly luciferase and Renilla luciferase ZBTB7A binding sites. Interestingly, these binding sites were were measured using the dual-luciferase reporter assay (Promega) significantly enriched for promoter region (13.8% vs. 1.1% back- and the results were normalized for Renilla activities. 0 ground) and 5 UTR region (8.9% vs. 0.4% background; Fig. 1C), suggesting that ZBTB7A may preferentially bind to the upstream Mouse xenografts or downstream sites near transcription start sites (TSS). Using a C4-2-tet-ZBTB7A xenografts were established in the flanks of motif enrichment analysis, we found that the previously known castrated male SCID mice by injecting 2 million cells mixed with ZBTB7A binding motif was significantly associated with ZBTB7A 50% Matrigel. Doxycycline-supplemented food was introduced at binding sites (Fig. 1D). We then clustered and ranked the tran- 6 weeks postinjection and tumor volume was measured by scription factors that may potentially co-occupy the ZBTB7A sites manual caliper using the formula V ¼ (W2 L)/2. Frozen sections based on their motif enrichment scores. As shown in Fig. 1E, the were examined to confirm that the samples used for RNA and top-ranked clusters were identified, and the possible factors protein extraction contain predominantly nonnecrotic tumor. All include POLR3A (a subunit of RNA polymerase III), TRIM28 animal experiments were approved by the UMass Boston Insti- (a Tripartite motif containing transcription cofactor), PDX1 (a tutional Animal Care and Use Committee and were performed in homeodomain transcription factor), NFKB1 (the DNA binding accordance with institutional and national guidelines. subunit of NF-kB), ING4 (a PHD-finger containing chromatin BETA analysis remodeling protein), and E2F2 (an E2F family transcription As previously described (6), binding and expression target factor). The POLR3A cluster (cluster 1) also includes a number analysis (BETA) was performed to assess the association of AR of nuclear hormone receptors. and ZBTB7A binding sites with the expression of AR-activated To study the effect of ZBTB7A binding on gene transcription in versus AR-repressed genes. BETA software package was used (with prostate cancer cells, we performed an RNA-seq analysis in VCaP default parameters) to integrate ChIP-seq of ZBTB7A and AR with cells (cultured in full serum condition) transfected with siRNA androgen-regulated gene expression profiling. The red and the targeting ZBTB7A or nontarget control (NTC) and then carried out purple lines represent the AR-activated and AR-repressed genes, BETA (15) to identify potential ZBTB7A directly regulated genes. respectively. The black dashed line indicates the nondifferentially As shown in Fig. 1F, chromatin binding of ZBTB7A was associated P ¼ 37 expressed genes as background. Genes are cumulated by the rank with both ZBTB7A-activated ( 10 ) and ZBTB7A-repressed P ¼ 21 on the basis of the regulatory potential score from high to low. genes ( 10 ), suggesting that ZBTB7A may have both tran- P-value represent the significance of difference in the AR-activated scriptional activator and repressor functions in prostate cancer or AR-repressed group compared with the nondifferentially cells. As the BETA analysis also ranked the potential for genes that expressed group by the Kolmogorov–Smirnov test. may be directly regulated by ZBTB7A (Supplementary Table S1), we next performed Kyoto Encyclopedia of Genes and Genomes Statistical analysis (KEGG) pathway analysis (using DAVID 6.8) on these potential Data in bar graphs represent mean SD of at least 3 biological direct targets of ZBTB7A. Interestingly, the activator activity of fi repeats. Statistical analysis was performed using Student t test by ZBTB7A was associated with speci c cancer types, including – comparing treatment versus vehicle control or otherwise as indi- prostate and non small cell lung cancers (Fig. 1G), suggesting cated. P value <0.05 () was considered to be statistically signif- that ZBTB7A may retain some tumor promoting activities in icant. For animal studies, 1-way ANOVA was performed for the prostate cancer cells through gene activation. The repressor func- tumor volume data measured at the final day of the treatments. tion of ZBTB7A was enriched for transcription and translation regulation, chemokine signaling pathway, and DNA replication, supporting its tumor suppressor role in prostate cancer cells. Results Interestingly, the suppression of chemokine signaling was also Characterization of ZBTB7A transcriptional program in supported by a recent study showing that ZBTB7A regulates the prostate cancer cells infiltration and composition of immune cells within prostate The zinc finger and BTB domain containing transcription cancer tumor through repressing the expression of a chemokine, repressors, including ZBTB7A and ZBTB16, were recently identi- CXCL5 (19). To further determine which signaling pathways are fied as critical tumor suppressors in prostate cancer cells. Using most impacted by ZBTB7A, we performed a gene set enrichment TCGA prostate cancer dataset (17), we found that over 6% of analysis (GSEA) on ZBTB7A-regulated genes. As seen in Fig. 1H, prostate cancer samples have deep deletion of ZBTB7A (1.2%) or although ZBTB7A-activated genes were not significantly

