Dorothy Teegarden

Background: Metabolic adaptability plays a critical role in supporting the growth and survival of metastatic breast cancer cells, and potentially supports site-specific metastasis. This study investigates the differential metabolism of glutamine and adaptability to varying glutamine levels of triple-negative breast cancer (TNBC) cells that metastasize to the lungs (metM-Wnt^Lung) or liver (metM-Wnt^Liver). Methods: The metastatic cell lines were exposed to varying in vitro glutamine concentrations (0.5, 2, and 4 mM). Cell viability, migration, ¹⁴C-glutamine uptake, mRNA abundance of metabolic enzymes, and ¹³C₅-glutamine cellular flux, were measured. Results: At an intermediate level of in vitro glutamine supplementation (2 mM), metM-Wnt^Lung cells exhibited greater glutamine uptake, catabolic enzyme expression, and flux of glutamine-derived carbon into the TCA cycle compared with metM-Wnt^Liver cells. Despite this, metM-Wnt^Liver cells were more viable and migratory than metM-Wnt^Lung cells under both higher (4 mM) and lower (0.5 mM) glutamine levels, suggesting metabolic adaptability. Exposure to ammonia upregulated ammonia-assimilating enzymes in metM-Wnt^Liver, but not metM-Wnt^Lung cells, plausibly mitigating ammonia toxicity in higher glutamine conditions. In glutamine-deprived conditions, the metM-Wnt^Lung cells maintained higher glutamine oxidation, but the metM-Wnt^Liver cells had higher total glutathione and GSH/GSSG ratios and enhanced resistance to oxidative stress, suggesting glutamate utilization toward glutathione synthesis. Additionally, metM-Wnt^Liver cells utilized glucose-derived carbons through pyruvate carboxylase (PC) to maintain TCA cycle activity, with elevated PC expression and M+3-labeled oxaloacetate enrichment to a greater extent than metM-Wnt^Lung cells. PC silencing in metM-Wnt^Liver cells enhanced cell viability under glutamine deprivation, which was reversed by inhibiting phosphoglycerate dehydrogenase (PHGDH, a key enzyme in de novo serine synthesis). We propose that PC activity compensates for low glutamine levels, including diverting glucose to adaptive pathways such as de novo serine synthesis. Conclusions: Overall, our findings demonstrate that metM-Wnt^Liver cells, but not metM-Wnt^Lung cells, exhibit glutamine-specific metabolic plasticity, characterized by the modulation of glutamine catabolism, enhanced ammonia metabolism and antioxidant defense, and support of glucose metabolism, which are associated with better cell survival and migration under glutamine excess and deprivation.

Background: Breast cancer is the most common cancer among women, and metastasis is the leading cause of mortality. It is still unknown how breast cancer cells metabolically adapt to successfully metastasize to different organs to survive adverse conditions, including varying nutrient availability. The purpose of this study is to elucidate the metabolic characteristics and glucose adaptation mechanisms of breast cancer cells that preferentially metastasize to the lungs or the liver. Methods: Using a Wnt-driven breast cancer model with preferential metastasis to lung (metM-Wnt ^Lung ) or liver (metM-Wnt ^Liver ), we measured ¹⁴ C-glucose uptake, ¹³ C ₆ -glucose metabolic flux, metabolic enzyme levels, and cell viability under normal (5 mM), high (25 mM), and low (1 or 0 mM) glucose conditions. Results: Under normal glucose conditions, metM-Wnt ^Lung cells were more glycolytic, exhibiting greater flux of ¹³ C ₆ -glucose-derived carbons into glycolytic intermediates, such as pyruvate and lactate. In contrast, metM-Wnt ^Liver cells favored oxidative phosphorylation, with higher levels of ¹³ C ₆ -glucose-derived carbons in tricarboxylic acid (TCA) cycle metabolites such as oxaloacetate indicative of higher pyruvate carboxylase (PC) activity. Exposure to high glucose reduced metM-Wnt ^Liver cell viability, with no effect on metM-Wnt ^Lung cells, suggesting better adaptability of metM-Wnt ^Lung cells to glucose excess. This was accompanied by increased PC activity and oxidative phosphorylation in metM-Wnt ^Lung cells, whereas metM-Wnt ^Liver cells shifted to a more glycolytic phenotype. Under glucose deprivation, metM-Wnt ^Lung cells were more viable than metM-Wnt ^Liver cells, suggesting that metM-Wnt ^Lung cells have better adaptability to glucose deprivation. Inhibiting phosphoenolpyruvate carboxykinase, a key enzyme in gluconeogenesis, reduced metM-Wnt ^Lung cell viability compared to metM-Wnt ^Liver cells. Similarly, inhibiting catabolism of glutamine, a gluconeogenic substrate, decreased metM-Wnt ^Lung cell viability compared to metM-Wnt ^Liver cells, indicating that metM-Wnt ^Lung cells rely on more on gluconeogenesis and glutamine metabolism under glucose deprivation. Conclusion: Our findings reveal that metM-Wnt ^Lung cells exhibit greater metabolic flexibility to glucose than metM-Wnt ^Liver cells by shifting from glycolysis to oxidative phosphorylation under high glucose conditions while utilizing gluconeogenesis and glutamine under glucose deprivation conditions.

