Abstract 5035: MDB-seq screening of muscle-invasive urothelial carcinoma revealed unpredicted novel tumor suppressor genes
Authors: Guida Elisa , Marchionni Luigi , Goldberg Rachel , Schoenberg Mark , Bivalaqua Trinity , Netto George J. , Moque Mohammad O. , Sidransky David 
Published: July 2016

Bladder cancer (BC) is among the most frequent cancers in the United States. More than 70% patients suffer from superficial disease called non-muscle invasive bladder cancer (NMIBC) which, after the first successful surgery, has favorable survival but high risk of recurrence; on the other hand, muscle-invasive urothelial carcinoma (MIBC) is less prevalent but typically associated with a relatively poor prognosis. Since BC has been largely described as a multifactorial disease, with some DNA mutational component and strong evidence supporting an important role performed by the exposure to environmental factors, mainly derived from tobacco smoking and occupational hazards, consequent epigenetic DNA alterations could play important pathological role and also be used as markers for detection and stratification of the disease. In order to discover new methylated genes we performed novel sequential high throughput Methyl-DNA Binding (MDB) proteins Pull-down followed by large genome sequencing of 5 primary MIBC (not subject to previous therapy nor BCG) versus their normal counterparts.

After Bioinformatic cross-analysis between the enriched sequences in our MDBseq experiment (corresponfing to 85 genes) and publicly available database (BC TCGA), we have come up to a list containing 16 hypermethylated genes in BC (our «consensus genes»). From the first unbiased analysis of this list, we have found some genes already found as important and/or hypermethylated in BC (as for example, CDKN2A, Sall3, Nkx6-2, CDO1), confirming the validity of our approach. Interestingly, we have observed high presence of transcription factors (homeobox, Paired, Zn fingers) and proteins involved in fundamental developmental processes. Among the newly hypermethylated genes in BC, we have chosen VIPR2 for further characterization. VIPR2 encodes the vasoactive intestinal peptide receptor VPAC2, a G-protein-coupled receptor that is expressed in a variety of tissues (expressed throughout the central nervous system and the periphery); it binds VIP14, activates cyclic AMP (cAMP)-signaling and PKA. The VIP-VPAC2 signaling pathway has been implicated in several neural and behavioral processes and is known to influence circadian rhythms and cognition. We first confirmed inverse correlation between diminished expression and promoter methylation in 11 BC cell lines (5637, HT-1376, J82, SW780, UM-UC-3, BFTC-905, BFTC-909, Scaber, RT4, T24) and a normal human urinary tract epithelium immortalized cell line SV-HUC-1 (HUC-1). Next, we determined that pharmacological unmasking with DNA methylation transferases (DNMTs) inhibitors (5-Aza-deoxycytidine) re-expressed the transcript in the cells analyzed. We are in the process of analyzing VIPR2 expression and promoter methylation in primary BC samples. After confirming in primary BC samples, we will further characterize the role of VIPR2 in bladder cancer pathogenesis.

Citation Format: Elisa Guida, Luigi Marchionni, Rachel Goldberg, Mark Schoenberg, Trinity Bivalaqua, George J. Netto, Mohammad O. Moque, David Sidransky. MDB-seq screening of muscle-invasive urothelial carcinoma revealed unpredicted novel tumor suppressor genes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5035.