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Abstract Background Leptin, an energy balance regulator secreted by adipocytes, increases metastatic potential of breast cancer cells. The impact on cancer cell metabolism remains unclear given that most studies of leptin and breast cancer cell metabolism utilize supraphysiological glucose concentrations. Methods Using two murine models of metastatic triple-negative breast cancer (TNBC) differing in genetic alterations (4T1: p53 and Pik3ca mutations; metM-Wntlung: increased Wnt signaling) and cultured in physiological (5 mM) glucose media, we tested the hypothesis that leptin increases migration of metastatic breast cancer cells through regulation of glucose metabolism. Results Our results showed that leptin treatment, compared with vehicle, increased cell migration in each cell line, with decreased leptin receptor (Ob-R) mRNA expression in 4T1, but not metM-Wntlung, cells. AMP-activated protein kinase (AMPK) was activated in 4T1 with leptin treatment but decreased in metM-Wntlung. Leptin decreased fatty acid synthase (Fasn) and carnitine palmitoyltransferase 1a (Cpt1a) mRNA expression in 4T1 cells but increased their expression in metM-Wntlung cells. Fatty acid oxidation was not necessary for leptin-induced migration in either cell line. Leptin increased palmitate synthesis from glucose in metM-Wntlung, but not 4T1 cells. Moreover, although leptin increased glucose transporter 1 (Glut1) mRNA expression in both cell lines and inhibition of glycolysis blocked leptin-induced migration in metM-Wntlung, but not 4T1 cells. Conclusion Taken together, these results demonstrate that at physiological glucose concentrations, leptin increases migration of 4T1 and metM-Wntlung cells via shared and distinct effects on energy metabolism, suggesting that the type of TNBC genetic alteration plays a role in differential metabolic regulation of leptin-induced migration.

Abstract 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. Graphical abstract

IntroductionObesity is a multi-factorial disease frequently associated with poor nutritional habits and linked to many detrimental health outcomes. Individuals with obesity are more likely to have increased levels of persistent inflammatory and metabolic dysregulation. The goal of this study was to compare four dietary patterns differentiated by macronutrient content in a postmenopausal model. Dietary patterns were high carbohydrate (HC), high fat (HF), high carbohydrate plus high fat (HCHF), and high protein (HP) with higher fiber.MethodsChanges in body weight and glucose levels were measured in female, ovariectomized C57BL/6 mice after 15 weeks of feeding. One group of five mice fed the HCHF diet was crossed over to the HP diet on day 84, modeling a 21-day intervention. In a follow-up study comparing the HCHF versus HP dietary patterns, systemic changes in inflammation, using an 80-cytokine array and metabolism, by untargeted liquid chromatography-mass spectrometry (LCMS)-based metabolomics were evaluated.ResultsOnly the HF and HCHF diets resulted in obesity, shown by significant differences in body weights compared to the HP diet. Body weight gains during the two-diet follow-up study were consistent with the four-diet study. On Day 105 of the 4-diet study, glucose levels were significantly lower for mice fed the HP diet than for those fed the HC and HF diets. Mice switched from the HCHF to the HP diet lost an average of 3.7 grams by the end of the 21-day intervention, but this corresponded with decreased food consumption. The HCHF pattern resulted in dramatic inflammatory dysregulation, as all 80 cytokines were elevated significantly in the livers of these mice after 15 weeks of HCHF diet exposure. Comparatively, only 32 markers changed significantly on the HP diet (24 up, 8 down). Metabolic perturbations in several endogenous biological pathways were also observed based on macronutrient differences and revealed dysfunction in several nutritionally relevant biosynthetic pathways.ConclusionOverall, the HCHF diet promoted detrimental impacts and changes linked to several diseases, including arthritis or breast neoplasms. Identification of dietary pattern-specific impacts in this model provides a means to monitor the effects of disease risk and test interventions to prevent poor health outcomes through nutritional modification.

Obesity promotes triple-negative breast cancer (TNBC), and effective interventions are urgently needed to break the obesity-TNBC link. Epidemiologic studies indicate that bariatric surgery reduces TNBC risk, while evidence is limited or conflicted for weight loss via low-fat diet (LFD) or calorie restriction (CR). Using a murine model of obesity-driven TNBC, we compared the antitumor effects of vertical sleeve gastrectomy (VSG) with LFD, chronic CR, and intermittent CR. Each intervention generated weight and fat loss and suppressed tumor growth relative to obese mice (greatest suppression with CR). VSG and CR regimens exerted both similar and unique effects, as assessed using multiomics approaches, in reversing obesity-associated transcript, epigenetics, secretome, and microbiota changes and restoring antitumor immunity. Thus, in a murine model of TNBC, bariatric surgery and CR each reverse obesity-driven tumor growth via shared and distinct antitumor mechanisms, and CR is superior to VSG in reversing obesity’s procancer effects.

