Activation of RHOA transcription in epithelial tumors may be caused by gene amplification and/or demethylation of the promoter region

RHOA is a small GTPase involved in morphogenesis, cell adhesion, and cell cycle control. RHOA (3p21.31) is a potential oncogene, causing cell malignant transformation. A study was made of the mechanisms activating RHOA transcription in several epithelial tumors. Semiquantitative RT-PCR revealed elevated transcription of RHOA in tumors (45 cases of breast cancer, renal cell carcinoma, and epithelial ovarian carcinoma; p < 10(-4)). For the first time transcriptional activation of RHOA in tumors was reliably associated with its amplification (p = 10(-7)). Digestion with four methyl-sensitive restriction enzymes (HpaII, HhaI, AciI, and Bsh1236I) and subsequent PCR revealed a change in methylation of the RHOA promoter region in 23 out of 45 tumors, hypomethylation being two times more frequent than hypermethylation. Demethylation of the RHOA promoter region was associated with a two- to tenfold increase in the transcription level of the gene. Thus, two mechanisms-amplification and demethylation of the promoter region-were shown to change the level of RHOA transcription in epithelial tumors.