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Figure 1. Functional characterization of ZBTB7A transcriptional activity in prostate cancer cells. A, Genetic alterations of ZBTB7A and ZBTB16 in TCGA prostate cancer cohort (retrieved in cBioPortal). B, The expression levels of ZBTB7A and ZBTB16 (normalized to GAPDH) in SU2C mCRPC cohort versus TCGA primary prostate cancer cohort. C, ZBTB7A ChIP-seq was done in VCaP cells cultured in full FBS and the genomic distribution of ZBTB7A binding is shown. D, A Bayesian DNA motif comparison method, BLiC (33), was applied on ZBTB7A ChIP-seq peaks (cutoff: P ¼ 1015). The ZBTB7A binding motif was found with z-score ¼50.87. E, Potential ZBTB7A co-occupied factors were identified in 6 clusters with high confidence z-score (< 40). Other identified factors with high similarity-score (>2.85) in each cluster are also listed. F, BETA was performed to assess the direct regulation of ZBTB7A target genes using the ChIP-seq analysis of ZBTB7A and the differential gene expression analysis (from RNA-seq) in VCaP cells transfected with siZBTB7A and nontarget control (confirmed by immunoblotting). G, The direct ZBTB7A-regulated genes identified from BETA analysis were subjected to KEGG pathway analysis by DAVID 6.8 to identify pathways associated with ZBTB7A activation or repression function. H, GSEA was carried out on ZBTB7A-regulated genes. associated with any known pathways, the expression of ZBTB7A- role in activating DNA replication and cell-cycle progression, repressed genes were highly enriched for the activation function of indicating ZBTB7A may function to suppress the activity of E2Fs. E2Fs, an oncogenic transcription factor family that plays critical Collectively, these global cistrome and transcriptome studies of

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ZBTB7A revealed its transcription factor activities in prostate impact of ZBTB7A on AR transcriptional activity. Because the cancer cells and linked its tumor suppressor function to the above global studies were done in VCaP cells under the full serum possible downregulation of E2F activity. condition, which contains substantial levels of testosterone and DHT (dihydrotestosterone, a potent form of testosterone) as well ZBTB7A chromatin binding is associated with transcriptional as other steroid hormones (20), we were able to perform com- repression activity of AR bined analyses with the previously published AR ChIP-seq and Our previous studies in multiple prostate cancer models and gene expression proﬁling datasets based on VCaP cells treated human prostate cancer samples have indicated that repressing with 10 nmol/L DHT (4, 16), we found that 30% of ZBTB7A E2F-activated DNA replication is the major transcriptional repres- binding peaks (5,875 of 17,691) were overlapped with AR bind- sion activity of AR (4, 6). Therefore, we next assessed the global ing sites (Fig. 2A) and these sites were highly enriched for

Figure 2. ZBTB7A chromatin binding is associated with transcriptional repression activity of AR. A, The Venn diagram shows overlap between previously identiﬁed AR binding sites in VCaP cells and ZBTB7A binding sites. B, The heatmap view for the signal intensities of the aligned reads from ChIP-ZBTB7A and ChIP-AR across all ZBTB7A binding sites (1kb).C, BETA analysis on AR-regulated genes with nearby AR and ZBTB7A unique or overlapping binding sites. D, ZBTB7A-repressed genes with overlapping AR and ZBTB7A binding sites were analyzed by KEGG pathway analysis. E, Motif enrichment analysis was carried out at the AR binding sites (3kb up/downstream of TSS) within AR-repressed gene (1.5-fold cutoff) loci. The potential factors within the top-ranked clusters are listed (z-score < 10, similarity score >2.85). AR motif was the most enriched motif within 600bp around TSS. F, ZBTB7A was immunoprecipitated in VCaP cells treated with or without 10 nmol/L DHT for 4 hours, followed by immunoblotting for AR and ZBTB7A. G, COS7 cells were cotransfected with V5-tagged ZBTB7A and HA-tagged AR fragments (NTD: 1-539 aa, NTDþDBD: 1-628 aa, DBDþLBD: 538-919 aa, and LBD: 662-919 aa). AR fragments were immunoprecipitated by anti-HA beads, followed by immunoblotting for V5 (V5-ZBTB7A).

