The biologically active form of vitamin D3 (cholecalciferol) is 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the ligand for the vitamin D receptor (VDR). In addition to its well characterized role in bone metabolism, vitamin D is recognized as a potent regulator of cell proliferation and differentiation. In mammary gland, 1,25(OH)2D3 opposes estrogen and progesterone stimulated proliferation and branching through VDR dependent pathways. 1,25(OH)2D3 is produced from 25(OH)D, the major circulating metabolite of vitamin D. The enzyme that catalyzes the production of 1,25(OH)2D3 is the vitamin D 1-hydroxylase (1-OHASE). The long term goal of the proposed studies is to validate the vitamin D 1-OHASE as a target for breast cancer prevention. Although initially thought to be exclusively expressed in kidney, the 1-OHASE has recently been localized to numerous extra-renal tissues, including epidermis, colon, and prostate gland. In preliminary experiments, we have demonstrated expression of 1-OHASE in non transformed human mammary epithelial cells, and we propose to confirm and extend these findings in the pilot studies described here. We hypothesize that normal breast tissue expresses 1-OHASE and generates 1,25(OH)2D3, which acts locally to inhibit growth and maintain differentiation of mammary epithelial cells. A corollary to this hypothesis is that deregulation of 1-OHASE and loss of 1,25(OH)2D3 mediated growth regulation may be associated with neoplastic progression.
Two specific aims have been designed to test this hypothesis.
In Specific Aim 1, we will examine the expression and regulation of the vitamin D 1-OHASE during normal development in mice (Specific Aim 1a) and in nontransformed human mammary cells in vitro.
In Specific Aim 2, we will assess the expression and activity of the vitamin D 1-OHASE as a function of neoplastic progression in tissues and tumors derived from MMTV-neu mice (Specific Aim 2a) and in progressively transformed human mammary epithelial cell lines (Specific Aim 2b). Techniques will include real time quantitative polymerase chain reaction (PCR), immunohistochemistry, laser capture microdissection and immunoblotting. The data on 1-OHASE expression and activity will be interpreted in relation to expression of the VDR and sensitivity to 25(OH)D mediated growth inhibition. These studies will provide important information regarding the expression and function of the vitamin D signaling pathway in normal mammary gland, which might have implications for breast cancer prevention.
