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

Author Manuscript Published OnlineFirst on August 23, 2019; DOI: 10.1158/0008-5472.CAN-19-0815 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

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

1 Center for Personalized Cancer Therapy, University of Massachusetts Boston, Boston, Massachusetts 02125,

USA; 2 Princess Margaret Cancer Center/University Health Network, Toronto, Ontario M5G1L7, Canada; 3

Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5S, Canada; 4 Beth Israel

Deaconess Medical Center, Boston, MA 02215

D.H., S.C., and W.H. contributed equally to this work.

Correspondence: Changmeng Cai, Center for Personalized Cancer Therapy, University of Massachusetts

Boston, Boston, Massachusetts 02125, USA, ([email protected]); Housheng Hansen He, Princess

Margaret Cancer Center/University Health Network, Toronto, Ontario M5G1L7, Canada,

([email protected])

Running Title: ZBTB7A Mediates AR Transcriptional Repression Activity

Key Words: ZBTB7A, Rb, E2F1, prostate cancer, androgen receptor, AR, androgen-deprivation therapy, high dose testosterone, DNA replication

ABSTRACT

Loss of expression of context-specific tumor suppressors is a critical event that facilitates the development of prostate cancer (PCa). Zinc finger and BTB domain containing transcriptional repressors, such as ZBTB7A and

ZBTB16, have been recently identified as tumor suppressors that play important roles in preventing PCa progression. In this study, we used combined ChIP-seq and RNA-seq analyses of PCa cells to identify direct

ZBTB7A-repressed genes which are enriched for transcriptional targets of E2F and identified that the androgen receptor (AR) played a critical role in the transcriptional suppression of these E2F targets. AR recruitment of the Retinoblastoma protein (Rb) was required to strengthen the E2F-Rb transcriptional repression complex. In addition, ZBTB7A was rapidly recruited to the E2F-Rb binding sites by AR and negatively regulated the transcriptional activity of E2F1 on DNA replication genes. Finally, ZBTB7A suppressed the growth of castration-resistant PCa (CRPC) in vitro and in vivo, and overexpression of ZBTB7A acted in synergy with high-dose testosterone treatment to effectively prevent the recurrence of CRPC. Overall, this study provides novel molecular insights of the role of ZBTB7A in CRPC cells and demonstrates globally its critical role in mediating the transcriptional repression activity of AR.

SIGNIFICANCE

ZBTB7A is recruited to the E2F-Rb binding sites by AR and negatively regulates the transcriptional activity of

E2F1 on DNA replication genes.

INTRODUCTION

Prostate cancer (PCa) is one of the most common cancers in men. The development of primary PCa depends on the activity of androgen receptor (AR), a ligand-dependent nuclear receptor transcription factor. The standard treatment of PCa is surgical or medical castration (known as androgen deprivation therapy, ADT) to reduce circulating androgens. However, patients invariably relapse into more aggressive castration-resistant prostate cancer (CRPC) with increased expression and restored activity of AR (1). Although CRPC can be further treated with more aggressive ADT using agents such as abiraterone and enzalutamide (2,3), the tumors still generally relapse within one year and a large portion of these relapsed tumors still express AR and AR

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 23, 2019; DOI: 10.1158/0008-5472.CAN-19-0815 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. regulated genes. While AR is known for its transcriptional activator function, it can also act as a transcriptional repressor to suppress the expression of a subset of genes, including AR itself, androgen synthetic genes, and genes mediating DNA replication and repair (4,5). Mechanistically, we have shown that AR globally recruits hypophosphorylated Retinoblastoma protein (Rb) to the promoters/enhancers of DNA replication gene loci and strengthens the activity of E2F-Rb suppressor complex (6). Therefore, loss of this tumor suppressor activity of

AR after ADT is likely to be one mechanism contributing to the progression to CRPC (1). This transcriptional repressor activity of AR also provides one of the mechanisms for the high-dose testosterone therapy in CRPC patients (7-10).

In addition to AR, the recently reported zinc finger and BTB domain containing transcription repressors, such as ZBTB7A and ZBTB16, can also function as tumor suppressors in preventing the progression of PCa

(11-14). ZBTB7A, also known as LRF/POKEMON, consists of a protein-protein interacting BTB domain at the

N-terminus and DNA binding zinc fingers at the C-terminus (13). Even though ZBTB7A has been identified as a proto-oncogene in other cancer types, a recent study using a transgenic mouse model indicates that it functions as a tumor suppressor in PCa and that loss of its expression can drive the development of aggressive invasive tumor in Pten-null prostate epithelial cells by bypassing the Pten-loss induced cellular senescence (15). Mechanistically, ZBTB7A was shown to repress the activity of SOX9, a proto-oncogene in

PCa (16), and to impair the SOX9 regulation of an RB-targeting miRNA, thus allowing cells to bypass the Pten- loss induced senescence (15). Although its tumor suppressor activity has been demonstrated in mouse PCa cells, the activities of ZBTB7A at the chromatin level in human PCa cells remain to be characterized. Using a combined analysis of the ZBTB7A cistrome and transcriptome, we have mapped the binding sites of ZBTB7A and identified direct ZBTB7A-regulated genes. Significantly, the direct ZBTB7A-repressed genes were enriched for the activation function of E2Fs, suggesting that ZBTB7A may function to repress their oncogenic activities in PCa cells.

Since our previous studies indicated AR can function as a transcriptional repressor to suppress DNA replication genes through enhancing the chromatin binding of Rb that reinforces the suppressor activity of E2F-

Rb complex (6), we next determined how ZBTB7A chromatin binding in PCa cells globally impacts the transcriptional activity of AR. By co-analyzing the previous reported AR cistrome database in PCa cells (4,16),

ZBTB7A and AR overlapping sites are significantly associated with the repression activity of AR on gene transcription. More importantly, we also show that ZBTB7A binding at those AR repression sites is rapidly increased upon androgen stimulation and the increased binding is highly associated with E2F-Rb binding, indicating that AR-recruited ZBTB7A may cooperate with Rb in regulating the transcriptional activity of E2Fs.

By co-immunoprecipitation assays, we demonstrated that ZBTB7A can physically interact with AR, Rb, and

E2F1, further indicating that ZBTB7A may be an additional component of the AR-Rb repressor complex.