Triple-negative breast cancer (TNBC) lacks targeted therapies, leaving cytotoxic chemotherapy as the current standard treatment. However, chemotherapy resistance remains a major clinical challenge. Increased insulin-like growth factor 1 signaling can potently blunt chemotherapy response, and lysosomal processes including the nutrient scavenging pathway autophagy can enable cancer cells to evade chemotherapy-mediated cell death. Thus, we tested whether inhibition of insulin receptor/insulin-like growth factor 1 receptor with the drug BMS-754807 and/or lysosomal disruption with hydroxychloroquine (HCQ) could sensitize TNBC cells to the chemotherapy drug carboplatin. Using in vitro studies in multiple TNBC cell lines, in concert with in vivo studies employing a murine syngeneic orthotopic transplant model of TNBC, we show that BMS-754807 and HCQ each sensitized TNBC cells and tumors to carboplatin and reveal that exogenous metabolic modulators may work synergistically with carboplatin as indicated by Bliss analysis. Additionally, we demonstrate the lack of overt in vivo toxicity with our combination regimens and, therefore, propose that metabolic targeting of TNBC may be a safe and effective strategy to increase sensitivity to chemotherapy. Thus, we conclude that the use of exogenous metabolic modulators, such as BMS-754807 or HCQ, in combination with chemotherapy warrants additional study as a strategy to improve therapeutic responses in women with TNBC.

Every cancer genome is unique, resulting in potentially near infinite cancer cell phenotypes and an inability to predict clinical outcomes in most cases. Despite this profound genomic heterogeneity, many cancer types and subtypes display a non-random distribution of metastasis to distant organs, a phenomenon known as organotropism. Proposed factors in metastatic organotropism include hematogenous versus lymphatic dissemination, the circulation pattern of the tissue of origin, tumor-intrinsic factors, compatibility with established organ-specific niches, long-range induction of premetastatic niche formation, and so-called “prometastatic niches” that facilitate successful colonization of the secondary site following extravasation. To successfully complete the steps required for distant metastasis, cancer cells must evade immunosurveillance and survive in multiple new and hostile environments. Despite substantial advances in our understanding of the biology underlying malignancy, many of the mechanisms used by cancer cells to survive the metastatic journey remain a mystery. This review synthesizes the rapidly growing body of literature demonstrating the relevance of an unusual cell type known as “fusion hybrid” cells to many of the hallmarks of cancer, including tumor heterogeneity, metastatic conversion, survival in circulation, and metastatic organotropism. Whereas the concept of fusion between tumor cells and blood cells was initially proposed over a century ago, only recently have technological advancements allowed for detection of cells containing components of both immune and neoplastic cells within primary and metastatic lesions as well as among circulating malignant cells. Specifically, heterotypic fusion of cancer cells with monocytes and macrophages results in a highly heterogeneous population of hybrid daughter cells with enhanced malignant potential. Proposed mechanisms behind these findings include rapid, massive genome rearrangement during nuclear fusion and/or acquisition of monocyte/macrophage features such as migratory and invasive capability, immune privilege, immune cell trafficking and homing, and others. Rapid acquisition of these cellular traits may increase the likelihood of both escape from the primary tumor site and extravasation of hybrid cells at a secondary location that is amenable to colonization by that particular hybrid phenotype, providing a partial explanation for the patterns observed in some cancers with regard to sites of distant metastases.

IntroductionAdvanced age and obesity are independent risk and progression factors for triple negative breast cancer (TNBC), which presents significant public health concerns for the aging population and its increasing burden of obesity. Due to parallels between advanced age- and obesityrelated biology, particularly adipose inflammation, we hypothesized that advanced age and obesity each accelerate mammary tumor growth through convergent, and likely interactive, mechanisms.MethodsTo test this hypothesis, we orthotopically transplanted murine syngeneic TNBC cells into the mammary glands of young normoweight control (7 months), young diet-induced obese (DIO), aged normoweight control (17 months), and aged DIO female C57BL/6J mice.ResultsHere we report accelerated tumor growth in aged control and young DIO mice, compared with young controls. Transcriptional analyses revealed, with a few exceptions, overlapping patterns of mammary tumor inflammation and tumor immunosuppression in aged control mice and young DIO mice, relative to young controls. Moreover, aged control and young DIO tumors, compared with young controls, had reduced abundance ofcytotoxic CD8 T cells. Finally, DIO in advanced age exacerbated mammary tumor growth, inflammation and tumor immunosuppression.DiscussionThese findings demonstrate commonalities in the mechanisms driving TNBC in aged and obese mice, relative to young normoweight controls. Moreover, we found that advanced age and DIO interact to accelerate mammary tumor progression. Given the US population is getting older and more obese, age- and obesity-related biological differences will need to be considered when developing mechanism-based strategies for preventing or controlling breast cancer.

