NRIP1 co-activates nuclear translocated FOXO3 to upregulate TFAM expression and promote radioresistance in non-small cell lung cancer.

Radioresistance remains a major obstacle in the treatment of non-small cell lung cancer (NSCLC). This study investigated the coordinated regulation of TFAM, FOXO3, and NRIP1 in NSCLC radioresistance. Radioresistant cell lines (A549-RR and H157-RR) were established to examine the effects of silencing these factors on cellular responses to radiation. In vivo, the impact of FOXO3 knockdown on tumor growth under irradiation was evaluated using A549-RR xenografts. Results show that TFAM expression was elevated in radioresistant cells, and its knockdown significantly restored radiosensitivity. ChIP-qPCR demonstrated direct FOXO3 binding to TFAM regulatory regions, establishing FOXO3 as an upstream transcriptional activator of TFAM. Silencing FOXO3 reduced TFAM expression and enhanced radiosensitivity, whereas LOM612, a FOXO nuclear relocator, promoted FOXO3 nuclear accumulation, upregulated TFAM, and reduced radiosensitivity. NRIP1 deficiency constrains FOXO3-dependent regulation of TFAM. Restoring NRIP1 selectively enhanced TFAM without affecting FOXO3 abundance, indicating its role as a coactivator. Co-immunoprecipitation confirmed FOXO3/NRIP1 interaction in NSCLC cells, with stronger interactions observed in radioresistant cells. Accordingly, NRIP1 silencing decreased TFAM levels and increased radiosensitivity. In vivo, FOXO3 knockdown markedly suppressed A549-RR tumor growth and improved radiotherapy response. Collectively, these findings indicate that nuclear accumulation of FOXO3 drives NSCLC radioresistance by transcriptionally upregulating TFAM, with NRIP1 enhancing this regulatory activity. Targeting FOXO3 may represent a promising strategy to enhance radiosensitivity in NSCLC.