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ZBTB7A Mediates AR Transcriptional Repression Activity

promoter binding (Supplementary Fig. S1). The binding intensity intensity of AR and Rb, suggesting that AR may directly recruit of ZBTB7A was also highly correlated with the intensity of AR ZBTB7A to those sites. Supportively, the binding intensity of binding (Fig. 2B), indicating a possible interaction of ZBTB7A and ZBTB7A at those AR and Rb overlapping sites was also similarly AR to coregulate gene transcription. We then performed BETA to androgen-induced (Supplementary Fig. S2A). We then conducted determine the global association of ZBTB7A binding with the AR BETA to determine whether AR recruited ZBTB7A and Rb are regulation of gene transcription. As seen in Fig. 2C, although AR associated with AR repression function. As shown in Fig. 3C repressive function appeared to associate with ZBTB7A unique and D, AR repression function was more significantly associated þ þ þ þ þ sites (P ¼ 10 16), it was more strongly correlated with AR and with AR /ZBTB7A sites (P ¼ 10 43) and AR /ZBTB7A /Rb ZBTB7A overlapping sites (P ¼ 10 39) and not with AR unique sites (P ¼ 10 31) than AR activation function (P ¼ 10 23 and sites (P ¼ 0.839). In contrast, AR activation function was asso- P ¼ 10 13, respectively). Moreover, the absence of either Rb þ þ þ þ ciated more strongly with ZBTB7A-absent AR binding sites (P ¼ (AR /ZBTB7A /Rb ) or ZBTB7A binding (AR /ZBTB7A /Rb ) 10 17), and was less significantly associated with ZBTB7A and AR at those AR repression sites significantly decreased their associa- overlapping sites (P ¼ 10 15) or ZBTB7A unique sites (P ¼ 10 7). tion with AR repression function (P ¼ 10 5 and P ¼ 10 9, Collectively, these global analyses of ZBTB7A and AR binding sites respectively), suggesting that AR recruitment of ZBTB7A and Rb strongly indicate that ZBTB7A may be involved in AR-mediated may be both required for the full repression activity of AR. transcriptional repression in prostate cancer cells. Because the previous study indicates that ZBTB7A may regulate We next examined the functions for the ZBTB7A-repressed Rb expression in transgenic mouse model (12), we first examined genes that have nearby AR and ZBTB7A overlapping sites (Sup- whether ZBTB7A upregulates Rb in prostate cancer cells. As shown plementary Table S2, ranking of genes based on BETA). As shown in Supplementary Fig. S2B, Rb protein expression was not affected in Fig. 2D, this subset of genes were enriched for pathways by knocking down ZBTB7A in VCaP cells, suggesting that ZBTB7A mediating translation and DNA replication and damage repair, does not regulate Rb in human CRPC cells. To further determine the latter of which was consistent with the previously reported whether ZBTB7A contributes to the AR-mediated repression major function of direct AR-repressed genes (4, 6). To further activity on DNA replication, we selected a panel of previously confirm that ZBTB7A can bind to AR occupied repression sites, we identified Rb-dependent AR-repressed DNA replication/repair performed a motif enrichment analysis on the AR binding sites genes, including MCMs (minichromosome maintenance com- adjacent to the androgen-repressed gene loci. Consistently, we plex genes), BLM (a Bloom syndrome RecQ like helicase gene), found that ZBTB7A binding motif was among the top-ranked FANCI (a Fanconi anemia complementation group gene), and motifs enriched at those AR repression sites (Fig. 2E). The E2F TK1 (a thymidine kinase gene; ref. 6), for the subsequent studies. binding motif was also top ranked, consistent with our previous The rapid increase of ZBTB7A binding by androgen-stimulation at studies showing AR interaction with E2F–Rb complex at the those previously identified AR-repressed promoters (6) was con- repression sites (4, 6). A previous study found that ZBTB7A can firmed using ChIP-qPCR (Fig. 3E). We next cloned the MCM7 directly interact with AR through the POZ domain of ZBTB7A and promoter (800bp fragment, containing AR, ZBTB7A, and E2F1 ligand-binding domain (LBD) of AR, and this interaction impairs binding sites) into a luciferase reporter to examine the effect of AR activation of PSA (21). Using a coimmunoprecipitation assay ZBTB7A on E2F1 activity. As seen in Fig. 3F, E2F1 significantly in VCaP cells, we confirmed that the endogenous ZBTB7A can induced MCM7-promoter activity, which can be suppressed by interact with AR and the interaction appeared to be enhanced by Rb. Importantly, the expression of ZBTB7A alone markedly down- androgen stimulation (Fig. 2F). We next sought to determine regulated the E2F1 activity and coexpression of ZBTB7A and Rb whether the LBD of AR is the domain responsible for AR inter- can further repress the E2F1 activity. These result suggest that action with ZBTB7A. Experiments in cells transiently overexpres- ZBTB7A and Rb may function in parallel to collaboratively sing AR fragments and ZBTB7A were then carried out and the suppress E2F1-mediate transcriptional activation. Interestingly, result indicated that the entire C-terminal domains, including overexpressing AR alone was not sufficient to suppress E2F1 both LBD and DNA-binding domain (DBD), may be required for activity (Fig. 3G), further suggesting that AR may indirectly repress the full interaction with ZBTB7A (Fig. 2G). Overall, these data E2F1 activity in PCa cells through recruitment of ZBTB7A and Rb. support the function of ZBTB7A in mediating AR-dependent Furthermore, we also demonstrated that doxycycline-induced transcriptional repression activity in prostate cancer cells through silencing of ZBTB7A in VCaP-tet-shZBTB7A stable cells (expres- a possible direct interaction with AR protein. sing tetracycline-regulated lentiviral shRNA against ZBTB7A) impaired the androgen-induced repression on these DNA repli- AR recruitment of ZBTB7A is required for the transcriptional cation genes (Fig. 3H). Collectively, these genomic and molecular repression function of AR on E2F-regulated genes studies indicated an important function of AR recruited ZBTB7A The current model on AR transcriptional repression activity by collaboration with Rb to repress E2F-regulated transcriptional based on our previous studies is that the rapid AR recruitment of activation of DNA replication. Rb can reinforce the E2F–Rb repressor complex to suppress the transcription of genes mediating DNA replication/repair and cell ZBTB7A mediates AR repression of PCAT-1 lncRNA cycle (6). Because the recruitment of Rb is one major event that The gene profiling database used to study androgen regulation mediates this specific activity of AR, we next determined whether in VCaP cells was based on Affymetrix gene microarrays (4), AR may also recruit ZBTB7A to those suppression sites. To test this which primarily detect protein coding genes. However, recent hypothesis, we performed additional ChIP-seq of ZBTB7A in studies have revealed important functions of noncoding RNAs, VCaP cells treated with or without 10 nmol/L DHT for 4 hours. particularly lncRNAs, in regulating gene transcription in tumor As seen in Fig. 3A and B, the binding intensity of ZBTB7A at those cells (22). Therefore, we sought to identify the androgen- ZBTB7A and AR overlapping sites was rapidly increased by the repressed lncRNAs in androgen-treated prostate cancer cells by short-term androgen treatment and correlated with the binding using RNA-seq. In VCaP cells treated with 10 nmol/L DHT for

