Association among plasma 1,25(OH)2D, ratio of 1,25(OH)2D to 25(OH)D, and prostate cancer aggressiveness

Abstract Background African‐American (AA) men tend to present with more aggressive prostate cancer (Gleason score >7) than European‐American (EA) men. Vitamin D and its metabolites are implicated in prostate cancer biology with vitamin D deficiency, indicated by its metabolite levels in serum or plasma, usually observed in AA men. Objective To determine if 1, 25‐dihydroxy vitamin D3 [1,25(OH)2D] plasma levels in AA and EA prostate cancer patients alter the risk of having aggressive prostate cancer. Design Research subjects from the North Carolina‐Louisiana Prostate Cancer Project (AA n = 435 and EA n = 532) were included. Plasma metabolites 1,25(OH)2D and 25‐hydroxyvitamin D3 [25(OH)D] were measured using liquid chromatography with tandem mass spectrophotometry. Research subjects were classified into low (Gleason sum < 7, stage T1‐T2, and Prostate‐specific antigen (PSA) < 9 ng/mL) or high (Gleason sum > 8 or Gleason sum = 7 with 4 + 3, or PSA > 20 ng/mL, or Gleason sum = 7 and stage T3‐T4) aggressive disease. Results Research subjects in the second and third tertiles of plasma levels of 1, 25(OH)2D had lower odds of high aggressive prostate cancer (AA [ORT2vsT1: 0.66, 95%CI: 0.39‐1.12; ORT3vsT1: 0.83, 95%CI: 0.49‐1.41] and EA [ORT2vsT1: 0.68, 95%CI: 0.41‐1.11; ORT3vsT1: 0.67, 95%CI: 0.40‐1.11]) compared with the first tertile, though confidence intervals included the null. Greater 1,25(OH)2D/25(OH)D molar ratios were associated with lower odds of high aggressive prostate cancer more evidently in AA (ORQ4vsQ1: 0.45, CI: 0.24‐0.82) than in EA (ORQ4vsQ1: 0.64, CI: 0.35‐1.17) research subjects. Conclusions The 1,25(OH)2D/25(OH)D molar ratio was associated with decreased risk of high aggressive prostate cancer in AA men, and possibly in EA men. Further studies analyzing vitamin D polymorphisms, vitamin D binding protein levels, and prostatic levels of these metabolites may be useful. These studies may provide a better understanding of the vitamin D pathway and its biological role underlying health disparities in prostate cancer.

Conclusions: The 1,25(OH) 2 D/25(OH)D molar ratio was associated with decreased risk of high aggressive prostate cancer in AA men, and possibly in EA men. Further studies analyzing vitamin D polymorphisms, vitamin D binding protein levels, and prostatic levels of these metabolites may be useful. These studies may provide a better understanding of the vitamin D pathway and its biological role underlying health disparities in prostate cancer.
prostate cancer, racial disparities, vitamin D

| INTRODUCTION
Vitamin D is absorbed primarily through the skin and converted to its metabolites by a family of hydroxylase enzymes that include CYP27B1 and CYP24A1. 1,2 Vitamin D is converted to 25-hydroxyvitamin D3 (25(OH)D) in the liver and to its active metabolite 1, 25-dihydroxy vitamin D3 (1,25(OH) 2 D) in the kidney. 1 1,25(OH) 2 D binds the vitamin D receptor (VDR) that allows VDR to bind to specific DNA motifs of its target genes, such as p21 and p27. 3,4 Alterations in components of vitamin D metabolism, which include polymorphisms in the hydroxylase enzymes, can lead to varying serum vitamin D metabolite levels in individuals from different ethnic cohorts. [5][6][7][8] African-American (AA) men and women usually present with low 25(OH)D levels compared with their European-American (EA) counterparts, [9][10][11] even though 1,25(OH) 2 D levels are similar by race. 12 Skin pigmentation, decreased consumption of dairy products and fortified foods, lack of vitamin D supplementation, and elevated body mass index (BMI) have been related to low 25(OH)D levels in AAs. 11,13 AA men are more likely to be diagnosed with and/or die from aggressive prostate cancer than EA men. 14 Differences in vitamin D metabolite levels have been suggested to play a role in prostate cancer disparities between AA and EA men. 15 19 or induces Rb1 20 that result in cell cycle arrest in the G1 phase, to reduce prostate cancer cell proliferation in vitro. 1,25(OH) 2 D also induces cell cycle arrest and senescence in an IL-1α-dependent manner in prostate progenitor epithelial/stem cells isolated from adult mice in vitro. 21 In vivo, dietary vitamin D or calcitriol supplementation reduced prostate cancer xenograft growth and increased 1,25(OH) 2 D serum levels. 22 In another study, disruption of CYP24A1 activity increased 1,25(OH) 2 D levels and reduced prostate cancer growth in vitro and in vivo. 23 However, the mechanism of action of 1,25(OH) 2 D was not defined well in these studies.
Most studies in prostate cancer measure vitamin D sufficient or deficient status in individuals by measuring 25(OH)D levels in the serum and not necessarily the active 1,25(OH) 2 D metabolite. [15][16][17][18] Our previous study showed that research subjects with the highest percentage of African ancestry had the lowest levels of 25(OH)D. 24 While high 25(OH)D was associated with increased odds of high aggressive prostate cancer among AAs in PCaP, the relationship was modified by calcium intake and high 25(OH)D coupled with high calcium intakes decreased the odds of having high aggressive prostate cancer in AA men. 24 In the current study, we investigated if the active metabolite 1,25(OH) 2 D levels and molar ratios of 1,25(OH) 2 D to 25(OH)D were associated with the risk of high aggressive prostate cancer in a racially diverse, case-only study.

| Data collection
A trained registered nurse conducted structured interviews, collected necessary specimens and measurements of research subjects at their home within 4 months of initial diagnosis, on average. Structured interviews were performed using a questionnaire that included a family history of prostate cancer, cancer screening history and vitamin supplementation among other variables listed in Table 1.
The National Cancer Institute Diet History Questionnaire modified to accommodate Cajun, and Creole foods were used to inform about food portion sizes and intake frequency for a list of 124 food items.
The Diet*Calc software was used to calculate the intake of nutrients and minerals including calcium and vitamin D.   (Table 2), and there was no evidence of a dose-response association.
T A B L E 4 Association between molar ratio of plasma 1,25(OH) 2 D to 25(OH)D and prostate cancer aggressiveness by race

| DISCUSSION
Our study shows that increased plasma 1,25(OH) 2   Our study is strengthened by the use of rapid case ascertainment to identify a population-based sample of approximately equal numbers of AA and EA men with newly-diagnosed prostate cancer.
Limitations include the use of only one measurement of plasma vitamin D metabolites that was collected after diagnosis, so we cannot rule out the possibility that disease status may have affected vitamin D metabolite concentrations. As in any observational study, residual or unmeasured confounding are possible limitations.

| CONCLUSIONS
Our study showed that higher levels of the 1,25(OH) 2 D/25(OH)D molar ratio, and possibly of the active 1,25(OH) 2 D metabolite, were inversely related to odds of high aggressive prostate cancer, particularly in AA men. But the complex nature of the vitamin D pathway, its cross-talk with stromal components and its dependence on other nutritional elements warrant careful analysis in relation to health disparities that occur in prostate cancer.