Abstract 4245: Silencing of the PMEPA1 gene, a key regulator of androgen receptor in prostate cancer.
Authors: Sharad Shashwat E. , Li Hua , Haffner Micheal , Yegnasubramanian Srinivasan , Ravindranth Lakshmi , Chen Yongmei , Srinivasan Alagarsamy , Petrovics Gyorgy , Srivastava Shiv , Dobi Albert 
Published: August 2014

Introduction and Objectives: PMEPA1 is an androgen inducible gene, abundantly expressed in prostate epithelial cells and controls androgen receptor (AR) protein levels by recruiting NEDD4-1 E3 ubiquitin ligase to degrade AR. PMEPA1 gene expression is either reduced or lost in two-thirds of prostate cancer (CaP). Methylation of the PMEPA1 promoter may contribute to PMEPA1 gene silencing. Previously we have shown that AR binds to an Androgen Responsive Element (ARE) in the proximal promoter of the PMEPA1 gene in a hormone-dependent manner. These results prompted us to investigate the methylation status of proximal ARE and intronic SP1 enhancer sites in cell lines and in laser capture micro-dissected (LCM) human prostate tumors.

Methods: Methylation status of the PMEPA1 promoter was evaluated in AR positive LNCaP, VCaP and LAPC4 cell lines and in LCM-enriched tumor cells using a previously validated method combining precipitation of methylated-DNA and methylation-sensitive restriction enzyme digestion (COMPARE-MS). Expression of PMEPA1 at protein and transcript levels was assessed by Western blot and Q-PCR, respectively in response to DNA methyl transferase inhibitor 5-aza-2’-deoxycitidine.

Results: In a panel of LCM derived genomic DNA (N=50), PMEPA1 promoter sequences were found to be hypermethylated in 44% of human prostate tumors (N=22). PMEPA1 methylation was consistent with loss of PMEPA1 gene expression. PMEPA1 methylation was also detected in androgen-sensitive VCaP, LNCaP and LAPC4 CaP cell lines. Western blot and Q-PCR analysis revealed that demethylation restores PMEPA1 expression in CaP cells. Evaluation of proximal promoter sequences of PMEPA1 suggested that DNA methylation may directly inhibit AR binding to the proximal ARE and to the downstream intronic SP1 enhancer site of PMEPA1 gene. PMEPA1 methylation status showed a trend towards association with race, with a very low (12%) incidence of PMEPA1 methylation in African Americans subjects.

Conclusions: PMEPA1 promoter regulatory sequences are hypermethylated in prostate cancer cell lines and in primary tumors suggesting that DNA methylation may contribute to the silencing of PMEPA1 gene in a significant subset of prostate tumors. These data, along with our earlier observation showing elevation of AR levels in response to the inhibition of PMEPA1 expression suggest a role of DNA methylation in the silencing of PMEPA1 gene and AR regulation in human CaP. Overall, reduced or eliminated PMEPA1 expression is likely to interfere with AR signaling and/ or other pathways in CaP.

Source of Funding: This study was supported by the NIH grant RO CA106653 to S.S.

Citation Format: Shashwat E. Sharad, Hua Li, Micheal Haffner, Srinivasan Yegnasubramanian, Lakshmi Ravindranth, Yongmei Chen, Alagarsamy Srinivasan, Gyorgy Petrovics, Shiv Srivastava, Albert Dobi. Silencing of the PMEPA1 gene, a key regulator of androgen receptor in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4245. doi:10.1158/1538-7445.AM2013-4245