The Transmembrane Protein TM4SF3 Interacts With AR and AR-V7 and is Recruited to AR Target Genes

Abstract

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.

MDM2 regulates the stability of AR, AR-V7, and TM4SF3 proteins in prostate cancer

“…3B). These data suggest that MDM2 may be involved in the ubiquitination of AR and TM4SF3, which we have previously shown to be disrupted by hormone treatment ( Khatiwada et al 2023 ). To examine the possibility that MDM2 affects ubiquitination, we monitored the ubiquitination of AR and TM4SF3, both of which were inhibited by MDM2i ( Fig.…”

“…Since the TM4SF3 interaction with AR leads to mutual stabilization of both proteins ( Bhansali et al 2016 , Khatiwada et al 2023 ), it is possible that MDM2 is indirectly affecting the TM4SF3 protein via its direct effect on AR. To address this, we compared the effect of MDM2i on TM4SF3 in LNCaP cells to PC-3 cells, which do not express AR ( Otsuka et al 2013 ).…”

“…Our lab has published that the proteasomal inhibitor MG132 blocked TM4SF3 degradation ( Bhansali et al 2016 ), suggesting for the first time that TM4SF3 is a target of ubiquitination. More recently, we have demonstrated that TM4SF3 is, in fact, polyubiquitinated ( Khatiwada et al 2023 ). We have also reported that TM4SF3 directly interacts with AR, and the complex is protected from proteasomal degradation by inhibiting ubiquitination of both proteins ( Khatiwada et al 2023 ).…”

“…More recently, we have demonstrated that TM4SF3 is, in fact, polyubiquitinated ( Khatiwada et al 2023 ). We have also reported that TM4SF3 directly interacts with AR, and the complex is protected from proteasomal degradation by inhibiting ubiquitination of both proteins ( Khatiwada et al 2023 ). These data led us to hypothesize that there may be a common E3 ligase that acts on both AR and TM4SF3, which is inhibited by the androgen-induced AR/TM4SF3 interaction.…”

“…Proteasomal degradation of TM4SF3, in contrast to AR and AR-V7, was unknown until our two recent studies, showing that MG132 can block TM4SF3 degradation in prostate cancer cells ( Bhansali et al 2016 ), and demonstrated TM4SF3 in vitro ubiquitination that is inhibited by association with either AR or AR-V7 ( Khatiwada et al 2023 ). In view of these findings, we set up a screen of E3 ligases known to act on AR ubiquitination to identify a common E3 ligase for all three proteins.…”