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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 bladder outlet obstrution secondary to 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 would be regulated by DNA methyltransferases leading to suppression of SRD5A2. METHODS: Benign prostatic tissues from wild-type mice at 3, 6 and 12 months of age were used. In addition, 96 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 cell line, BPH-1. To determine the effect of CpG methylation, SRD5A2 promoter-luciferase constructs were methylated in vitro using M.SssI methylase. Statistical analyses were performed with JMP Pro version 11 (SAS Institute Inc., Cary, NC). RESULTS: We analyzed 96 human BPH samples from transurethral resection of prostate (TURP) surgeries and found that absence of SRD5A2 was closely associated with hypermethylation of the SRD5A2 promoter region. We found that methylation of SRD5A2 was regulated by DNA methytransferase 1 (DNMT1) and the cytokines, TNF-alpha, NF-κB and IL-6, regulated DNMT1protein expression and thereby indirectly affected SRD5A2 promoter methylation and gene expression. Suppression of cytokines inhibited activity of DNMT1, while over-expression of p65 or treatment with TNF-alpha or IL-6 up-regulated DNMT1. In addition, we found that methylation of the SRD5A2 promoter and concomitant decrease in protein expression were closely associated with patient's increasing age (P CONCLUSIONS: Expression of SRD5A2 is not static and ubiquitous in benign adult prostate tissues. We show that SRD5A2 expression is lacking in many benign human adult prostate tissues. Methylation of SRD5A2 promoter region, which is regulated by DNMT1, accounts for absence of SRD5A2 expression in many adult human prostate tissues. Methylation and expression of SRD5A2 may be used as a gene signature to tailor therapies for more effective treatment of prostatic diseases. Citation Format: Ge Rongbin, Zongwei Wang, Seth Bechis, Alexander Otsetov, Shengyu Hua, Shulin Wu, Chin-Lee Wu, Shahin Tabatabaei, Aria Olumi. Variable expression of 5-alpha reductase 2 in the aging adult prostate is regulated by DNA methylation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1052. doi:10.1158/1538-7445.AM2015-1052

BACKGROUND Metformin, one of the most commonly used medications for treatment of type 2 diabetes, has emerged as a potential anticancer agent. The molecular mechanisms associated with the antitumor effect of metformin are still poorly understood. In this study, we show that Metformin represses cancer cells via alternate pathways in N-Cadherin Wild type and N-Cadherin deficient cells. METHODS Cell viability and apoptosis were determined by MTT, TUNEL and FACS. Various stable cell lines were generated in vitro and then injected into both flanks of nude mice to generate xenografts. Mice were treated with i.p. doxorubicin (every 5 days × 4 cycles with 4 mg/kg) or p.o.metformin (200 ug/ml, diluted in the drinking water). Xenograft tumor volume was measured every 5 days. RESULTS We found that metformin represses N-cadherin, independent of AMPK, in wild-type N-cadherin cancer cells. Ectopic expression of N-cadherin rendered cells resistant to metformin. Silencing or over-expression of AMPK did not alter sensitivity of cancer cells to metformin in N-cadherin wild-type cells. In metformin-resistant cancer cells, N-cadherin level did not change with metformin treatment. Suppression of N-cadherin changed the phenotype of metformin-resistant to metformin-sensitive cells. We also demonstrated that NF-kB is a downstream molecule of N-cadherin and metformin regulated NF-kB signaling via inhibiting N-cadherin, rather than direct modulation of NF-kB. Silencing of NF-kB in resistant cells sensitized the cells to metformin. We also found that metformin down-regulated N-cadherin and TWIST1 concurrently. Ectopic expression of TWIST1 regulated N-cadherin/NF-kB signaling and made cells more resistant to metformin. Suppression of TWIST1 sensitized resistant cells to metformin. Metformin inhibited growth of tumor xenografts, and high levels of N-cadherin made tumors more resistant to metformin, while suppression of N-cadherin made tumors more sensitive. In N-cadherin deficient cancer cells, metformin activated AMPK. Suppression of AMPK compromised metformin-mediated inhibition of NF-kB and changed the cells to a more metformin-resistant phenotype. In the deficient cells, Ectopic expression of N-cadherin attenuated AMPK's ability to suppress NF-kB, leading to a more metformin resistant phenotype. Similarly, a specific AMPK inhibitor, compound C, inhibited the therapeutic effects of metformin. Similar to the in vitro findings, suppression of AMPK in xenografts rendered tumors resistant to metformin. CONCLUSIONS: We suggest that metformin's anti-cancer therapeutic effect in N-cadherin wild-type cells may be mediated through repression of the TWIST/N-cadherin/NF-kB signaling pathway, independent of AMPK. However, in N-cadherin deficient cells, metformin activates AMPK. Elucidating the molecular pathways responsible for metformin's anti-cancer effect may help delineate its role for cancer therapies. Citation Format: Ge Rongbin, Zongwei Wang, Jijun Li, Aria Olumi. Metformin represses cancer cells via alternate pathways in N-Cadherin wild-type and N-Cadherin-deficient cells. [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 2765. doi:10.1158/1538-7445.AM2014-2765

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