Abstract B6: Epigenetic regulation of the androgen receptor regulator PMEPA1 in prostate cancer cells
Authors: Sharad Shashwat , Li Hua , Haffner Michael C. , Yegnasubramanian Srinivasan , Petrovics Gyorgy , Shiv Srivastava , Dobi Albert 
Published: May 2013

Introduction and Objective: Prostate cancer (CaP) is the most common noncutaneous malignancy in men in the U.S. Epigenetics may play important roles in CaP tumorigenesis. PMEPA1 is an androgen-inducible gene expressed in the prostate epithelium and shown to control the androgen receptor (AR) protein levels by recruiting the NEDD4-1 E3 ubiquitin ligase to AR. PMEPA1 gene expression is either reduced or lost in two-third of prostate tumors. In cell culture models of CaP, DNA methylation has been shown to play a role in epigenetic inactivation of PMEPA1. These results prompted us to investigate the methylation status of PMEPA1 in cells line models of CaP and laser capture micro-dissected (LCM) CaP specimens.

Methods: Androgen responsive LNCaP (harboring mutant AR), VCaP and LAPC4 (both harboring wild type AR), and AR signaling negative DU-145 and PC-3, were treated with the DNA methyl transferase inhibitor 5-aza-2′-deoxycitidine (decitabine) for two weeks with 0, 2.5 and 5 uM of decitabine. Methylation status of PMEPA1 was evaluated by COMPARE-MS (Combination of methylated-DNA precipitation and methylation-sensitive restriction enzymes) method using methyl DNA binding protein-mediated enrichment followed by quantitative PCR. Expression of PMEPA1 at protein and transcript levels were assessed by Western blot and Q-PCR, respectively. Genomic DNAs from LCM CaP specimens were analyzed for methylation status of PMEPA1. As the control, DNA methylation status and expression of GSTP1 was evaluated.

Results: DNA methylation of the promoter downstream sequences of the PMEPA1 gene was detected in VCaP, LNCaP and LAPC4 androgen inducible CaP cell lines. Western blot and Q-PCR analysis revealed significantly induction of PMEPA1 expression in response to decitabine treatment in the evaluated androgen responsive cells. In AR signaling negative DU-145 cells, Western blot and Q-PCR assays revealed induction of PMEPA1 protein and transcript, respectively, after decitabine treatment. In contrast, there was no detectable PMEPA1 protein in PC-3 cells, although Q-PCR showed decitabine induced increased expression of the PMEPA1 transcript. In a panel of LCM derived genomic DNA PMEPA1 was methylated in 13/25 specimens (52%), whereas, GSTP1 was methylated in 19/25 specimens (76%).

Conclusion: PMEPA1 promoter downstream sequences are methylated in CaP cell lines and primary tumors suggesting that methylation contributes to the silencing of PMEPA1 gene in a subset of CaP. These data along with our earlier observation showing elevated AR levels by the loss of PMEPA1 expression, provide an intriguing role of methylation in a negative feedback regulation of AR in CaP.

Citation Format: Shashwat Sharad, Hua Li, Michael C. Haffner, Srinivasan Yegnasubramanian, Gyorgy Petrovics, Srivastava Shiv, Albert Dobi. Epigenetic regulation of the androgen receptor regulator PMEPA1 in prostate cancer cells [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr B6.