Hypoxia-mediated repression of pyruvate carboxylase drives immunosuppression.

BACKGROUND: Metabolic plasticity mediates breast cancer survival, growth, and immune evasion during metastasis. However, how tumor cell metabolism is influenced by and feeds back to regulate breast cancer progression are not fully understood. We identify hypoxia-mediated suppression of pyruvate carboxylase (PC), and subsequent induction of lactate production, as a metabolic regulator of immunosuppression. METHODS: We used qPCR, immunoblot, and reporter assays to characterize repression of PC in hypoxic primary tumors. Steady state metabolomics were used to identify changes in metabolite pools upon PC depletion. In vivo tumor growth and metastasis assays were used to evaluate the impact of PC manipulation and pharmacologic inhibition of lactate transporters. Immunohistochemistry, flow cytometry, and global gene expression analyzes of tumor tissue were employed to characterize the impact of PC depletion on tumor immunity. RESULTS: PC is essential for metastatic colonization of the lungs. In contrast, depletion of PC in tumor cells promotes primary tumor growth. This effect was only observed in immune competent animals, supporting the hypothesis that repression of PC can suppress anti-tumor immunity. Exploring key differences between the pulmonary and mammary environments, we demonstrate that hypoxia potently downregulated PC. In the absence of PC, tumor cells produce more lactate and undergo less oxidative phosphorylation. Inhibition of lactate metabolism was sufficient to restore T cell populations to PC-depleted mammary tumors. CONCLUSIONS: We present a dimorphic role for PC in primary mammary tumors vs. pulmonary metastases. These findings highlight a key contextual role for PC-directed lactate production as a metabolic nexus connecting hypoxia and antitumor immunity.

FGFR1 Signaling Facilitates Obesity-Driven Pulmonary Outgrowth in Metastatic Breast Cancer.

Survival of dormant, disseminated breast cancer cells contributes to tumor relapse and metastasis. Women with a body mass index greater than 35 have an increased risk of developing metastatic recurrence. Herein, we investigated the effect of diet-induced obesity (DIO) on primary tumor growth and metastatic progression using both metastatic and systemically dormant mouse models of breast cancer. This approach led to increased PT growth and pulmonary metastasis. We developed a novel protocol to induce obesity in Balb/c mice by combining dietary and hormonal interventions with a thermoneutral housing strategy. In contrast to standard housing conditions, ovariectomized Balb/c mice fed a high-fat diet under thermoneutral conditions became obese over a period of 10 weeks, resulting in a 250% gain in fat mass. Obese mice injected with the D2.OR model developed macroscopic pulmonary nodules compared with the dormant phenotype of these cells in mice fed a control diet. Analysis of the serum from obese Balb/c mice revealed increased levels of FGF2 as compared with lean mice. We demonstrate that serum from obese animals, exogenous FGF stimulation, or constitutive stimulation through autocrine and paracrine FGF2 is sufficient to break dormancy and drive pulmonary outgrowth. Blockade of FGFR signaling or specific depletion of FGFR1 prevented obesity-associated outgrowth of the D2.OR model. IMPLICATIONS: Overall, this study developed a novel DIO model that allowed for demonstration of FGF2:FGFR1 signaling as a key molecular mechanism connecting obesity to breakage of systemic tumor dormancy and metastatic progression.

1α,25-dihydroxyvitamin D reduction of MCF10A-ras cell viability in extracellular matrix detached conditions is dependent on regulation of pyruvate carboxylase.

