Promoter hypomethylation as potential confounder of Ras gene overexpression and their clinical significance in subsets of urothelial carcinoma of bladder

Abstract

Overexpression of normal Ras and its aberrant CpG island methylation in the promoter regions have been shown to direct cells for uncontrolled abnormal growth and bladder tumor formation and therefore, fetched recent attention as a marker of diagnosis and prognosis to predict the biological behavior of urothelial carcinoma of bladder (UCB). Methylation pattern at CpG islands of the promoter regions of rat sarcoma (Ras) gene homologues namely Kristen-Ras (K-Ras), Harvey (H-Ras), and Neuroblastoma (N-Ras) were examined by methylation specific polymerase chain reaction (MSP). Real time-quantitative polymerase chain reaction (RT-qPCR) was done to determine transcriptomic expressions of these Ras isoforms in the prospective series of 42 NMIBC (non-muscle invasive bladder cancer) and 45 MIBC (muscle invasive bladder cancer) biopsies. CpG loci in H-Ras and K-Ras were observed to be more hypomethylated in MIBC, whereas more hypomethylation in N-Ras was noted in NMIBC. Strong association of hypomethylation index with tumor stage, grade, type and size validate them it as marker of diagnosis in UCB patients. Differential overexpression of H-Ras, N-Ras and K-Ras genes in NMIBC and MIBC and their association with patients' demographics identify them as important diagnostic markers in pathogenesis of UCB. Given the reported ability of promoter hypomethylation to activate Ras expression, correlation studies examined positive significant association between hypomethylation index and expression. Study concludes that promoter hypomethylation of N-Ras and K-Ras could be a potential confounder of their increased expression in NMIBC. Biological significance of simultaneous presence of higher expression and promoter hypomethylation of Ras gene isoforms in MIBC is difficult to resolve in a given cohort of patients.

Keywords: Clinical variables; Promoter hypomethylation; Ras genes; Transcriptomic expression; Urothelial carcinoma of bladder.