Abstract 667: Prognostic value and functional impact of aberrant GABRE∼miR-452∼miR-224 promoter methylation in prostate cancer.
Authors: Kristensen Helle , Haldrup Christa , Mortensen Martin M. , Borre Michael , Høyer Søren , Wild Peter J. , Arsov Christian , Goering Wolfgang , Visakorpi Tapio , Ørntoft Torben F. , Sørensen Karina D. 
Published: August 2014

Abstract
Prostate cancer (PC) is a major cause of morbidity and mortality among men in Western countries. Currently available tools for PC diagnosis and prognosis are suboptimal and novel biomarkers are urgently needed. This study investigated the regulation, function, and PC biomarker potential of a genomic locus encompassing miR-224, miR-452, and their host gene gamma-aminobutyric acid (GABA) A receptor family epsilon subunit (GABRE). First, mRNA and miRNA expression profiling data for three independent patient sets, including a total of 80 nonmalignant and 281 PC tissue samples, showed significant downregulation of all three genes in PC. Next, we asked if this locus was silenced in PC by aberrant hypermethylation of its promoter-associated CpG island. Indeed, bisulfite sequencing of 11 nonmalignant and 21 PC tissue samples showed frequent cancer-specific hypermethylation, which was successfully validated in an independent set of 35 nonmalignant and 245 PC tissue samples by methylation-specific qPCR. Notably, GABRE promoter methylation distinguished PC from nonmalignant prostate with 96% sensitivity and 94% specificity (AUC=0.98). Consistent with an epigenetic silencing mechanism, methylation levels correlated inversely with GABRE, miR-224, and miR-452 expression in both clinical samples and prostatic cell lines.
By univariate cox regression analysis, high GABRE methylation was significantly (p=0.011, p=0.015, p=0.002) associated with PSA recurrence after radical prostatectomy in three independent PC patient cohorts (n=186, n=129, n=148). Moreover, in multivariate models, GABRE methylation added significant independent prognostic value to established clinicopathological predictors in two cohorts (p=0.031 and p=0.020) and was borderline significant in the smaller third cohort (p=0.073). In line with the significant adverse prognostic value of GABRE promoter methylation, functional studies in vitro suggested that silencing of this locus may lead to increased malignancy of PC cells. Thus, ectopic expression of miR-224 and miR-452 mimics, respectively, inhibited proliferation, migration, and invasion of two PC cell lines (PC3 and DU145) with undetectable endogenous levels of both miRNAs. We are currently analyzing post-transfection microarray gene expression profiling data to identify target genes mediating the tumor suppressive functions of miR-224 and miR-452; results will be presented at the conference.
In conclusion, our results showed that the GABRE∼miR-452∼miR-224 locus is frequently silenced in PC by aberrant promoter hypermethylation and, furthermore, is a new promising epigenetic candidate biomarker for PC diagnosis and prognosis. Also, the tumor suppressive functions of miR-224 and miR-452 demonstrated in two PC cell lines may suggest this locus as a potential target for epigenetic therapy of PC.
Citation Format: Helle Kristensen, Christa Haldrup, Martin M. Mortensen, Michael Borre, Søren Høyer, Peter J. Wild, Christian Arsov, Wolfgang Goering, Tapio Visakorpi, Torben F. Ørntoft, Karina D. Sørensen. Prognostic value and functional impact of aberrant GABRE∼miR-452∼miR-224 promoter methylation 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 667. doi:10.1158/1538-7445.AM2013-667