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Development of oncologic conditions is often accompanied by inadequate vitamin D status. The chemoprevention ability of this molecule is of high interest for breast cancer, the most common malignancy in women worldwide. Because current effective vitamin D analogues, including the naturally occurring active metabolite 1,25-dihydroxycholecalciferol (1,25(OH)2D), frequently cause hypercalcemia at pharmacologic doses, the development of safer molecules for clinical chemopreventive use is essential. This study examines whether exogenously supplied prohormone 25-hydroxycholecalciferol (25(OH)D) can delay tumor progression in vivo without hypercalcemic effects. A low vitamin D diet (25 IU/kg) in the non-immunodeficient MMTV-PyMT mouse model of metastatic breast cancer revealed a significant acceleration of mammary neoplasia compared with normal diet (1,000 IU/kg). Systemic perfusion of MMTV-PyMT mice with 25(OH)D or 1,25(OH)2D delayed tumor appearance and significantly decreased lung metastasis, and both metabolites reduced Ki-67, cyclin D1, and ErbB2 levels in tumors. Perfusion with 25(OH)D caused a 50% raise in tumor 1,25(OH)2D levels, indicating good tumor penetration and effective activation. Importantly, in contrast with 1,25(OH)2D, perfusion with 25(OH)D did not cause hypercalcemia. In vitro treatment of cultured MMTV-PyMT mammary tumor cells with 25(OH)D inhibited proliferation, confirming local activation of the prohormone in this system. This study provides an in vivo demonstration in a non-immunodeficient model of spontaneous breast cancer that exogenous 25(OH)D delays neoplasia, tumor growth, and metastasis, and that its chemoprevention efficacy is not accompanied by hypercalcemia. Cancer Prev Res; 8(2); 120–8. ©2014 AACR.

Supplementary Table 1: Continuous perfusion of MMTV-PyMT mice with 25(OH)D increases local production of 1,25(OH)2D in breast tumor tissue without elevating blood calcemia. Measurements were made at sacrifice (8 weeks of treatment). Results are expressed as mean {plus minus} S.E.M. P value < 0.05. Supplementary Figure 1. Flow cytometry: The MMTV-PyMT cells in culture at passage 3 were near 100% viable as tested by exclusion of Fixable Viability Dye staining (Affymetrix Ebioscience). (A) Unstained, (B) stained. (C) Cells were uniformly positive for CK8 (a modulator of cell adhesion/growth dependent signal transduction in breast tumor cells and a marker of cells of epithelial origin (38)), illustrating the exclusion of connective tissue and the invasive potential of the cultured tumor cells. Supplementary Figure 2. Serial measurements of blood calcium concentrations: Calcium levels were consistently elevated during perfusion with 1,25(OH)2D, whereas treatment with 25(OH)D had no effect. Mice were bled once a week and plasma calcium levels determined as described in "Materials and Methods" section. Results represent means {plus minus} SE of calcium concentrations of mice from each group. Blue squares: control; black triangles: mice perfused with 1,25(OH)2D; green circles: mice perfused with 25(OH)D. * represent values significantly different from same-week controls.