1,25-dihydroxyvitamin D3 (1,25D) and its cognate nuclear receptor, the vitamin D receptor (VDR), regulate calcium homeostasis via transcriptional activation of target genes. 1,25D is also known to mediate growth arrest and apoptosis in a variety of cancer cell lines and tumors, however, the role of the VDR and the role of transcriptional activation in this process remain unclear. To investigate the role of the VDR in mediating the anti-cancer properties of 1,25D, two murine mammary tumor cell lines with differential VDR expression were characterized.
WT145 cells, which express transcriptionally functional VDR protein, were growth inhibited and rendered apoptotic by 1,25D and synthetic vitamin D analogs. In contrast, KO240 cells, which express no detectable VDR protein or mRNA, were neither growth inhibited nor rendered apoptotic by 1,25D, at doses as high as 1ƒÝM. Collectively, these data indicate that functional VDR protein is required for the anti-cancer effects of 1,25D and structurally related vitamin D based therapeutics in vitro.
To determine whether the anti-cancer actions of vitamin D are mediated via the VDR in vivo, nude mice bearing tumors derived from WT145 or KO240 cells were treated with EB1089, a synthetic analog of 1,25D, or placebo for six weeks. An untreated subset of tumor-bearing mice was exposed to ultraviolet (UV) light, to activate endogenous 1,25D production. Both EB1089 and UV light exposure decreased volume of WT145 tumors through decreased tumor cell proliferation and increased tumor cell apoptosis. No effects of either EB1089 or UV treatments were observed in KO240 tumors, indicating that the vitamin D pathway mediates its anti-tumor effects in vivo via tumor-cell VDR.
KO240 cells stably expressing wild-type VDR and VDR point mutants from hereditary rickets patients were created and characterized, to further examine the mechanism of VDR in growth regulation. KO cells stably expressing VDR were growth inhibited by 1,25D and its structural analogs, indicating that the vitamin D growth regulatory pathway could be reconstituted in VDR null cells. Cells expressing mutant VDRs were differentially affected by 1,25D and analogs; VDR that lack DNA binding ability were growth inhibited by physiological doses of 1,25D while ligand binding domain mutants were not. This suggests that the anti-cancer effects of 1,25D, while VDR mediated, are in part mediated via novel, DNA-independent mechanisms.
