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. 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 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 1 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. 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 2 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), 3 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. we show that a significant portion of ZBTB7A binding sites overlap with AR binding sites and that these 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. 4 Downloaded from