Intratumoral Hypoxia Triggers Mitochondrial BHLHE40 ROS Sensing Pathway to Promote Radioresistance in Triple‐Negative Breast Cancer

Intratumoral hypoxia is a hallmark of triple-negative breast cancer (TNBC) and induces complex biological responses, including treatment resistance and mitochondrial production of reactive oxygen species (ROS). However, the direct mechanisms through which hypoxia-induced ROS are sensed and contribute to therapeutic resistance remain elusive. ROS can regulate the function of transcription factors through oxidation of cysteine thiol groups, but the compartmentalization limits their physical interaction with transcription factors. Here, BHLHE40, a transcription factor traditionally recognized for its nuclear function, is identified as a novel mitochondrial sensor of hypoxia-induced ROS. Mitochondrial BHLHE40 experiences ROS-dependent oxidation of cysteine thiol groups and forms disulfide-linked homodimers. In addition to post-translational modification that regulates BHLHE40 protein levels, hypoxia also increases BHLHE40 mRNA levels through hypoxia-inducible factors (HIFs)-dependent transcriptional activation. These dual mechanisms of modulating BHLHE40 ensure its rapid elevation during the early stage of hypoxia. Functionally, BHLHE40 plays a critical role in hypoxia-induced radioresistance through transcriptional activation of cellular antioxidant systems and inhibition of cytotoxic effects mediated by irradiation-generated ROS. This study reveals a previously unrecognized role of BHLHE40 in sensing and regulating ROS in response to hypoxia, and highlights its potential as a therapeutic target to overcome hypoxia-promoted radioresistance in TNBC.