Simple SummaryUlcerative colitis, a form of inflammatory bowel disease, is a major risk factor for developing colorectal cancer. Patients with ulcerative colitis often present with low serum folate levels due to the adverse effect of maintenance therapies and/or intestinal malabsorption and require folic acid supplementation. While folic acid supplementation confers protection against sporadic colorectal cancer when given prior to colon tumor formation, it can promote cancer formation when administered to mice with early colonic lesions. Few studies have focused on the impact of folic acid on colorectal cancer risk in patients with ulcerative colitis. In this study, folic acid supplementation created a hypomethylated field within the inflamed non-neoplastic colonic mucosa of mice with colitis and promoted the formation of colitis-associated tumors. These data suggest that caution should be taken when recommending folic acid supplements to patients with ulcerative colitis who are at high risk of colorectal cancer.AbstractPurpose: The purpose of this study was to assess the effect of folic acid (FA) supplementation on colitis-associated colorectal cancer (CRC) using the azoxymethane/dextran sulfate sodium (AOM/DSS) model. Methods: Mice were fed a chow containing 2 mg/kg FA at baseline and randomized after the first DSS treatment to receive 0, 2, or 8 mg/kg FA chow for 16 weeks. Colon tissue was collected for histopathological evaluation, genome-wide methylation analyses (Digital Restriction Enzyme Assay of Methylation), and gene expression profiling (RNA-Seq). Results: A dose-dependent increase in the multiplicity of colonic dysplasias was observed, with the multiplicity of total and polypoid dysplasias higher (64% and 225%, respectively) in the 8 mg FA vs. the 0 mg FA group (p< 0.001). Polypoid dysplasias were hypomethylated, as compared to the non-neoplastic colonic mucosa (p< 0.05), irrespective of FA treatment. The colonic mucosa of the 8 mg FA group was markedly hypomethylated as compared to the 0 mg FA group. Differential methylation of genes involved in Wnt/β-catenin and MAPK signaling resulted in corresponding alterations in gene expression within the colonic mucosa. Conclusions: High-dose FA created an altered epigenetic field effect within the non-neoplastic colonic mucosa. The observed decrease in site-specific DNA methylation altered oncogenic pathways and promoted colitis-associated CRC.