Abstract 29: Molecular epidemiology of Gc-globulin isotype, circulating 25(OH)D levels, and uptake of vitamin D metabolites in colon cancer cells.

Associations between low circulating vitamin D metabolite concentrations and risk for colorectal neoplasia are demonstrated by numerous studies. Gc-globulin (GC) is the transport protein for vitamin D metabolites and isotypes of the protein, based on two nonsynonymous polymorphisms (rs7041 and rs4588), have different frequencies by race and ethnicity. The phenotype groups are 1F\_1F, 1F\_1S, 1S\_1S, 1F\_2, 1S\_2 and 2\_2. There is evidence that the GC isotypes have varying affinities for 25(OH)D and 1α,25(OH)2D, which may affect delivery of vitamin D metabolites at the cellular level. Furthermore, epidemiologic studies have demonstrated higher circulating 25(OH)D in individuals with copies of the 1S allele. Significantly different levels of vitamin D target gene transcription in monocytes treated with 25(OH)D and serum containing known GC isotype have also been observed. However, there is not yet evidence that uptake of 1α,25(OH)2D varies with GC isotype or evidence for variation in uptake of either metabolite in colon cancer cell lines, specifically. We therefore conducted an epidemiological association study of GC isotype and circulating vitamin D metabolite concentrations, as well as experiments to investigate vitamin D metabolite uptake in colon cancer cells. The VDR-RXR mammalian-two-hybrid (M2H) and vitamin D responsive element (VDRE)-based luciferase assay systems were employed for the latter study. HCT-116 cells were dosed with ethanol vehicle or various concentrations of 1α,25(OH)2D or 25(OH)D in the presence of plasma from individuals with known GC isotype. Luciferase assays were then conducted to measure metabolite uptake and activation of the VDR pathway in these cells. The epidemiological analysis demonstrated statistically significantly lower 25(OH)D for Caucasian participants with at least one copy of the GC_2 allele (p < 0.01) compared to the 1F or 1S phenotypes. The cellular-level data showed overall statistically significant differences in uptake of 1α,25(OH)2D by GC isotype (p < 0.001) as well as across 1α,25(OH)2D concentrations (10−8 to 5×10−10 M) (p<0.001). Overall, the 1F\_1F, 1F\_1S and 1F\_2 isotypes produced the greatest induction of the luciferase reporter gene in the M2H system; while the 2\_2, 1S\_2 and 1S\_1S isotypes displayed lower activity. Similar results for 1α,25(OH)2D were obtained using the VDRE assay system. The induction by 25(OH)D was statistically significantly associated with GC isotype in the VDRE-based system (p<0.001), but not with the M2H system (p = 0.36). However, stratification by concentration demonstrated that system activation was significantly different by GC isotype for all 25(OH)D concentrations in the VDRE system (p<0.01), but only the two highest concentrations of 5×10−7 M and 2×10−7 M with the M2H assay (p< 0.05). The induction by genotype varied more with 25(OH)D, though the 2_2 isotype again exhibited consistently lower activation. These results suggest that GC isotype alters the delivery of 1α,25(OH)2D and, potentially to a lesser extent, 25(OH)D to colon cells. These results may help identify additional populations at risk for disease related to the vitamin D endocrine system. Alternate methodologies for cellular-level experiments and repeated evaluation in epidemiological studies are necessary, especially to account for variation by race/ethnicity. Studying Gc-globulin phenotype in combination with circulating metabolite levels may improve our understanding of vitamin D deficiency and its relation to health outcomes. Citation Format: Elizabeth A. Hibler, Elizabeth T. Jacobs, Peter W. Jurutka. Molecular epidemiology of Gc-globulin isotype, circulating 25(OH)D levels, and uptake of vitamin D metabolites in colon cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Post-GWAS Horizons in Molecular Epidemiology: Digging Deeper into the Environment; 2012 Nov 11-14; Hollywood, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(11 Suppl):Abstract nr 29.