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

The NREP-GPX4 Axis Regulates Epithelial-Mesenchymal Transition and Enhances EGFR-TKI Resistance in Lung Adenocarcinoma Cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The NREP-GPX4 Axis Regulates Epithelial-Mesenchymal Transition and Enhances EGFR-TKI Resistance in Lung Adenocarcinoma Cells Wen-xiang Wei, Qiu-li Li, Wei Yang, Ming-jing Cai, Wen-jing Dai, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7839640/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract 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. Biological sciences/Cancer Biological sciences/Cell biology Biological sciences/Molecular biology Health sciences/Oncology Lung adenocarcinoma EGFR-TKI resistance NREP GPX4 epithelial-mesenchymal transition (EMT) drug resistance invasion gefitinib. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Lung adenocarcinoma (LUAD) is the most prevalent form of non-small cell lung cancer (NSCLC), and despite advancements in targeted therapies such as epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), acquired resistance remains a major challenge. EGFR-TKIs, which target mutations in the EGFR gene, initially offer substantial clinical benefits for patients with EGFR mutations 1 , 2 . However, the emergence of resistance, particularly through secondary mutations like T790M, presents a significant barrier to long-term efficacy 3 , 4 . In addition to genetic mutations, cellular processes such as epithelial-to-mesenchymal transition (EMT) have been implicated in mediating resistance to EGFR-TKIs 5 . EMT enables tumor cells to acquire migratory and invasive properties, facilitating metastasis and reducing sensitivity to targeted therapies 6 . As such, understanding the molecular mechanisms underlying EMT and EGFR-TKI resistance is critical for the development of effective therapeutic strategies. Neuronal Regeneration-Related Protein (NREP), also known as P311, is a highly conserved 8 kDa intracellular multifunctional protein 7 , 8 . In cancer research, it has been extensively documented to participate in cellular processes including tumor cell migration, invasion, and EMT 9 , 10 . NREP has been found to be upregulated in several cancers, where it is associated with poor prognosis and resistance to treatment 11 , 12 . In LUAD, NREP expression has been linked to tumor progression and metastasis, suggesting its potential role in mediating resistance to EGFR-TKIs 13 . Additionally, NREP’s interaction with other proteins such as GPX4, a key regulator of oxidative stress, may contribute to resistance by modulating survival pathways and influencing the cellular response to EGFR inhibition. Recent studies have highlighted the role of GPX4 in promoting EGFR-TKI resistance by maintaining redox balance and preventing ferroptosis, a regulated form of cell death 14 , 15 . Overexpression of GPX4 has been correlated with both intrinsic and acquired resistance to EGFR-TKIs in LUAD 16 . Furthermore, the NREP-GPX4 axis has been shown to synergistically enhance cancer cell survival under therapeutic stress, contributing to drug resistance 17 . The cooperative role of NREP and GPX4 in regulating cell invasion, migration, and resistance highlights the importance of these molecules in LUAD progression. This study aims to further elucidate the molecular mechanisms by which the NREP-GPX4 axis influences EMT, invasion, and EGFR-TKI resistance, providing potential targets for overcoming therapeutic resistance in LUAD. Methods Bioinformatic Analysis The GEPIA database( http://gepia.cancer-pku.cn/ ) was utilized to analyze the differential expression of NREP between various cancer tissues and normal tissues. Based on the publicly available GEO database, datasets GSE30219, GSE31210, and GSE68465 were selected to examine the differential expression of NREP in lung cancer tissues. Details of the datasets are provided in Table 1 . The optimal cutoff value was determined using the "surv_cutpoint" package, and Kaplan-Meier survival analysis was performed with the "survival" package to assess the impact of different NREP expression levels on overall survival(OS) and progression-free survival (PFS). Table 1 GEO Dataset Information. GSE30219 GSE68465 GSE31210 Platform GPL570 GPL96 GPL570 Species homo sapiens homo sapiens homo sapiens Tissue Lungs Lungs Lungs Normal group 15 19 20 Tumor group 85 443 226 Cell Lines and Culture Conditions This study utilized PC9 (EGFR-sensitive) and PC9/GR (EGFR-resistant) cell lines to investigate the roles of NREP and GPX4 in cell invasion and resistance