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Figure 3. AR recruitment of ZBTB7A is required for the transcriptional repression activity of AR on E2F-regulated genes. A, ZBTB7A ChIP-seq was done in VCaP cells treated with or without 10 nmol/L DHT for 4 hours in hormone-depleted media. The heatmap view for the signal intensities of aligned reads across all ZBTB7A binding sites (3 kb) is shown and the ZBTB7A bindings were compared with Rb and AR bindings in DHT-treated VCaP cells. B, The mean signal density of AR, ZBTB7A, and Rb bindings centered at ZBTB7A binding sites (3 kb). C, The Venn diagram illustrating overlap among AR, ZBTB7A, and Rb binding sites (DHT-stimulated). D, BETA analysis was used to assess the correlation of AR, ZBTB7A, and Rb binding sites with the expression of AR-activated genes (red) and AR-repressed genes (blue) over static background (black). E, ChIP-qPCR for ZBTB7A binding at indicated promoters in VCaP cells treated with vehicle or 10 nmol/L DHT for 1 hour. F and G, MCM7 promoter (800bp)–driven luciferase report activity was measured in HEK293 cells transfected with E2F1, Rb, and/or ZBTB7A (F) in full serum, or E2F1 and/or AR in presence or absenceof10nmol/LDHT(G). H, VCaP cells stably expressing tetracycline-regulated lentiviral shRNA against ZBTB7A (VCaP-tet-shZBTB7A) were established and ZBTB7A expression was examined by immunoblotting in cells pretreated with 0.1 mg/mL doxycycline for 2 days and then with 10 nmol/L DHT for 24 hours. The mRNA expressions of a panel of Rb mediated AR-repressed genes were measured. , P < 0.05.

