Methylation of NOTCH genes in normal and at-risk colorectal epithelium

Abstract

Introduction Colorectal cancer (CRC) arises from genetic defects in stem cells. NOTCH signalling plays a key role in stem cell replication control. NOTCH-related genes are overexpressed in CRC. The mechanism for this is not known but could include epigenetic activation of NOTCH oncogenes via promoter hypomethylation. Methylation can be modulated by environmental stimuli including dietary factors such as butyrate, produced by bacterial fermentation of non-digestible carbohydrates in the colon. Butyrate exerts potent anti-oncogenic effects in the colorectal mucosa. Methods Participants were recruited at endoscopy and included those at normal risk of CRC (n=75), or higher risk of CRC because of previous adenomatous polyps (n=28) or ulcerative colitis (n=12). Participants provided 9 rectal biopsies. Normal risk participants were randomised to resistant starch (Hi-maize 260) or polydextrose supplementation in a 2x2 factorial placebo controlled trial for 50 days. Methylation of several CpG sites in the promoters of JAG1 (NOTCH pathway ligand) and RBP-J (NOTCH intracellular activator) was quantified using pyrosequencing. Results For JAG1 there was trend towards lower methylation at all CpG sites in those at higher CRC risk. Methylation at RBP-J CpG 11 was lower in polyp patients than in controls (18.0(1.5) vs. 23.6% (0.8), p=0.011). At JAG1 CpG 4, methylation increased following polydextrose supplementation compared to placebo (3.1(0.4) vs. 1.7%(0.4), p=0.009). A similar, but non-significant, trend was observed at other CpG sites for JAG1. Conclusions DNA methylation of NOTCH signalling genes is altered in macroscopically normal colorectal mucosa of patients at higher CRC risk. The observed changes in JAG1 methylation after polydextrose supplementation are consistent with a protective effect against carcinogenesis.