Colorectal cancer (CRC) is a significant health burden especially among African Americans (AA). Epidemiological studies have correlated low serum vitamin D with CRC risk, and, while hypovitaminosis D is more common and more severe in AA, the mechanisms by which vitamin D modulates CRC risk and how these differ by race are not well understood. Active vitamin D (1α,25(OH)2D3) has chemoprotective effects primarily through transcriptional regulation of target genes in the colon. We hypothesized that transcriptional response to 1α,25(OH)2D3 differs between AA and European Americans (EA) irrespective of serum vitamin D and that regulatory variants could impact transcriptional response. We treated ex vivo colon cultures from 34 healthy subjects (16 AA and 18 EA) with 0.1 µM 1α,25(OH)2D3 or vehicle control for 6 hours and performed genome-wide transcriptional profiling. We found 8 genes with significant differences in transcriptional response to 1α,25(OH)2D3 between AA and EA with definitive replication of inter-ethnic differences for uridine phosphorylase 1 (UPP1) and zinc finger-SWIM containing 4 (ZSWIM4). We performed expression quantitative trait loci (eQTL) mapping and identified response cis-eQTLs for ZSWIM4 as well as for histone deacetylase 3 (HDAC3), the latter of which showed a trend toward significant inter-ethnic differences in transcriptional response. Allele frequency differences of eQTLs for ZSWIM4 and HDAC3 accounted for observed transcriptional differences between populations. Taken together, our results demonstrate that transcriptional response to 1α,25(OH)2D3 differs between AA and EA independent of serum 25(OH)D levels . We provide evidence in support of a genetic regulatory mechanism underlying transcriptional differences between populations for ZSWIM4 and HDAC3. Further work is needed to elucidate how response eQTLs modify vitamin D response and whether genotype and/or transcriptional response correlate with chemopreventive effects. Relevant biomarkers, such as tissue-specific 1α,25(OH)2D3 transcriptional response, could identify individuals likely to benefit from vitamin D for CRC prevention as well as elucidate basic mechanisms underlying CRC disparities.

We found vitamin D to increase expression of UPP1, leading to reduce uridine-induced DNA damage, in colon cells and organoids. A polymorphism in UPP1 found more frequently in African Americans than European Americans reduced UPP1 expression upon cell exposure to 1α,25(OH)D. Differences in expression of UPP1 in response to vitamin D could contribute to the increased risk of CRC in African Americans.

The vitamin D related pathway has been evaluated in carcinogenesis but its genetic contribution remains poorly understood. We examined single-nucleotide polymorphisms (SNPs) in the vitamin D related pathway genes using data from a genome-wide association study (GWAS) of breast cancer in the African Diaspora that included 3,686 participants (1,657 cases). Pathwayand gene-level analyses were conducted using the adaptive rank truncated product test. Odds ratios (OR) and 95% confidence intervals (CI) were estimated at SNP-level. After stringent Bonferroni corrections, we observed no significant association between variants in the vitamin D pathway and breast cancer risk at the pathway-, gene-, or SNP-level. In addition, no association was found for either the reported signals from GWASs of vitamin D related traits, or the SNPs within vitamin D receptor (VDR) binding regions. Furthermore, a decrease in genetically predicted 25(OH)D levels by Mendelian randomization was not associated with breast cancer (p 5 0.23). However, an association for breast cancer with the pigment synthesis/metabolism pathway almost approached significance (pathway-level p 5 0.08), driven primarily by a nonsense SNP rs41302073 in TYRP1, with an OR of 1.54 (95% CI 5 1.24-1.91, p adj 5 0.007). In conclusion, we found no evidence to support an association between vitamin D status and breast cancer risk in women of African ancestry, suggesting that vitamin D is unlikely to have significant effect on breast carcinogenesis. Interestingly, TYRP1 might be related to breast cancer through a non-vitamin D relevant mechanism but further studies are needed.In vitro and in vivo studies strongly suggested that vitamin D status may have a protective effect against breast cancer because of its role in regulating cell proliferation, differentiation and apoptosis. 1,2 Epidemiology studies also reported that individuals with dark skin pigmentation like African American women have vitamin D deficiency, 3,4 and women of African ancestry experience higher breast cancer-specific mortality 5 and are more likely to present with estrogen receptor negative (ER-) tumors. 6 However, the correlation between vitamin D status and breast cancer is still unclear in population studies. 7,8

Active vitamin D, 1α,25(OH)2D3, is a nuclear hormone with roles in colonic homeostasis and carcinogenesis; yet, mechanisms underlying these effects are incompletely understood. Human organoids are an ideal system to study genomic and epigenomic host-environment interactions. Here, we use human colonic organoids to measure 1α,25(OH)2D3 responses on genome-wide gene expression and chromatin accessibility over time. Human colonic organoids were cultured and treated in triplicate with 100 nM 1α,25(OH)2D3 or vehicle control for 4 h and 18 h for chromatin accessibility, and 6 h and 24 h for gene expression. ATAC- and RNA-sequencing were performed. Differentially accessible peaks were analyzed using DiffBind and edgeR; differentially expressed genes were analyzed using DESeq2. Motif enrichment was determined using HOMER. At 6 h and 24 h, 2,870 and 2,721 differentially expressed genes, respectively (false discovery rate, FDR < 5%), were identified with overall stronger responses with 1α,25(OH)2D3. Similarly, 1α,25(OH)2D3 treatment led to stronger chromatin accessibility especially at 4 h. The vitamin D receptor (VDR) motif was strongly enriched among accessible chromatin peaks with 1α,25(OH)2D3 treatment accounting for 30.5% and 11% of target sequences at 4 h and 18 h, respectively (FDR < 1%). A number of genes such as CYP24A1, FGF19, MYC, FOS, and TGFBR2 showed significant transcriptional and chromatin accessibility responses to 1α,25(OH)2D3 treatment with accessible chromatin located distant from promoters for some gene regions. Assessment of chromatin accessibility and transcriptional responses to 1α,25(OH)2D3 yielded new observations about vitamin D genome-wide effects in the colon facilitated by application of human colonic organoids. This framework can be applied to study host-environment interactions between individuals and populations in the future.

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