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.

Twist1, a basic helix-loop-helix transcription factor that regulates a number of genes involved in epithelial-to-mesenchymal transition (EMT), is upregulated in prostate cancer. Androgen regulation of Twist1 has been reported in a previous study. However, the mechanism of androgen regulation of the Twist1 gene is not understood because the Twist1 promoter lacks androgen receptor (AR)-responsive elements. Previous studies have shown that the Twist1 promoter has putative binding sites for PEA3 subfamily of ETS transcription factors. Our lab has previously identified Ets Variant 1 (ETV1), a member of the PEA3 subfamily, as a novel androgen-regulated gene that is involved in prostate cancer cell invasion through unknown mechanism. In view of these data, we hypothesized that androgen-activated AR upregulates Twist1 gene expression via ETV1. Our data confirmed the published work that androgen positively regulates Twist1 gene expression and further showed that this positive effect was directed at the Twist1 promoter. The positive effect of androgen on Twist1 gene expression was abrogated upon disruption of AR expression by siRNA or of AR activity by Casodex. More importantly, our data show that disruption of ETV1 leads to significant decrease in both androgen-mediated upregulation as well as basal level of Twist1, which we are able to rescue upon re-expression of ETV1. Indeed, we are able to show that ETV1 mediates the androgen upregulation of Twist1 by acting on the proximal region of Twist1 promoter. Additionally, our data show that Twist1 regulates prostate cancer cell invasion and EMT, providing a possible mechanism by which ETV1 mediates prostate cancer cell invasion. In conclusion, in this study we report Twist1 as an indirect target of AR and androgen regulation through ETV1.

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.

Androgen Receptor (AR) is essential for the development and progression of prostate cancer (PCa) from the primary stage to the usually lethal stage known as castration-resistant prostate cancer (CRPC). Constitutively active Androgen Receptor splice variants (AR-Vs) lacking the ligand- binding domain are responsible for the abnormal activation of AR and acquired resistance to AR- targeting drugs occurring in CRPC. Among the 20 AR-Vs reported, AR-V7 is the best characterized and is expressed in about 20% of CRPC patients. Our lab has recently identified a novel mechanism of AR regulation via the transmembrane protein TM4SF3 (Transmembrane 4 superfamily 3). Our published data show that AR interacts with TM4SF3 in an androgen-dependent manner, resulting in the nuclear localization and mutual stabilization of both proteins. We have more recently learned that TM4SF3 also interacts with AR-V7 through the N-terminus, which is the same in AR and AR-V7. In view of these results and the importance of AR in prostate cancer, we hypothesize that the TM4SF3 interaction with either AR or AR-V7 prevents the ubiquitination of TM4SF3 and AR or AR-V7 and thus promotes their protein stability and leads to elevated survival and growth of prostate cancer cells. To test this, we have explored the ubiquitination of all three proteins and observed for the first-time ubiquitination of TM4SF3 and AR-V7 in prostate cancer cells. AR and TM4SF3 ubiquitination was reduced when both proteins are co-expressed in the presence of androgen and AR-V7 ubiquitination occurred only when AR is co-expressed. To further show the importance of TM4SF3 in the enhanced stability of both AR proteins, we have used the de-palmitoylating agent 2-BP (2-Bromopalmitate) in prostate cancer cells. As expected, 2-BP treatment resulted in markedly reduced levels of endogenous TM4SF3, and interestingly both AR and AR-V7, leading to the cytotoxicity of LNCaP cells, which express AR, and CWR-22Rv1 cells, which express both AR and AR-V7. Using a Bimolecular Fluorescence Complementation (BiFC) assay, we have mapped the AR interaction region to a 21-amino acid sequence found in the N-terminus and designed a peptide, AT1, based on this sequence. Peptide AT1 reduced the interaction of TM4SF3 with either AR or AR-V7 and caused attenuated levels of endogenous TM4SF3, AR, and AR-V7 in LNCaP or CWR-22Rv1, resulting in significant reduction in the growth of both prostate cancer cells. Future studies will focus on identification of ubiquitination sites on TM4SF3 and AR-V7 and E3 ligases acting on the proteins, elucidation of possible TM4SF3 role(s) on AR and AR-V7 nuclear functions, and analysis of the importance of the TM4SF3 interaction with AR or AR-V7 in prostate tumorigenesis. Citation Format: Prabesh Khatiwada, Mamata Malla, Lirim Shemshedini. TM4SF3 interaction with AR or AR-V7 is important for the survival of prostate cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2072.