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ZBTB7A Mediates AR Transcriptional Repression Activity

24 hours, 111 lncRNAs were identified as androgen-induced ZBTB7A suppresses CRPC tumor growth in vitro genes and 245 lncRNAs were identified as androgen-repressed Although loss of Zbtb7a expression in conjunction with loss of genes (Supplementary Table S3). Among the identified androgen- Pten in mouse prostate epithelial cells promote prostate cancer induced lncRNA subset, PCAT-14 and PCAT-18 have been previ- development (12), it is not clear whether ZBTB7A can similarly ously suggested as biomarkers for predicting prostate cancer function as a tumor suppressor in human CRPC cells. Although outcomes (23, 24). PRCAT47 (also called ARLNC1) was another VCaP cells, which were derived from CRPC bone metastases, were recently identified androgen-upregulated gene that functions to used as the primary model for the mechanistic studies, these cells stabilize the AR transcript and enhances AR signaling and prostate cannot directly form xenograft tumors in castrated mice and it is cancer tumor growth (25). Among the androgen-repressed difficult to use this model for the subsequent functional studies. lncRNA subset, PCAT-29 has been reported previously as an Therefore, to further study the tumor suppressor role of ZBTB7A androgen-repressed tumor suppressor gene in prostate cancer in vitro and in vivo, we selected a well-known LNCaP-derived CRPC cells (26). Importantly, we identified PCAT-1 (prostate cancer– model, C4-2 cells, to generate the stable cell line that can indu- associated transcript 1) as a novel androgen-repressed gene, cibly overexpress ZBTB7A. Similar to VCaP cells, AR is overex- which promotes prostate cancer by mechanisms such as stabiliz- pressed and androgen can suppress cell growth through an ing MYC protein, repressing BRCA2, and activating AKT (27–29). Rb-dependent mechanism in C4-2 cells (6). More importantly, Therefore, suppressing the expression of this oncogenic lncRNA in these cells AR also similarly suppresses the expression of the may be an important activity for AR to act as a tumor suppressor in majority of DNA-replication genes that are androgen-repressed in prostate cancer. VCaP cells and this repression activity of AR is partially impaired We next examined androgen-regulation of PCAT-1 expression by silencing of Rb (Supplementary Fig. S4). C4-2 cells express in prostate cancer cells. As seen in Supplementary Fig. S3A, PCAT- comparable level of ZBTB7A as in VCaP cells and high-passage 1 expression was significantly decreased by DHT treatment in LNCaP cells, which are also resistant to ADT (31), although the VCaP cells and this suppression activity was abolished when cells AR-negative PC-3 cells express higher level of ZBTB7A (Fig. 4A). were treated with an HDAC1 inhibitor (mocetinostat), indicating Importantly, ZBTB7A was also recruited by AR to the promoter a critical function of HDAC1 that may be required for AR repres- regions of the DNA replication genes and this chromatin recruit- sion on PCAT-1. Interestingly, although PCAT-1 was not clearly ment can be impaired by AR antagonist treatment (enzalutamide) androgen-repressed in androgen-dependent LNCaP prostate can- or AR-targeted siRNA (Supplementary Fig. S5A and S5B). Silenc- cer cells, which express less AR than VCaP cells (4), it was ing ZBTB7A in C4-2 cells increased cell proliferation in hormone- significantly repressed by androgen in AR-overexpressing LNCaP reduced condition and compromised the antiproliferative effect (LN-AR) cells (Supplementary Fig. S3B), suggesting that the of DHT in C4-2 cells (Fig. 4B–D), indicating that the endogenous repression on PCAT-1 requires high levels of AR expression. ZBTB7A is required for tumor suppressor function of AR in To assess the androgen regulation of PCAT-1 in vivo, we examin- CRPC cells. ed its expression in a previously established VCaP xenograft To further study the effect of restoring ZBTB7A expression in tumor progression model (30). In this model, we have clearly CRPC cells, we established a C4-2 stable cell line that express demonstrated the restoration of the expression of PSA and tetracycline-regulated V5-tagged ZBTB7A (Fig. 4E). First, induced TMPRSS2-ERG due to regained AR activation activity and the overexpression of ZBTB7A further decreased the mRNA and increased expression of AR, AKR1C3 (an androgen synthesis protein expression levels of the DNA replication genes that gene), and DNA replication genes due to loss of AR repression were suppressed by DHT treatment (Fig. 4F and G), consistent activity in the castration-resistant stage of the tumor (4, 6, 30). As with the effect of silencing ZBTB7A in VCaP cells. Second, using a seen in Supplementary Fig. S3C, the expression of PCAT-1 was V5 pull-down assay we also demonstrated that AR, Rb, and rapidly increased in at least a portion of xenograft tumors upon HDAC1 can all interact with ZBTB7A (Fig. 4H), suggesting that castration and the increased level was retained in the relapsed ZBTB7A may be a component of the AR–Rb suppressor complex. tumors, suggesting ADT may alleviate the AR repression on Conversely, we also immunoprecipitated endogenous E2F1 or Rb PCAT-1 and hence result in the increased expression of PCAT-1 in these cells and found that the V5-tagged ZBTB7A can be in CRPC. coimmunoprecipitated with these proteins (Fig. 4I), indicating We next determined whether ZBTB7A is involved in the AR- that ZBTB7A can interact with E2F1. However, the interaction of mediated repression of PCAT-1. Through examining the AR and E2F1 with Rb was not significantly affected by overexpression of ZBTB7A ChIP-seq results, we identified AR and ZBTB7A chroma- ZBTB7A, suggesting that the repression activity of ZBTB7A on tin binding sites in the PCAT-1 locus. Interestingly, although the E2F1 may not be mediated through Rb. Consistently, silencing or identified AR binding site (named S1) was very close to the overexpressing ZBTB7A had little effect on Rb binding to the ZBTB7A binding site (named S2), these 2 peaks were not exactly promoters of DNA replication genes (Supplementary Fig. S6A– overlapping. Using ChIP-qPCR analysis, we showed that AR S6C), further suggesting that ZBTB7A can suppress E2F activity chromatin binding was significantly stimulated by DHT treat- without increasing chromatin binding of Rb–E2F repressor com- ment on S1 and weakly increased on S2 site (Supplementary plex. Nonetheless, induced expression of ZBTB7A by using lower Fig. S3D, left). However, ZBTB7A binding was increased by DHT dose of doxycycline to minimize any toxicity effect (Supplemen- on the S2 site (Supplementary Fig. S3D, middle), suggesting that tary Fig. S7) also led to decreased cell growth, and more impor- AR binding at S1 site may increase the recruitment of ZBTB7A at S2 tantly could act in synergy with DHT treatment, particularly with site. Moreover, we also found that silencing ZBTB7A impaired the lower doses of DHT (1 nmol/L; Fig. 4J). Consistent with the effect AR repression activity on PCAT-1 transcript in VCaP cells (Sup- on growth, overexpression of ZBTB7A also enhanced the supplementary Fig. S3E), indicating that ZBTB7A contributes to the pression effect of 1 nmol/L DHT on DNA replication genes full repression activity of AR on PCAT-1. (Fig. 4K).