An emerging hallmark of cancer is cellular metabolic reprogramming to adapt to varying cellular environments. Throughout the process of metastasis cancer cells gain anchorage independence which confers survival characteristics when detached from the extracellular matrix (ECM). Previous work demonstrates that the bioactive metabolite of vitamin D, 1α,25-dihydroxyvitamin D (1,25[OH]2D), suppresses cancer progression, potentially by suppressing the ability of cells to metabolically adapt to varying cellular environments such as ECM detachment. The purpose of the present study was to determine the mechanistic bases of the effects of 1,25(OH)2D on cell survival in ECM-detached conditions. Pretreatment of MCF10A-ras breast cancer cells for 3 d with 1,25(OH)2D reduced the viability of cells in subsequent detached conditions by 11%. Enrichment of 13C5-glutamine was reduced in glutamate (21%), malate (30%), and aspartate (23%) in detached compared to attached MCF10A-ras cells. Pretreatment with 1,25(OH)2D further reduced glutamine flux into downstream metabolites glutamate (5%), malate (6%), and aspartate (10%) compared to detached vehicle treated cells. Compared to attached cells, detachment increased pyruvate carboxylase (PC) mRNA abundance and protein expression by 95% and 190%, respectively. Consistent with these results, 13C6-glucose derived M+3 labelling was shown to preferentially replenish malate and aspartate, but not citrate pools, demonstrating increased PC activity in detached cells. In contrast, 1,25(OH)2D pretreatment of detached cells reduced PC mRNA abundance and protein expression by 63% and 56%, respectively, and reduced PC activity as determined by decreased 13C6-glucose derived M+3 labeling in citrate (8%) and aspartate (50%) pools, relative to vehicle-treated detached cells. While depletion of PC with doxycycline-inducible shRNA reduced detached cell viability, PC knockdown in combination with 1,25(OH)2D treatment did not additionally affect the viability of detached cells. Further, PC overexpression improved detached cell viability, and inhibited the effect of 1,25(OH)2D on detached cell survival, suggesting that 1,25(OH)2D mediates its effects in detachment through regulation of PC expression. These results suggest that inhibition of PC by 1,25(OH)2D suppresses cancer cell anchorage independence.

Hypoxia-Mediated ATF4 Induction Promotes Survival in Detached Conditions in Metastatic Murine Mammary Cancer Cells.

Regions of hypoxia are common in solid tumors and drive changes in gene expression that increase risk of cancer metastasis. Tumor cells must respond to the stress of hypoxia by activating genes to modify cell metabolism and antioxidant response to improve survival. The goal of the current study was to determine the effect of hypoxia on cell metabolism and markers of oxidative stress in metastatic (metM-Wntlung) compared with nonmetastatic (M-Wnt) murine mammary cancer cell lines. We show that hypoxia induced a greater suppression of glutamine to glutamate conversion in metastatic cells (13% in metastatic cells compared to 7% in nonmetastatic cells). We also show that hypoxia increased expression of genes involved in antioxidant response in metastatic compared to nonmetastatic cells, including glutamate cysteine ligase catalytic and modifier subunits and malic enzyme 1. Interestingly, hypoxia increased the mRNA level of the transaminase glutamic pyruvic transaminase 2 (Gpt2, 7.7-fold) only in metM-Wntlung cells. The change in Gpt2 expression was accompanied by transcriptional (4.2-fold) and translational (6.5-fold) induction of the integrated stress response effector protein activating transcription factor 4 (ATF4). Genetic depletion ATF4 demonstrated importance of this molecule for survival of hypoxic metastatic cells in detached conditions. These findings indicate that more aggressive, metastatic cancer cells utilize hypoxia for metabolic reprogramming and induction of antioxidant defense, including activation of ATF4, for survival in detached conditions.

Increased Ammonium Toxicity in Response to Exogenous Glutamine in Metastatic Breast Cancer Cells.

Several cancers, including breast cancers, show dependence on glutamine metabolism. The purpose of the present study was to determine the mechanistic basis and impact of differential glutamine metabolism in nonmetastatic and metastatic murine mammary cancer cells. Universally labeled 13C5-glutamine metabolic tracing, qRT-PCR, measures of reductive-oxidative balance, and exogenous ammonium chloride treatment were used to assess glutamine reprogramming. Results show that 4 mM media concentration of glutamine, compared with 2 mM, reduced viability only in metastatic cells, and that this decrease in viability was accompanied by increased incorporation of glutamine-derived carbon into the tricarboxylic acid (TCA) cycle. While increased glutamine metabolism in metastatic cells occurred in tandem with a decrease in the reduced/oxidized glutathione ratio, treatment with the antioxidant molecule N-acetylcysteine did not rescue cell viability. However, the viability of metastatic cells was more sensitive to ammonium chloride treatment compared with nonmetastatic cells, suggesting a role of metabolic reprogramming in averting nitrogen cytotoxicity in nonmetastatic cells. Overall, these results demonstrate the ability of nonmetastatic cancer cells to reprogram glutamine metabolism and that this ability may be lost in metastatic cells.

Vitamin D regulation of energy metabolism in cancer.

