Maintaining reductive-oxidative (redox) balance is an essential feature in breast cancer cell survival, with cellular metabolism playing an integral role in maintaining redox balance through its supply of reduced NADPH. In the present studies, the effect of 1,25-dihydroxyvitamin D (1,25(OH)2D) on redox balance was investigated in early stages of breast cancer. Treatment with 1,25(OH)2D promoted oxidative stress in MCF10A-ras and MCF10A-ErbB2 breast epithelial cells, as measured by the decreased ratios of NADPH/NADP+ and reduced to oxidized glutathione (GSH/GSSG). The mRNA and protein expression of the enzyme pyruvate carboxylase (PC) was downregulated with 1,25(OH)2D treatment, suggesting a potential mechanism. Genetic depletion of PC in MCF10A-ras cells resulted in a decreased ratio of NADPH/NADP+ and GSH/GSSG, with 1,25(OH)2D treatment having no further effect. Mutation analysis confirmed the presence and functionality of a vitamin D response element in the PC gene promoter region. Collectively, these results provide evidence that 1,25(OH)2D promotes oxidative stress in early breast cancer progression through transcriptional downregulation of PC.

Breast cancer is the second most common cancer among women in the US. The active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)2D), is proposed to inhibit cellular processes and to prevent breast cancer. The current studies investigated the effect of 1,25(OH)2D on glutamine metabolism during cancer progression employing Harvey-ras oncogene transformed MCF10A human breast epithelial cells (MCF10A-ras). Treatment with 1,25(OH)2D significantly reduced intracellular glutamine and glutamate levels measured by nuclear magnetic resonance (NMR) by 23 ± 2% each. Further, 1,25(OH)2D treatment reduced glutamine and glutamate flux, determined by [U-13C5] glutamine tracer kinetics, into the TCA cycle by 31 ± 0.2% and 17 ± 0.4%, respectively. The relative levels of mRNA and protein abundance of the major glutamine transporter, solute linked carrier family 1 member A5 (SLC1A5), was significantly decreased by 1,25(OH)2D treatment in both MCF10A-ras cells and MCF10A which overexpress ErbB2 (HER-2/neu). Consistent with these results, glutamine uptake was reduced by 1,25(OH)2D treatment and the impact was eliminated with the SLC1A5 inhibitor L-γ-Glutamyl-p-nitroanilide (GPNA). A consensus sequence to the vitamin D responsive element (VDRE) was identified in silico in the SLC1A5 gene promoter, and site-directed mutagenesis analyses with reporter gene studies demonstrate a functional negative VDRE in the promoter of the SLC1A5 gene. siRNA-SLC1A5 transfection in MCF10A-ras cells significantly reduced SLC1A5 mRNA expression as well as decreased viable cell number similar to 1,25(OH)2D treatment. SLC1A5 knockdown also induced an increase in apoptotic cells in MCF10A-ras cells. These results suggest 1,25(OH)2D alters glutamine metabolism in MCF10A-ras cells by inhibiting glutamine uptake and utilization, in part through down-regulation of SLC1A5 transcript abundance. Thus, 1,25(OH)2D down-regulation of the glutamine transporter, SLC1A5, may facilitate vitamin D prevention of breast cancer.

Breast cancer is the most common cancer among women. Emerging research indicates that modifying lifestyle factors during pregnancy may convey long-term health benefits to offspring. This study was designed to determine whether maternal exercise during pregnancy leads to reduced mammary tumorigenesis in female offspring. Pregnant rats were randomly assigned to exercised and sedentary groups, with the exercised group having free access to a running wheel and the sedentary group housed with a locked wheel during pregnancy. Female pups from exercised or sedentary dams were weaned at 21 days of age and fed a high fat diet without access to a running wheel. At 6 weeks, all pups were injected with the carcinogen N-methyl-N-nitrosourea (MNU). Mammary tumor development in all pups was monitored for 15 weeks. Pups from exercised dams had a substantially lower tumor incidence (42.9%) compared to pups from sedentary dams (100%). Neither tumor latency nor histological grade differed between the two groups. These data are the first to demonstrate that exercise during pregnancy potentiates reduced tumorigenesis in offspring. This study provides an important foundation towards developing more effective modes of behavior modification for cancer prevention.

typically a single-chain variable fragment derived from a monoclonal antibody against tumor-associated cell surface antigens. The transmembrane domain of a CAR is commonly from CD28, which provides stability to the CAR. The intracellular signaling domain is generally comprised of a CD3ζ structure, costimulatory molecules and/or cytokine expression cassettes, for enhanced downstream signaling and T-cell function [1].

