Abstract 2072: TM4SF3 interaction with AR or AR-V7 is important for the survival of prostate cancer cells
Authors: Khatiwada Prabesh , Malla Mamata , Shemshedini Lirim 
Published: September 2020

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.