Abstract 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 β 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.

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.

The worldwide prevalence of overweight and obesity has tripled since 1975. In the United States, the percentage of adults who are obese exceeds 42.5%. Individuals with obesity often display multiple metabolic perturbations, such as insulin resistance and persistent inflammation, which can suppress the immune system. These alterations in homeostatic mechanisms underlie the clinical parameters of metabolic syndrome, an established risk factor for many cancers, including breast cancer. Within the growth-promoting, proinflammatory milieu of the obese state, crosstalk between adipocytes, immune cells and breast epithelial cells occurs via obesity-associated hormones, angiogenic factors, cytokines, and other mediators that can enhance breast cancer risk and/or progression. This review synthesizes evidence on the biological mechanisms underlying obesity-breast cancer links, with emphasis on emerging mechanism-based interventions in the context of nutrition, using modifiable elements of diet alone or paired with physical activity, to reduce the burden of obesity on breast cancer.

Cancer cells experience unique and dynamic shifts in their metabolic function in order to survive, proliferate, and evade growth inhibition in the resource-scarce tumor microenvironment. Therefore, identification of pharmacological agents with potential to reprogram cancer cell metabolism may improve clinical outcomes in cancer therapy. Cancer cells also often exhibit an increased dependence on the process known as autophagy, both for baseline survival and as a response to stressors such as chemotherapy or a decline in nutrient availability. There is evidence to suggest that this increased dependence on autophagy in cancer cells may be exploitable clinically by combining autophagy modulators with existing chemotherapies. In light of the increased metabolic rate in cancer cells, interest is growing in approaches aimed at “starving” cancer through dietary and pharmacologic interventions that reduce availability of nutrients and pro-growth hormonal signals known to promote cancer progression. Several dietary approaches, including chronic calorie restriction and multiple forms of fasting, have been investigated for their potential anti-cancer benefits, yielding promising results in animal models. Induction of autophagy in response to dietary energy restriction may underlie some of the observed benefit. However, while interventions based on dietary energy restriction have demonstrated safety in clinical trials, uncertainty remains regarding translation to humans as well as feasibility of achieving compliance due to the potential discomfort and weight loss that accompanies dietary restriction. Further induction of autophagy through dietary or pharmacologic metabolic reprogramming interventions may enhance the efficacy of autophagy inhibition in the context of adjuvant or neo-adjuvant chemotherapy. Nonetheless, it remains unclear whether therapeutic agents aimed at autophagy induction, autophagy inhibition, or both are a viable therapeutic strategy for improving cancer outcomes. This review discusses the literature available for the therapeutic potential of these approaches.

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.

Triple negative breast cancer: Obesity and metabolism fuel disease spread Metabolic changes contribute to the metastatic potential of triple negative breast cancer (TNBC), a mouse study shows. Stephen Hursting and colleagues from the University of North Carolina at Chapel Hill, USA, established metastatic mouse TNBC cells driven by Wnt-1, a signaling protein that’s highly active in this aggressive subtype of breast cancer. In a lab dish, these cells showed signs of increased invasiveness; and when transplanted into mice, the cells readily formed tumors that metastasized to the lungs. Obese mice experienced more aggressive tumor growth and spread than normal-weight animals. Gene expression analyses revealed that TNBC cells with metastatic potential have an energetic leg-up over their non-metastatic counterparts in the face of obesity-induced metabolic changes, suggesting that targeting metabolic perturbations could help reduce the burden of metastatic TNBC, particularly for obese women.