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Figure 4. ZBTB7A suppresses CRPC tumor growth in vitro. A, Immunoblotting for ZBTB7A in four CRPC cell lines. B, Immunoblotting for ZBTB7A expression in C4-2 cells stably expressing lentiviral shRNA against ZBTB7A or nontarget-control (shNTC). C, Cell proliferation (by counting live cells) was measured in the ZBTB7A-silencing cells versus control C4-2 cells. D, Cell proliferation was also measured in these cell lines treated with 10 nmol/L DHT for 2days.E, Immunoblotting for V5 expression in C4-2-tet-ZBTB7A cells stimulated with 0 to 1 mg/mL doxycycline for 2 days. F and G, The expressions of a panel of AR-repressed DNA replication genes were examined in C4-2-tet-ZBTB7A cells treated with 0.25 mg/mL doxycycline and 10 nmol/L DHT for 2 days. The mRNA expression was measured by qRT-PCR (F) and the protein expression was measured by immunoblotting (G). H, V5-ZBTB7A was immunoprecipitated using V5-antibody-condugated beads in C4-2-tet-ZBTB7A cells stimulated by 0.25 mg/mL doxycycline for 2 days, followed by immunoblotting for AR, Rb, HDAC1, and ZBTB7A. I, E2F1 or Rb was immunoprecipitated in C4-2-tet-ZBTB7A cells stimulated by 0.25 mg/mL doxycycline for 2 days, followed by immunoblotting for ZBTB7A, Rb, and E2F1. J, Cell proliferation was measured in C4-2-tet-ZBTB7A cells treated with 0.1 mg/mL doxycycline and with 0 to 100 nmol/L DHT for 0 to 6 days. K, The expressions of a panel of AR-repressed DNA replication genes were examined in C4-2-tet-ZBTB7A cells treated with 0.1 mg/mL doxycycline and 1 nmol/L DHT for 2 days. , P < 0.05.