Lipid accumulation is associated with breast cancer metastasis. However, the mechanisms underlying how breast cancer cells increase lipid stores and their functional role in disease progression remain incompletely understood. Herein we quantified changes in lipid metabolism and characterized cytoplasmic lipid droplets in metastatic versus non-metastatic breast cancer cells. ¹⁴C-labeled palmitate was used to determine differences in fatty acid (FA) uptake and oxidation. Despite similar levels of palmitate uptake, metastatic cells increase lipid accumulation and oxidation of endogenous FAs compared to non-metastatic cells. Isotope tracing also demonstrated that metastatic cells support increased de novo lipogenesis by converting higher levels of glutamine and glucose into the FA precursor, citrate. Consistent with this, metastatic cells displayed increased levels of fatty acid synthase (FASN) and de novo lipogenesis. Genetic depletion or pharmacologic inhibition of FASN reduced cell migration, survival in anoikis assays, and in vivo metastasis. Finally, global proteomic analysis indicated that proteins involved in proteasome function, mitotic cell cycle, and intracellular protein transport were reduced following FASN inhibition of metastatic cells. Overall, these studies demonstrate that breast cancer metastases accumulate FAs by increasingde novo lipogenesis, storing TAG as cytoplasmic lipid droplets, and catabolizing these stores to drive several FAO-dependent steps in metastasis.

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-Wnt^lung: 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-Wnt^lung, cells. AMP-activated protein kinase (AMPK) was activated in 4T1 with leptin treatment but decreased in metM-Wnt^lung. Leptin decreased fatty acid synthase (Fasn) and carnitine palmitoyltransferase 1a (Cpt1a) mRNA expression in 4T1 cells but increased their expression in metM-Wnt^lung cells. Fatty acid oxidation was not necessary for leptin-induced migration in either cell line. Leptin increased palmitate synthesis from glucose in metM-Wnt^lung, 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-Wnt^lung, but not 4T1 cells. Conclusion: Taken together, these results demonstrate that at physiological glucose concentrations, leptin increases migration of 4T1 and metM-Wnt^lung 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.

Introduction: Metabolic adaptability, including glucose metabolism, enables cells to survive multiple stressful environments. Glycogen may serve as a critical storage depot to provide a source of glucose during times of metabolic demand during the metastatic cascade; therefore, understanding glycogen metabolism is critical. Our goal was to determine mechanisms driving glycogen accumulation and its role in metastatic (MCF10CA1a) compared to nonmetastatic (MCF10A-ras) human breast cancer cells.Methodology: ¹³C₆-glucose flux analysis in combination with inhibitors of the gluconeogenic pathway via phosphoenolpyruvate carboxykinase (PCK), the anaplerotic enzyme pyruvate carboxylase (PC), and the rate-limiting enzyme of the pentose phosphate pathway (PPP) glucose 6-phosphate dehydrogenase (G6PD). To determine the requirement of glycogenolysis for migration or survival in extracellular matrix (ECM) detached conditions, siRNA inhibition of glycogenolysis (liver glycogen phosphorylase, PYGL) or glycophagy (lysosomal enzyme α-acid glucosidase, GAA) enzymes was utilized.Results: Metastatic MCF10CA1a cells had 20-fold greater glycogen levels compared to non-metastatic MCF10A-ras cells. Most glucose incorporated into glycogen of the MCF10CA1a cells was in the five ¹³C-containing glucose (M+5) instead of the expected M+6 glycogen-derived glucose moiety, which occurs through direct glucose conversion to glycogen. Furthermore, ¹³C₆-glucose in glycogen was quickly reduced (~50%) following removal of ¹³C-glucose. Incorporation of ¹³C₆-glucose into the M+5 glucose in the glycogen stores was reduced by inhibition of PCK, with additional contributions from flux through the PPP. Further, inhibition of PC reduced total glycogen content. However, PCK inhibition increased total unlabeled glucose accumulation into glycogen, suggesting an alternative pathway to glycogen accumulation. Inhibition of the rate-limiting steps in glycogenolysis (PYGL) or glycophagy (GAA) demonstrated that both enzymes are necessary to support MCF10CA1a, but not MCF10A-ras, cell migration. GAA inhibition, but not PYGL, reduced viability of MCF10CA1a cells, but not MCF10A-ras, in ECM detached conditions.Conclusion: Our results indicate that increased glycogen accumulation is primarily mediated through the gluconeogenesis pathway and that glycogen utilization is required for both migration and ECM detached survival of metastatic MCF10CA1a cells. These results suggest that glycogen metabolism may play an important role in the progression of breast cancer metastasis.

Background/Objectives: Excess adiposity is associated with a higher risk of breast cancer metastasis and mortality. Evidence suggests that dietary vitamin D inhibits breast cancer metastasis. However, the mechanistic link between vitamin D’s regulation of adipocyte metabolism and metastasis has not been previously investigated. Therefore, the purpose of these experiments was to examine the effect of the active form of vitamin D, 1α,25-dihydroxyvitamin D (1,25(OH)₂D), on adipocyte release of bioactive compounds and whether the impact on adipocytes leads to inhibition of breast cancer cell migration, an important step of metastasis. Methods: Differentiated 3T3-L1 adipocytes were treated with 1,25(OH)₂D for two days, followed by either harvesting the adipocytes or collecting adipocyte-conditioned media without 1,25(OH)₂D. A transwell migration assay was conducted with vehicle- or 1,25(OH)₂D-conditioned media. In order to explore the mechanism underlying effects on breast cancer metastatic capability, the mRNA expression of leptin, adiponectin, insulin-like growth factor (IGF-1), interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1) was measured in adipocytes following either vehicle or 1,25(OH)₂D treatment. Results: Conditioned media from 1,25(OH)₂D-treated adipocytes inhibited the migration of metastatic MDA-MB-231 breast cancer cells compared to conditioned media from vehicle-treated adipocytes. Treatment of adipocytes with 1,25(OH)₂D decreased mRNA expression of leptin, adiponectin, IGF-1, IL-6, and MCP-1. Consistent with mRNA expression, concentrations of leptin, adiponectin, IGF-1, and IL-6 in adipocyte-conditioned media were decreased with 1,25(OH)₂D treatment, although MCP-1 remained unchanged. Conclusions: In summary, these results suggest that 1,25(OH)₂D alters adipocyte secretions to prevent breast cancer metastasis.

Pemigatinib blocks FGF2-induced signaling and 3D outgrowth of D2.OR cells via inhibition of FGFR1.

These are the figure legends for Supplementary figures 1 and 2.

Paracrine FGF2 can induce the outgrowth of D2.OR cells in 3D culture.

Pemigatinib blocks FGF2-induced signaling and 3D outgrowth of D2.OR cells via inhibition of FGFR1.