Abstract 1363: Epigenetic inactivation of VGF in urothelial cell carcinoma and its potential as a non-invasive biomarker using urine
Authors: Hayashi Masamichi , Bernert Heike , Kagohara Luciane Tsukamoto , Brait Mariana , Schoenberg Mark , Bivalacqua Trinity , Netto George , Koch Wayne , Sidransky David , Hoque Mohammad O. 
Published: May 2015

Background: Urothelial Cell Carcinoma (UCC) presents as a very heterogeneous disease which cannot sufficiently be characterized with the currently known genetic and epigenetic markers. To identify new epigenetic markers for UCC we scrutinized the methylation status of 19 genes that has been shown to be cancer specific methylated genes in other solid tumors in our comprehensive genome-wide discovery approach.

Methods: We performed promoter methylation of 19 genes (CAMK4, CKMT1b, FKBP4, GALE, HOXB5, HPN, KRT14, LPAR2, MAL, NBR1, NDP, NF1, PDLIM3, PHKA2, PVRL1, RGS4, SGK1, VGF and ZMYM2) that are related to key molecular pathways involved in urothelial carcinogenesis including MAPK pathway or PI3K/AKT pathway. The methods we used includes bisulfite sequencing, methylation specific PCR and quantitative methylation specific PCR(QMSP). All the 19 genes and/or a particular gene were evaluated in urothelial cancer cell lines, primary tumor tissues and urine sediments.

Results: By bisulfite sequencing, methylation frequencies for CAMK4, CKMT1b, FKBP4, GALE, HOXB5, HPN, KRT14, LPAR2, MAL, NBR1, NDP, NF1, PDLIM3, PHKA2, PVRL1, RGS4, SGK1, VGF and ZMYM2 were 33.3%, 42.9%, 71.4%, 100.0%, 42.9%, 71.4%, 85.7%, 28.6%, 100.0%, 16.7%, 42.9%, 100.0%, 33.3%, 16.7%, 16.7%, 33.3%, 16.7%, 57.1% and 57.1% respectively in urothelial cancer cell lines. Based on methylation frequencies and known functional evidence, CAMK4, HOXB5, MAL, RGS4, VGF and ZMYM2 were selected for re-expression analysis after 5-Aza-2′-deoxycytidine treatment of UCC cell lines. We also found that promoter methylation of these 6 genes were inversely correlated with gene expression. Since VGF gene was found to be completely methylated in the representative cell lines, we further performed QMSP analysis on 19 primary UCC tissues of different stages and grades with paired normal. Quantitative methylation values were higher in almost all the primary UCC tissues than the paired normal (student's t-test, P=0.005). In an independent cohort of 35 low grade UCC primary tissues, we performed QMSP assay. Twelve out of thirty-five (34.3%) of low grade UCC cases displayed VGF methylation that indicate VGF could be a marker for determining initiation of neoplastic transformation. To explore the potential of methylated VGF as a biomarker for non-invasive detection of UCC, we tested 20 urine samples from UCC cases and 20 urine samples from subjects without any known neoplastic diseases. VGF showed significantly high frequency of QMSP positive status (Fisher's exact test, P=0.020) in urine from UCC cases (8/20) compared to controls (1/20).

Conclusion: Our selected genes from genome-wide screening approach successfully identified UCC specific methylated genes that can be determined in primary tissues and urine. Further studies are needed to understand the role of VGF in urothelial carcinogenesis and its potential for the detection of low grade UCC using urine.

Citation Format: Masamichi Hayashi, Heike Bernert, Luciane Tsukamoto Kagohara, Mariana Brait, Mark Schoenberg, Trinity Bivalacqua, George Netto, Wayne Koch, David Sidransky, Mohammad O. Hoque. Epigenetic inactivation of VGF in urothelial cell carcinoma and its potential as a non-invasive biomarker using urine. [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 1363. doi:10.1158/1538-7445.AM2014-1363