ER-phagy drives resistance to mitochondria-targeted therapy in breast cancer.

Endoplasmic reticulum stress and ER-phagy are emerging regulators of cancer cell adaptation to metabolic and oxidative stress, yet their integration with mitochondrial dysfunction remains poorly understood. Here, we identify ER-phagy as a previously unrecognized adaptive response to ISOXUS, an isoxazole derivative of usnic acid with selective anticancer activity. ISOXUS, a mitochondrial respiratory complex II inhibitor, induces bioenergetic collapse, reactive oxygen species accumulation, and extensive ER-derived vacuolization. Using integrated transcriptomic and metabolomic analyses, we demonstrate that ISOXUS selectively triggers ER-phagy in mitochondria-dependent MCF-7 breast cancer cells, but not in more glycolytic triple-negative MDA-MB-231 cells, revealing a cell-type-specific stress adaptation program. ER-phagy induction is associated with upregulation of the ER-phagy receptor FAM134B and depends on ER stress signalling, as pharmacological ER stress inhibition suppresses this process. Multi-omics profiling uncovers coordinated repression of mitochondrial gene expression together with activation of ER-centered metabolic pathways, including amino acid metabolism, the tricarboxylic acid cycle, and one-carbon folate metabolism. Notably, we also identify UFMylation-related genes (CDK5RAP3, DDRGK1) as novel candidates involved in ER-phagy induced by ISOXUS. Moreover, mitochondrial inhibitors, rotenone and oligomycin, unexpectedly promote, while antioxidant a-tocopherol blocks ISOXUS-induced ER-phagy, and all compounds partially improve cell viability under ISOXUS treatment, implicating ROS-driven ER-phagy as a cytoprotective mechanism. Integrated analyses further reveal activation of the integrated stress response (ISR), dominated by the PERK-ATF4 axis, driving glutamine-dependent metabolic reprogramming and suppression of apoptosis-related pathways. The late-stage autophagy inhibition lowered the glutathione synthesis after ISOXUS treatment. Collectively, our findings uncover a previously unappreciated mitochondria-ER-ISR axis that governs metabolic adaptation to ISOXUS and identifies ER-phagy as a potential therapeutic vulnerability in breast cancer.