Vitamin D exerts anti-cancer effects in recent clinical trials and preclinical models. The actions of vitamin D are primarily mediated through its hormonal form, 1,25-dihydroxyvitamin D (1,25(OH)2 D). Previous literature describing in vitro studies has predominantly focused on the anti-tumourigenic effects of the hormone, such as proliferation and apoptosis. However, recent evidence has identified 1,25(OH)2 D as a regulator of energy metabolism in cancer cells, where requirements for specific energy sources at different stages of progression are dramatically altered. The literature suggests that 1,25(OH)2 D regulates energy metabolism, including glucose, glutamine and lipid metabolism during cancer progression, as well as oxidative stress protection, as it is closely associated with energy metabolism. Mechanisms involved in energy metabolism regulation are an emerging area in which vitamin D may inhibit multiple stages of cancer progression. LINKED ARTICLES: This article is part of a themed issue on New avenues in cancer prevention and treatment (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.12/issuetoc.

Proteomic Characterization of Cytoplasmic Lipid Droplets in Human Metastatic Breast Cancer Cells.

One of the characteristic features of metastatic breast cancer is increased cellular storage of neutral lipid in cytoplasmic lipid droplets (CLDs). CLD accumulation is associated with increased cancer aggressiveness, suggesting CLDs contribute to metastasis. However, how CLDs contribute to metastasis is not clear. CLDs are composed of a neutral lipid core, a phospholipid monolayer, and associated proteins. Proteins that associate with CLDs regulate both cellular and CLD metabolism; however, the proteome of CLDs in metastatic breast cancer and how these proteins may contribute to breast cancer progression is unknown. Therefore, the purpose of this study was to identify the proteome and assess the characteristics of CLDs in the MCF10CA1a human metastatic breast cancer cell line. Utilizing shotgun proteomics, we identified over 1500 proteins involved in a variety of cellular processes in the isolated CLD fraction. Interestingly, unlike other cell lines such as adipocytes or enterocytes, the most enriched protein categories were involved in cellular processes outside of lipid metabolism. For example, cell-cell adhesion was the most enriched category of proteins identified, and many of these proteins have been implicated in breast cancer metastasis. In addition, we characterized CLD size and area in MCF10CA1a cells using transmission electron microscopy. Our results provide a hypothesis-generating list of potential players in breast cancer progression and offers a new perspective on the role of CLDs in cancer.

Pyruvate carboxylase and cancer progression.

Pyruvate carboxylase (PC) is a mitochondrial enzyme that catalyzes the ATP-dependent carboxylation of pyruvate to oxaloacetate (OAA), serving to replenish the tricarboxylic acid (TCA) cycle. In nonmalignant tissue, PC plays an essential role in controlling whole-body energetics through regulation of gluconeogenesis in the liver, synthesis of fatty acids in adipocytes, and insulin secretion in pancreatic ß cells. In breast cancer, PC activity is linked to pulmonary metastasis, potentially by providing the ability to utilize glucose, fatty acids, and glutamine metabolism as needed under varying conditions as cells metastasize. PC enzymatic activity appears to be of particular importance in cancer cells that are unable to utilize glutamine for anaplerosis. Moreover, PC activity also plays a role in lipid metabolism and protection from oxidative stress in cancer cells. Thus, PC activity may be essential to link energy substrate utilization with cancer progression and to enable the metabolic flexibility necessary for cell resilience to changing and adverse conditions during the metastatic process.

Transdisciplinary Obesity Prevention Research Sciences (TOPRS) Curriculum Increases Knowledge About Complex Causes and Consequences of Obesity for Undergraduate Students.

Most researchers and public health officials would agree that the causes and consequences of obesity are complex and multi-faceted. However, curricula designed to address these complexities are limited and often guided by a single discipline. The purpose of the Transdisciplinary Obesity Prevention Research Sciences (TOPRS) program was to develop a "flip-the-classroom" curriculum on obesity prevention across multiple disciplines such that students would gain an appreciation of the complex origins of obesity. The curriculum is based on the 6 C's model (cell, child, clan, community, country, culture) that proposes a cell-to-society approach to obesity. Twenty video micro-lectures were developed and students were tested on content knowledge pre- and post-viewing. The curriculum was administered at three university sites to 74 undergraduate students across 23 declared majors from 2014-2016. There were significant gains in knowledge about the causes and consequences of obesity. Recommendations are offered to adopt this curriculum in undergraduate and other educational settings.