Furthermore, we carried out in vitro and in vivo studies to examine the effects of overexpression of ZBTB7A on

CRPC tumor growth and results show that overexpression of ZBTB7A in CRPC cells significantly reduced the cancer development, and that overexpression of ZBTB7A can synergize with high-dose testosterone therapy in treating CRPC. Overall, this study has provided novel insights into the tumor suppressor activity of ZBTB7A in

PCa cells and identified ZBTB7A as a critical mediator required for AR-dependent transcriptional repression activity.

MATERIALS AND METHODS

Cell lines and cell culture: The VCaP and C4-2 cell lines were purchased from ATCC. All the cell lines were recently authenticated using short tandem repeat (STR) profiling by DDC Medical and tested for mycoplasma contamination (negative result) by using MycoAlert mycoplasma detection kit (Lonza). VCaP cells were cultured in DMEM medium with 10% FBS (fetal bovine serum, Gibco). VCaP-tet-shZBTB7A (tetracycline- inducible ZBTB7A silencing) cells were maintained in DMEM medium with 10% tetracycline-free FBS. C4-2 and C4-2-shZBTB7A (stable ZBTB7A silencing) cells were cultured in RPMI-1640 medium supplemented with

2% FBS plus 8% CSS (charcoal-dextran stripped FBS, Gibco). C4-2-tet-ZBTB7A (tetracycline-inducible

ZBTB7A overexpressing) cells were maintained in RPMI-1640 medium with 2% tetracycline-free FBS plus 8%

CSS. For androgen stimulation assays, cells were generally grown to 50-60% confluence in medium containing

5% CSS for 2-3 days (d) and then treated with DHT or inhibitors for indicated time.

Chromatin immunoprecipitation (ChIP): For preparation of ChIP, dispensed cells were formalin fixed, lysed, and sonicated to break the chromatin into 500–800 bp fragments, followed by immunoprecipitation. The qPCR analysis was carried out using the SYBR Green method on the QuantStudio 3 Real-time PCR system (Thermo

Fisher Scientific). The primers for MCM7-pro, BLM-pro, FANCI-pro, and TK1-pro were previously listed (6).

RT-PCR and immunoblotting: The expression of genes was measured using real-time RT-PCR analyses with Taqman one-step RT-PCR reagents (Thermo Fisher Scientific) and results were normalized to co- amplified GAPDH. The primer and probe sets for the following genes: MCM2, MCM7, FANCI, BLM, TK1,

PCAT1, and GAPDH were purchased as inventoried mix from Applied Biosystems at Thermo Fisher. For immunoblotting, cells were lysed with RIPA buffer with protease inhibitors (Thermo Fisher Scientific) and anti-

ZBTB7A (Bethyl), anti-AR PG21 (Millipore), anti-Rb, anti-E2F1 (Cell Signaling), anti-V5, anti-HA (Sigma), anti-

HDAC1, anti-GAPDH, or anti--actin (Abcam) antibodies were used. Immunoblotting results shown are representative of at least 3 independent experiments.

RNAi and transfection: siRNAs against ZBTB7A and non-target control (NTC) were purchased from

Dharmacon and transfected into cells using lipofectamine 2000 (Thermo Fisher). C4-2-shZBTB7A cells were generated using lentiviral shRNA against ZBTB7A or NTC (Dharmacon). VCaP-tet-shZBTB7A cells were generated using tetracycline-inducible lentiviral shRNA against ZBTB7A or NTC (Dharmacon). C4-2-tet-

ZBTB7A cells were generated by stable infection with lenti-virus containing tetracycline-inducible V5-tagged

ZBTB7A cDNA in C4-2 cells. COS-7 cells cultured in DMEM with 10% FBS were transiently transfected with plasmids expressing HA-tagged AR 1-539aa, AR 1-628aa, AR 538-919aa, or AR 662-919aa for overnight, followed by immunoprecipitation assay.

ChIP-Seq and RNA-Seq: For ChIP-seq, VCaP cells were fixed and then lysed, and the chromatin was sheared to 300-500 bp in size using the Bioruptor sonicator (Diagenode). The chromatin was incubated with

ZBTB7A antibody bound to protein G beads (Thermo Fisher) for overnight. After immunoprecipitation, samples were reversely crosslinked in 65°C water bath, and DNA was extracted with QIAquick PCR purification kit

(Qiagen). The ChIP-Seq libraries were prepared using ThruPlex DNA-seq 48D Kit (Rubicon Genomics) and then sequenced using Illumina HiSeq 2500. For RNA-seq, VCaP cells transfected with siZBTB7A or siNTC were harvested for RNA extraction using RNeasy mini kits (Qiagen), followed by RNA-Seq library preparation with TruSeq Stranded RNA LT Kit (Illumina).

For data analysis, ChIP-seq raw reads were aligned to hg19 using bwa (version 0.7.2-r351). The resulted sam files are converted to bam with samtools [version 0.1.18 (r982:295)]. MACS2 (version

2.0.10.20131216) was used to call peak on the bam files. bedGraph files containing signal per million reads produced from MACS2 was converted to bigwig files with ucsctool kit (315). The R package ChIPpeakAnno

(version 3.10.1) was used for analyzing peak intervals. deepTools (version 2.4.1) was used to extract and visualize signal from bigwig files. The RNA-seq differential gene expression analysis was performed using

TopHat pipeline on Galaxy. The GEO accession for ChIP-seq and RNA-seq is GSE123091.

Cell proliferation assay: C4-2-shNTC and C4-2-shZBTB7A cells were maintained in RPMI-1640 supplemented with 2% FBS plus 8% CSS. After DHT treatments, cells were stained with Muse Count &

Viability Assay kit for 5 minutes and then counted by Muse® Cell Analyzer (EMD Millipore).

Luciferase reporter assay: HEK293 cells were transfected with a Firefly luciferase reporter construct containing ~800bp promoter of MCM7 gene together with a Renilla luciferase reporter construct for 24h prior to the treatments. The activities of Firefly luciferase and Renilla luciferase were measured using the dual- luciferase reporter assay (Promega) and the results were normalized for Renilla activities.

Mouse xenografts: C4-2-tet-ZBTB7A xenografts were established in the flanks of castrated male SCID mice by injecting ~2 million cells mixed with 50% Matrigel. Doxycycline-supplemented food was introduced at ~6 weeks post-injection and tumor volume was measured by manual caliper using the formula V= (W2xL)/2.

