NUAK1 silencing enhances radiotherapy-induced ferroptosis in locally advanced rectal cancer by impairing Nrf2-driven transcription of GPX4

Radiotherapy resistance is a primary cause of treatment failure in locally advanced rectal cancer (LARC). Tumor cells can evade radiotherapy-induced ferroptosis by upregulating the antioxidant protein GPX4. NUAK1 is highly expressed in colorectal cancer and associated with poor prognosis, but its role in regulating radiosensitivity remains to be elucidated. Radiosensitivity was assessed in vitro and in vivo using CCK-8 assays, clonogenic survival assays, and a nude mouse xenograft model. Lipid peroxidation levels were quantified via flow cytometry with C11-BODIPY probe and malondialdehyde (MDA) assay. Ferroptosis-specific mitochondrial morphology was examined by transmission electron microscopy. Nuclear translocation of Nrf2 was detected by nucleocytoplasmic separation experiments and Western blot, while GPX4 promoter and antioxidant response element (ARE) transcriptional activities were measured using dual-luciferase reporter assays. The contribution of ferroptosis was assessed using cell death inhibitors. The Nrf2-specific inhibitor ML385 was utilized to investigate the functional dependency between NUAK1 and Nrf2. Immunohistochemistry (IHC) was used to analyze the correlation between NUAK1 protein expression and radiotherapy response in LARC patients. NUAK1 knockdown significantly enhanced radiotherapy-induced ferroptosis and radiosensitivity, as evidenced by increased lipid peroxidation, altered mitochondrial morphology, and reduced clonogenic survival. Mechanically, NUAK1 deficiency impaired Nrf2 nuclear translocation and transcriptional activity after radiation, leading to downregulation of GPX4 expression. The Nrf2 inhibitor ML385 completely abrogated NUAK1 overexpression-induced GPX4 upregulation and radioprotection. Clinical sample analysis revealed that high NUAK1 expression was correlated with radioresistance and poor tumor regression. This study is the first to elucidate the critical role of the NUAK1-Nrf2-GPX4 signaling axis in LARC radioresistance. These findings not only reveal a novel mechanism by which tumor cells evade radiotherapy-induced ferroptosis but also provide a potential biomarker and combination therapeutic target for overcoming clinical radioresistance.