CYB5R1 drives stemness and malignant progression via STAM2/STAT3 in chemotherapy-enriched triple-negative breast cancer

Triple-negative breast cancer (TNBC) frequently develops chemoresistance, but the mechanisms supporting tumor persistence remain incompletely understood. We integrated single-cell transcriptomic data with clinical cohort analyses to identify genes associated with chemotherapy exposure and prognosis. Malignant epithelial populations enriched after chemotherapy occupied terminal branches of the inferred pseudotime trajectory and exhibited higher stemness scores. CYB5R1 was enriched in these populations and associated with poor survival. Functional experiments showed that CYB5R1 promoted proliferation, migration, invasion, epithelial–mesenchymal transition (EMT), and stem cell-like properties in TNBC cells. CYB5R1 depletion suppressed these phenotypes and reduced STAM2 expression and STAT3 phosphorylation. STAM2 re-expression partially restored STAT3 phosphorylation and malignant phenotypes, supporting its role as a downstream mediator of CYB5R1. CYB5R1 depletion also increased cellular sensitivity to gemcitabine and selumetinib. Combined treatment produced greater antitumor effects than either agent alone in vitro and in vivo. These findings support a role for CYB5R1 in TNBC progression and EMT- and stemness-associated phenotypes, mediated partly through STAM2/STAT3 signaling. They also support further evaluation of gemcitabine plus selumetinib in TNBC and investigation of CYB5R1 as a candidate biomarker of prognosis and drug sensitivity.