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ZBTB7A Mediates AR Transcriptional Repression Activity

Figure 5. Overexpression of ZBTB7A delays the recurrence of CRPC tumor treated by high-dose testosterone. A, C4-2-tet-ZBTB7A xenograft tumors were established and passaged in castrated male SCID mice. A cohort of 12 mice (at 6 weeks post-injection) was randomly divided into two arms fed with regular diet or doxycycline- supplemented diet and the development of xenograft tumors was monitored for over 4 weeks. B, The C4-2-tet-ZBTB7A CRPC xenograft tumors were established (50 mm3) prior to the treatments. A cohort of 24 mice was randomly divided into four arms: (i) on regular diet with vehicle injection (cotton oil); (ii) on regular diet with testosterone treatment (10 mg/kg via i.p. injection every day); (iii) on doxycycline-supplemented diet with vehicle injection (cotton oil); and (iv) on doxycycline-supplemented diet with the same testosterone treatment, and the development of xenograft tumors was monitored for over 5 weeks. C, The current model for the ZBTB7A-mediated AR transcriptional repression activity.

Overexpression of ZBTB7A delays the recurrence of CRPC progression. Despite the limited tissue materials in treatment tumor treated by high-dose testosterone groups, we were able to extract a small amount of RNA to examine Finally, we assessed the effect of ZBTB7A overexpression on the effect of the combination treatment on a few DNA replication CRPC tumor growth in vivo. As seen in Fig. 5A, induced over- genes. As shown in Supplementary Fig. S9, although the expres- expression of ZBTB7A (prior to the establishment of xenograft sion levels of the DNA replication genes may be restored in the tumor) significantly delayed the development of C4-2 CRPC doxycycline- or testosterone-treated group, they appeared to xenograft tumor, suggesting a critical and potent tumor suppres- remain repressed in the combination treatment group. Overall, sor function of ZBTB7A in CRPC cells. The doxycycline supple- these in vitro and in vivo studies clearly demonstrated the critical mented food had no toxic effect on the health of mice and the tumor suppressor function of ZBTB7A in CRPC cells and sug- growth of C4-2 xenograft tumors (Supplementary Fig. S8A and gested a potential therapeutic strategy by enhancing ZBTB7A S8B). We then determined whether overexpression of ZBTB7A can expression or activity to improve the efficacy of the high-dose enhance the efficacy of high-dose testosterone treatment in this testosterone therapy in CRPC patients. The model for ZBTB7A- CRPC model. For this experiment, we allowed the CRPC xenograft mediated transcriptional repression activity of AR on a subset of tumor to establish (50 mm3) prior to the treatments. As seen E2F-regulated DNA replication genes was summarized in Fig. 5C. in Fig. 5B, although the induction of ZBTB7A expression or the treatment of testosterone markedly suppressed the CRPC tumor growth, the tumors began to relapse after 1 month of treat- Discussion ments. Significantly, the combination treatment was able to delay Loss of expression of zinc finger and BTB domain containing the recurrence of the CRPC tumor, suggesting that overexpression transcription factors, such as ZBTB7A and ZBTB16, are commonly of ZBTB7A can synergize with testosterone to suppress CRPC seen in prostate cancer tumors, and their expression is decreased