Breast cancer is the second most common cancer among women in the US. Genetics, environment, as well as dietary factors are thought to have significant impacts on breast cancer risk. Epidemiological evidence supports a role for vitamin D in protection against breast cancer. 1,25 dihydroxyvitamin D (1,25(OH)2D), the active form of vitamin D, is proposed to regulate cellular processes that are involved in breast cancer progression. Metabolic reprogramming of glucose, termed the Warburg effect, as well as increased glutamine uptake and metabolism for energy use is a characteristic of many cancer cells. The current studies were designed to investigate the effect of 1,25(OH)2D on glutamine metabolism in mammary cells during an early stage of cancer progression. We employed untransformed (MCF10A) and ras oncogene transformed MCF10A human breast epithelial cells (MCF10A-ras). Intracellular glutamine and glutamate levels, determined by nuclear magnetic resonance, were both reduced with 1,25(OH)2D by 23% in MCF10A-ras cells. However, 1,25(OH)2D decreased intracellular glutamine level in MCF10A cells by only 9%, with no effect on intracellular glutamate level in MCF10A cells. Glutamine and glutamate flux into the TCA cycle were determined using [U-13C5] L-glutamine and gas chromatography-mass spectrometry. Treatment with 1,25(OH)2D decreased glutamine flux into the TCA cycle by 18% and 31% in MCF10A and MCF10A-ras cells, respectively, and decreased glutamate flux into the TCA cycle by 13% and 17% in MCF10A and MCF10A-ras cells, respectively. The mRNA and protein expression of the major glutamine transporter, solute linked carrier family 1 member A5 (SLC1A5), was significantly decreased by 1,25(OH)2D in MCF10A-ras cells. Consistent with these results, glutamine uptake was reduced by 1,25(OH)2D, and the SLC1A5 inhibitor L-γ-Glutamyl-p-nitroanilide (GPNA) inhibited the effect of 1,25(OH)2D on glutamine uptake in both MCF10A and MCF10A-ras cells. The mRNA level of the glutamine transporter, SLC1A5, was also decreased by 1,25(OH)2D in MCF10A which overexpress ErbB2 (HER-2/neu) and in malignant MCF10CA1a cells. These results suggest 1,25(OH)2D may alter glutamine metabolism in MCF10A-ras cells by inhibiting glutamine uptake and utilization, in part through down-regulation of SLC1A5 transcript abundance. The 1,25(OH)2D down-regulation of the glutamine transporter, SLC1A5, may play a role in vitamin D prevention of breast cancer. Note: This abstract was not presented at the meeting. Citation Format: Xuanzhu Zhou, Wei Zheng, Fariba Tayyari, G.A.Nagana Gowda, Daniel Raftery, Shawn S. Donkin, Brian Bequette, Dorothy Teegarden. 1,25-Dihydroxyvitamin D regulation of glutamine metabolism in Harvey-ras transformed MCF10A human breast epithelial cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1200. doi:10.1158/1538-7445.AM2015-1200

Breast cancer is the second most common cancer among women in the US. The active form of vitamin D, 1α,25 dihydroxyvitamin D (1,25(OH)2D), is proposed to inhibit cellular processes that may prevent breast cancer. Possible pro-oxidative effects of 1,25(OH)2D in breast cancer cells which may selectively promote cell death have been proposed. Glutamic-oxaloacetic transaminase 1 (GOT1) is an essential transaminase in glutamine metabolism. GOT1 converts aspartate into oxaloacetate in the cytoplasm, which is then subsequently converted to malate and pyruvate. NADPH is produced during the last step of this process and increased NADPH/NADP+ ratio has the potential to maintain cellular redox balance. Therefore, it is of interest to study 1,25(OH)2D regulation of GOT1 during cancer progression to understand the role of vitamin D in regulating oxidative stress as a mechanism to prevent breast cancer. We employed untransformed MCF10A human breast epithelial cells (MCF10A), as well as Harvey ras-oncogene or ErbB2 (HER-2/neu) oncogene transformed cells (MCF10A-ras and MCF10A-ErbB2) as cell models for early cancer progression. Results showed that 1,25(OH)2D significantly decreased GOT1 mRNA expression at 48 hours in MCF10A-ras cells and MCF10A-ErbB2 cells by 35 ± 8.8% and 49 ± 10%, respectively, and GOT1 protein expression at 72 hours by 51 ± 4.9% and 51 ± 16%, respectively. The susceptibility of MCF10A-ras cells to hydrogen peroxide induced cell death was significantly increased by 1,25(OH)2D, as determined by measuring viable cell number using MTT assay. We further showed that 96-hour 1,25(OH)2D treatment significantly reduced cellular NADPH/NADP+ ratio by 30 ± 2.8% and GSH/GSSG ratio by 34 ± 2.7% in MCF10A-ras cells. Collectively, these results support that 1,25(OH)2D inhibits GOT1 expression and has pro-oxidative effects in oncogene transformed MCF10A cell lines, which may contribute to its role in preventing breast cancer development. Citation Format: Xuanzhu Zhou, Tomasz Wilmanski, Dorothy Teegarden. Effect of 1,25-Dihydroxyvitamin D in regulating glutamic-oxaloacetic transaminase 1 (GOT1) and redox balance during breast cancer progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1047.

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Studies were designed to investigate the effect of 1,25 dihydroxyvitamin D (1,25D) on glutamine metabolism during cancer progression employing untransformed (10A) and ras oncogene transformed MCF10A human breast epithelial cells (ras). Glutamine restriction reduced cell number in both cell lines, and 1,25D ameliorated these reductions. Intracellular glutamine and glutamate levels, determined by NMR, were increased 25% in ras cells compared to 10A cells, and 1,25D reduced these levels by 23% in ras cells. Fractional glutamine and glutamate flux, utilizing U13C5‐L‐glutamine, into the TCA cycle was increased in ras cells by 21% and 18%, respectively, compared to 10A cells. 1,25D decreased glutamine flux into the TCA cycle by 18% and 31% in 10A and ras cells, respectively, and decreased glutamate flux into the TCA cycle by 13% and 17% in 10A and ras cells, respectively. The mRNA expression of the glutamine transporter, solute linked carrier family 1 member A5 (SLC1A5), was decreased by 34% in ras compared to 10A cells. 1,25D further decreased SLC1A5 mRNA expression in ras by 45%, but had no effect in 10A cells. These results suggest 1,25D may alter glutamine metabolism in ras cells by inhibiting glutamine uptake and utilization, in part through down‐regulation of SLC1A5. Grant Funding Source: Supported by NIH R25CA128770 and NCI CCSG CA23168