Epidemiological investigations have demonstrated that low serum levels of the vitamin D metabolite 25(OH)D, the precursor molecule for the hormone 1,25(OH)2D, are associated with risk of colorectal neoplasia, though results have been equivocal, and little is known about sex- or colorectal sub-site-specific relationships for these biomarkers. From a functional standpoint, anti-proliferative, pro-differentiating, and growth inhibitory effects of 1,25(OH)2D have been shown in colorectal cancer cell lines. Thus, there is compelling evidence that active vitamin D metabolites play a role in reducing the risk for colorectal neoplasia, but the best approach for attaining optimal levels of 1,25(OH)2D at the cellular level are not known, and the functional effects of genetic variation in the vitamin D receptor (VDR) or in primary enzymes responsible for metabolism of 1,25(OH)2Dat the tissue level are poorly understood. Preliminary evidence indicates that SNPs in VDR may influence circulating concentrations of 1,25(OH)2D;further, two enzymes, CYP27B1 and CYP24A1, are of particular importance in metabolic homeostasis of this molecule. Although there has been research into the functionality of and epidemiological associations of the VDR, there are few studies of the molecular or epidemiological effects of genetic variation in these two key enzymes. Further, the mechanisms through which vitamin D may exert its chemopreventive effects are unknown, though work by our group as well as others suggests that APC, b-catenin, and 1,25(OH)2D-VDR represent a molecular crossroads of intracellular regulators of colonic cell proliferation. The specific aims of the proposed work are to 1) Measure circulating 25(OH)D and 1,25(OH)2D concentrations among participants in clinical trials of colorectal adenoma recurrence, and assess the association between these variables and the odds of adenoma recurrence; 2) Genotype study participants for variation in VDR, CYP27B1 and CYP24A1 and evaluate the association between SNPs and odds for colorectal adenoma recurrence and circulating concentrations of vitamin D metabolites; 3) Elucidate the functional effects of selected polymorphisms in the CYP27B1 and CYP24A1 genes in human Caco-2 colonic cells; 4) Investigate molecular crosstalk between VDR (and VDR polymorphic variants) and 2-catenin, as well as the functional influence of APC on these signal transduction pathways. Secondary aims include investigation of sex-stratified associations between vitamin D metabolites and recurrence of adenomas and advanced adenomas, as well as assessment of whether these associations vary by colorectal sub-site. This work has the potential to offer a cost-effective preventative measure for colorectal carcinogenesis through improving vitamin D status, as well as to illuminate novel molecular evidence for the functional effects of genetic variation in key enzymes of vitamin D metabolism, and for the elucidation of signaling pathways, including APC and b-catenin, that likely modulate the actions of vitamin D metabolites and VDR. PUBLIC HEALTH RELEVANCE: The proposed work has the potential to offer a cost-effective preventative measure for colorectal carcinogenesis through improving vitamin D status, as well as providing molecular evidence for the functional effects of genetic variation in key enzymes in the vitamin D pathway, and for the pathways through which vitamin D metabolites are acting, including via APC/B-catenin.