25-hydroxyvitamin D-3 1 alpha-hydroxylase expression in breast cancer and use of non-1 alpha-hydroxylated vitamin D analogue

Introduction The cytochrome P450 mitochondrial enzyme 25-hydroxyvitamin D-3 1 alpha-hydroxylase (1 alpha-hydroxylase) of renal tubule cells hydroxylates the major circulating form of vitamin D (25(OH) D-3) to the active systemic hormone 1,25(OH)(2)D-3. Local production of 1,25( OH)(2)D-3 appears to occur also at other sites where 1 alpha-hydroxylase is expressed for autocrine/ paracrine regulation. To reduce risks of hypercalcemia during treatment with vitamin D, we have previously suggested use of non-1 alpha-hydroxylated vitamin D analogues to target tissues where 1 alpha-hydroxylase is expressed, including the parathyroid glands in secondary hyperparathyroidism. The present study was undertaken to examine expression of 1 alpha-hydroxylase in breast cancer and to investigate whether a non-1 alpha-hydroxylated vitamin D analogue displayed biological function. In addition, expression of the 25-hydroxyvitamin D-3 24-hydroxylase (24-hydroxylase) and the vitamin D receptor (VDR) was investigated. Methods The expression of 1 alpha-hydroxylase, 24-hydroxylase and VDR was investigated in breast cancer specimens ( n = 19) and normal breast tissues ( n = 10) by immunohistochemistry and/or RT-PCR. Consecutive cryosections of 6 mu m essentially free of immune cells were used in the analyses. The effect of vitamin D analogues on transcriptional activation was analyzed in transiently transfected MCF-7 breast cancer cells. Results 1 alpha-hydroxylase protein was demonstrated in 79% and 100% of breast cancer specimens and normal breast, respectively. The overall relative mRNA levels of 1 alpha-hydroxylase and 24-hydroxylase in normal breast compared to breast tumors were: 1 alpha-hydroxylase, 1 +/- 0.07 versus 0.7 +/- 0.05, respectively ( p < 0.001); 24-hydroxylase, 1 +/- 0.08 verus 2.1 +/- 0.2, respectively ( p < 0.001). The VDR was expressed in 95% of the tumors as expected, with mRNA levels of 1 +/- 0.09 and 1.4 +/- 0.12 ( p < 0.05) in breast cancer and normal breast, respectively. The ketoconazole- sensitive transcription activation potential of the non-1 alpha-hydroxylated vitamin D analogue prodrug of EB1089 (EB1285) was demonstrated in MCF-7 cells, which express 1 alpha-hydroxylase. The activity of EB1285 was about 20% of 1,25(OH)(2)D-3. Conclusion These results demonstrate nearly normal expression levels of 1 alpha-hydroxylase, 24-hydroxylase and VDR in the majority of investigated breast cancer specimens. A non-1 alpha-hydroxylated vitamin D analogue displayed activity in breast cancer cells. Such analogues may present future therapeutic options for proliferative disorders where 1 alpha-hydroxylase is expressed.