Abstract B21: Epigenetic and post-transcriptional regulation of the kallikrein gene family as a novel panel of prostate cancer biomarkers
Authors: Olkhov-Mitsel Ekaterina , Kwast Theodorus Van der , Kron Ken , Ozcelik Hilmi , Briollais Laurent , Fleshner Neil , Diamandis Eleftherios , Zlotta Alexandre , Bapat Bharati 
Published: August 2014

Prostate cancer (PCa), the most commonly diagnosed cancer in North American men, is currently detected using a serum test for prostate-specific antigen (PSA), a protein encoded by kallikrein 3 (KLK3). However, PSA testing has limited sensitivity and specificity leading to over-diagnosis of PCa. Therefore, improved biomarkers are urgently needed to identify aggressive vs. indolent PCa. Other members of the 15-member KLK gene family, which encode for serine proteases, are likely candidates since their expression is altered in PCa. Emerging evidence has shown that epigenetic mechanisms such as DNA methylation and aberrant microRNA (miRNA) expression may regulate KLK expression in carcinogenesis. Importantly, these marks are potentially promising biomarkers since they can be detected in tissue and serum samples of PCa patients. Studies to date have not examined how DNA methylation and miRNA regulation mechanisms cooperate to influence KLK regulation. Using genome-wide CpG island DNA methylation profiling, we identified tumor-specific hypermethylation of KLK6 and KLK10 genes in PCa which we validated using methylation specific quantitative real-time PCR (MethyLight). We demonstrated that KLK10 DNA methylation was significantly associated with pathological stage and ERG oncogene expression status in 2 independent cohorts of PCa patients (Cohort I, n=150 and II, n=124, P-value =0.007, 0.031, P-value =0.001, P&lt;0.001, respectively). Further, in cohort II, low KLK10 DNA methylation was associated with biochemical recurrence in univariate and multivariate analyses (P-values=0.046, 0.028, respectively, HR=2.11, 95% CI: 1.10-4.02). A similar trend for KLK6 DNA methylation was observed. The results suggest that KLK6 and KLK10 DNA methylation distinguishes organ confined from locally invasive PCa and may have prognostic value. To better understand the regulation of this protease family and potentially PCa pathogenesis, we have performed in silico analysis to identify a panel of miRNAs implicated in KLK regulation. Notably, we found mir-137 was predicted to target KLK10 and was hypomethylated by genome-wide methylation array profiling. Among the 21 CpG island microarray probes representing the mir-137 genomic region, 3 were significantly differentially methylated in high grade vs. low grade PCas and 5 were significantly differentially methylated between ERG positive and negative cases (P-values&lt;0.05). We then measured mir-137 expression in PCa patients from cohort I using quantitative real time PCR and determined relative quantities of miRNA using 2-ΔCt method after normalization to U6 snRNA. The expression of mir-137 was 37% higher in PCa tissue vs. normal (P-value=0.28). Further, the proportion of cases with high mir-137 expression, based on a third quartile threshold, were significantly different between Gleason Score (GS) ≥8 vs. GS ≤7 and ERG positive vs. negative expression status (P-values=0.035, 0.013, respectively). These findings highlight the complexity of epigenetic regulatory systems and pave the way for further investigation of miRNA regulation of the KLK gene family. The association of KLK10 methylation and mir-137 profiles with ERG expression status further characterizes molecular subtypes of PCa, which will allow for potentially establishing a diagnostic and/or prognostic model of clinical significance for PCa.

Citation Format: Ekaterina Olkhov-Mitsel, Theodorus Van der Kwast, Ken Kron, Hilmi Ozcelik, Laurent Briollais, Neil Fleshner, Eleftherios Diamandis, Alexandre Zlotta, Bharati Bapat. Epigenetic and post-transcriptional regulation of the kallikrein gene family as a novel panel of prostate cancer biomarkers. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B21.