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in more aggressive CRPC (see Fig. 1). However, the biological Furthermore, we have demonstrated in vitro and in vivo that functions of these genes in prostate cancer cells remain to be ZBTB7A is a potent tumor suppressor, which can markedly repress identified. ZBTB16 is a classic AR regulated gene and it can CRPC tumor growth. More importantly, we have demonstrated function as a tumor suppressor in prostate cancer cells through that overexpression of ZBTB7A can enhance the growth suppres- inhibiting the MAPK pathways (14). In contrast, ZBTB7A is not sive activity of high-dose androgen treatment on CRPC cells regulated by AR and was previously known as an oncogene in in vitro and in vivo. Based on our results (see Fig. 4J), even lower many cancer types but recently reported to play a tumor suppres- concentration of androgen treatment can suppress prostate cancer sor role in prostate cancer. The loss of Zbtb7a expression in cell proliferation when ZBTB7A is expressed in high levels. This conjunction with loss of Pten in mouse prostate epithelial cells finding may provide an explanation of why a subset of prostate was previously shown to drive the development of aggressive cancer or CRPC tumors have to select for the decrease or loss of prostate cancer (12). This tumor suppressive activity of ZBTB7A ZBTB7A expression in order to escape from AR-mediated growth- was largely attributed to maintaining the expression of Rb, a suppression activity. Significantly, our study also provides a critical cell cycle regulator that mediates G1–S transition, through rationale to therapeutically enhance the high-dose testosterone the negative regulation of SOX9 activity on activating an Rb- treatment in CRPC (currently in phase II clinical trials; ref. 10) targeting miRNA. In this study, our integrated ZBTB7A cistrome through elevating the expression or activity of ZBTB7A. Future and transcriptome analyses have indicated that ZBTB7A directly study is clearly needed to identify the actionable targets that are represses the E2F-regulated genes, consistent with those previous involved in regulating ZBTB7A expression or activity in CRPC findings. More importantly, our mechanistic study of ZBTB7A has cells. demonstrated that ZBTB7A can repress DNA replication and cell- cycle progression through a very distinct mechanism by which Disclosure of Potential Conflicts of Interest ZBTB7A was recruited by AR to the E2F binding sites and sup- S.P. Balk is a consultant at Sanofi, Kronos, Constellation Pharamceuticals, presses its transcriptional activation function. We have previously and Radius, and has provided expert testimony for Atellas. No potential fl reported this transcriptional repressor activity of AR and revealed con icts of interest were disclosed by the other authors. its critical biological function on repressing DNA replication and Authors' Contributions cell cycle (4). We have further shown in another study that this Conception and design: D. Han, S. Chen, S. Gao, S.P. Balk, H.H. He, C. Cai transcriptional repressor activity of AR was through enhancing Rb – Development of methodology: D. Han, J.N. Owiredu, H.H. He, C. Cai chromatin binding and thus strengthening the Rb E2F repressor Acquisition of data (provided animals, acquired and managed patients, complex (6). Importantly, a recent study using castration-resistant provided facilities, etc.): D. Han, W. Han, J.N. Owiredu, M. Li, H.H. He LuCaP PDX models also indicates that the most robust molecular Analysis and interpretation of data (e.g., statistical analysis, biostatistics, phenotype for the high-dose testosterone treatment is the sup- computational analysis): D. Han, S. Chen, W. Han, S. Gao, J.N. Owiredu, M. Li, pression of E2F transcriptional output (32). In this study, we S.P. Balk, H.H. He, C. Cai Writing, review, and/or revision of the manuscript: D. Han, S. Chen, W. Han, report that AR can rapidly recruit ZBTB7A to those AR and E2F–Rb S. Gao, S.P. Balk, H.H. He, C. Cai overlapping sites within DNA replication genes and this recruit- Administrative, technical, or material support (i.e., reporting or organizing ment can further enhance the transcriptional repression of the data, constructing databases): D. Han, M. Li, H.H. He target genes. Mechanistically, we have also demonstrated that Study supervision: H.H. He, C. Cai ZBTB7A can directly interact with E2F1 and negatively regulate its transcriptional activity (see Figs. 3F and 4I). Because ZBTB7A Acknowledgments was known to recruit HDACs (also see Fig. 4H), this transcrip- This work was supported by grants from NIH (R00 CA166507 and R01 tional corepressor function of ZBTB7A on AR may be through CA211350 to C. Cai and P01 CA163227 to S.P. Balk), DOD (W81XWH-15-1- 0554 to S. Gao and W81XWH-16-1-0445 to C. Cai), CIHR (142246, 152863, strengthening the recruitment of HDACs that can deacetylate 152864, and 159567 to H.H. He), Prostate Cancer Canada (RS2016-1022 histone 3 lysine 27 and thus represses gene transcription activated and TAG2018-2061 to H.H. He), NSERC (498706 to H.H. He), Terry Fox by E2F. New Investigator Award (1069 to H.H. He), and Princess Margaret Cancer In addition to DNA replication genes, we have also identified Foundation (to H.H. He). We thank Dr. Jill A. Macoska and Susan C. Patalano the lncRNA PCAT-1 as a novel AR-repressed gene in prostate (Genomics Core, University of Massachusetts Boston) for assistance and cancer cells. One of the major functions of this oncogenic lncRNA guidance of high-throughput sequencing. in prostate cancer cells is to regulate MYC oncoprotein (27). The costs of publication of this article were defrayed in part by the payment of Therefore, ZBTB7A may suppress MYC activity through transcrip- page charges. This article must therefore be hereby marked advertisement in PCAT-1 tionally repressing expression. Overall, this study iden- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. tified a biologically important lncRNA as a novel repression target of AR and demonstrated the role of ZBTB7A in mediating this Received March 10, 2019; revised July 9, 2019; accepted August 20, 2019; repression process. published first August 23, 2019.

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ZBTB7A Mediates the Transcriptional Repression Activity of the Androgen Receptor in Prostate Cancer

Dong Han, Sujun Chen, Wanting Han, et al.

Cancer Res 2019;79:5260-5271. Published OnlineFirst August 23, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-19-0815

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