Tetrandrine stabilizes NCOA4 to promote ferritinophagy-mediated ferroptosis and enhance antitumor immunity in NSCLC

Abstract Background Natural products are an important source of anticancer agents. Tetrandrine is a natural bisbenzylisoquinoline alkaloid, but how it regulates ferritinophagy and ferroptosis in non-small cell lung cancer (NSCLC) and affects antitumor immunity remains unclear. This study investigated its mechanism of action and potential for combination with anti-PD-1 therapy. Methods Antitumor activity was evaluated in NSCLC cell lines, patient-derived lung adenocarcinoma organoids, and mouse tumor models. Proteomic and transcriptomic analyses, pharmacological interventions, and gene overexpression and knockdown experiments were used to investigate ferritinophagy and ferroptosis. Target-binding, protein stability, and ubiquitination assays examined the regulation of nuclear receptor coactivator 4 (NCOA4). Co-immunoprecipitation, proximity ligation assays, structural prediction, and truncation and deletion analyses characterized the NCOA4–TAX1BP1 interaction. Tumor cell–PBMC co-cultures and LLC and CMT167 syngeneic tumor models were used to assess T-cell responses and combination treatment. Results Tetrandrine reduced NSCLC cell and organoid viability and increased intracellular Fe²⁺ accumulation and lipid peroxidation. Ferroptosis inhibitors partially attenuated these effects. Tetrandrine promoted lysosomal ferritin degradation and directly bound NCOA4, increasing its stability. HERC2 downregulation and reduced K48-linked NCOA4 ubiquitination contributed to this stabilization. Tetrandrine also enhanced the NCOA4–TAX1BP1 interaction. Deletion of NCOA4 residues 540–560 weakened this interaction and attenuated tetrandrine-induced iron accumulation, lipid peroxidation, and growth inhibition. In co-culture and animal models, tetrandrine increased markers of CD8⁺ T-cell activation or effector function, with some effects attenuated by iron chelation. Combined tetrandrine and anti-PD-1 treatment produced greater tumor suppression than either monotherapy in both syngeneic models. Conclusions Tetrandrine stabilizes NCOA4 and enhances its interaction with TAX1BP1, promoting ferritinophagy and subsequent ferroptosis. Its antitumor activity is accompanied by enhanced CD8⁺ T-cell effector responses in the tumor microenvironment and improved efficacy of anti-PD-1 treatment, supporting further investigation of tetrandrine for NSCLC therapy.