Molecular Actions of the Vitamin D Receptor in Mammary and Skin Carcinogenesis

Vitamin D has been linked to breast cancer risk in animal and human studies, suggesting that the active metabolite 1,25-Dihydroxyvitamin D (1,25D) might protect breast cells from transformation. In human mammary epithelial (HME-hTERT) cells, which express vitamin D receptor (VDR), 1,25D exerts anti proliferative and pro-differentiating effects, but the molecular mechanisms that mediate these actions are unknown. In previous studies we used genomic profiling to classify 1,25D regulated networks in HME-hTERT cells that may contribute to the anti-cancer effects of vitamin D. Through this approach we detected a 60-fold increase in the cytokine CD14 in HME-hTERT cells treated with vitamin D. CD14 is a component of the innate immune system which also functions in apoptotic cell clearance and mammary gland remodeling. The studies described in the first part of this thesis were designed to confirm and extend this preliminary data. HME-hTERT cells, which were used to generate the preliminary data, and HME-PR cells, a transformed derivative, and HC11 cells, mouse mammary epithelial cells, were cultured and treated with vitamin D metabolites (25-hydroxyvitamin D and 1,25-dihydroxyvitamin D). Doses ranged from 0-100nM, and duration of treatment was 24h. qPCR was used to analyze the expression of CD14 and related immune response genes. These studies determined the extent to which 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D alter the synthesis or secretion of cytokines in mammary cells (both human and mouse) and how transformation alters the basal and vitamin D-induced expression of these cytokines. Our data suggest that vitamin D regulation of cytokines is altered during the process of carcinogenesis and that the specific targets of VDR that regulate immune responses differ in human and mouse cells. In addition to effects on immune responses, the anti-cancer effects of vitamin D have been linked to modulation of the extraceullular matrix. In previous studies, the Welsh lab identified Has2 (hyaluronic acid synthase 2) as a vitamin D regulated gene in invasive murine mammary cancer cells. Has2 is the rate-limiting enzyme in the synthesis of hyaluronic acid (HA), which accumulates in the extracellular matrix. Has2 over-expression and HA synthesis have been linked to cancer cell invasion and metastasis in vitro and abnormal skin phenotypes in vivo. The abnormal skin phenotype associated with Has2 over-expression is highly similar to skin changes reported in VDR knockout (VDRKO) mice, suggesting that Has2 regulation by VDR may have functional consequences in vivo. In the second part of this thesis project, we further investigated the link between VDR and Has2 expression/function in skin and mammary tissue of mice. Tissues from wildtype and VDRKO mice of different ages were sectioned and processed for Has2 (immunofluorescence), HA (HABP binding assay) and proteoglycans (alcian blue staining). Additional in vitro studies were conducted to establish western blotting and PCR assays for study of this pathway. Our data suggest that vitamin D and the VDR physiologically regulate Has2 and HA production in vivo, especially in the epidermis