Epigenetic inactivation of VGF associated with Urothelial Cell Carcinoma and its potential as a non-invasive biomarker using urine

Masamichi Hayashi,Mohammad O Hoque,George J Netto,Trinity Bivalacqua,David Sidransky,Mariana Brait,Wayne Koch,Leonel Maldonado,Luciane Tsukamoto Kagohara,Heike Bernert,Mark Schoenberg

doi:10.18632/oncotarget.1949

Abstract

To identify new epigenetic markers and further characterize Urothelial Cell Carcinoma (UCC), we tested the promoter methylation (PM) status of 19 genes previously identified as cancer specific methylated genes in other solid tumors. We used bisulfite sequencing, methylation specific PCR and quantitative methylation specific PCR (QMSP) to test the PM status of 19 genes in urothelial cancer cell lines. Among the 19 genes tested, VGF was found to be completely methylated in several UCC cell lines. VGF QMSP analysis showed that methylation values of almost all the primary 19 UCC tissues were higher than the paired normal tissues (P=0.009). In another cohort, 12/35 (34.3%) of low grade UCC cases displayed VGF methylation. As a biomarker for non-invasive detection of UCC, VGF showed a significantly higher frequency of methylation in urine from UCC cases (8/20) compared to controls (1/20) (P=0.020). After treatment of cell lines with 5-Aza-2'-deoxycytidine, VGF was robustly re-expressed. Forced expression of VGF in bladder cancer cell lines inhibited cell growth. Selection of candidates from genome-wide screening approach in other solid tumors successfully identified UCC specific methylated genes.

Highlights

2.7 million people are assessed to be diagnosed with, or have a history of urothelial cell carcinoma (UCC) [1]
The methylation frequency and patterns of each of the 19 genes were evaluated by bisulfite sequencing in 6 to 7 bladder cancer cell lines
Based on the later criteria, methylation positive bladder cancer cell lines were ranged from 16.7% to 100%

Summary

Introduction

2.7 million people are assessed to be diagnosed with, or have a history of urothelial cell carcinoma (UCC) [1]. Of all UCC, the majority (75–85%) presents as non-musclewww.impactjournals.com/oncotarget invasive bladder cancer (NMIBC) confined to the mucosa [stage Ta in 70%; carcinoma in situ (CIS) in 10%] or to the sub-mucosa (stage T1 in 20%) [3,4,5]. These NMIBC lesions recur quite frequently (70-80%) presenting a significant problem in the overall management and monitoring of the disease [5]. In order to manage UCC in a cost effective manner, it is required to understand the biology of this disease and to establish tumor specific biomarkers for early cancer detection and monitoring. To identify new epigenetic markers and further characterize Urothelial Cell Carcinoma (UCC), we tested the promoter methylation (PM) status of 19 genes previously identified as cancer specific methylated genes in other solid tumors

Methods

Results

Conclusion