Redox regulation of EGFR activation by thioredoxin reductase 3 drives resistance to EGFR inhibitors in triple-negative breast cancer.

Although 40-70% of TNBC cases overexpress EGFR, clinical responses to EGFR-targeted therapies have been minimal. This poor efficacy may result from intrinsic resistance mechanisms, inactive EGFR signaling, or reduced EGFR localization on the plasma membrane. To identify genetic determinants of EGFR inhibitor resistance, we performed a genome-wide CRISPR/Cas9 knockout screen in MDA-MB-231 cells. The screen revealed that loss of the redox-regulating enzyme Thioredoxin Reductase 3 (TXNRD3) sensitized TNBC cells to the EGFR inhibitor erlotinib. Functional validation showed that both siRNA-induced knockdown and pharmacological inhibition of TXNRD3 with the FDA-approved drug auranofin significantly enhanced the cytotoxic effects of EGFR inhibitors in EGFR-high TNBC cells. Mechanistically, TXNRD3 depletion or inhibition increased intracellular reactive oxygen species (ROS), leading to oxidation-dependent activation and phosphorylation of EGFR (Y1068) and subsequent activation of downstream signaling pathways in TNBC cells that otherwise lack active EGFR. The combined treatment of auranofin and EGFR inhibitors triggered GSDME-mediated pyroptosis in a ROS-dependent manner. Importantly, the combination of auranofin with erlotinib exhibited potent anti-tumor efficacy in vivo in both MDA-MB-231 xenograft and 4T1.2 syngeneic TNBC models. Collectively, our findings identify TXNRD3 as a redox-dependent regulator of EGFR activity and drug response in TNBC and demonstrate that auranofin-mediated TXNRD3 inhibition can re-activate EGFR signaling, thereby sensitizing TNBC tumors to EGFR-targeted therapy. This study provides a mechanistic rationale for repurposing auranofin in combination with EGFR inhibitors as a novel therapeutic strategy for EGFR-high TNBCs.