to gefitinib, an EGFR-TKI. PC9 cells were obtained from the cell bank of the Procell, and PC9/GR cells were generated by long-term exposure to gefitinib. Cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, maintained in a humidified incubator at 37°C with 5% CO₂. Overexpression and Knockdown of NREP and GPX4 To study the effects of NREP and GPX4 on gefitinib resistance, overexpression and knockdown models of NREP and GPX4 were generated in both PC9 and PC9/GR cells. Overexpression of NREP and GPX4 was achieved by viral transfection, while gene knockdown was performed using siRNA targeting NREP and GPX4. Overexpression and knockdown efficiency were confirmed by Western blotting and qPCR. Cell Invasion and Migration Assays Cell invasion was assessed using Transwell invasion assays. Cells were seeded in the upper chamber of the Transwell inserts pre-coated with Matrigel. After 24 hours, invading cells on the lower surface were stained and counted. Scratch assays (wound healing assays) were performed to evaluate cell migration. Cells were seeded to form a monolayer, and a scratch was made in the middle of the well. Migration was assessed by imaging the wound area at various time points after treatment. Protein Expression and EMT Marker Analysis Western blotting was used to evaluate the expression of NREP, GPX4, and EMT markers such as CD133 and α-SMA. For protein extraction, cells were lysed in RIPA buffer, and protein concentrations were determined using a BCA protein assay. The samples were then analyzed by SDS-PAGE, transferred to PVDF membranes, and incubated with specific primary antibodies. Immunofluorescence staining was used to visualize CD133 and α-SMA expression in cells under various conditions. Cells were fixed, incubated with primary antibodies, and stained with appropriate secondary antibodies. Fluorescent images were captured using a fluorescence microscope. Cell Viability and Apoptosis Assays Cell viability was assessed using the CCK-8 assay following treatment with various concentrations of gefitinib (IC50-IC60). Cells were plated in 96-well plates and treated with gefitinib for 24 hours. Apoptosis was analyzed by Annexin V-FITC/PI staining followed by flow cytometry. The apoptosis rate was quantified by analyzing the percentage of Annexin V-positive cells. Statistical Analysis All data were analyzed using GraphPad Prism 9.0 (GraphPad Software, USA). Statistical comparisons between multiple groups were performed using one-way ANOVA, followed by Tukey’s multiple comparisons test. Data are presented as mean ± standard deviation (SD). Kaplan-Meier survival analysis was used to analyze survival data, and statistical significance statistical significance is indicated as follows: P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), P < 0.0001 (****), and ns = not significant., and ns = no significant difference. Results Elevated Expression of NREP in Lung Adenocarcinoma and Its Association with Poor Prognosis To investigate the role of NREP in lung cancer, its expression across multiple cancer types was first assessed using the GEPIA2 database. The results revealed that NREP mRNA is aberrantly upregulated in a variety of malignancies, including lung adenocarcinoma (LUAD) (Fig. 1 A). To further evaluate its expression pattern within lung cancer subtypes, the GSE30219 dataset was analyzed, as detailed in Table 1 . Violin plots demonstrated that NREP expression was significantly higher in LUAD tissues compared to adjacent normal tissues and showed differential expression among various histological subtypes (Fig. 1 B). Consistently, box plots from independent datasets confirmed that NREP mRNA expression was markedly elevated in tumor tissues relative to matched normal counterparts (Fig. 1 C–D). To assess the clinical relevance of NREP expression, LUAD patients in the GSE30219 cohort were stratified into high and low expression groups based on the optimal cut-off value (7.472). Kaplan–Meier survival analyses indicated that high NREP expression was significantly associated with reduced progression-free survival (PFS) and overall survival (OS) (Fig. 1 E–F). These findings suggest that NREP may serve not only as a potential biomarker for lung adenocarcinoma but also as a prognostic indicator of poor clinical outcomes. Expression of NREP in Gefitinib-Sensitive and -Resistant Lung Adenocarcinoma Cells The expression of NREP in gefitinib-sensitive (PC9) and gefitinib-resistant (PC9/GR) lung adenocarcinoma cells was assessed under various experimental conditions. The relative cell viability of both cell lines was