Frozen sections were examined to confirm that the samples used for RNA and protein extraction contain predominantly non-necrotic tumor. All animal experiments were approved by the UMass Boston Institutional

Animal Care and Use Committee and were performed in accordance with institutional and national guidelines.

BETA analysis: As previously described (6), BETA (Binding and Expression Target Analysis) was performed to assess the association of AR and ZBTB7A binding sites with the expression of AR-activated versus - repressed genes. BETA software package was used (with default parameters) to integrate ChIP-seq of

ZBTB7A and AR with androgen-regulated gene expression profiling. The red and the purple lines represent the

AR-activated and AR-repressed genes, respectively. The black dashed line indicates the non-differentially expressed genes as background. Genes are cumulated by the rank on the basis of the regulatory potential score from high to low. p-value represent the significance of difference in the AR-activated or AR-repressed group compared with the non-differentially expressed group by the Kolmogorov-Smirnov test.

Statistical analysis: Data in bar graphs represent mean±SD of at least 3 biological repeats. Statistical analysis was performed using Student’s t-test by comparing treatment versus vehicle control or otherwise as indicated. p-value<0.05 (*) was considered to be statistically significant. For animal studies, one-way ANOVA was performed for the tumor volume data measured at the final day of the treatments.

RESULTS

Characterization of ZBTB7A transcriptional program in PCa cells

The Zinc finger and BTB domain containing transcription repressors, including ZBTB7A and ZBTB16, were recently identified as critical tumor suppressors in PCa cells. Using TCGA PCa dataset (17), we found that over 6% of PCa samples have deep deletion of ZBTB7A (1.2%) or ZBTB16 (5%) (Fig. 1A), indicating the critical role of this gene family in PCa development. We then compared the expression levels of ZBTB7A and

ZBTB16 in clinical cohorts of primary PCa versus metastatic CRPC. As shown in Fig. 1B, expression levels of

ZBTB7A and ZBTB16 were higher in the primary PCa cohort (TCGA) than in the CRPC cohort (SU2C) (18), suggesting that their expression is decreased during CRPC development. While the transcriptional repression activity of ZBTB16 in PCa cells has been described (14), the activity of ZBTB7A on chromatin has not yet been determined. Therefore, we next examined its chromatin binding in PCa cells. ChIP-seq analysis of ZBTB7A in

VCaP cells (an AR-amplified CRPC cell line) cultured in full serum condition identified 17,691 high confidence

1C), suggesting that ZBTB7A may preferentially bind to the upstream or downstream sites near transcription start sites (TSSs). Using a motif enrichment analysis, we found that the previously known ZBTB7A binding motif was significantly associated with ZBTB7A binding sites (Fig. 1D). We then clustered and ranked the transcription factors that may potentially co-occupy the ZBTB7A sites based on their motif enrichment scores.

As shown in Fig. 1E, the top ranked clusters were identified, and the possible factors include POLR3A (a subunit of RNA polymerase III), TRIM28 (a Tripartite motif containing transcription cofactor), PDX1 (a homeodomain transcription factor), NFKB1 (the DNA binding subunit of NFB), ING4 (a PHD-finger containing chromatin remodeling protein), and E2F2 (an E2F family transcription factor). The POLR3A cluster (cluster 1) also includes a number of nuclear hormone receptors.

To study the effect of ZBTB7A binding on gene transcription in PCa cells, we performed an RNA-seq analysis in VCaP cells (cultured in full serum condition) transfected with siRNA targeting ZBTB7A or nontarget control (NTC) and then carried out Binding and Expression Target Analysis (BETA) (15) to identify potential

ZBTB7A directly regulated genes. As shown in Fig. 1F, chromatin binding of ZBTB7A was associated with both

ZBTB7A-activated (p=10-37) and -repressed genes (p=10-21), suggesting that ZBTB7A may have both transcriptional activator and repressor functions in PCa cells. As the BETA analysis also ranked the potential for genes that may be directly regulated by ZBTB7A (Supplementary Table S1), we next performed KEGG pathway analysis (using DAVID 6.8) on these potential direct targets of ZBTB7A. Interestingly, the activator activity of ZBTB7A was associated with specific cancer types, including prostate and non-small cell lung cancers (Fig. 1G), suggesting that ZBTB7A may retain some tumor promoting activities in PCa cells through gene activation. The repressor function of ZBTB7A was enriched for transcription and translation regulation, chemokine signaling pathway, and DNA replication, supporting its tumor suppressor role in PCa cells.

Interestingly, the suppression of chemokine signaling was also supported by a recent study showing that

ZBTB7A regulates the infiltration and composition of immune cells within PCa tumor through repressing the expression of a chemokine, CXCL5 (19). To further determine which signaling pathways are most impacted by

ZBTB7A, we performed a gene set enrichment analysis (GSEA) on ZBTB7A-regulated genes. As seen in Fig.

1H, while ZBTB7A-activated genes were not significantly associated with any known pathways, the expression of ZBTB7A-repressed genes were highly enriched for the activation function of E2Fs, an oncogenic transcription factor family that plays critical role in activating DNA replication and cell cycle progression, indicating ZBTB7A may function to suppress the activity of E2Fs. Collectively, these global cistrome and transcriptome studies of ZBTB7A revealed its transcription factor activities in PCa cells and linked its tumor suppressor function to the possible downregulation of E2F activity.

ZBTB7A chromatin binding is associated with transcriptional repression activity of AR

Our previous studies in multiple PCa models and human PCa samples have indicated that repressing E2F- activated DNA replication is the major transcriptional repression activity of AR (4,6). Therefore, we next assessed the global impact of ZBTB7A on AR transcriptional activity. Since the above global studies were done in VCaP cells under the full serum condition, which contains substantial levels of testosterone and DHT

(dihydrotestosterone, a potent form of testosterone) as well as other steroid hormones (20), we were able to perform combined analyses with the previously published AR ChIP-seq and gene expression profiling datasets based on VCaP cells treated with 10nM DHT (4,16), we found that ~30% of ZBTB7A binding peaks (5,875 out of 17,691) were overlapped with AR binding sites (Fig. 2A) and these sites were highly enriched for promoter binding (Supplementary Fig. S1). The binding intensity of ZBTB7A was also highly correlated with the intensity of AR binding (Fig. 2B), indicating a possible interaction of ZBTB7A and AR to co-regulate gene transcription.