Abstract Calorie restriction (CR) extends lifespan and has been shown to reduce age-related diseases including cancer, diabetes, and cardiovascular and neurodegenerative diseases in experimental models. Recent translational studies have tested the potential of CR or CR mimetics as adjuvant therapies to enhance the efficacy of chemotherapy, radiation therapy, and novel immunotherapies. Chronic CR is challenging to employ in cancer patients, and therefore intermittent fasting, CR mimetic drugs, or alternative diets (such as a ketogenic diet), may be more suitable. Intermittent fasting has been shown to enhance treatment with both chemotherapy and radiation therapy. CR and fasting elicit different responses in normal and cancer cells, and reduce certain side effects of cytotoxic therapy. Findings from preclinical studies of CR mimetic drugs and other dietary interventions, such as the ketogenic diet, are promising for improving the efficacy of anticancer therapies and reducing the side effects of cytotoxic treatments. Current and future clinical studies will inform on which cancers, and at which stage of the cancer process, CR, fasting, or CR mimetic regimens will prove most effective.

In this study, a new compound, 4-(N)-docosahexaenoyl 2′, 2′-difluorodeoxycytidine (DHA-dFdC), was synthesized and characterized. Its antitumor activity was evaluated in cell culture and in mouse models of pancreatic cancer. DHA-dFdC is a poorly soluble, pale yellow waxy solid, with a molecular mass of 573.3 Da and a melting point of about 96°C. The activation energy for the degradation of DHA-dFdC in an aqueous Tween 80–based solution is 12.86 kcal/mol, whereas its stability is significantly higher in the presence of vitamin E. NCI-60 DTP Human Tumor Cell Line Screening revealed that DHA-dFdC has potent and broad-spectrum antitumor activity, especially in leukemia, renal, and central nervous system cancer cell lines. In human and murine pancreatic cancer cell lines, the IC50 value of DHA-dFdC was up to 105-fold lower than that of dFdC. The elimination of DHA-dFdC in mouse plasma appeared to follow a biexponential model, with a terminal phase t1/2 of about 58 minutes. DHA-dFdC significantly extended the survival of genetically engineered mice that spontaneously develop pancreatic ductal adenocarcinoma. In nude mice with subcutaneously implanted human Panc-1 pancreatic tumors, the antitumor activity of DHA-dFdC was significantly stronger than the molar equivalent of dFdC alone, DHA alone, or the physical mixture of them (1:1, molar ratio). DHA-dFdC also significantly inhibited the growth of Panc-1 tumors orthotopically implanted in the pancreas of nude mice, whereas the molar equivalent dose of dFdC alone did not show any significant activity. DHA-dFdC is a promising compound for the potential treatment of cancers in organs such as the pancreas.

Obesity is associated with increased risk and poor prognosis of many types of cancers. Several obesity-related host factors involved in systemic metabolism can influence tumor initiation, progression, and/or response to therapy, and these have been implicated as key contributors to the complex effects of obesity on cancer incidence and outcomes. Such host factors include systemic metabolic regulators including insulin, insulin-like growth factor 1, adipokines, inflammation-related molecules, and steroid hormones, as well as the cellular and structural components of the tumor microenvironment, particularly adipose tissue. These secreted and structural host factors are extrinsic to, and interact with, the intrinsic metabolic characteristics of cancer cells to influence their growth and spread. This review will focus on the interplay of these tumor cell–intrinsic and extrinsic factors in the context of energy balance, with the objective of identifying new intervention targets for preventing obesity-associated cancer.

We tested the hypothesis that obesity reversal by calorie restriction (CR) versus treadmill exercise (EX) differentially modulates adipose gene expression using 48 female C57BL/6 mice administered a diet-induced obesity (DIO) regimen for 8 weeks, then randomized to receive for 8 weeks either: (1) a control (AIN-76A) diet, fed ad libitum (DIO control); (2) a 30% CR regimen; (3) a treadmill EX regimen (with AIN-76A diet fed ad libitum); or (4) continuation of the DIO diet. Relative to the DIO controls, both CR and EX reduced adiposity by 35–40% and serum leptin levels by 80%, but only CR increased adiponectin and insulin sensitivity. Gene expression microarray analysis of visceral white adipose tissue revealed 209 genes responsive to both CR and EX, relative to the DIO group. However, CR uniquely altered expression of an additional 496 genes, whereas only 20 were uniquely affected by EX. Of the genes distinctly responsive to CR, 17 related to carbohydrate metabolism and glucose transport, including glucose transporter (GLUT) 4. Chromatin immunoprecipitation assays of the Glut4 promoter revealed that, relative to the DIO controls, CR significantly increased histone 4 acetylation, suggesting epigenetic regulation may underlie some of the differential effects of CR versus EX on the adipose transcriptome.