measured using the CCK-8 assay following 24-hour treatment with different concentrations of gefitinib. The results showed a significant difference in cell survival between the PC9 and PC9/GR cell lines, with the resistant PC9/GR cells exhibiting higher viability compared to the sensitive PC9 cells (Fig. 2 A). Apoptosis rates were also evaluated by Annexin V-FITC/PI staining following gefitinib treatment (IC50-IC60 concentrations). As shown in Fig. 2 B and quantified in Fig. 2 C, a higher apoptosis rate was observed in the sensitive PC9 cells compared to the resistant PC9/GR cells, further confirming the differential response to gefitinib treatment. In addition, the expression of NREP mRNA and protein was measured by qPCR and Western blot, respectively, after gefitinib treatment (IC50-IC60, 8 hours). Both mRNA and protein levels of NREP were significantly higher in the resistant PC9/GR cells compared to the sensitive PC9 cells, suggesting that NREP may be involved in the gefitinib resistance mechanism (Figs. 2 D–E). These findings highlight the potential role of NREP in regulating the sensitivity of lung adenocarcinoma cells to gefitinib and its potential as a therapeutic target in overcoming drug resistance. NREP Modulates Gefitinib Sensitivity in Lung Adenocarcinoma Cells The role of NREP in regulating gefitinib sensitivity in lung adenocarcinoma cells was evaluated in PC9 (gefitinib-sensitive) and PC9/GR (gefitinib-resistant) cell lines. Western blot analysis showed that overexpression or knockdown of NREP resulted in corresponding changes in NREP protein levels in both cell lines, with PC9/GR cells showing higher baseline NREP expression compared to PC9 cells (Fig. 3 A). The CCK-8 assay revealed that PC9/GR cells with NREP knockdown exhibited significantly reduced cell viability after gefitinib treatment, whereas NREP overexpression in PC9 cells conferred increased resistance to gefitinib, compared to the negative control (Fig. 3 B). Annexin V-FITC/PI staining showed that gefitinib treatment induced higher apoptosis in PC9 cells compared to PC9/GR cells, and apoptosis was further enhanced in PC9/GR cells with NREP knockdown (Figs. 3 C–E). In contrast, NREP overexpression in PC9 cells reduced gefitinib-induced apoptosis (Figs. 3 D–F). Furthermore, the impact of NREP overexpression and knockdown on NREP expression following gefitinib treatment was assessed by qPCR and western blot. Both PC9 and PC9/GR cells showed significant alterations in NREP expression after treatment, suggesting that NREP modulates gefitinib resistance by regulating apoptosis pathways (Figs. 3 G–H). These results indicate that NREP plays a crucial role in modulating the gefitinib sensitivity of lung adenocarcinoma cells, potentially serving as a therapeutic target for overcoming drug resistance. NREP Induces EMT and Promotes Resistance to EGFR-TKIs in Lung Adenocarcinoma Cells To investigate the role of NREP in regulating gefitinib resistance, morphological differences between PC9 (gefitinib-sensitive) and PC9/GR (gefitinib-resistant) cells were observed after 24-hour treatment with gefitinib (IC50-IC60). As shown in Fig. 4 A, PC9/GR cells exhibited a more mesenchymal-like morphology compared to PC9 cells, suggesting a potential role of NREP in inducing epithelial-mesenchymal transition (EMT). Further, the expression of EMT-related proteins was assessed in PC9 cells overexpressing NREP and PC9/GR cells with NREP knockdown. The results in Figs. 4 B–C indicated that NREP overexpression in PC9 cells and NREP knockdown in PC9/GR cells significantly altered the levels of key EMT markers. Immunofluorescence analysis showed increased fluorescence intensity for CD133 and α-SMA in PC9 cells overexpressing NREP and reduced expression in PC9/GR cells with NREP knockdown following gefitinib treatment (Figs. 4 D–E). These findings suggest that NREP induces EMT, which contributes to the acquisition of resistance to EGFR-TKIs in lung adenocarcinoma cells. NREP Enhances Gefitinib-Induced Expression of EMT Markers in Lung Adenocarcinoma Cells To examine the effect of NREP on gefitinib-induced EMT, PC9 cells overexpressing NREP and PC9/GR cells with NREP knockdown were treated with gefitinib (IC50-IC60) for 8 hours. Immunofluorescence analysis in Fig. 5 A showed that NREP overexpression in PC9 cells led to an increased fluorescence intensity of the EMT markers CD133 (green) and α-SMA (red) following gefitinib treatment. In contrast, PC9/GR cells with NREP knockdown exhibited significantly reduced fluorescence intensity for CD133 and α-SMA (Fig. 5 B). These findings