1,25-Dihydroxyvitamin D Regulation of Glutamine Metabolism in Human Breast Cancer Prevention

Xuanzhu Zhou, Purdue University

Abstract

Breast cancer is the most common cancer and the second leading cause of cancer death among US women. Many epidemiological studies suggest vitamin D is a dietary factor which has cancer preventive effects. The bioactive form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)2D), is proposed to exert anti-cancer effects through vitamin D receptor (VDR) mediated transcriptional activity. However, the mechanisms through which 1,25(OH)2D prevents breast cancer progression are not fully understood. Cancer cells exhibit many distinct characteristics including altered energy metabolism and oxidative stress regulation. Hallmarks of cancer cells include increased glycolysis rate and decreased utilization of glucose carbon in the TCA cycle for energy production (the Warburg effect) and then less ATP generated from glucose. Therefore, glutamine is proposed to be an alternative source for ATP generation to support rapid cancer cell growth and proliferation in cancer cells. Previous studies from our laboratory demonstrate that breast epithelial cells with H-ras oncogene transformation have upregulated glucose metabolism and 1,25(OH)2D inhibits glucose metabolism in these cells. However, alterations in glutamine metabolism during cancer progression as well as the mechanisms by which 1,25(OH)2D regulates glutamine metabolism are not fully understood. In addition, glutamine is involved in glutathione (GSH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) generation, thus glutamine metabolism plays a role in oxidative stress regulation. Data from our laboratory shows that 1,25(OH)2D has pro-oxidative effects in cells with H-ras transformation. Thus, we are also interested in studying the effect of 1,25(OH)2D on regulating oxidative stress, with respect to glutamine metabolism, in cells in early breast cancer progression. We employed MCF10A human breast epithelial cells and H-ras or HER2/neu/ErbB2 oncogene transformed MCF10A (MCF10A- ras or MCF10A-ErbB2) cells as a model of mammary cells in early cancer progression. We hypothesized that glutamine is essential for the proliferation of oncogene transformed cells, and 1,25(OH)2D inhibits glutamine uptake and metabolism in these cells. In addition, we hypothesize that 1,25(OH)2D increases the susceptibility of cells with oncogene transformation to oxidative stress induced cell death, thus preventing breast cancer development. To study the mechanisms and role of glutamine metabolism regulation by 1,25(OH)2D, we assessed the dependency of untransformed or oncogene transformed MCF10A cells on glutamine with or without 1,25(OH) 2D pretreatment. We showed that oncogene transformed MCF10A cells, but not untransformed MCF10A cells, are dependent on glutamine for optimal survival. We further measured the intracellular glutamine metabolite levels, using nuclear magnetic resonance (NMR) spectroscopy, in MCF10A and MCF10A-ras cells with or without 1,25(OH)2D treatment for four days. We also determined the incorporation of glutamine into TCA cycle intermediates employing stably labeled glutamine. Our results demonstrated that MCF10A-ras cells have upregulated glutamine metabolism compared to untransformed cells and 1,25(OH)2D reduced the intracellular levels of glutamine as well as glutamine flux into the TCA cycle for energy production in MCF10A- ras cells. We also measured 1,25(OH)2D regulation of glutamine uptake using tritium labeled glutamine and demonstrated that 1,25(OH) 2D inhibited glutamine uptake in MCF10A-ras and MCF10A- ErbB2. Further, we showed that glutamine transporter SLC1A5 mRNA and protein abundance was significantly reduced by 1,25(OH)2D in oncogene transformed MCF10A cells. Importantly, a functional VDRE on SLC1A5 promoter was identified using luciferase reporter gene and site-directed mutagenesis assays. These results suggest that ras oncogene transformation is sufficient to induce glutamine metabolism reprogramming, and 1,25(OH) 2D inhibits glutamine uptake and metabolism through transcriptionally regulating glutamine transporter SLC1A5 expression. To understand 1,25(OH) 2D regulation of oxidative stress during early breast cancer progression, we tested the response of MCF10A, MCF10A-ras and MCF10A- ErbB2 cells to oxidative stress with or without 1,25(OH)2D treatment. Results showed that 1,25(OH)2D increased sensitivity of oncogene transformed cells to oxidative stress-induced cell death and decreased cellular NADPH/NADP+ and GSH/GSSG ratio, suggesting pro-oxidative effects of 1,25(OH)2D. In addition, we demonstrated that 1,25(OH) 2D inhibited the mRNA and protein abundance of GOT1 in both MCF10A- ras and MCF10A-ErbB2 cells. The addition of oxaloacetate (OAA), a downstream metabolite of GOT1, prevented 1,25(OH)2D mediated increase in cell susceptibility to hydrogen peroxide induced cell death. However, GOT1 over-expression did not alter the effect of 1,25(OH)2D on inducing cell death under oxidative stress conditions. Collectively, these results support that 1,25(OH)2D has pro-oxidative effects in oncogene transformed MCF10A cell lines, which may contribute to its role in preventing against breast cancer development. However, these pro-oxidative effects of 1,25(OH)2D are not likely through regulating GOT1 expression. In summary, these studies demonstrate the importance of glutamine in energy metabolism and oxidative stress regulation in cells in early progression to cancer, as well as 1,25(OH)2D regulation of glutamine metabolism. These findings propose novel mechanisms by which 1,25(OH)2D may prevent breast cancer progression.

Degree

Ph.D.

Advisors

Teegarden, Purdue University.

Subject Area

Cellular biology|Nutrition|Oncology

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