Deficiency and Excess of Folic Acid Intake Promote Colorectal Carcinogenesis in AOM/DSS-Treated Mice: Roles in Uracil...

Background/Objectives: Colorectal cancer (CRC) is a leading cause of cancer-related mortality worldwide, yet the association between folic acid (FA) intake and CRC risk remains controversial. This study investigated the effects of varying dietary FA levels on colorectal carcinogenesis and the underlying mechanisms. Methods: BALB/c mice were fed diets containing FA at <0.1, 2.0, 6.0, 8.0, or 20.0 mg/kg for 14 weeks. After 4 weeks, colorectal tumorigenesis was induced using the azoxymethane/dextran sulfate sodium (AOM/DSS) protocol. Tumor multiplicity, maximum tumor diameter, tumor volume, colorectal length, histopathology, and cell proliferation were assessed. Mechanistic assessments included uracil misincorporation, thymidylate synthase (TS), telomere attrition, genome-wide DNA methylation, RAP1 signaling, immune-related markers, and inflammatory cytokines in colorectal tissues. Results: Both FA deficiency (<0.1 mg/kg) and excess (8.0/20.0 mg/kg) increased colorectal tumor burden, with increased tumor number, larger maximum diameter, greater tumor volume, shortened colorectal length, and enhanced cell proliferation, whereas the 6.0 mg/kg diet group showed the lowest tumor burden. FA deficiency reduced TS expression, elevated deoxyuridine monophosphate (dUMP) levels, decreased deoxythymidine monophosphate (dTMP) levels, increased uracil misincorporation, and exacerbated telomere attrition, as evidenced by shortened telomeres and increased damage. In contrast, excessive FA intake induced Rap1 GTPase-activating protein (RAP1GAP) hypermethylation, reduced Rap1GAP expression, enhanced RAP1 activity, and upregulated programmed death-ligand 1 (PD-L1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) expression. Conclusions: Dietary FA can exhibit a U-shaped association with colorectal carcinogenesis, with protective effects observed within an optimal range. FA deficiency and excess may drive tumor development through distinct molecular pathways involving uracil misincorporation-induced telomere attrition and DNA methylation-mediated immunosuppression, respectively.