Prostate cancer (PCa) is one of the most common cancers in men. Primary PCa depends on androgen for growth and survival by binding to the C-terminal ligand-binding domain of androgen receptor (AR), a transcriptional factor that regulates multiple gene expression. 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 a more aggressive castration-resistant prostate cancer (CRPC) and AR remains important in this stage of disease.

Although AR is well known for its transcriptional activator function, it can also suppress the expression of a subset of genes, including AR itself, multiple androgen synthetic genes, and a group of DNA replication genes. 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 Rb-E2F suppressor complex. This novel activity of AR provides one molecular basis for treating CRPC patients with high level androgens. Importantly, we have also shown in vitro that this tumor-suppressor function of AR can be enhanced by CDK4/6 inhibitors, which block Rb phosphorylation. Although most CRPC are Rb-positive, recent sequencing studies indicate that ~10-15% of CRPC showed Rb loss in their chromosome. Therefore, we have generated tetracycline-inducible Rb-silencing CRPC cell lines to further examine the impact of Rb-loss on the transcriptional repression activity of AR. Consistently, we have found that Rb-silencing globally impaired the AR repression on DNA replication genes. Furthermore, we have also examined the AR-independent function of Rb and found that Rb-loss led to the activation of neuroendocrine genes.

To further understand the mechanisms of how AR performs its transcriptional repression activity, we analyzed AR ChIP-seq data and androgen-regulated gene profile. This analysis resulted in the identification of ZBTB7A binding motif as the most enriched motif within AR repression sites. ZBTB7A, also known as LRF/POKEMON, is a zinc finger and BTB domain containing transcription repressor consisting of a protein-protein interacting BTB domain at N-terminus and DNA binding zinc fingers at C-terminus. Even though ZBTB7A has been identified as a proto-oncogene, a recent study using transgenic mouse models shows that it functions as a tumor suppressor in driving PCa progression with PTEN-null background. Therefore, we hypothesize that ZBTB7A might play a key role in regulating the Rb-dependent transcriptional repressor activity of AR.

To globally determine this function of ZBTB7A, we carried out ZBTB7A ChIP-seq to identify high confident binding peaks. Motif analysis on these peaks indicated the marked enrichment of binding motif of ZBTB family members, suggesting that these binding sites are specific for ZBTB7A binding. Significantly, ~30% of these ZBTB7A binding sites overlapped with AR binding sites, particularly at promoter region. Using the unbiased Binding and Expression Target Analysis, we further found that these ZBTB7A and AR cobinding sites were significantly associated with the expression of AR-repressed genes. More importantly, the binding of ZBTB7A at these sites highly associated with Rb binding and silencing ZBTB7A impaired the AR repression activity on the DNA replication genes, suggesting that ZBTB7A may cooperate with Rb in regulating AR repressor activity. By coimmunoprecipitation assays, we found that ZBTB7A physically interacts with AR and Rb and these interactions are enhanced by androgen stimulation, suggesting that ZBTB7A is an additional component in the AR repressor complex. Furthermore, by additional ChIP-seq analyses we have also found that ZBTB7A binding at those AR repression sites was rapidly increased upon androgen stimulation, further indicating that AR may recruit ZBTB7A to the chromatin and ZBTB7A and Rb may cooperatively mediate the transcriptional activity of AR.

This study provides novel insights in the molecular basis of transcriptional repression function of AR and may have significant therapeutic impact on enhancing current androgen therapy to treat CRPC patients by enhancing the expression or activity of ZBTB7A.

Citation Format: Wanting Han, Dong Han, Sujun Chen, Shuai Gao, Housheng He, Changmeng Cai. Rb and ZBTB7A cooperatively mediate the transcriptional repression function of androgen receptor [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A066.