Exquisite Specificity of a PMEPA1 Isoform in Regulation of the Androgen Receptor in Prostate Cancer
Discipline: Biological Sciences
Subcategory: Cancer Research
Session: 1
Room: Marriott Balcony A
Natashia Benjamin - University of the District of Columbia
Co-Author(s): Shashwat Sharad, Center for Prostate Disease Research, Department of Surgery, Uniformed Services University of the Health Sciences, MD; Alagarsamy Srinivasan, Center for Prostate Disease Research, Department of Surgery, Uniformed Services University of the Health Sciences, MD; Brandy Huderson, University for the District of Columbia, D.C; Albert Dobi, Center for Prostate Disease Research, Department of Surgery, Uniformed Services University of the Health Sciences, MD and John P. Murtha Cancer Center, Walter Reed National Military, MD; Shiv Srivastava, Center for Prostate Disease Research, Department of Surgery, Uniformed Services University of the Health Sciences, MD and John P. Murtha Cancer Center, Walter Reed National Military, MD; Hua Li, Center for Prostate Disease Research, Department of Surgery, Uniformed Services University of the Health Sciences, MD
The prostate trans-membrane protein, androgen-induced1 (PMEPA1), an androgen responsive gene with prostate abundance was originally discovered in the CPDR lab. There are 3 major isoforms of PMEPA1 gene (a, b and c) detected in CaP cells. The PMEPA1, as a NEDD4 (E3 ubiqitin ligase) binding protein, degrades the androgen receptor (AR) through a negative feedback loop in prostate cancer (CaP) cells. Decreased PMEPA1 results in increased AR protein and activated AR signaling is frequently associated with prostate tumorigenesis. In this study, we investigate the protein-protein interaction between AR, PMEPA1 isoforms and NEDD4, to test the hypothesis that the presence of one of the main isoforms is important for the degradation of the androgen receptor. The research is important as it aids in improving or finding a new approach in CaP therapeutic development. HEK293 cells were transfected with expression plasmids of PMEPA1 isoforms, AR or NEDD4 antibodies. The protein binding between AR, NEDD4 and PMEPA1 isoforms were detected using co-immunoprecipitation (IP) methods. Among the PMEPA1 isoforms only the androgen responsive PMEPA1-b isoform bound to AR (wild-type and T877A mutated). The deletion of transmembrane domain at N-terminus of PMEPA1-b interrupted the binding between AR and PMEPA1-b. PMEPA1-b is the only PMEPA1 isoform directly binds to AR. The transmembrane domain of PMEPA1-b protein is crucial to mediate binding between AR and PMEPA1. These results underscore the promise of the PMEPA1-b driven AR degradation as a new approach in CaP therapeutic development. Future research involves investigating the protein interaction in the final stages of CaP. References: 1) Linda Xu, et al. “A novel androgen-regulated gene, PMEPA1, located on chromosome 20q13 exhibits high level expression in prostate “Genomics, 2000; 66: 257–263 2) Linda Xu, et al. “PMEPA1, an androgen-regulated NEDD4-binding protein, exhibits cell growth inhibitory function and decreased expression during prostate cancer progression.” Cancer Research, 2003; 63: 4299-4304 3) Hongyun Li, et al. “A feedback loop between the androgen receptor and a NEDD4-binding protein, PMEPA1, in prostate cancer cells.” J Biol Chem, 2008; 283(43):28988-28995. 4) Hua Li, et al. ‘Silencing of PMEPA1 results in the acceleration of the prostate cancer cells: P-TEN, AR, and NEDD4.’ Oncotarget, 2015; 6(17):15137-15149.
Funder Acknowledgement(s): NB was supported by the Historically Black Colleges and Universities (HBCU) Summer Undergraduate Training Program in Prostate Cancer, DoD/PCRP Training Award to SS and Deepak Kumar and BH. Sources of support: This research in part was supported by CPDR core funding: HJF/CPDR-USU HU0001-17-2-0019
Faculty Advisor: Brandy Huderson, brandy.huderson@udc.edu
Role: The processes after transfection of antibodies within the HEK-293 cells were done by me. These include, the immunoblotting process as well as the development of the film. Equal amounts of antibodies were loaded in the wells, then the gels were run at a constant voltage for an hour and half. After transfer unto a filter paper membrane, membranes were blotted with the necessary primary and secondary antibodies, for a specific time frame(primary antibodies left on membranes overnight while secondary left for an hour). Finally, the films were developed and analyzed.