1,25-Dihydroxyvitamin D regulates lipid metabolism and glucose utilization in differentiated 3T3-L1 adipocytes.

It is well established that adipose tissue can both store and metabolize vitamin D. The active form of vitamin D, 1,25 dihydroxyvitamin D [1,25(OH)2D], regulates adipocyte differentiation and inflammation, highlighting the multifaceted role that vitamin D plays in adipose tissue physiology. However, there is limited evidence regarding vitamin D regulation of mature adipocyte lipid metabolism. We hypothesize that 1,25(OH)2D alters lipid and glucose metabolism in differentiated 3T3-L1 adipocytes to reduce triacylglycerol (TAG) accumulation. In this study, 1,25(OH)2D (10 nmol/L) stimulated a 21% reduction in TAG accumulation in differentiated 3T3-L1 adipocytes after 4 days (P = .01) despite a significant increase in fatty acid uptake (P < .01). Additionally, 1,25(OH)2D stimulated a 2.5-fold increase in 14CO2 production from [1-14C] palmitic acid (P < .01), indicative of an elevated rate of fatty acid ß-oxidation, while stimulating a 9% reduction in de novo fatty acid synthesis (P = .03). Interestingly, d-[U-13C]glucose incorporation into fatty acids was reduced by 30% in response to 1,25(OH)2D (P < .01), indicating a reduced contribution of glucose as a substrate for de novo lipogenesis. Consistent with these findings, mRNA expression of the anaplerotic enzyme pyruvate carboxylase was reduced by 41% (P < .01). In summary, 1,25(OH)2D stimulated fatty acid oxidation and reduced TAG accumulation in differentiated adipocytes. Furthermore, 1,25(OH)2D reduced glucose utilization as a substrate for fatty acid synthesis potentially by downregulating pyruvate carboxylase and stimulating glucose disposal as glycerol. Collectively, these 1,25(OH)2D-induced changes in lipid metabolism and glucose utilization may contribute to the reduction in TAG accumulation and be protective against excessive fat mass accumulation and associated metabolic disorders.

Pyruvate carboxylase supports the pulmonary tropism of metastatic breast cancer.

BACKGROUND: Overcoming systemic dormancy and initiating secondary tumor grow under unique microenvironmental conditions is a major rate-limiting step in metastatic progression. Disseminated tumor cells encounter major changes in nutrient supplies and oxidative stresses compared to the primary tumor and must demonstrate significant metabolic plasticity to adapt to specific metastatic sites. Recent studies suggest that differential utilization of pyruvate sits as a critical node in determining the organotropism of metastatic breast cancer. Pyruvate carboxylase (PC) is key enzyme that converts pyruvate into oxaloacetate for utilization in gluconeogenesis and replenishment of the TCA cycle. METHODS: Patient survival was analyzed with respect to gene copy number alterations and differential mRNA expression levels of PC. Expression of PC was analyzed in the MCF-10A, D2-HAN and the 4 T1 breast cancer progression series under in vitro and in vivo growth conditions. PC expression was depleted via shRNAs and the impact on in vitro cell growth, mammary fat pad tumor growth, and pulmonary and non-pulmonary metastasis was assessed by bioluminescent imaging. Changes in glycolytic capacity, oxygen consumption, and response to oxidative stress were quantified upon PC depletion. RESULTS: Genomic copy number increases in PC were observed in 16-30% of metastatic breast cancer patients. High expression of PC mRNA was associated with decreased patient survival in the MCTI and METABRIC patient datasets. Enhanced expression of PC was not recapitulated in breast cancer progression models when analyzed under glucose-rich in vitro culture conditions. In contrast, PC expression was dramatically enhanced upon glucose deprivation and in vivo in pulmonary metastases. Depletion of PC led to a dramatic decrease in 4 T1 pulmonary metastasis, but did not affect orthotopic primary tumor growth. Tail vein inoculations confirmed the role of PC in facilitating pulmonary, but not extrapulmonary tumor initiation. PC-depleted cells demonstrated a decrease in glycolytic capacity and oxygen consumption rates and an enhanced sensitivity to oxidative stress. CONCLUSIONS: Our studies indicate that PC is specifically required for the growth of breast cancer that has disseminated to the lungs. Overall, these findings point to the potential of targeting PC for the treatment of pulmonary metastatic breast cancer.

