Elizabeth A. Hibler

Vitamin D and breast cancer recurrence in the Women's Healthy Eating and Living (WHEL) Study

BACKGROUND There is a paucity of research evaluating the relation between vitamin D and recurrence of breast cancer after treatment. OBJECTIVE This study was designed to evaluate the associations between circulating concentrations of 25-hydroxyvitamin D [25(OH)D] and dietary, supplemental, and total intake of vitamin D and recurrent or new breast cancer events within the Womens Healthy Eating and Living (WHEL) Study. DESIGN A prospective cohort study design (n = 3085) was used to evaluate the relation between dietary, supplemental, and total vitamin D intake and recurrent breast cancer, and a nested case-control study with 512 matched pairs was used for analysis of the association between 25(OH)D and breast cancer recurrence. RESULTS No relation between 25(OH)D and breast cancer recurrence was observed. Compared with women with serum concentrations of 25(OH)D ≥ 30 ng/mL, adjusted odds ratios (95% CI) for breast cancer recurrence were 1.14 (0.57, 2.31) for those with concentrations < 10 ng/mL, 1.00 (0.68-1.48) for concentrations ≥ 10 and < 20 ng/mL, and 1.05 (0.76, 1.47) for concentrations ≥ 20 and < 30 ng/mL. No significant associations were observed when analyses were stratified by pre- and postmenopausal status or for local, regional, or distant recurrence or death. Vitamin D intake was not related to breast cancer recurrence overall, although for premenopausal women there was a significant inverse association between dietary vitamin D intake and recurrence (P for trend = 0.02). CONCLUSION These results do not provide support for a relation between concentrations of 25(OH)D after treatment and the recurrence of breast cancer. This trial is registered at clinicaltrials.gov for the WHEL Study as NCT00003787.

Genetic Polymorphisms in Vitamin D Receptor VDR/RXRA Influence the Likelihood of Colon Adenoma Recurrence

Low circulating levels of vitamin D affect colorectal cancer risk. The biological actions of the hormonal form of vitamin D, 1,25(OH)(2)D(3), are mediated by the vitamin D receptor (VDR), which heterodimerizes with retinoid X receptors (RXR). Using a single nucleotide polymorphism (SNP) tagging approach, we assessed the association between genetic variations in RXRA and VDR and odds of recurrent (metachronous) colorectal neoplasia in a pooled population of two studies. A total of 32 tag SNPs in RXRA and 42 in VDR were analyzed in 1,439 participants. A gene-level association was observed for RXRA and any (P = 0.04) or proximal (P = 0.03) metachronous neoplasia. No gene-level associations were observed for VDR, nor was any single SNP in VDR related to any metachronous adenoma after correction for multiple comparisons. In contrast, the association between RXRA SNP rs7861779 and proximal metachronous neoplasia was of borderline statistical significance [odds ratio (OR), 0.68; 95% confidence interval (95% CI), 0.53-0.86; unadjusted P = 0.001; adjusted P = 0.06], including when observed independently in each individual study. Haplotypes within linkage blocks of RXRA support an approximately 30% reduction in odds of metachronous neoplasia arising in the proximal colon among carriers of specific haplotypes, which was strongest (OR(proximal), 0.67; 95% CI, 0.52-0.86) for carriers of a CGGGCA haplotype (rs1805352, rs3132297, rs3132296, rs3118529, rs3118536, and rs7861779). Our results indicate that allelic variation in RXRA affects metachronous colorectal neoplasia, perhaps of particular importance in the development of proximal lesions.

CYP24A1 and CYP27B1 Polymorphisms Modulate Vitamin D Metabolism in Colon Cancer Cells