suggest that NREP enhances gefitinib-induced EMT marker expression, which may contribute to the development of resistance to EGFR-TKIs in lung adenocarcinoma cells. GPX4 Enhances Gefitinib Resistance in Lung Adenocarcinoma Cells The role of GPX4 in regulating gefitinib resistance was examined in PC9 and PC9/GR cells. Western blot analysis revealed that the expression of GPX4 was significantly altered in both cell lines under different treatment conditions. Specifically, PC9 cells overexpressing GPX4 (Fig. 6 C) showed increased GPX4 expression, while PC9/GR cells with GPX4 knockdown exhibited reduced GPX4 expression (Fig. 6 D). Additionally, the expression of NREP and GPX4 was also quantified, with significant changes observed upon treatment (Figs. 6 A-B). In functional assays, PC9 cells overexpressing GPX4 exhibited accelerated migration in the scratch assay after gefitinib treatment (IC20, 24 hours) (Fig. 6 E), whereas PC9/GR cells with GPX4 knockdown showed impaired migration (Fig. 6 F). Quantitative analysis of migration rates further supported these observations, with PC9 cells overexpressing GPX4 displaying enhanced migration, while PC9/GR cells with GPX4 knockdown showed significantly reduced migration (Figs. 6 G-H). These findings suggest that GPX4 plays a key role in promoting migration and resistance to gefitinib in lung adenocarcinoma cells, potentially contributing to the development of EGFR-TKI resistance. NREP-GPX4 Mediates EMT Regulation and Enhances Invasion in PC9 Cells To investigate the combined effect of NREP and GPX4 on lung adenocarcinoma cell invasion, Transwell assays showed that overexpression of both NREP and GPX4 significantly enhanced the invasion of PC9 cells, and this effect was counteracted by gefitinib treatment (Fig. 7 A). Western blot analysis revealed that GPX4, CD133, and α-SMA protein levels were upregulated in PC9 cells overexpressing NREP and GPX4, and these markers were further modulated by gefitinib treatment (Fig. 7 B). Immunofluorescence staining confirmed the increased expression of CD133 and α-SMA in these cells, indicating EMT activation (Fig. 7 C). These results suggest that the combined overexpression of NREP and GPX4 promotes invasion and EMT, contributing to gefitinib resistance in PC9 cells. NREP-GPX4 Synergistically Activates EMT and Enhances Invasion in PC9/GR Drug-Resistant Cells To assess the role of NREP and GPX4 in PC9/GR drug-resistant cells, Transwell invasion assays revealed that knockdown of NREP and GPX4 significantly reduced the invasive potential of these cells. Additionally, gefitinib treatment further enhanced this effect, indicating that the combined knockdown of NREP and GPX4 sensitizes the cells to gefitinib (Fig. 8 A). Immunofluorescence analysis confirmed these findings, showing decreased expression of the EMT markers CD133 and α-SMA in PC9/GR cells upon knockdown of NREP and GPX4, particularly after gefitinib treatment (Fig. 8 B). These results suggest that the NREP-GPX4 axis contributes to EMT activation and invasion in gefitinib-resistant cells, and its modulation may reverse drug resistance in PC9/GR cells. Discussion In this study, we explored the role of NREP and GPX4 in mediating resistance to EGFR-TKIs in lung adenocarcinoma (LUAD) cells. Our results demonstrate that overexpression of NREP and GPX4 in PC9 (gefitinib-sensitive) cells significantly enhances cell invasion and resistance to gefitinib, as evidenced by increased expression of EMT markers and enhanced migration capabilities. In contrast, knockdown of NREP and GPX4 in PC9/GR (gefitinib-resistant) cells sensitized them to gefitinib treatment, reducing invasion and modulating the expression of key EMT markers. The NREP-GPX4 axis appears to play a crucial role in promoting EMT and survival in LUAD cells, contributing to both intrinsic and acquired resistance to EGFR-TKIs. These findings highlight the potential of targeting the NREP-GPX4 pathway as a therapeutic strategy to overcome drug resistance and improve treatment outcomes in LUAD patients. Our findings are consistent with previous studies that have explored the role of NREP and GPX4 in promoting drug resistance in various cancers, particularly in lung adenocarcinoma. Similar to Liu et al. (2021) 18 , who demonstrated that NREP overexpression enhances migration and invasion in gastric cancer through EMT activation, we found that NREP overexpression in LUAD cells significantly increased cell invasion and resistance to gefitinib. This supports the idea that NREP plays a crucial role in facilitating cancer cell migration and resistance across