Inhibition of pyruvate carboxylase by 1α,25-dihydroxyvitamin D promotes oxidative stress in early breast cancer progression.

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.

1α,25-dihydroxyvitamin D inhibits de novo fatty acid synthesis and lipid accumulation in metastatic breast cancer cells through down-regulation of pyruvate carboxylase.

Both increased de novo fatty acid synthesis and higher neutral lipid accumulation are a common phenotype observed in aggressive breast cancer cells, making lipid metabolism a promising target for breast cancer prevention. In the present studies, we demonstrate a novel effect of the active metabolite of vitamin D, 1α,25-dihydroxyvitamin D (1,25(OH)2D) on lipid metabolism in malignant breast epithelial cells. Treatment of MCF10CA1a breast epithelial cells with 1,25(OH)2D (10 nM) for 5 and 7 days decreased the level of triacylglycerol, the most abundant form of neutral lipids, by 20%(±3.9) and 50%(±5.9), respectively. In addition, 1,25(OH)2D treatment for 5 days decreased palmitate synthesis from glucose, the major fatty acid synthesized de novo (48%±5.5 relative to vehicle). We have further identified the anaplerotic enzyme pyruvate carboxylase (PC) as a target of 1,25(OH)2D-mediated regulation and hypothesized that 1,25(OH)2D regulates breast cancer cell lipid metabolism through inhibition of PC. PC mRNA expression was down-regulated with 1,25(OH)2D treatment at 2 (73%±6 relative to vehicle) and 5 (56%±8 relative to vehicle) days. Decrease in mRNA abundance corresponded with a decrease in PC protein expression at 5 days of treatment (54%±12 relative to vehicle). Constitutive overexpression of PC in MCF10CA1a cells using a pCMV6-PC plasmid inhibited the effect of 1,25(OH)2D on both TAG accumulation and de novo palmitate synthesis from glucose. Together, these studies demonstrate a novel mechanism through which 1,25(OH)2D regulates lipid metabolism in malignant breast epithelial cells.

Maternal high fructose and low protein consumption during pregnancy and lactation share some but not all effects on early-life growth and metabolic programming of rat offspring.

Maternal nutritional stress during pregnancy acts to program offspring metabolism. We hypothesized that the nutritional stress caused by maternal fructose or low protein intake during pregnancy would program the offspring to develop metabolic aberrations that would be exacerbated by a diet rich in fructose or fat during adult life. The objective of this study was to characterize and compare the fetal programming effects of maternal fructose with the established programming model of a low-protein diet on offspring. Male offspring from Sprague-Dawley dams fed a 60% starch control diet, a 60% fructose diet, or a low-protein diet throughout pregnancy and lactation were weaned onto either a 60% starch control diet, 60% fructose diet, or a 30% fat diet for 15 weeks. Offspring from low-protein and fructose-fed dam showed retarded growth (P<.05) at weaning (50.3, 29.6 vs 59.1±0.8 g) and at 18 weeks of age (420, 369 vs 464±10.9 g). At 18 weeks of age, offspring from fructose dams expressed greater quantities (P<.05) of intestinal Pgc1a messenger RNA compared with offspring from control or low-protein dams (1.31 vs 0.89, 0.85; confidence interval, 0.78-1.04). Similarly, maternal fructose (P=.09) and low-protein (P<.05) consumption increased expression of Pgc1a in offspring liver (7.24, 2.22 vs 1.22; confidence interval, 2.11-3.45). These data indicate that maternal fructose feeding is a programming model that shares some features of maternal protein restriction such as retarded growth, but is unique in programming of selected hepatic and intestinal transcripts.

1α,25-Dihydroxyvitamin D Inhibits the Metastatic Capability of MCF10CA1a and MDA-MB-231 Cells in an In Vitro Model of Breast to Bone Metastasis.