Vitamin D is a well-studied agent for cancer chemoprevention and treatment. Its chief circulating metabolite, 25-hydroxyvitamin D, is converted into the active hormone 1,25-dihydroxyvitamin D (1,25D) by the cytochrome P450 enzyme CYP27B1 in kidney and other tissues. 1,25D is then deactivated by CYP24A1 and ultimately catabolized. Colorectal carcinoma cells express CYP27B1 and CYP24A1 that locally regulate 1,25D with potential implications for its impact on carcinogenesis. While 1,25D inhibits cancer growth, the effects of polymorphic variations in genes encoding proteins involved in 1,25D homeostasis are poorly understood. Using an RXR-VDR mammalian two-hybrid (M2H) biologic assay system, we measured vitamin D metabolite uptake and activation of the vitamin D receptor (VDR) pathway in colon cancer cells that expressed one of five CYP27B1 single-nucleotide polymorphisms (SNP) or four CYP24A1 SNPs. Compared with the wild-type control, four of five CYP27B1 SNPs reduced enzymatic activity, whereas one (V166L) increased activity. For CYP24A1, all tested SNPs reduced enzyme activity. Quantitative real-time PCR analyses supported the results of M2H experiments. The observed SNP-directed variation in CYP functionality indicated that vitamin D homeostasis is complex and may be influenced by genetic factors. A comprehensive understanding of 1,25D metabolism may allow for a more personalized approach toward treating vitamin D-related disorders and evaluating risk for carcinogenesis.

Polymorphic variation in the GC and CASR genes and associations with vitamin D metabolite concentration and metachronous colorectal neoplasia.

Background: Vitamin D levels and calcium intake have been associated with risk of colorectal neoplasia, and genetic variation in vitamin D pathway genes may affect circulating vitamin D metabolite concentrations and/or risk for colorectal lesions. This study evaluated associations between polymorphic variation in the Gc-globulin (GC) and calcium-sensing receptor (CASR) and odds for metachronous colorectal neoplasia and vitamin D metabolite concentrations. Methods: Participants from the Ursodeoxycholic Acid (UDCA) and Wheat Bran Fiber (WBF) trials (n = 1,439) were analyzed using a single-nucleotide polymorphism (SNP) tagging approach, with a subset (n = 404) of UDCA trial participants for whom vitamin D metabolite concentrations were also available. A total of 25 GC and 35 CASR tagSNPs were evaluated using multiple statistical methods. Results: Principal components analyses did not reveal gene-level associations between GC or CASR and colorectal neoplasia; however, a significant gene-level association between GC and 25(OH)D concentrations (P < 0.01) was observed. At the individual SNP level and following multiple comparisons adjustments, significant associations were observed between seven GC (rs7041, rs222035, rs842999, rs1155563, rs12512631, rs16846876, and rs1746825) polymorphisms and circulating measures of 25(OH)D (adjusted P < 0.01) and CASR SNP rs1042636 and proximal colorectal neoplasia (adjusted P = 0.01). Conclusions: These results show a possible association between variation in CASR and odds of colorectal neoplasia as well as the potential role of variation in GC with circulating 25(OH)D concentrations. Impact: Additional research is warranted to determine the mechanism of GC genotype in influencing 25(OH)D concentrations and to further elucidate the role of CASR in colorectal neoplasia. Cancer Epidemiol Biomarkers Prev; 21(2); 368–75. ©2011 AACR.

Association between polymorphic variation in VDR and RXRA and circulating levels of vitamin D metabolites.

The vitamin D metabolite 1,25(OH)2D is the bioactive ligand of the vitamin D receptor (VDR). VDR forms a heterodimer with the retinoid X receptors (RXRs) that when bound to ligand influences the transcriptional control of genes that regulate circulating levels of vitamin D metabolites. Whether genetic variation in VDR or RXRA affects circulating levels of 1,25(OH)2D or 25(OH)D has not been established. We used a single nucleotide polymorphism (SNP) tagging approach to evaluate the association between SNPs in VDR and RXRA and serum levels of 1,25(OH)2D and 25(OH)D. A total of 42 tagSNPs in VDR and 32 in RXRA were analyzed in a sample of 415 participants. Principal components analyses revealed a gene-level association between RXRA and serum 1,25(OH)2D concentrations (P=0.01), but not 25(OH)D. No gene-level association was found for VDR with either serum biomarker. At the single-SNP level, a significant positive trend was observed for increasing 1,25(OH)2D levels with each additional copy of the A allele for RXRA SNP rs9409929 (P-trend=0.003). After a multiple comparisons adjustment, no individual SNP in VDR or RXRA was significantly associated with either outcome. These results demonstrate an association between genetic variation in RXRA and 1,25(OH)2D serum concentrations.

