Abstract 2626: Exquisite specificity of PMEPA1 isoforms in regulation of androgen signaling through AR protein degradation in prostate cancer
Authors: Sharad Shashwat , Benjamin Natashia , Sztupinszki Zsófia , Szallasi Zoltan , Srivastava Shiv , Rosner Inger L. , Cullen Jennifer , Li Hua , Dobi Albert 
Published: August 2019

Introduction: Prostate Transmembrene Protein, androgen Induced 1 (PMEPA1) gene was defined as an androgen and TGF-β responsive gene to inhibit androgen receptor (AR) and TGF-β signaling via negative feedback loops. Our previous studies identified two functionally distinct PMEPA1 isoforms: androgen responsive PMEPA1-b and TGF-β responsive PMEPA1-a. These two isoforms share high homology within the intracellular domains including PY1 and PY2 motifs essential for binding to E3 ubiqintin ligase, NEDD4. It is still unclear which PMEPA1 isoform with distinct N-terminus regulates AR protein stability in CaP cells.

Methods: An immunoprecipitation (IP) assay was used to study the protein-protein interactions between AR, PMEPA1 isoforms and NEDD4. HEK293 cells were used for co-transfection of the expression vectors harboring PMEPA1 isoforms, AR and NEDD4. RNA Seq data from TCGA datasets were utilized to analyze the correlation between transcript levels of both PMEPA1 isoforms and clinical outcomes including Gleason scores, overall survival and progression free survival of cancers of prostate, breast, lung and colon.

Results: Only PMEPA1-b isoform bound to wild-type and T877A mutated AR, directly. In contrast, PMEPA1 isoforms a, b and c bound to E3 ubiquintin ligase NEDD4. As expected, only PMEPA1-b bound to AR and NEDD4 for tri-complex interactome. Interestingly, the direct binding between AR and NEDD4 was not detected, which highlighted the adaptor function of PMEPA1-b in mediating proteasome-mediated AR degradation. Furthermore, the deletion of the transmembrane domain compromised binding of PMEPA1-b with AR. The TGF-β responsive PMEPA1-a was not found to bind to AR or form AR/NEDD/PMEPA1 complex. Consistently, ectopic PMEPA1-b down-regulated AR protein, inhibited AR signaling, and suppressed cell growth of LNCaP cells. But PMEPA1-a was not detected to interfere with AR signaling in CaP cells. Clinically, a higher ratio of PMEPA1-a/b was correlated with higher Gleason scores in CaP. And higher expression of PMEPA1-a and b was associated with more aggressive lung cancer and colon cancer. All of these findings highly suggest that the biological functions of PMEPA1 isoforms on TGF-β or AR signaling are tissue specific.

Conclusions: Our study highlighted PMEPA1 isoform b as an essential docking platform by interacting with AR at N-terminus and NEDD4 at C-terminus for mediating the protein degradation of AR by NEDD4. Additionally, the transmembrane domain at N-terminus of PMEPA1-b protein was essential for NEDD4 dependent AR degradation. These findings might help guide a novel anti-AR strategy in development of anti-CaP therapy. RNA seq data analysis further underscored the tissue-specificity of division of labor among PMEPA1 isoforms in tum

Citation Format: Shashwat Sharad, Natashia Benjamin, Zsófia Sztupinszki, Zoltan Szallasi, Shiv Srivastava, Inger L. Rosner, Jennifer Cullen, Hua Li, Albert Dobi. Exquisite specificity of PMEPA1 isoforms in regulation of androgen signaling through AR protein degradation in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2626.