Abstract 5566: Functional new insights into the regulation of the androgen receptor by PMEPA1/NEDD4-1 in prostate cancer
Authors: Li Hua , Sharad Shashwat , Ravindranath Lakshmi , Haffner Michael , Heidenberg Daniel , Yegnasubramanian Srinivasan , Chen Yongmei , Mohamed Ahmed , Srinivasan Alagarsamy , Petrovics Gyorgy , Srivastava Shiv 
Published: May 2015

Abstract
Introduction: Androgen receptor (AR) stability has been shown to be regulated by MDM2 E3 ubiquitin ligase. Our data suggest a role for PMEPA1 and NEDD4-1 in the regulation of AR protein in prostate cancer cells. The AR inducible PMEPA1 recruits NEDD4-1, an E3 ubiquitin ligase, to degrade AR. Decreased or absent PMPEA1 was observed in more than 65% of prostate tumors. PMEPA1 depleted transfectant is established in LNCaP cells with a shRNA lentivirus system and shown elevated AR protein levels, AR signaling and increased cell growth. In the current study, we investigated the growth and AR signaling of PMEPA1 depleted prostate cancer cells in the presence of AR inhibitors. Also we investigated the role of PMEPA1 silencing by DNA methylation in the regulation of AR.
Methods: PMEPA1 depleted LNCaP cells were treated with AR inhibitors (MDV3100 or bicalutamide) in the presence or absence of the synthetic androgen R1881. Cell growth was monitored by cell counting, BrdU incorporation, colony plating and soft agar assays. Cell cycle was analyzed with propidium iodide staining based flowcytometry. AR, PMEPA1, PSA proteins were assessed by immunoblot assay. Methylation of the PMEPA1 gene was evaluated in LCM-enriched tumor samples by combing precipitation of methylated-DNA and methylation-sensitive restriction enzyme digestion (COMPARE-MS). The transcript levels of PMEPA1 gene was assessed by qRT-PCR.
Results: PMEPA1 depleted LNCaP cells are more resistance to growth inhibition by MDV3100 or bicalutamide in both hormone and hormone-depleted conditions, which demonstrated stronger BrdU incorporation abilities, cell plating efficiency and colony formation capacities in soft agar under MDV3100 or bicalutamid treatment compared to controls. The half life of AR was increased, and more cells in S-phase and less apoptotic cells in PMEPA1 depleted LNCaP cells. Methylation of PMEPA1 gene and mRNA levels showed significant correlation in prostate tumors (n=77, P=0.001). The higher frequency of PMEPA1 gene methylation was noticed in Caucasian American patients.
Conclusions: PMEPA1 depleted prostate cancer cells develop increased resistance to AR inhibitors through enhanced AR signaling. Silencing of PMEPA1 may contribute to AR activation in prostate cancer. These data highlight that PMEPA1 deficiency may confer resistance to AR inhibitors during prostate cancer progression.
This study is supported by the NIH grant (R01CA106653) to SS.
Citation Format: Hua Li, Shashwat Sharad, Lakshmi Ravindranath, Michael Haffner, Daniel Heidenberg, Srinivasan Yegnasubramanian, Yongmei Chen, Ahmed Mohamed, Alagarsamy Srinivasan, Gyorgy Petrovics, Shiv Srivastava. Functional new insights into the regulation of the androgen receptor by PMEPA1/NEDD4-1 in prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5566. doi:10.1158/1538-7445.AM2014-5566