Inhibition of Breast Cancer Metastasis and Regulation of Lipid Metabolism by the Active Vitamin D Metabolite 1α,25-Dihydroxyvitamin D3
Breast cancer continues to be one of the most commonly diagnosed cancers in the United States, with 1 in 8 women expected to develop invasive breast cancer during her lifetime. Metastatic breast cancer in particular is a debilitating disease accounting for over 40,000 deaths each year. Therefore, it is imperative to identify effective preventive compounds against breast cancer metastasis that may prolong survival of breast cancer patients. Epidemiological evidence suggests that vitamin D may protect against metastatic breast cancer. However, the mechanisms orchestrating vitamin D’s antimetastatic effect are unclear and necessitate futher investigation. In the present studies, the effect of 1α,25 dihydroxyvitamin D (1,25(OH)2D), the bioactive form of vitamin D, on metastatic breast cancer capability was investigated using an in vitro (rMET) model that accurately recapitulates multiple steps of breast to bone metastasis, the most common metastatic site in breast cancer patients. Treatment with 1,25(OH)2D (10 nM) in the rMET model significantly inhibited the metastatic potential of MCF10CA1a and MDA-MB-231 breast epithelial cells. 1,25(OH)2D further inhibited the ability of MCF10CA1a epithelial cells to adapt to the drastically different bone environment recapitulated in the rMET model, suggesting 1,25(OH)2D may inhibit both the migration of cells away from the primary tumor as well as their ability to colonize secondary organs. Epithelial-mesenchymal transition is an essential feature of metastatic breast cancer cells characterized by loss of epithelial cell-cell junction proteins (E-cadherin) and acquisition of mesenchymal markers (N-cadherin). Invesitigation into the effect of 1,25(OH)2D E- and N-cadherin expression in MCF10CA1a breast epithelial cells demonstrated an inhibitory effect of 1,25(OH)2D on EMT. E-cadherin mRNA and protein expression were both upregulated while N-cadherin mRNA was downregulated with 1,25(OH)2D treatment in this cell line. breast cancer cells undergo multiple genetic and phenotypic changes towards their progression to a metastatic phenotype. Lipid metabolic reprograming characterized by upregulated de novo fatty acid synthesis as well as increased neutral lipid accumulation is a comon characteristic observed in metastatic breast cancer cells, making it a promising therapeutic target in metastatic breast cancer prevention. Therefore, the effect of 1,25(OH)2D on metastatic breast cancer lipid metabolism was investigated using MCF10CA1a breast epithelial cells. 1,25(OH)2D treatment drastically inhibited de novo fatty acid synthesis, as measured by [U]-13C-glucose incorporation into palmitate and stearate through liquid chromatography coupled mass spectrometry. Furthermore, measurement of neutral lipid accumulation using a triacylglycerol colorimetric assay as well as confocal microscopy demonstrated that treatment with 1,25(OH)2D inhibited neutral lipid levels in metastatic breast cancer cells in a time dependent manner. The anaplerotic enzyme pyruvate carboxylase (PC) emerged as a target of 1,25(OH)2D-mediated regulation. PC expression was decreased with 1,25(OH)2D treatment and its overexpression reversed the effect of 1,25(OH)2D on de novo fatty acid synthesis in MCF10CA1a cells. PC overexpression further promoted a metastatic phenotype as measured by a Boyden Chamber migration assay in MCF10CA1a and MCF10A-Harvey ras oncogene transfected cells, a model of early breast cancer progression. Together these studies suggest that PC is an essential enzyme in breast cancer metastasis and that 1,25(OH)2D may potentially inhibit metastasis through downregulation of PC expression. PC was shown to play an important role in maintaining redox balance in pancreatic β-cells. We therefore hypothesized that PC downregulation by 1,25(OH)2D may promote oxidative stress. To test this hypothesis, the effect of 1,25(OH)2D on oxidative stress was investigated in MCF10A-Harvey ras oncogene transfected breast epithelial cells. 1,25(OH)2D increased reactive oxygen species as well as cell susceptibility to hydrogen peroxide induced cell death. Treatment with 1,25(OH)2D further depleted reduced glutathione levels, demonstrating a pro-oxidative effect of vitamin D in early breast cancer progression. Treatment with the mitochondrial pyruvate carrier inhibitor UK-5099 mimicked the effect of 1,25(OH)2D on GSH levels, with 1,25(OH)2D having no further effect. Administration of oxaloacetate (2 mM), the product of pyruvate metabolism in the mitochondria by PC, rescued 1,25(OH)2D treated cells from hydrogen peroxide induced cell death. Collectively, the present studies provide novel insight into vitamin D mediated mechanisms in early and later breast cancer prevention, potentially through the downregulation of the mitochondrial enzyme PC. Results of the present investigation provide additional evidence for a protective role of vitamin D against breast cancer development and metastasis.
Teegarden, Purdue University.
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