Plasma 25-hydroxyvitamin D(3) and bladder cancer risk according to tumor stage and FGFR3 status: a mechanism-based epidemiological study

Abstract

Background: Previous evidence suggests that 25-hydroxyvitamin D(3) [25(OH)D(3)] protects against several cancers. However, little is known regarding urothelial bladder cancer (UBC). We analyzed the association between plasma 25(OH)D(3) and overall risk of UBC, as well as according to stage and FGFR3 molecular subphenotypes.

Methods: Plasma concentrations of 25(OH)D(3) in 1125 cases with UBC and 1028 control subjects were determined by a chemiluminescence immunoassay. FGFR3 mutational status and expression in tumor tissue were assessed. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by logistic regression adjusting for potential confounders. Analyses were further stratified by tumor invasiveness and grade, FGFR3 expression, and smoking status. Cell proliferation was measured in human UBC cell lines cultured with 1α,25-dihydroxyvitamin D(3).

Results: A statistically significantly increased risk of UBC was observed among subjects presenting the lowest concentrations of 25(OH)D(3) (OR(adj) = 1.83; 95% CI = 1.19 to 2.82; P = .006), showing a dose-response effect (P (trend) = .004). The association was stronger for patients with muscle-invasive tumors, especially among low-FGFR3 expressers (OR(adj) = 5.94; 95% CI = 1.72 to 20.45; P = .005). The biological plausibility of these associations is supported by the fact that, in vitro, 1α,25-dihydroxyvitamin D(3) upregulates FGFR3 expression in UBC cell lines with low levels of wild-type FGFR3.

Conclusion: These findings support a role of vitamin D in the pathogenesis of UBC and show that 25(OH)D(3) levels are associated with FGFR3 expression in the tumor. Because FGFR3 mutation and overexpression are markers of better outcome, our findings suggest that individuals with low levels of plasma 25(OH)D(3) may be at high risk of more aggressive forms of UBC.

Figure 1.

Effects of vitamin D on proliferation and expression of p21, p27, and FGFR3 in urothelial bladder cancer (UBC) cells. Vitamin D induces growth arrest and an upregulation of p21, p27, and FGFR3 in UBC cells. A) Phase contrast microscopy of MGH-U4 and RT112 cells treated with 1α,25-dihydroxyvitamin D₃ [1α,25 (OH)₂D₃] (100nM) or vehicle for 72 hours (scale bar = 100 µm). B) 1α,25(OH)₂D₃ treatment inhibits proliferation of UBC cells in vitro. C) Western blot analysis showing induction of the CDK inhibitors p21 and p27 in MGH-U4 cells treated with 1α,25(OH)₂D₃ at different time points. Tubulin was used as a loading control. Values indicate fold-change referred to zero time point. D) Western blot analysis showing changes in FGFR3 in MGH-U4 and RT112 cells treated with 1α,25(OH)₂D₃ at the indicated time points. E) Quantitative reverse-transcription polymerase chain reaction analysis showing the upregulation of FGFR3 messenger RNA levels in MGH-U4 and RT112 cells treated with 10nM 1α,25(OH)₂D₃ (gray bars). Values were normalized to HPRT and referred to expression at time zero (black bars). Comparisons with P less than .05 are indicated with an asterisk. Error bars represent standard deviation.

Figure 2.

Odds ratios (OR) and 95% confidence intervals for the association between plasma 25-hydroxyvitamin D₃ [25(OH)D₃] and bladder cancer risk by smoking status (A) and by season of blood collection (B). Estimates are adjusted for age, sex, region, smoking status, and season of blood collection, when appropriate.

Figure 3.

Odds ratios (ORs) and 95% confidence intervals (CIs) for the association between plasma 25-hydroxyvitamin D₃ [25(OH)D₃] levels and risk of low- and high-grade non-muscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC) in relationship with tumor FGFR3 expression levels. Estimates are adjusted for age, sex, region, and smoking status. *Likelihood-ratio test P for the comparison between the odds ratio of MIBC in the low- and high-FGFR3-expression groups = .26.