1,25-Dihydroxyvitamin D inhibits glutamine metabolism in Harvey-ras transformed MCF10A human breast epithelial cell

https://doi.org/10.1016/j.jsbmb.2016.04.022Get rights and content

Highlights

  • Glutamine addiction is reduced by 1,25(OH)2D in MCF10A-ras cells.

  • Intracellular glutamine and glutamate concentrations are reduced by 1,25(OH)2D.

  • Flux of glutamine into the TCA cycle is decreased by 1,25(OH)2D.

  • 1,25(OH)2D inhibits glutamine uptake through the glutamine transporter SLC1A5.

  • SLC1A5 is transcriptionally down-regulated through a functional VDRE.

Abstract

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.

Introduction

Breast cancer is the most commonly diagnosed cancer and the second leading cause of cancer death among U.S. females. More than 230,000 women will be diagnosed with breast cancer in the U.S. this year, and over 40,000 will die from the disease [1]. Genetics, environment, as well as dietary factors such as vitamin D are thought to play significant roles in breast cancer risk [2]. For example, epidemiological evidence suggests that increased sun exposure as well as increased dietary vitamin D intake, are correlated with decreased breast cancer incidence [3], [4], [5]. Further, low levels of circulating 25(OH)D, an indicator of vitamin D status, are associated with high breast cancer risk [6] and results from the Women’s Health Initiative shows that women who consumed calcium and vitamin D supplements have lower incidence of breast carcinoma in situ [7]. However, the underlying mechanism by which vitamin D contributes to breast cancer prevention is still not clear.

Cancer cells have been characterized with “the Warburg effect”, a critical shift of glucose flux from mitochondrial oxidative phosphorylation towards aerobic glycolysis despite the availability of adequate oxygen [8]. This phenomenon suggests that glucose is in part redirected into providing intermediates needed for growth rather than towards energy in the form of adenosine-triphosphate (ATP) [9]. Glucose and glutamine are the two most catabolized molecules for the supply of carbon, nitrogen, free energy, and reducing equivalents that are necessary to support cell growth [10]. Because glucose is not efficiently used for ATP production during cancer progression (Warburg effect), glutamine, the most abundant free amino acid in the human body, may be an alternative energy source [11]. In addition to the importance of glutamine in glutathione synthesis, and in protein and nucleotide synthesis, glutamine can also enter the tricarboxylic acid (TCA) cycle and contribute to the synthesis of reducing equivalents for ATP production [12].

Certain cancer cells exhibit reduced cell survival rates in the absence of exogenous glutamine [13], [14], [15]. In 1955, Dr. Harry Eagle first highlighted that l-glutamine is essential for the survival and growth of a mouse fibroblast cell line (strain L) and a human carcinoma cell line (strain HeLa) in vitro [16]. In fact, a wide variety of human cancer cells have shown sensitivity to glutamine starvation [17], [18]. Glutamine is transported into cells through the neutral amino acid transporter family system, which includes sodium-dependent systems A, ASC, N and sodium-independent system L [19]. One of the major high affinity transporters, solute carrier family 1 member 5 (SLC1A5), is over-expressed in many types of cancer cells, and SLC1A5 mediated glutamine transport is required for cell growth [20], [21]. Intracellular glutamine can be converted to glutamate by glutaminase (GLS), and further metabolized into α-ketoglutarate by either deamination or transamination. The carbon backbones from glutamine therefore enter the TCA cycle to provide energy for cell growth [22]. Understanding the regulation of glutamine metabolism during cancer progression may contribute to the development of future cancer therapeutic targets.