Concentrations of the Vitamin D Metabolite 1,25(OH)2D and Odds of Metabolic Syndrome and its Components

AIM Few epidemiological studies have investigated the association between circulating concentrations of the active vitamin D metabolite 1,25(OH)2D and metabolic syndrome. We sought to determine whether blood levels of 1,25(OH)2D are associated with metabolic syndrome and its individual components, including waist circumference, triglycerides, blood pressure, and glucose, and high-density lipoprotein. We also investigated these associations for the more abundant precursor vitamin D metabolite, 25(OH)D. METHODS Participants from two completed clinical trials of colorectal neoplasia with available metabolic syndrome data and blood samples for measurement of 1,25(OH)2D (n=1048) and 25(OH)D (n=2096) were included. Cross-sectional analyses of the association between concentrations of 1,25(OH)2D, 25(OH)D, metabolic syndrome, and its components were conducted. RESULTS A statistically significant inverse association was observed for circulating concentrations of 1,25(OH)2D and metabolic syndrome, with adjusted ORs (95% CIs) of 0.73 (0.52-1.04) and 0.52 (0.36-0.75) for the second and third tertiles of 1,25(OH)2D, respectively (p-trend <0.001). Significant inverse relationships were also observed between 1,25(OH)2D and high triglycerides (p-trend <0.001), and low high-density lipoprotein (p-trend <0.001). For 25(OH)D concentrations, significant inverse associations were found for metabolic syndrome (p-trend <0.01), high waist circumference (p-trend <0.04) and triglyceride levels (p-trend <0.01). Participants with 25(OH)D ≥30 ng/ml and in the highest tertile of 1,25(OH)2D demonstrated significantly lower odds of metabolic syndrome, with an OR (95% CI) of 0.38 (0.19-0.75) compared to those in the lowest category for both metabolites. CONCLUSION These results provide new evidence that the relatively rarely-studied active hormonal form of vitamin D, 1,25(OH)2D, is associated with metabolic syndrome and its components, and confirm prior findings for 25(OH)D. The finding that 1,25(OH)2D is related to high-density lipoprotein, while 25(OH)D is not, suggests that there may be an independent mechanism of action for 1,25(OH)2D in relation to metabolic dysregulation.

Association between circulating concentrations of 25(OH)D and colorectal adenoma: A pooled analysis

The relationship between the biomarker of vitamin D status, 25(OH)D, and the risk for colorectal neoplasia is suggestive but equivocal. Questions remain regarding whether there are differential associations between 25(OH)D and colorectal adenoma by gender, colorectal subsite or features of baseline and recurrent adenomas. We sought to investigate the relationship between 25(OH)D and both baseline and recurrent adenoma characteristics. Our study was conducted among 2,074 participants in a pooled population of two clinical intervention trials of colorectal adenoma recurrence. A cross‐sectional analysis of 25(OH)D and baseline adenoma characteristics and a prospective study of recurrent adenomas and their characteristics were conducted. There was a statistically significant inverse association between the concentrations of 25(OH)D and the presence of three or more adenomas at baseline. Compared to participants with 25(OH)D levels of <20 ng/mL, the adjusted odds ratios (ORs) (95% condifdence intervals [CIs]) were 0.99 (0.70–1.41) for those with concentrations of ≥20 and <30 ng/mL, and 0.73 (0.50–1.06) among participants with levels of ≥30 ng/mL (p‐trend = 0.05). Baseline villous histology was also significantly inversely related to 25(OH)D levels (p‐trend = 0.04). Conversely, 25(OH)D concentrations were not associated with overall colorectal adenoma recurrence, with ORs (95% CIs) of 0.91 (0.71–1.17) and 0.95 (0.73–1.24; p‐trend = 0.85). These findings support the concept that the relationship between vitamin D and colorectal neoplasia may vary by stage of adenoma development.

Prospective changes in global DNA methylation and cancer incidence and mortality

Background:Methylation of repetitive elements Alu and LINE-1 in humans is considered a surrogate for global DNA methylation. Previous studies of blood-measured Alu/LINE-1 and cancer risk are inconsistent.Methods:We studied 1259 prospective methylation measurements from blood drawn 1–4 times from 583 participants from 1999 to 2012. We used Cox regression to evaluate time-dependent methylation as a biomarker for cancer risk and mortality, and linear regression to compare mean differences in methylation over time by cancer status and analyse associations between rate of methylation change and cancer.Results:Time-dependent LINE-1 methylation was associated with prostate cancer incidence (HR: 1.38, 95% CI: 1.01–1.88) and all-cancer mortality (HR: 1.41, 95% CI: 1.03–1.92). The first measurement of Alu methylation (HR: 1.39, 95% CI: 1.08–1.79) was associated with all-cancer mortality. Participants who ultimately developed cancer had lower mean LINE-1 methylation than cancer-free participants 10+ years pre-diagnosis (P<0.01). Rate of Alu methylation change was associated with all-cancer incidence (HR: 3.62, 95% CI: 1.09–12.10).Conclusions:Our results add longitudinal data on blood Alu and LINE-1 methylation and cancer, and potentially contribute to their use as early-detection biomarkers. Future larger studies are needed and should account for the interval between blood sample collection and cancer diagnosis.

