The NREP-GPX4 Axis Regulates Epithelial-Mesenchymal Transition and Enhances EGFR-TKI Resistance in Lung Adenocarcinoma Cells

Background: Lung adenocarcinoma (LUAD) is a leading cause of cancer-related deaths worldwide. Despite the advent of targeted therapies, such as epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), the development of resistance remains a significant challenge. Neuronal Regeneration-Related Protein (NREP) has been implicated in promoting drug resistance in various cancers, but its role in EGFR-TKI resistance in LUAD remains underexplored. Additionally, glutathione peroxidase 4 (GPX4), a key regulator of oxidative stress, may interact with NREP to influence drug resistance mechanisms. This study investigates the role of NREP and GPX4 in regulating EGFR-TKI resistance in LUAD cells. Methods: In this study, we used gefitinib-sensitive (PC9) and gefitinib-resistant (PC9/GR) LUAD cell lines to explore the combined effects of NREP and GPX4. Overexpression and knockdown of NREP and GPX4 were achieved using plasmid transfection and siRNA, respectively. Cell viability was assessed using CCK-8 assays. Apoptosis was analyzed by flow cytometry, and invasion was evaluated through Transwell assays. The expression of EMT markers (CD133, α-SMA) and GPX4 was determined by Western blotting and immunofluorescence. The effects of NREP and GPX4 on cell migration and EMT induction were assessed under gefitinib treatment conditions. Results: Overexpression of NREP and GPX4 in PC9 cells enhanced resistance to gefitinib, promoted EMT, and increased invasion, whereas knockdown of NREP and GPX4 in PC9/GR cells sensitized them to gefitinib and reduced invasion. Western blot and immunofluorescence analysis showed upregulation of EMT markers (CD133, α-SMA) and GPX4 in NREP overexpressing cells, while knockdown of NREP and GPX4 led to decreased expression of these markers. Additionally, NREP and GPX4 synergistically activated EMT and invasion in drug-resistant cells. Conclusions: The NREP-GPX4 axis plays a pivotal role in mediating EMT and promoting EGFR-TKI resistance in LUAD. Targeting this axis may offer a novel therapeutic strategy to overcome resistance to EGFR inhibitors and improve treatment outcomes in LUAD patients.