Abstract 2313: Dynamic expression of 5-alpha reductase 2 in aging prostate is regulated by DNA methyltransferase 1

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA BACKGROUND: 5-alpha reductase type 2 (SRD5A2), an enzyme that is critical for prostatic development and growth, is utilized as an inhibitory target by Finasteride for patients with BPH. However, we have found that many aging benign prostate tissues do not express the enzyme. Since the SRD5A2 promoter contains a CpG island, we hypothesized that somatic methylation of the promoter may account for absence of SRD5A2 expression. METHODS: Benign prostatic tissues from wild-type mice at 3, 6 and 12 months of age were used. In addition, prostate samples from human male patients who were treated by TURP for bladder outlet obstruction secondary to BPH were used. Methylation of SRD5A2 promoter was assessed using Methylated CpG Island Recovery Assay (MIRA). DNMT1 siRNA and 5-AZA-C were used to determine the methylation status of SRD5A2 in benign prostatic cells (BPH-1). SRD5A2 promoter-luciferase constructs were methylated in vitro using M.SssI methylase. RESULTS: Benign prostatic tissues from wild-type mice of different ages (3 months, 6 months and 12 months, n=6 per group) were harvested. We found that expression of SRD5A2 and DNMT1, 3a, 3b were not significantly changed in mice at the age of 3 and 6 months. However, 33% of mice at age of 12 months (2/6) had low levels of SRD5A2 with concomitant high DNMT1 protein levels. Consistent with reduced expression of SRD5A2 and increased DNMT1, the methylation of SRD5A2 promoter was increased as assayed by MIRA. Moreover, silencing DNMT1 with siRNA or exposure of BPH-1 cells to 5-AZA-C led to re-expression of SRD5A2. We used SRD5A2 promoter-luciferase constructs that were methylated in vitro using M.SssI methylase. When the methylated SRD5A2 promoter constructs were ectopically expressed in BPH-1 cells, we found that methylation of the SRD5A2 promoter caused reduction of luciferase expression by 80%, suggesting that methylation of the SRD5A2 promoter regulates the expression of SRD5A2. Since age-associated increased IL-6 has been shown to regulate DNMT1, we next evaluated expression of IL-6 in aging prostate tissue. We found that expression of IL-6 and its downstream molecule, phosphorylated-STAT3, correlated with expression of DNMT1. Concurrent with our animal and in-vitro studies, we analyzed 80 benign human prostate samples from TURPs and found that 24/80 (30%) of did not express the SRD5A2 protein with concurrent methylation of SRD5A2 promoter, suggesting that methylation and absence of of SRD5A2 protein expression are closely linked (p=0.0006, Fisher's exact test). This data suggests that expression of SRD5A2 is highly variable in human adult prostate tissue. CONCLUSIONS: Expression of SRD5A2 in aging prostates is a dynamic process that is regulated by methylation of SRD5A2 promoter. DNMT1 regulates the methylation and expression of SRD5A2. Dynamic and variable expression of SRD5A2 has implications for management of BPH and chemopreventive strategies for prostate cancer. Citation Format: Ge Rongbin, Zongwei Wang, Chin-Lee Wu, Shahin Tabatabaei, Aria Olumi. Dynamic expression of 5-alpha reductase 2 in aging prostate is regulated by DNA methyltransferase 1. [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 2313. doi:10.1158/1538-7445.AM2014-2313