We then performed BETA to determine the global association of ZBTB7A binding with the AR regulation of gene transcription. As seen in Fig. 2C, while AR repressive function appeared to associate with ZBTB7A unique sites (p=10-16), it was more strongly correlated with AR and ZBTB7A overlapping sites (p=10-39) and not with AR unique sites (p=0.839). In contrast, AR activation function was associated more strongly with ZBTB7A- absent AR binding sites (p=10-17), and was less significantly associated with ZBTB7A and AR overlapping sites

(p=10-15) or ZBTB7A unique sites (p=10-7). Collectively, these global analyses of ZBTB7A and AR binding sites strongly indicate that ZBTB7A may be involved in AR-mediated transcriptional repression in PCa cells.

We next examined the functions for the ZBTB7A-repressed genes that have nearby AR and ZBTB7A overlapping sites (Supplementary Table S2, ranking of genes based on BETA). As shown in Fig. 2D, this

To further confirm that ZBTB7A can bind to AR occupied repression sites, we performed a motif enrichment analysis on the AR binding sites adjacent to the androgen-repressed gene loci. Consistently, we found that

ZBTB7A binding motif was among the top ranked motifs enriched at those AR repression sites (Fig. 2E). The

E2F binding motif was also top ranked, consistent with our previous studies showing AR interaction with E2F-

Rb complex at the repression sites (4,6). A previous study found that ZBTB7A can directly interact with AR through the POZ domain of ZBTB7A and ligand-binding domain (LBD) of AR, and this interaction impairs AR activation of PSA (21). Using a coimmunoprecipitation assay in VCaP cells, we confirmed that the endogenous

ZBTB7A can interact with AR and the interaction appeared to be enhanced by androgen stimulation (Fig. 2F).

We next sought to determine whether the LBD of AR is the domain responsible for AR interaction with ZBTB7A.

Experiments in cells transiently overexpressing AR fragments and ZBTB7A were then carried out and the result indicated that the entire C-terminal domains, including both LBD and DNA binding domain (DBD), may be required for the full interaction with ZBTB7A (Fig. 2G). Overall, these data support the function of ZBTB7A in mediating AR-dependent transcriptional repression activity in PCa cells through a possible direct interaction with AR protein.

AR recruitment of ZBTB7A is required for the transcriptional repression function of AR on E2F- regulated genes

The current model on AR transcriptional repression activity based on our previous studies is that the rapid AR recruitment of Rb can reinforce the E2F-Rb repressor complex to suppress the transcription of genes mediating DNA replication/repair and cell cycle (6). Since the recruitment of Rb is one major event that mediates this specific activity of AR, we next determined whether AR may also recruit ZBTB7A to those suppression sites. To test this hypothesis, we performed additional ChIP-seq of ZBTB7A in VCaP cells treated with or without 10nM DHT for 4 hours (h). As seen in Fig. 3A and B, the binding intensity of ZBTB7A at those

ZBTB7A and AR overlapping sites was rapidly increased by the short-term androgen treatment and correlated with the binding intensity of AR and Rb, suggesting that AR may directly recruit ZBTB7A to those sites.

Supportively, the binding intensity of ZBTB7A at those AR and Rb overlapping sites was also similarly androgen-induced (Supplementary Fig. S2A). We then conducted BETA to determine whether AR recruited

ZBTB7A and Rb are associated with AR repression function. As shown in Fig. 3C and 3D, AR repression function more significantly associated with AR+/ZBTB7A+ sites (p=10-43) and AR+/ZBTB7A+/Rb+ sites (p=10-31) than AR activation function (p=10-23 and p=10-13, respectively). Moreover, the absence of either Rb

(AR+/ZBTB7A+/Rb-) or ZBTB7A binding (AR+/ZBTB7A-/Rb+) at those AR repression sites significantly decreased their association with AR repression function (p=10-5 and p=10-9, respectively), suggesting that AR recruitment of ZBTB7A and Rb may be both required for the full repression activity of AR.

Since the previous study indicates that ZBTB7A may regulate Rb expression in transgenic mouse model (12), we first examined whether ZBTB7A upregulates Rb in PCa cells. As shown in Supplementary Fig.

S2B, Rb protein expression was not affected by knocking down ZBTB7A in VCaP cells, suggesting that

ZBTB7A does not regulate Rb in human CRPC cells. To further determine whether ZBTB7A contributes to the

AR-mediated repression activity on DNA replication, we selected a panel of previously identified Rb-dependent

AR-repressed DNA replication/repair genes, including MCMs (minichromosome maintenance complex genes),

BLM (a Bloom syndrome RecQ like helicase gene), FANCI (a Fanconi anemia complementation group gene), and TK1 (a thymidine kinase gene) (6), for the subsequent studies. The rapid increase of ZBTB7A binding by androgen-stimulation at those previously identified AR-regulated promoters/enhancers (6) was confirmed using

ChIP-qPCR (Fig. 3E). We next cloned the MCM7 promoter (~800bp fragment, containing AR, ZBTB7A, and

E2F1 binding sites) into a luciferase reporter to examine the effect of ZBTB7A on E2F1 activity. As seen in Fig.

3F, E2F1 significantly induced MCM7-promoter activity, which can be suppressed by Rb. Importantly, the expression of ZBTB7A alone markedly downregulated the E2F1 activity and coexpression of ZBTB7A and Rb can further repress the E2F1 activity. This result suggests that ZBTB7A and Rb may function in parallel to collaboratively suppress E2F1-mediate transcriptional activation. Interestingly, overexpressing AR alone was not sufficient to suppress E2F1 activity (Fig. 3G), further suggesting that AR may indirectly repress E2F1 activity in PCa cells through recruitment of ZBTB7A and Rb. Furthermore, we also demonstrated that doxycycline-induced silencing of ZBTB7A in VCaP-tet-shZBTB7A stable cells (expressing tetracycline- regulated lentiviral shRNA against ZBTB7A) impaired the androgen-induced repression on these DNA

ZBTB7A mediates AR repression of PCAT-1 lncRNA

The gene profiling database used to study androgen regulation in VCaP cells was based on Affymetrix gene microarrays (4), which primarily detect protein coding genes. However, recent studies have revealed important functions of non-coding RNAs, particularly lncRNAs, in regulating gene transcription in tumor cells (22).

