Mammary adipocytes bioactivate 25‐hydroxyvitamin D3 and signal via vitamin D3 receptor,modulating mammary epithelial cell growth

The vitamin D₃ receptor (VDR) is present in all microenvironments of the breast, yet it is hypothesized to signal through the epithelium to regulate hormone induced growth and differentiation. However, the influence or contribution of the other microenvironments within the breast that express VDR, like the breast adipose tissue, are yet to be investigated. We hypothesized that the breast adipocytes express the signaling components necessary to participate in vitamin D₃ synthesis and signaling via VDR, modulating ductal epithelial cell growth and differentiation. We utilized human primary breast adipocytes and VDR wild type (WT) and knockout (KO) mice to address whether breast adipocytes participate in vitamin D₃‐induced growth regulation of the ductal epithelium. We report in this study that breast primary adipocytes express VDR, CYP27B1 (1α‐hydroxylase, 1α‐OHase), the enzyme that generates the biologically active VDR ligand, 1α,25‐dihydroxyvitamin D₃ (1,25D₃), and CYP24 (24‐hydroxylase, 24‐OHase), a VDR‐1,25D₃ induced target gene. Furthermore, the breast adipocytes participate in bioactivating 25‐hydroxyvitamin D₃ (25D₃) to the active ligand, 1,25D₃, and secreting it to the surrounding microenvironment. In support of this concept, we report that purified mammary ductal epithelial fragments (organoids) from VDR KO mice, co‐cultured with WT breast adipocytes, were growth inhibited upon treatment with 25D₃ or 1,25D₃ compared to vehicle alone. Collectively, these results demonstrate that breast adipocytes bioactivate 25D₃ to 1,25D₃, signal via VDR within the adipocytes, and release an inhibitory factor that regulates ductal epithelial cell growth, suggesting that breast adipose tissue contributes to vitamin D₃‐induced growth regulation of ductal epithelium. J. Cell. Biochem. 112: 3393–3405, 2011. © 2011 Wiley Periodicals, Inc.     

Tumor suppressor gene p16/INK4A/CDKN2A‐dependent regulation into and out of the cell cycle in a spontaneous canine model of breast cancer

p16/INK4A/CDKN2A is an important tumor suppressor gene that arrests cell cycle in G1 phase inhibiting binding of CDK4/6 with cyclin D1, leaving the Rb tumor suppressor protein unphosphorylated and E2F bound and inactive. We hypothesized that p16 has a role in exit from cell cycle that becomes defective in cancer cells. Well characterized p16‐defective canine mammary cancer cell lines (CMT28, CMT27, and CMT12), derived stably p16‐transfected CMT cell clones (CMT27A, CMT27H, CMT28A, and CMT28F), and normal canine fibroblasts (NCF), were used to investigate expression of p16 after serum starvation into quiescence followed by re‐feeding to induce cell cycle re‐entry. The parental CMT cell lines used lack p16 expression either at the mRNA or protein expression levels, while p27 and other p16‐associated proteins, including CDK4, CDK6, cyclin D1, and Rb, were expressed. We have successfully demonstrated cell cycle arrest and relatively synchronous cell cycle re‐entry in parental CMT12, CMT28 and NCF cells as well as p16 transfected CMT27A, CMT27H, CMT28A, and CMT28F cells and confirmed this by ³H‐thymidine incorporation and flow cytometric analysis of cell cycle phase distribution. p16‐transfected CMT27A and CMT27H cells exited cell cycle post‐serum‐starvation in contrast to parental CMT27 cells. NCF, CMT27A, and CMT28F cells expressed upregulated levels of p27 and p16 mRNA, post‐serum starvation, as cells exited cell cycle and entered quiescence. Because quiescence and differentiation are associated with increased levels of p27, our data demonstrating that p16 was upregulated along with p27 during quiescence, suggests a potential role for p16 in maintaining these non‐proliferative states. J. Cell. Biochem. 114: 1355–1363, 2013. © 2012 Wiley Periodicals, Inc.