Associations between vitamin D-binding protein isotypes, circulating 25(OH)D levels, and vitamin D metabolite uptake in colon cancer cells.

Vitamin D metabolites have been extensively studied as cancer chemopreventive agents. Gc-globulin (GC) isotypes, based on rs7041 and rs4588 diplotypes, have varying affinities for 1α,25-dihydroxyvitamin D (1,25(OH)2D) and 25-hydroxyvitamin D (25(OH)D), which may affect circulating metabolite concentration as well as delivery at the cellular level. We evaluated associations between GC isotype and circulating vitamin D metabolite concentrations in 403 ursodeoxycholic acid (UDCA) clinical trial participants. Metabolite uptake was evaluated in human colon cancer (HCT-116) cells treated with ethanol vehicle, 1,25(OH)2D, or 25(OH)D, and with plasma from individuals with known GC isotype. Mammalian-2-hybrid and vitamin D–responsive element-based luciferase assays were used to measure the vitamin D receptor pathway activation as a marker for metabolite uptake. Regression analysis demonstrated significantly lower serum 25(OH)D concentration for clinical trial participants with 1F_2, 1S_2, or 2_2 isotypes (P < 0.01) compared with 1S_1S. Consistent with these in vivo observations, cellular data revealed that 25(OH)D uptake varied less by GC isotype only at the higher concentration tested (P = 0.05), while 1,25(OH)2D uptake differed markedly by GC isotype across concentration and assay (P < 0.01). The 1F_1S and 1F_2 isotypes produced the greatest reporter gene induction with 1,25(OH)2D treatment and, while activation varied less with 25(OH)D, the 2_2 isotype demonstrated increased induction at the lower concentration. These results suggest that vitamin D metabolite concentration and delivery to colon cells may vary not only by GC isotype, but also that certain isotypes may more effectively deliver 1,25(OH)2D versus 25(OH)D. Overall, these results may help identify populations at risk for cancer and potential recipients of targeted chemoprevention. Cancer Prev Res; 7(4); 426–34. ©2014 AACR.

Physical activity, sedentary behavior, and vitamin D metabolites

Physical activity is associated with circulating 25-hydroxyvitamin D (25(OH)D). However, the influence of activity and/or sedentary behavior on the biologically active, seco-steroid hormone 1α,25-dihydroxyvitamin D (1,25(OH)2D) is unknown. We conducted a cross-sectional analysis among ursodeoxycholic acid (UDCA) randomized trial participants (n=876) to evaluate associations between physical activity, sedentary behavior, and circulating vitamin D metabolite concentrations. Continuous vitamin D metabolite measurements and clinical thresholds were evaluated using multiple linear and logistic regression models, mutually adjusted for either 1,25(OH)2D or 25(OH)D and additional confounding factors. A statistically significant linear association between 1,25(OH)2D and moderate-vigorous physical activity per week was strongest among women (β (95% CI): 3.10 (1.51-6.35)) versus men (β (95% CI): 1.35 (0.79-2.29)) in the highest tertile of activity compared to the lowest (p-interaction=0.003). Furthermore, 25(OH)D was 1.54ng/ml (95% CI 1.09-1.98) higher per hour increase in moderate-vigorous activity (p=0.001) and odds of sufficient 25(OH)D status was higher among physically active participants (p=0.001). Sedentary behavior was not significantly associated with either metabolite in linear regression models, nor was a statistically significant interaction by sex identified. The current study identified novel associations between physical activity and serum 1,25(OH)2D levels, adjusted for 25(OH)D concentrations. These results identify the biologically active form of vitamin D as a potential physiologic mechanism related to observed population-level associations between moderate-vigorous physical activity with bone health and chronic disease risk. However, future longitudinal studies are needed to further evaluate the role of physical activity and vitamin D metabolites in chronic disease prevention.