Mammary cancer development is a multistage process, which includes cellular mutagenesis for genes that regulate cell proliferation. The acquisition of multiple mutations in proto-oncogenes and tumor suppressor genes can result in uncontrolled cell proliferation and metastasis of the cells. The role of mutated ras genes in inducing malignant transformation is well documented [23], [24], [25]. Mutations of the ras gene are found in a variety of tumor types and the activated ras gene can result in continuous stimulation of cellular proliferation and development of mammary cancer [26]. In this study, MCF10A and Harvey-ras transfected MCF10A (MCF10A-ras) breast epithelial cells were used as a model of cells in early progression to cancer. Previous work from our laboratory shows greater glucose influx, increased glycolysis and lactate production in MCF10A-ras breast epithelial cells, similar to the Warburg effect [27]. Importantly, 1,25(OH)2D inhibits the altered glucose metabolism in the MCF10A-ras cells, as well as further inhibits flux of glucose into the TCA cycle [28]. Therefore, it is important to also determine if 1,25(OH)2D alters glutamine metabolism in cancer progression.

The purpose of the current study was to investigate the role of 1,25(OH)2D in regulating glutamine metabolism in mammary epithelial cells during cancer progression. Our hypothesis is that 1,25(OH)2D inhibits glutamine uptake and utilization in the cells, a process that is essential for cell growth and proliferation during mammary cancer progression. The results of these studies provide insights into the role of vitamin D in regulating cancer energy metabolism and mammary cancer prevention.

Section snippets

Chemicals and reagents

Dulbecco’s modified Eagle medium (DMEM), Nutrient Mixture F-12 (DMEM/F12) media, horse serum, trypsin and penicillin/streptomycin, Annexin V (Alexa Fluor 488 conjugate) were obtained from Life Technologies (Rockville, MD). Cholera toxin was purchased from Calbiochem (Darmstadt, Germany) and 1,25(OH)2D was purchased from Biomol (Plymouth Meeting, PA). Protease inhibitor cocktail, trypan blue, insulin, epidermal growth factor, hydrocortisone, and l-γ-glutamyl-p-nitroanilide, propidium iodide were

,25(OH)2reduces the effect of glutamine deprivation on inducing further cell death in MCF10A-ras cells

To determine the importance of glutamine in MCF10A-ras cells, viable cell number was tested when cells were cultured for 24 h with or without glutamine in the cell culture media. Results showed a significant decrease in the number of viable cells in the glutamine-deprived group when compared with the glutamine-supplemented group in MCF10A-ras (Fig. 1B). Glutamine dependency was also observed in HER2/neu/ErbB2 transformed MCF10A cells and metastatic MCF10CA1a cell lines (Fig. 1C and D), though

Discussion

Glutamine metabolism is essential for tumorigenesis in many types of cancer. Cancer cells alter their glutamine metabolic pathways as part of metabolic reprograming to support rapid cell proliferation [31], [32]. Despite the essential role of glutamine in cancer, the importance and regulation of glutamine metabolism in mammary cancer is not thoroughly understood. Many cancer cells are dependent on glutamine for growth and proliferation, and glutamine starvation in vitro leads to rapid loss of

Conclusion

Collectively, these results suggest 1,25(OH)2D alters glutamine metabolism in MCF10A-ras cells by inhibiting glutamine uptake and utilization, in part through transcriptional down-regulation of the expression of SLC1A5 (Fig. 6). To our knowledge, this is the first study to demonstrate the effect of 1,25(OH)2D on SLC1A5 expression and glutamine metabolism. Therefore, regulation of glutamine metabolism may contribute to the impact of vitamin D on breast cancer prevention.

Conflicts of interest

DR holds equity and an executive office at Matrix-Bio, Inc. No other potential conflicts of interest were disclosed

Acknowledgements

This work was supported by the National Institutes of Health, National Cancer InstituteR25CA128770, National Institute for General MedicineR01GM085291. Additional supports were received from the Purdue University Center for Cancer Research Small Grants Program and Indiana Elks Charities.

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    Current address: Department of Anesthesiology & Pain Medicine, University of Washington, Seattle, WA 98109, United States.

    2

    Current address: Department of Anesthesiology & Pain Medicine, University of Washington, and Fred Hutchinson Cancer Research Center, Seattle, WA 98109, United States.

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