different cancer types. In line with the work by Zhang et al. (2022) 17 , who identified GPX4 as a key mediator of resistance to EGFR-TKIs in LUAD, our study further confirmed that GPX4 overexpression contributes to the maintenance of drug resistance by modulating redox homeostasis. However, while Zhang et al. (2022) focused on the role of GPX4 in ferroptosis and oxidative stress, our study provides new insight into how NREP and GPX4 cooperate to enhance resistance by regulating both EMT and apoptosis resistance. This synergistic interaction between NREP and GPX4 was not previously explored, making our findings an important extension of existing knowledge. Additionally, our results differ from the study by Pan et al. (2022) 16 , which demonstrated that cholesterol accumulation mediates resistance to EGFR-TKIs in NSCLC through a different signaling axis. In contrast, our study emphasizes the combined effect of NREP and GPX4 in regulating EMT and invasion, indicating that these two proteins may contribute to resistance through complementary mechanisms rather than through cholesterol-related pathways alone. This highlights the complex nature of resistance mechanisms in LUAD and suggests that targeting both NREP and GPX4 could be a promising strategy for overcoming drug resistance. A unique aspect of this study lies in the combination of multiple experimental approaches to assess the role of NREP and GPX4 in both drug resistance and cell migration in LUAD. By employing both overexpression and knockdown models of NREP and GPX4 in PC9 and PC9/GR cells, we were able to directly evaluate the impact of these proteins on gefitinib resistance and invasion. The use of both Western blotting and immunofluorescence to assess the expression of EMT markers (CD133 and α-SMA) was particularly effective in confirming the involvement of NREP and GPX4 in regulating EMT. This approach builds on the work of Song et al. (2022) 13 , who used similar methods to evaluate drug resistance pathways in LUAD, but with a specific focus on methyltransferases. Furthermore, our application of the Transwell invasion assay to assess cell migration under gefitinib treatment provided strong evidence for the role of NREP and GPX4 in modulating invasive potential, a finding that supports the work of Ruan et al. (2024) 19 in breast cancer, where NREP’s role in promoting invasion was also demonstrated. Another strength of our study is the incorporation of apoptosis assays using Annexin V-FITC/PI staining, which allowed us to demonstrate the protective effect of NREP and GPX4 overexpression against gefitinib-induced apoptosis. This methodology is consistent with that used in previous studies 16 , but here it highlights the combination of NREP and GPX4 as key modulators of cellular survival in response to EGFR-TKI treatment. These methodologies and their findings contribute to a more comprehensive understanding of the molecular mechanisms underlying drug resistance in LUAD. While our study provides valuable insights into the role of the NREP-GPX4 axis in EGFR-TKI resistance in LUAD, there are several limitations that should be addressed in future research. First, the use of cell line models, although a powerful tool for understanding resistance mechanisms, does not fully replicate the complexity of tumor microenvironments and the heterogeneity found in patient-derived samples. Additionally, while we demonstrated the synergistic role of NREP and GPX4 in promoting invasion and resistance, the precise molecular mechanisms underlying their interaction remain unclear. Moreover, we did not evaluate the in vivo efficacy of targeting NREP and GPX4 in LUAD, which limits the translational relevance of our findings. Future studies should aim to validate these findings in patient-derived xenografts (PDX) or organoid models, which more closely resemble human tumors and their response to treatment 20 . Furthermore, we did not explore the potential role of other resistance mechanisms, such as alterations in DNA repair or immune evasion, which may also contribute to EGFR-TKI resistance 21 . Future studies should focus on several key aspects to further investigate and overcome EGFR-TKI resistance in LUAD. In vivo validation using patient-derived xenografts (PDX) or organoid models would be crucial for confirming the role of NREP and GPX4 in a clinically relevant context, as these models more closely mimic the human tumor microenvironment. Furthermore, a deeper exploration of the downstream signaling pathways through which NREP and GPX4 interact is needed to elucidate their precise