Triple-negative breast cancer (TNBC) is an aggressive cancer subtype with limited treatment options. There is an emerging interest in blocking antioxidants for cancer therapy, but how antioxidants promote cancer growth is unclear. Glutathione (GSH) is the most abundant antioxidant in the body and our previous work has shown that GSH promotes tumorigenesis of triple-negative breast cancers (TNBC). It is generally assumed that GSH acts intracellularly as an antioxidant in cancer cells. However, our preliminary studies show that blocking intracellular GSH synthesis does not impede TNBC growth. These surprising results suggest an alternative mechanism where extracellular GSH supports breast tumor growth. The overarching goal of this proposal is to determine how extracellular GSH promotes tumor growth. It is known that extracellular GSH is present in plasma but cells cannot import GSH. Instead, GSH is metabolized by gamma-glutamyl transferase (GGT1) to produce a glutamyl- dipeptide and cysteinylglycine, which yields cystine and glycine. Indeed, we find that ablation of GSH synthesis in vivo not only lowers circulating GSH but also reduces the levels of cysteinylglycine, cysteine, and glycine in tissues. Further, we show that supplementation with GSH and cysteinylglycine can rescue TNBC growth upon cystine depletion in GGT1-dependent and -independent manners, respectively. Together, these preliminary data suggest an alternative mechanism where GSH functions as a circulating source of metabolites rather than as a direct antioxidant. In this proposal, we describe experiments that will test the hypothesis that the catabolism of extracellular GSH by tumor GGT1 supports TNBC growth. In Aim 1, we will elucidate the impact of extracellular GSH on TNBC growth. In Aim 2, we identify the reliance of TNBC on GGT1-mediated GSH catabolism. In Aim 3, we will determine the mechanisms by which cysteinylglycine supplies cysteine for TNBC growth. Our research will challenge the paradigm of antioxidant function in cancer by describing a novel mechanism of GSH function as a circulating source of amino acids. Further, these studies have the potential to reveal a completely new set of unrealized targets and therapeutic strategies for TNBC.