Glutamine and Cystine Dependence in Triple-Negative Breast Cancer: Metabolic Interplay and Therapeutic Implications

Abstract

Triple-negative breast cancer (TNBC) accounts for 10–20% of all breast cancers and is characterised by aggressive growth, high recurrence rates, and limited treatment options due to the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Despite advancements in breast cancer research, current therapies for TNBC remain inadequate, emphasising the urgent need for novel treatment strategies. One promising approach is targeting TNBC’s altered metabolic state, which differs significantly from that of luminal breast cancer—a subtype expressing one or more hormone receptors (ER and/or PR) and generally exhibiting a less aggressive phenotype. Notably, TNBC cells display a distinct metabolic profile, particularly a strong dependence on glutamine and cystine. This study investigates the metabolic interplay between glutamine and cystine dependence in TNBC cells compared to luminal breast cancer cells. To model high glutamine reliance in TNBC, Hs578Ts(i)8, an invasive subclone of the parental Hs578T cell line was used. Compared to Hs578T cells, Hs578Ts(i)8 cells exhibited increased proliferation, migration, and invasion, along with a heightened oxygen consumption rate primarily driven by glutamine anaplerosis, which is crucial for energy production. Firstly, TNBC cells’ dependence on extracellular glutamine and cystine was confirmed; a correlation between SLC7A11 (the cystine-glutamate antiporter) expression and glutamine reliance was also identified. Notably, Hs578Ts(i)8 cells exhibited the highest SLC7A11 expression among TNBC cell lines. Public database analysis (HMS LINCS) further identified SUM159 cells, another high-SLC7A11-expressing TNBC cell line, which also demonstrated strong glutamine dependence associated with increased glutamine-driven oxygen consumption. U13C5-glutamine labelling and Gas Chromatography-Mass Spectrometry (GC-MS) confirmed that TNBC cells preferentially utilize glutamine to fuel the tricarboxylic acid (TCA) cycle. Supplementation with membrane-permeable TCA cycle intermediates, α-ketoglutarate and succinate, rescued TNBC cells from glutamine deprivation, indicating that their reliance on extracellular glutamine is primarily due to its role in TCA cycle metabolism. Interestingly, both cystine restriction and SLC7A11 inhibition rescued TNBC cells from glutamine deprivation, whereas cystine deprivation alone significantly reduced TNBC cell survival. Further investigation revealed distinct rescue mechanisms between glutamine and cystine deprivation. Under glutamine deprivation, cystine depletion restored intracellular glutamate and TCA cycle intermediates, thereby maintaining energy production. Conversely, under cystine deprivation, glutamine-starved TNBC cells were rescued by the glutamate dehydrogenase (GLUD1) inhibitor R162. This effect was mediated by reduced reactive oxygen species (ROS) accumulation, suppression of glutaminolysis-induced oxidative stress, and activation of mitophagy, ultimately lowering lipid peroxidation levels. In conclusion, this study identifies extracellular glutamine and cystine reliance as a key metabolic feature of TNBC. It further demonstrates that TNBC cells can adapt to single-nutrient deprivation through compensatory metabolic mechanisms. These findings deepen our understanding of TNBC metabolism and provide insights into potential metabolic vulnerabilities that could be exploited for targeted therapy.