Therefore, we sought to identify the androgen-repressed lncRNAs in androgen-treated PCa cells by using

RNA-seq. In VCaP cells treated with 10nM DHT for 24h, 111 lncRNAs were identified as androgen-induced genes and 245 lncRNAs were identified as androgen-repressed genes (Supplementary Table S3). Amongst the identified androgen-induced lncRNA subset, PCAT-14 and PCAT-18 have been previously suggested as biomarkers for predicting PCa outcomes (23,24). PRCAT47 (also called ARLNC1) was another recently identified androgen-upregulated gene that functions to stabilize the AR transcript and enhances AR signaling and PCa tumor growth (25). Amongst the androgen-repressed lncRNA subset, PCAT-29 has been reported previously as an androgen-repressed tumor suppressor gene in PCa cells (26). Importantly, we identified

PCAT-1 (prostate cancer associated transcript 1) as a novel androgen-repressed gene, which promotes PCa by mechanisms such as stabilizing MYC protein, repressing BRCA2, and activating AKT (27-29). Therefore, suppressing the expression of this oncogenic lncRNA may be an important activity for AR to act as a tumor suppressor in PCa.

We next examined androgen-regulation of PCAT-1 expression in PCa cells. As seen in Supplementary

Fig. S3A, PCAT-1 expression was significantly decreased by DHT treatment in VCaP cells and this suppression activity was abolished when cells were treated with an HDAC1 inhibitor (mocetinostat), indicating a critical function of HDAC1 which may be required for AR repression on PCAT-1. Interestingly, while PCAT-1 was not clearly androgen-repressed in androgen-dependent LNCaP PCa cells, which express less AR than

CRPC VCaP cells (4), it was significantly repressed by androgen in AR-overexpressing LNCaP (LN-AR) cells

(Supplementary Fig. S3B), suggesting that the repression on PCAT-1 requires high levels of AR expression.

To assess the androgen regulation of PCAT-1 in vivo, we examined its expression in a previously established

VCaP xenograft tumor progression model (30). In this model, we have clearly demonstrated the restoration of the expression of PSA and TMPRSS2-ERG due to regained AR activation activity and the increased expression of AR, AKR1C3 (an androgen synthesis gene), and DNA replication genes due to loss of AR repression activity in the castration-resistant stage of the tumor (4,6,30). As seen in Supplementary Fig. S3C, the expression of PCAT-1 was rapidly increased in at least a portion of xenograft tumors upon castration and the increased level was retained in the relapsed tumors, suggesting ADT may alleviate the AR repression on

PCAT-1 and hence result in the increased expression of PCAT-1 in CRPC.

We next determined whether ZBTB7A is involved in the AR-mediated repression of PCAT-1. Through examining the AR and ZBTB7A ChIP-seq results, we identified AR and ZBTB7A chromatin binding sites in the

PCAT-1 locus. Interestingly, although the identified AR binding site (named S1) was very close to the ZBTB7A binding site (named S2), these two peaks were not exactly overlapping. Using ChIP-qPCR analysis, we showed that AR chromatin binding was significantly stimulated by DHT treatment on S1 and weakly increased on S2 site (Supplementary Fig. S3D, left panel). However, ZBTB7A binding was increased by DHT on the S2 site (Supplementary Fig. S3D, middle panel), suggesting that AR binding at S1 site may increase the recruitment of ZBTB7A at S2 site. Moreover, we also found that silencing ZBTB7A impaired the AR repression activity on PCAT-1 transcript in VCaP cells (Supplementary Fig. S3E), indicating that ZBTB7A contributes to the full repression activity of AR on PCAT-1.

ZBTB7A suppresses CRPC tumor growth in vitro

Although loss of Zbtb7a expression in conjunction with loss of Pten in mouse prostate epithelial cells promote

PCa development (12), it is not clear whether ZBTB7A can similarly function as a tumor suppressor in human

CRPC cells. Although VCaP cells, which were derived from CRPC bone metastases, were used as the primary model for the mechanistic studies, these cells cannot directly form xenograft tumors in castrated mice and it is difficult to use this model for the subsequent functional studies. Therefore, to further study the tumor suppressor role of ZBTB7A in vitro and in vivo we selected a well-known LNCaP-derived CRPC model, C4-2 cells, to generate the stable cell line that can inducibly overexpress ZBTB7A. Similar to VCaP cells, AR is

More importantly, in these cells AR also similarly suppresses the expression of the majority of DNA-replication genes that are androgen-repressed in VCaP cells and this repression activity of AR is partially impaired by silencing of Rb (Supplementary Fig. S4). C4-2 cells express comparable level of ZBTB7A as in VCaP cells and high-passage LNCaP cells, which are also resistant to ADT (31), while the AR-negative PC-3 cells express higher level of ZBTB7A (Fig. 4A). Importantly, ZBTB7A was also recruited by AR to the promoter regions of the

DNA replication genes and this chromatin recruitment can be impaired by AR antagonist treatment

(enzalutamide) or AR targeted siRNA (Supplementary Fig. S5A and B). Silencing ZBTB7A in C4-2 cells increased cell proliferation in hormone-reduced condition and compromised the anti-proliferative effect of DHT in C4-2 cells (Fig. 4B-D), indicating that the endogenous ZBTB7A is required for tumor suppressor function of

AR in CRPC cells.

To further study the effect of restoring ZBTB7A expression in CRPC cells, we established a C4-2 stable cell line that express tetracycline-regulated V5-tagged ZBTB7A (Fig. 4E). Firstly, induced overexpression of

ZBTB7A further decreased the mRNA and protein expression levels of the DNA replication genes that were suppressed by DHT treatment (Fig. 4F and G), consistent with the effect of silencing ZBTB7A in VCaP cells.

Secondly, using a V5 pull-down assay we also demonstrated that AR, Rb, and HDAC1 can all interact with

ZBTB7A (Fig. 4H), suggesting that ZBTB7A may be a component of the AR-Rb suppressor complex.

Conversely, we also immunoprecipitated endogenous E2F1 or Rb in these cells and found that the V5-tagged

ZBTB7A can be coimmunoprecipitated with these proteins (Fig. 4I), indicating that ZBTB7A can interact with

E2F1. However, the interaction of E2F1 with Rb was not significantly affected by overexpression of ZBTB7A, suggesting that the repression activity of ZBTB7A on E2F1 may not be mediated through Rb. Consistently, silencing or overexpressing ZBTB7A has little effect on Rb binding to the promoters of DNA replication genes

(Supplementary Fig. S6A-C), further suggesting that ZBTB7A can suppress E2F activity without increasing chromatin binding of Rb-E2F repressor complex. Nonetheless, induced expression of ZBTB7A by using lower dose of doxycycline to minimize any toxicity effect (Supplementary Fig. S7) also led to decreased cell growth, and more importantly could act in synergy with DHT treatment, particularly with lower doses of DHT (1nM) (Fig.

