Mitochondrial Glutathione Import Enables Breast Cancer Metastasis via Integrated Stress Response Signaling

Abstract Cancer cells require substantial metabolic adaptations to metastasize to distant organs, but the metabolites essential for successful colonization remain poorly defined. In this study, we used a mitochondrial metabolomics approach to compare primary and metastatic breast cancer cells. This analysis revealed accumulation of mitochondrial glutathione (GSH) during lung metastasis, driven by elevated expression of SLC25A39, a mitochondrial GSH transporter. Loss of SLC25A39 impairs metastatic colonization in genetic screens, cell line models, and patient-derived xenografts, without affecting primary tumor growth. Mitochondrial GSH import is specifically required during early colonization and functions independently of its canonical antioxidant role. CRISPR activation screens identified ATF4, a stress-induced transcription factor, as a bypass mechanism that restores metastatic potential in SLC25A39-deficient cells. Mechanistically, SLC25A39 is required for optimal ATF4 activation during metastasis and under hypoxia, linking mitochondrial GSH availability to integrated stress response signaling. These findings identify mitochondrial GSH as a necessary and limiting metabolite for metastatic progression. Significance: Mitochondrial GSH import via SLC25A39 is essential for early metastatic colonization in breast cancer, linking metabolic adaptation to stress response signaling. Targeting this pathway may uncover a therapeutic vulnerability specific to metastasis without affecting primary tumor growth.