Discovery of aberrant expression of R-RAS by cancer-linked DNA hypomethylation in gastric cancer using microarrays.

Although hypomethylation was the originally identified epigenetic change in cancer, it was overlooked for many years in preference to hypermethylation. Recently, gene activation by cancer-linked hypomethylation has been rediscovered. However, in gastric cancer, genome-wide screening of the activated genes has not been found. By using microarrays, we identified 1,383 gene candidates reactivated in at least one cell line of eight gastric cancer cell lines after treatment with 5-aza-2'deoxycytidine and trichostatin A. Of the 1,383 genes, 159 genes, including oncogenes ELK1, FRAT2, R-RAS, RHOB, and RHO6, were further selected as gene candidates that are silenced by DNA methylation in normal stomach mucosa but are activated by DNA demethylation in a subset of gastric cancers. Next, we showed that demethylation of specific CpG sites within the first intron of R-RAS causes activation in more than half of gastric cancers. Introduction of siRNA into R-RAS-expressing cells resulted in the disappearance of the adhered cells, suggesting that functional blocking of the R-RAS-signaling pathway has great potential for gastric cancer therapy. Our extensive gene list provides other candidates for this class of oncogene.