4J). Consistent with the effect on growth, overexpression of ZBTB7A also enhanced the suppression effect of

1nM DHT on DNA replication genes (Fig. 4K).

Overexpression of ZBTB7A delays the recurrence of CRPC tumor treated by high-dose testosterone

Finally, we assessed the effect of ZBTB7A overexpression on CRPC tumor growth in vivo. As seen in Fig. 5A, induced overexpression of ZBTB7A (prior to the establishment of xenograft tumor) significantly delayed the development of C4-2 CRPC xenograft tumor, suggesting a critical and potent tumor suppressor function of

ZBTB7A in CRPC cells. The doxycycline supplemented food had no toxic effect on the health of mice and the growth of C4-2 xenograft tumors (Supplementary Fig. S8A and B). We then determined whether overexpression of ZBTB7A can enhance the efficacy of high-dose testosterone treatment in this CRPC model.

For this experiment, we allowed the CRPC xenograft tumor to establish (~50mm3) prior to the treatments. As seen in Fig. 5B, while 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 treatments. Significantly, the combination treatment was able to delay the recurrence of the CRPC tumor, suggesting that overexpression of ZBTB7A can synergize with testosterone to suppress CRPC progression. Despite the limited tissue materials in treatment groups, we were able to extract a small amount of RNA to examine the effect of the combination treatment on a few DNA replication genes. As shown in Supplementary Fig. S9, while the expression levels of the DNA replication genes may be restored in the doxycycline- or testosterone-treated group, they appeared to remain repressed in the combination treatment group. Overall, these in vitro and in vivo studies clearly demonstrated the critical tumor suppressor function of ZBTB7A in CRPC cells and suggested a potential therapeutic strategy by enhancing ZBTB7A expression or activity to improve the efficacy of the high-dose testosterone therapy in CRPC patients. The model for ZBTB7A-mediated transcriptional repression activity of AR on a subset of E2F-regulated DNA replication genes was summarized in Fig. 5C.

DISCUSSION

Loss of expression of zinc finger and BTB domain containing transcription factors, such as ZBTB7A and

ZBTB16, are commonly seen in PCa tumors, and their expression is decreased in more aggressive CRPC

(see Fig. 1). However, the biological functions of these genes in PCa cells remain to be identified. ZBTB16 is a classic AR regulated gene and it can function as a tumor suppressor in PCa cells through inhibiting the MAPK pathways (14). In contrast, ZBTB7A is not regulated by AR and was previously known as an oncogene in many cancer types but recently reported to play a tumor suppressor role in PCa. The loss of Zbtb7a expression in conjunction with loss of Pten in mouse prostate epithelial cells was previously shown to drive the development of aggressive PCa (12). This tumor suppressive activity of ZBTB7A was largely attributed to maintaining the expression of Rb, a critical cell cycle regulator that mediates G1/S transition, through the negative regulation of

SOX9 activity on activating an Rb-targeting miRNA. In this study, our integrated ZBTB7A cistrome and transcriptome analyses have indicated that ZBTB7A directly represses the E2F-regulated genes, consistent with those previous findings. More importantly, our mechanistic study of ZBTB7A has demonstrated that

ZBTB7A can repress DNA replication and cell cycle progression through a very distinct mechanism by which

ZBTB7A was recruited by AR to the E2F binding sites and suppresses its transcriptional activation function.

We have previously reported this transcriptional repressor activity of AR and revealed its critical biological function on repressing DNA replication and cell cycle (4). We have further shown in another study that this transcriptional repressor activity of AR was through enhancing Rb chromatin binding and thus strengthening the Rb-E2F repressor complex (6). Importantly, a recent study using castration-resistant LuCaP PDX models also indicates that the most robust molecular phenotype for the high-dose testosterone treatment is the suppression of E2F transcriptional output (32). In this study, we report that AR can rapidly recruit ZBTB7A to those AR and E2F-Rb overlapping sites within DNA replication genes and this recruitment can further enhance the transcriptional repression of the target genes. Mechanistically, we have also demonstrated that ZBTB7A can directly interact with E2F1 and negatively regulate its transcriptional activity (see Fig. 3F and 4I). Since

ZBTB7A was known to recruit HDACs (also see Fig. 4H), this transcriptional corepressor function of ZBTB7A on AR may be through strengthening the recruitment of HDACs that can deacetylate histone 3 lysine 27 and thus represses gene transcription activated by E2F.

In addition to DNA replication genes, we have also identified the lncRNA PCAT-1 as a novel AR- repressed gene in PCa cells. One of the major functions of this oncogenic lncRNA in PCa cells is to regulate

MYC oncoprotein (27). Therefore, ZBTB7A may suppress MYC activity through transcriptionally repressing

PCAT-1 expression. Overall, this study identified a biologically important lncRNA as a novel repression target of AR and demonstrated the role of ZBTB7A in mediating this repression process.

Furthermore, we have demonstrated in vitro and in vivo that ZBTB7A is a potent tumor suppressor, which can markedly repress CRPC tumor growth. More importantly, we have demonstrated that overexpression of ZBTB7A can enhance the growth suppressive activity of high-dose androgen treatment on

CRPC cells in vitro and in vivo. Based on our results (see Fig. 4J), even lower concentration of androgen treatment can suppress PCa cell proliferation when ZBTB7A is expressed in high levels. This finding may provide an explanation of why a subset of PCa or CRPC tumors have to select for the decrease or loss of

ZBTB7A expression in order to escape from AR-mediated growth-suppression activity. Significantly, our study also provides a rationale to therapeutically enhance the high-dose testosterone treatment in CRPC (currently in phase II clinical trials) (10) through elevating the expression or activity of ZBTB7A. Future study is clearly needed to identify the actionable targets that are involved in regulating ZBTB7A expression or activity in CRPC cells.

ACKNOWLEDGMENTS

This work is supported by grants from NIH (R00 CA166507 and R01 CA211350 to C. Cai, P01 CA163227 to S.