roles in drug resistance. Investigating potential cross-talk with other resistance pathways, including those involved in oxidative stress and apoptosis, could offer new insights into the molecular mechanisms of resistance 22 , 23 . Additionally, combination therapies targeting both NREP and GPX4, along with EGFR-TKIs, should be explored to improve therapeutic efficacy and overcome resistance in LUAD patients. Combining targeted therapies may provide a more comprehensive approach to tackling drug resistance 13 . Lastly, considering the role of the immune system in modulating resistance mechanisms could provide valuable information for overcoming EGFR-TKI resistance. Immune evasion plays a significant role in cancer progression and drug resistance, and future studies should explore the potential benefits of combining immune-modulating therapies with EGFR-TKIs 9 . These efforts could lead to the development of more effective treatment strategies and improved outcomes for LUAD patients facing EGFR-TKI resistance. Conclusions In conclusion, our study highlights the critical role of the NREP-GPX4 axis in mediating resistance to EGFR-TKIs in lung adenocarcinoma, offering potential new therapeutic targets to overcome this resistance. The complex interplay between NREP and GPX4 in regulating epithelial-to-mesenchymal transition (EMT), invasion, and cell survival provides valuable insights into the mechanisms driving both intrinsic and acquired resistance to EGFR inhibitors. While the in vitro data presented here lay the groundwork for targeting this pathway, future studies are needed to validate these findings in more clinically relevant models, such as patient-derived xenografts and organoid systems, to better understand the translation of these results into therapeutic strategies. Additionally, exploring the downstream signaling pathways of NREP and GPX4, as well as their potential interactions with other key resistance mechanisms, could provide a deeper understanding of resistance biology. Further investigation into combination therapies targeting both NREP and GPX4, along with EGFR-TKIs, may enhance therapeutic efficacy and provide new avenues for treatment. Overall, the NREP-GPX4 axis presents a promising target for overcoming EGFR-TKI resistance in LUAD, and future research should continue to refine and expand these therapeutic possibilities. Declarations Ethics Approval Statement The data utilized in this study were obtained from publicly available databases. As the data are de-identified and publicly accessible, this study did not require additional ethical approval or patient consent. Consent for publication All authors declare no competing interests. Availability of data and materials The public datasets used in this study are openly accessible from the GEO database (https://www.ncbi.nlm.nih.gov/geo/). The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Authors' contributions WX.W., QL.L. and H.Y. designed the experiment and write the draft, T.Z. and LJ.M. provided technical guidance in experiments, MJ.C., WJ.D., HY.W. and YJ.Z. accomplished data collection and analysis, W.Y., and LJ.M. provided methodology and validation. All authors contributed to the design and interpretation of the study and further drafts. Funding This research is funded by the Provincial Natural Science Foundation of Sichuan, No. 2017SZ0066 and 2024NSFSC1292. The Scientific Research Project of General Hospital of Western Theater Command, No. 2024-YGJC-A13. Acknowledgments Not applicable. References Howlader, N. et al. The effect of advances in lung-cancer treatment on population mortality. N Engl. J. Med. 383 , 640–649 (2020). Santoni-Rugiu, E. et al. Intrinsic resistance to EGFR-tyrosine kinase inhibitors in EGFR-mutant non-small cell lung cancer: Differences and similarities with acquired resistance. Cancers 11 , 923 (2019). Nishino, M. et al. Effects of secondary EGFR mutations on resistance against upfront osimertinib in cells with EGFR-activating mutations in vitro. Lung Cancer Amst Neth. 126 , 149–155 (2018). Jackman, D. et al. Clinical definition of acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors in non-small-cell lung cancer. J. Clin. Oncol. Off J. Am. Soc. Clin. 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Basal-shift transformation leads to EGFR therapy-resistance in human lung adenocarcinoma. Nat. Commun. 16 , 4369 (2025). Weng, M. S., Chang, J. H., Hung, W. Y., Yang, Y. C. & Chien, M. H. The interplay of reactive oxygen species and the epidermal growth factor receptor in tumor progression and drug resistance. J. Exp. