In Vitro Glutamine Adaptability in Murine Liver- but Not Lung-Metastasizing Triple-Negative Breast Cancer Cells

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-WntLung) or liver (metM-WntLiver). 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-WntLung cells exhibited greater glutamine uptake, catabolic enzyme expression, and flux of glutamine-derived carbon into the TCA cycle compared with metM-WntLiver cells. Despite this, metM-WntLiver cells were more viable and migratory than metM-WntLung 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-WntLiver, but not metM-WntLung cells, plausibly mitigating ammonia toxicity in higher glutamine conditions. In glutamine-deprived conditions, the metM-WntLung cells maintained higher glutamine oxidation, but the metM-WntLiver cells had higher total glutathione and GSH/GSSG ratios and enhanced resistance to oxidative stress, suggesting glutamate utilization toward glutathione synthesis. Additionally, metM-WntLiver 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-WntLung cells. PC silencing in metM-WntLiver 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-WntLiver cells, but not metM-WntLung 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.