Balk), DOD (W81XWH-15-1-0554 to S. Gao, and W81XWH-16-1-0445 to C. Cai), CIHR (142246, 152863,

152864, and 159567 to H. He), Prostate Cancer Canada (RS2016-1022 and TAG2018-2061 to H. He),

NSERC (498706 to H. He), Terry Fox New Investigator Award (1069 to H. He) and Princess Margaret Cancer

Foundation to H. He). We thank Dr. Jill A. Macoska and Susan C. Patalano (Genomics Core, University of

Massachusetts Boston) for assistance and guidance of high-throughput sequencing.

CONFLIC OF INTREST

The authors declare no conflict of interest

REFERENCS

1. Yuan X, Cai C, Chen S, Chen S, Yu Z, Balk SP. Androgen receptor functions in castration-resistant

prostate cancer and mechanisms of resistance to new agents targeting the androgen axis. Oncogene

2014;33:2815-25

2. de Bono JS, Logothetis CJ, Molina A, Fizazi K, North S, Chu L, et al. Abiraterone and increased survival in

metastatic prostate cancer. N Engl J Med 2011;364:1995-2005

3. Green SM, Mostaghel EA, Nelson PS. Androgen action and metabolism in prostate cancer. Mol Cell

Endocrinol 2012;360:3-13

4. Cai C, He HH, Chen S, Coleman I, Wang H, Fang Z, et al. Androgen receptor gene expression in prostate

cancer is directly suppressed by the androgen receptor through recruitment of lysine-specific demethylase

1. Cancer Cell 2011;20:457-71

5. Zhao JC, Yu J, Runkle C, Wu L, Hu M, Wu D, et al. Cooperation between Polycomb and androgen

receptor during oncogenic transformation. Genome Res 2012;22:322-31

6. Gao S, Gao Y, He HH, Han D, Han W, Avery A, et al. Androgen Receptor Tumor Suppressor Function Is

Mediated by Recruitment of Retinoblastoma Protein. Cell Rep 2016;17:966-76

7. Schweizer MT, Antonarakis ES, Wang H, Ajiboye AS, Spitz A, Cao H, et al. Effect of bipolar androgen

therapy for asymptomatic men with castration-resistant prostate cancer: Results from a pilot clinical study.

Science translational medicine 2015;7:269ra2

8. Schweizer MT, Wang H, Luber B, Nadal R, Spitz A, Rosen DM, et al. Bipolar Androgen Therapy for Men

With Androgen Ablation Naive Prostate Cancer: Results From the Phase II BATMAN Study. Prostate

2016;76:1218-26

9. Lam HM, Corey E. Supraphysiological Testosterone Therapy as Treatment for Castration-Resistant

Prostate Cancer. Front Oncol 2018;8:167

10. Teply BA, Wang H, Luber B, Sullivan R, Rifkind I, Bruns A, et al. Bipolar androgen therapy in men with

metastatic castration-resistant prostate cancer after progression on enzalutamide: an open-label, phase 2,

multicohort study. Lancet Oncol 2018;19:76-86

11. Lunardi A, Ala U, Epping MT, Salmena L, Clohessy JG, Webster KA, et al. A co-clinical approach identifies

mechanisms and potential therapies for androgen deprivation resistance in prostate cancer. Nat Genet

2013;45:747-55

12. Wang G, Lunardi A, Zhang J, Chen Z, Ala U, Webster KA, et al. Zbtb7a suppresses prostate cancer

through repression of a Sox9-dependent pathway for cellular senescence bypass and tumor invasion. Nat

Genet 2013;45:739-46

13. Liu XS, Haines JE, Mehanna EK, Genet MD, Ben-Sahra I, Asara JM, et al. ZBTB7A acts as a tumor

suppressor through the transcriptional repression of glycolysis. Genes Dev 2014;28:1917-28

14. Hsieh CL, Botta G, Gao S, Li T, Van Allen EM, Treacy DJ, et al. PLZF, a tumor suppressor genetically lost

in metastatic castration-resistant prostate cancer, is a mediator of resistance to androgen deprivation

therapy. Cancer Res 2015;75:1944-8

15. Wang S, Sun H, Ma J, Zang C, Wang C, Wang J, et al. Target analysis by integration of transcriptome and

ChIP-seq data with BETA. Nature protocols 2013;8:2502-15

16. Cai C, Wang H, He HH, Chen S, He L, Ma F, et al. ERG induces androgen receptor-mediated regulation of

SOX9 in prostate cancer. J Clin Invest 2013;123:1109-22

17. Cancer Genome Atlas Research N. The Molecular Taxonomy of Primary Prostate Cancer. Cell

2015;163:1011-25

18. Robinson D, Van Allen EM, Wu YM, Schultz N, Lonigro RJ, Mosquera JM, et al. Integrative clinical

genomics of advanced prostate cancer. Cell 2015;161:1215-28

19. Bezzi M, Seitzer N, Ishikawa T, Reschke M, Chen M, Wang G, et al. Diverse genetic-driven immune

landscapes dictate tumor progression through distinct mechanisms. Nat Med 2018;24:165-75

20. Song W, Khera M. Physiological normal levels of androgen inhibit proliferation of prostate cancer cells in

vitro. Asian J Androl 2014;16:864-8

21. Cui J, Yang Y, Zhang C, Hu P, Kan W, Bai X, et al. FBI-1 functions as a novel AR co-repressor in prostate

cancer cells. Cell Mol Life Sci 2011;68:1091-103

22. Mouraviev V, Lee B, Patel V, Albala D, Johansen TE, Partin A, et al. Clinical prospects of long noncoding

RNAs as novel biomarkers and therapeutic targets in prostate cancer. Prostate Cancer Prostatic Dis

2016;19:14-20

23. Crea F, Watahiki A, Quagliata L, Xue H, Pikor L, Parolia A, et al. Identification of a long non-coding RNA as

a novel biomarker and potential therapeutic target for metastatic prostate cancer. Oncotarget 2014;5:764-

24. White NM, Zhao SG, Zhang J, Rozycki EB, Dang HX, McFadden SD, et al. Multi-institutional Analysis

Shows that Low PCAT-14 Expression Associates with Poor Outcomes in Prostate Cancer. Eur Urol

2017;71:257-66

25. Zhang Y, Pitchiaya S, Cieslik M, Niknafs YS, Tien JC, Hosono Y, et al. Analysis of the androgen receptor-

regulated lncRNA landscape identifies a role for ARLNC1 in prostate cancer progression. Nat Genet

2018;50:814-24

26. Malik R, Patel L, Prensner JR, Shi Y, Iyer MK, Subramaniyan S, et al. The lncRNA PCAT29 inhibits

oncogenic phenotypes in prostate cancer. Mol Cancer Res 2014;12:1081-7

27. Prensner JR, Chen W, Han S, Iyer MK, Cao Q, Kothari V, et al. The long non-coding RNA PCAT-1

promotes prostate cancer cell proliferation through cMyc. Neoplasia 2014;16:900-8

28. Prensner JR, Chen W, Iyer MK, Cao Q, Ma T, Han S, et al. PCAT-1, a long noncoding RNA, regulates

BRCA2 and controls homologous recombination in cancer. Cancer Res 2014;74:1651-60

29. Shang Z, Yu J, Sun L, Tian J, Zhu S, Zhang B, et al. LncRNA PCAT1 activates AKT and NF-kappaB

signaling in castration-resistant prostate cancer by regulating the PHLPP/FKBP51/IKKalpha complex.

Nucleic Acids Res 2019;47:4211-25

30. Cai C, Wang H, Xu Y, Chen S, Balk SP. Reactivation of androgen receptor-regulated TMPRSS2:ERG

gene expression in castration-resistant prostate cancer. Cancer Res 2009;69:6027-32

31. Igawa T, Lin FF, Lee MS, Karan D, Batra SK, Lin MF. Establishment and characterization of androgen-

independent human prostate cancer LNCaP cell model. Prostate 2002;50:222-35

32. Lam HM, Nguyen HM, Labrecque MP, Brown LG, Coleman IM, Gulati R, et al. Durable Response of

Enzalutamide-resistant Prostate Cancer to Supraphysiological Testosterone Is Associated with a

Multifaceted Growth Suppression and Impaired DNA Damage Response Transcriptomic Program in

Patient-derived Xenografts. Eur Urol 2019

33. Habib N, Kaplan T, Margalit H, Friedman N. A novel Bayesian DNA motif comparison method for clustering

and retrieval. PLoS Comput Biol 2008;4:e1000010

FIGURE LEGENDS

Figure 1. Functional characterization of ZBTB7A transcriptional activity in PCa cells

(A) Genetic alterations of ZBTB7A and ZBTB16 in TCGA PCa cohort (retrieved in cBioPortal). (B) The expression levels of ZBTB7A and ZBTB16 (normalized to GAPDH) in SU2C mCRPC cohort versus TCGA primary PCa cohort. (C) ZBTB7A ChIP-Seq was done in VCaP cells cultured in full FBS and the genomic distribution of ZBTB7A binding was shown. (D) A Bayesian DNA motif comparison method, BLiC (33), was applied on ZBTB7A ChIP-Seq peaks (cutoff: p=10-15). The ZBTB7A binding motif was found with z-score=-

50.87. (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 were 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 non- target 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) Gene set enrichment analysis (GSEA) was carried out on

ZBTB7A regulated genes.

Figure 2. ZBTB7A chromatin binding is associated with transcriptional repression activity of AR

(A) The Venn diagram showed 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 (±1 kb). (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

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 23, 2019; DOI: 10.1158/0008-5472.CAN-19-0815 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 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 were 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 10nM DHT for 4h, 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, NTD+DBD: 1-628 aa,

DBD+LBD: 538-919 aa, and LBD: 662-919 aa). AR fragments were immunoprecipitated by anti-HA beads, followed by immunoblotting for V5 (V5-ZBTB7A).

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 10nM DHT for 4h in hormone-depleted media. The heatmap view for the signal intensities of aligned reads across all ZBTB7A binding sites (±3 kb) was 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 nM DHT for 1h. (F, G) MCM7- promoter (~800bp) driven luciferase report activity was measured in HEK293 cells transfected with (F) E2F1,

Rb, and/or ZBTB7A in full serum, or (G) E2F1 and/or AR in presence or absence of 10nM DHT. (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 0.1µg/ml doxycycline for 2d and then with10nM DHT for 24h. The mRNA expressions of a panel of Rb mediated AR- repressed genes were measured.

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 non-target-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 10nM DHT for 2d. (E) Immunoblotting for V5 expression in C4-2-tet-ZBTB7A cells stimulated with 0-1µg/ml doxycycline for 2d. (F, G) The expressions of a panel of AR-repressed DNA replication genes were examined in C4-2-tet-ZBTB7A cells treated with 0.25 µg/ml doxycycline and 10nM DHT for 2d. (F) The mRNA expression was measured by qRT-PCR and (G) the protein expression was measured by immunoblotting. (H) V5-ZBTB7A was immunoprecipitated using V5-antibody- condugated beads in C4-2-tet-ZBTB7A cells stimulated by 0.25µg/ml doxycycline for 2d, followed by immunoblotting for AR, Rb, HDAC1, and ZBTB7A. (I) E2F1 or Rb was immunoprecipitated in C4-2-tet-ZBTB7A cells stimulated by 0.25µg/ml doxycycline for 2d, followed by immunoblotting for ZBTB7A, Rb, and E2F1. (J)

Cell proliferation was measured in C4-2-tet-ZBTB7A cells treated with 0.1 µg/ml doxycycline and with 0-10nM

DHT for 0-6d. (K) The expressions of a panel of AR-repressed DNA replication genes were examined in C4-2- tet-ZBTB7A cells treated with 0.1 µg/ml doxycycline and 1nM DHT for 2d.

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) were 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 (~50mm3) prior to the treatments. A cohort of

24 mice were randomly divided into four arms: (1) on regular diet with vehicle injection (cotton oil); (2) on regular diet with testosterone treatment (10mg/kg via i.p. injection every day); (3) on doxycycline-supplemented diet with vehicle injection (cotton oil); and (4) 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.

ZBTB7A mediates the transcriptional repression activity of the androgen receptor in prostate cancer

Changmeng Cai, Dong Han, Sujun Chen, et al.

Cancer Res Published OnlineFirst August 23, 2019.

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

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2019/08/23/0008-5472.CAN-19-0815.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet been Manuscript edited.

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/early/2019/08/23/0008-5472.CAN-19-0815. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.