Prostate cancer transitions from an early treatable form to the lethal castration-resistant prostate cancer (CRPC). Androgen receptor (AR) and constitutively active AR splice variants, like AR-V7, may be major drivers of CRPC. Our lab recently identified a novel mechanism of AR regulation via the transmembrane protein TM4SF3 (Transmembrane 4 superfamily 3), which exhibits a physical interaction, nuclear co-localization, and mutual stabilization with AR. Here we have mapped the interaction domains within AR and TM4SF3 and discovered that TM4SF3 also physically interacts with AR-V7, regulating its protein stability and the viability of CRPC cells expressing AR-V7. Ubiquitination of TM4SF3 and AR-V7 was detected for the first time and TM4SF3 interaction with either AR or AR-V7 resulted in mutual de-ubiquitination of both proteins, showing that mutual stabilization results from de-ubiquitination. Interestingly, nuclear TM4SF3 was co-recruited to the promoters of AR- and AR-V7-regulated genes and required for their expression, showing that TM4SF3 interaction is critical for their transcriptional functions. The results collectively show the multiple critical regulatory functions of TM4SF3 on AR or AR-V7 in prostate cancer cells.

Prostate cancer starts as a treatable hormone-dependent disease, but often ends in a drug-resistant form called castration-resistant prostate cancer (CRPC). Despite the development of the anti-androgens Enzalutamide and Abiraterone for CRPC, which target the Androgen Receptor (AR), drug resistance usually develops within 6 months and metastatic CRPC (mCRPC) leads to lethality. EZH2, found with SUZ12, EED, and RbAP48 in Polycomb Repressive Complex 2 (PRC2), has emerged as an alternative target for the treatment of deadly mCRPC. Unfortunately, drugs targeting EZH2 have shown limited efficacy in mCRPC. To address these failures, we have developed novel, dual-acting peptide inhibitors of PRC2 that uniquely target the SUZ12 protein component, resulting in the inhibition of both PRC2 canonical and noncanonical functions in prostate cancer. These peptides were found to inhibit not only the EZH2 methylation activity, but also block its positive effect on AR gene expression in prostate cancer cells. Since the peptide effect on AR levels is transcriptional, the inhibitory peptides can block the expression of both full-length AR and its splicing variants, including AR-V7 that plays a significant role in the development of drug resistance. This dual mode action provides the peptides with the capability to kill Enzalutamide-resistant CRPC cells. These peptides are also more cytotoxic to prostate cancer cells than the combination of Enzalutamide and an EZH2 inhibitory drug, which was recently suggested to be an effective treatment of mCRPC disease. Our data show that such a dual-acting therapeutic approach can be more effective than the existing front-line drug therapies for treating deadly mCRPC.