Breast cancer metastasis to the bone continues to be a major health problem, with approximately 80% of advanced breast cancer patients expected to develop bone metastasis. Although the problem of bone metastasis persists, current treatment options for metastatic cancer patients are limited. In this study, we investigated the preventive role of the active vitamin D metabolite, 1α,25-dihydroxyvitamin D (1,25(OH)2D), against the metastatic potential of breast cancer cells using a novel three-dimensional model (rMET) recapitulating multiple steps of the bone metastatic process. Treatment of MCF10CA1a and MDA-MB-231 cells inhibited metastasis in the rMET model by 70% (±5.7%) and 21% (±6%), respectively. In addition, 1,25(OH)2D treatment decreased invasiveness (20 ± 11% of vehicle) and decreased the capability of MCF10CA1a cells to survive in the reconstructed bone environment after successful invasion through the basement membrane (69 ± 5% of vehicle). An essential step in metastasis is epithelial-mesenchymal transition (EMT). Treatment of MCF10CA1a cells with 1,25(OH)2D increased gene (2.04 ± 0.28-fold increase) and protein (1.87 ± 0.20-fold increase) expression of E-cadherin. Additionally, 1,25(OH)2D treatment decreased N-cadherin gene expression (42 ± 8% decrease), a marker for EMT. Collectively, the present study suggests that 1,25(OH)2D inhibits breast cancer cell metastatic capability as well as inhibits EMT, an essential step in the metastatic process.

Stilbenoids remodel the DNA methylation patterns in breast cancer cells and inhibit oncogenic NOTCH signaling through epigenetic regulation of MAML2 transcriptional activity.

DNA hypomethylation was previously implicated in cancer progression and metastasis. The purpose of this study was to examine whether stilbenoids, resveratrol and pterostilbene thought to exert anticancer effects, target genes with oncogenic function for de novo methylation and silencing, leading to inactivation of related signaling pathways. Following Illumina 450K, genome-wide DNA methylation analysis reveals that stilbenoids alter DNA methylation patterns in breast cancer cells. On average, 75% of differentially methylated genes have increased methylation, and these genes are enriched for oncogenic functions, including NOTCH signaling pathway. MAML2, a coactivator of NOTCH targets, is methylated at the enhancer region and transcriptionally silenced in response to stilbenoids, possibly explaining the downregulation of NOTCH target genes. The increased DNA methylation at MAML2 enhancer coincides with increased occupancy of repressive histone marks and decrease in activating marks. This condensed chromatin structure is associated with binding of DNMT3B and decreased occupancy of OCT1 transcription factor at MAML2 enhancer, suggesting a role of DNMT3B in increasing methylation of MAML2 after stilbenoid treatment. Our results deliver a novel insight into epigenetic regulation of oncogenic signals in cancer and provide support for epigenetic-targeting strategies as an effective anticancer approach.

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

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-(13)C5] 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.

Altered glucose metabolism in Harvey-ras transformed MCF10A cells.

Metabolic reprogramming that alters the utilization of glucose including the "Warburg effect" is critical in the development of a tumorigenic phenotype. However, the effects of the Harvey-ras (H-ras) oncogene on cellular energy metabolism during mammary carcinogenesis are not known. The purpose of this study was to determine the effect of H-ras transformation on glucose metabolism using the untransformed MCF10A and H-ras oncogene transfected (MCF10A-ras) human breast epithelial cells, a model for early breast cancer progression. We measured the metabolite fluxes at the cell membrane by a selective micro-biosensor, [(13)C6 ]glucose flux by (13)C-mass isotopomer distribution analysis of media metabolites, intracellular metabolite levels by NMR, and gene expression of glucose metabolism enzymes by quantitative PCR. Results from these studies indicated that MCF10A-ras cells exhibited enhanced glycolytic activity and lactate production, decreased glucose flux through the tricarboxylic acid (TCA) cycle, as well as an increase in the utilization of glucose in the pentose phosphate pathway (PPP). These results provide evidence for a role of H-ras oncogene in the metabolic reprogramming of MCF10A cells during early mammary carcinogenesis.

International breast cancer and nutrition: a model for research, training and policy in diet, epigenetics, and chronic disease prevention.

This article summarizes presentations from the International Breast Cancer and Nutrition workshop held during the ASN Scientific Sessions and Annual Meeting at Experimental Biology 2014 in San Diego, CA, on 28 April 2014. An international collaboration was described among teams from low-, middle-, and high-income countries addressing environmental factors, especially diet, and epigenetic interactions that affect the risk of chronic disease. Speakers addressed opportunities and challenges involved in this type of international collaboration, assessing diet and nutritional status across a wide range of cultures, and research tools and discoveries from this group.

Maternal exercise during pregnancy reduces risk of mammary tumorigenesis in rat offspring.

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. Mammary tumor development in all pups was monitored for 15 weeks. Pups from exercised dams had a substantially lower tumor incidence (42.9%) compared with 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.