Colorectal cancer (CRC) is characterized by genome-wide alterations to DNA methylation that influence gene expression and genomic stability. Less is known about the extent to which methylation is disrupted in the earliest stages of CRC development. In this study we have combined laser-capture microdissection (LCM) with reduced representation bisulfite sequencing (RRBS) to identify cancer-associated DNA methylation changes in human aberrant crypt foci (ACF), the earliest putative precursor to CRC. Using this approach, methylation profiles have been generated for 10 KRAS-mutant ACF and 10 CRCs harboring a KRAS mutation, as well as matched samples of normal mucosa. Of 811 differentially methylated regions (DMRs) identified in ACF, 537 (66%) were hypermethylated and 274 (34%) were hypomethylated. DMRs located within intergenic regions were heavily enriched for AP-1 transcription factor binding sites and were frequently hypomethylated. Furthermore, gene ontology (GO) analysis demonstrated that DMRs associated with promoters were enriched for genes involved in intestinal development, including homeobox genes and targets of the Polycomb repressive complex 2 (PRC2). Consistent with their role in the earliest stages of colonic neoplasia, 75% of the loci harboring methylation changes in ACF were also altered in CRC samples, though the magnitude of change at these sites was lesser in ACF. While aberrant promoter methylation was associated with altered gene expression in CRC, this was not the case in ACF, suggesting the insufficiency of methylation changes to modulate gene expression in early colonic neoplasia. Together, these data demonstrate that DNA methylation changes, including significant hypermethylation, occur more frequently in early colonic neoplasia than previously believed, and identify epigenomic features of ACF that may provide new targets for cancer chemoprevention or lead to the development of new biomarkers for CRC risk.

The role of folate one-carbon metabolism in colorectal cancer development is controversial, with nutritional intervention studies producing conflicting results. It has been reported that ApcMin/+ mice maintained on a diet deficient in the methyl donors folic acid, methionine, choline and vitamin B12, and supplemented with homocysteine, show a >95% reduction in intestinal tumor development. The present study extends these findings and shows that tumor protection afforded by dietary methyl donor deficiency (MDD) is long-lasting. After eleven weeks of MDD, tumor protection persisted for at least an additional seven weeks of methyl donor repletion (22.2 ± 3.5 vs 70.2 ± 4.6 tumors per mouse; P<0.01). Sustained tumor protection was associated with a reduction in intestinal crypt length (26%, P<0.01), crypt cell division and crypt fission, and an increase in apoptosis of both normal crypts and tumors (4.9- and 3.2-fold, respectively, P<0.01). MDD also caused a significant reduction in the number of Dclk1-positive cells in the intestine (62%, P<0.01), a long-lived crypt cell with cancer stem cell potential. Several undesirable effects associated with methyl donor restriction (e.g., reduced body weight gain) were shown to be transient and readily reversible following methyl donor repletion. Taken together, these results indicate that even temporary dietary methyl donor restriction in adenoma-prone mice can induce persistent changes to the intestinal epithelium and provide long-lasting tumor protection. These data also suggest that transient reductions in dietary methyl donor consumption should be considered when studying the impact of folate on colon cancer risk in humans.

Familial adenomatous polyposis (FAP) is a genetic disorder characterized by the development of hundreds of polyps throughout the colon. Without prophylactic colectomy, most individuals with FAP develop colorectal cancer at an early age. Treatment with EPA in the free fatty acid form (EPA-FFA) has been shown to reduce polyp burden in FAP patients. Since high-purity EPA-FFA is subject to rapid oxidation, a stable form of EPA compound has been developed in the form of magnesium l-lysinate bis-eicosapentaenoate (TP-252). We assessed the chemopreventive efficacy of TP-252 on intestinal tumor formation using ApcΔ14/+ mice and compared it with EPA-FFA. TP-252 was supplemented in a modified AIN-93G diet at 1, 2 or 4% and EPA-FFA at 2.5% by weight and administered to mice for 11 weeks. We found that administration of TP-252 significantly reduced tumor number and size in the small intestine and colon in a dose-related manner and as effectively as EPA-FFA. To gain further insight into the cancer protection afforded to the colon, we performed a comprehensive lipidomic analysis of total fatty acid composition and eicosanoid metabolites. Treatment with TP-252 significantly decreased the levels of arachidonic acid (AA) and increased EPA concentrations within the colonic mucosa. Furthermore, a classification and regression tree (CART) analysis revealed that a subset of fatty acids, including EPA and docosahexaenoic acid (DHA), and their downstream metabolites, including PGE3 and 14-hydroxy-docosahexaenoic acid (HDoHE), were strongly associated with antineoplastic activity. These results indicate that TP-252 warrants further clinical development as a potential strategy for delaying colectomy in adolescent FAP patients.

Our understanding of the role of folate one-carbon metabolism in colon carcinogenesis remains incomplete. Previous studies indicate that a methyl donor deficient (MDD) diet lacking folic acid, choline, methionine, and vitamin B12 is associated with long-lasting changes to the intestinal epithelium and sustained tumor protection in Apc-mutant mice. However, the metabolic pathways by which the MDD diet effects these changes are unknown. Colon samples harvested from Apc Δ14/+ mice fed the MDD diet for 18 weeks were profiled using a GC-MS and LC-MS/MS metabolomics platform. Random forest and pathway analyses were used to identify altered metabolic pathways and associated gene expression changes were analyzed by RT-PCR. Approximately 100 metabolites impacted by the MDD diet were identified. As expected, metabolites within the methionine cycle, including methionine (−2.9-fold, P<0.001) and betaine (−3.3-fold, P<0.001), were reduced. Elevated homocysteine (110-fold, P<0.001) was associated with increased flux through the transsulfuration pathway. Unexpectedly, levels of deoxycholic acid (−4.5-fold, P<0.05) and several other secondary bile acids were reduced. There were also unexpected reductions in the levels of carnitine (−2.0-fold, P<0.01) and a panel of acylcarnitines involved in fatty acid β-oxidation. Finally, metabolites involved in redox balance, including ascorbate and hypotaurine, were found to be persistently elevated. These findings provide clues to the molecular changes underlying MDD-mediated tumor protection and identify regulatable metabolic pathways that may provide new targets for colon cancer prevention and treatment.