1,25-dihydroxyvitamin D3 (D3) has potent antitumor effects in prostate cancer (CaP). As a result, D3 is being evaluated for CaP prevention and treatment. Efforts in our laboratory have been directed towards utilizing vitamin D for prevention of CaP in vivo, using transgenic adenocarcinoma of mouse prostate (TRAMP) model. Our results indicate that short term or chronic administration of vitamin D to TRAMP mice prevents androgen dependent CaP progression. However, vitamin D did not prevent hormone refractory CaP in castrated TRAMP mice. We have observed that TRAMP mice chronically treated with vitamin D eventually become insensitive to the antitumor effects of vitamin D. Therefore, we hypothesize that CaP cells become insensitive to vitamin D via deregulation of key molecular signaling pathways. D3 reduced tumor burden over time in TRAMP mice treated with 20μg/kg D3 or vehicle from 4 to ∼30 wks-of-age. However, all mice eventually developed vitamin D insensitive (VDI) tumors following chronic treatment with D3. Primary cell cultures were generated from prostate tumors of control (vitamin D naïve) and D3 (VDI) mice. In vitro, VDI cells were less sensitive to D3 based on reduced growth inhibition and inhibition of DNA synthesis as measured by MTT and BrDU incorporation assays. VDI cells were also less sensitive to growth inhibition by vitamin analogs, paricalcitol and EB1089. CYP24-LUC reporter activity assay was used to demonstrate that VDI cells were less responsive to vitamin D-mediated gene transcriptional activity compared to naïve cells. Vitamin D receptor (VDR) was expressed equally in naïve and VDI cells following treatment with D3 as measured by immunofluorescence. This suggests that insensitivity of VDI cells to vitamin D may not be attributable to lack of VDR, which is a key mediator of the downstream effects of vitamin D. One mechanism of vitamin D insensitivity in androgen dependent CaP may be altered gene transcriptional activity, such as coregulator expression. Interestingly, contrary to our observations that D3 prevents androgen dependent CaP, D3 (20μg/kg) did not prevent hormone refractory CaP in castrated TRAMP mice when administered either before or after castration. However, ∼16% of mice developed hypercalcemia irrespective of treatment group. H&E staining and histological grading of prostate tissues revealed that these mice developed poorly differentiated grade 6 tumors. Immunoradiometric assay was used to measure parathyroid hormone-related protein (PTHrP), which is elevated in hypercalcemia associated with various malignancies. PTHrP was elevated in the sera of hypercalcemic mice (73.3 ± 29.3pg/ml), however, it was undetectable in non-tumor bearing mice. PTHrP mRNA was also detectable by RT-PCR in tumors of hypercalcemic mice. Our studies indicate that there is a correlation between hypercalcemia and elevated PTHrP levels in castrated TRAMP mice that develop poorly differentiated CaP.
[Proc Amer Assoc Cancer Res, Volume 47, 2006]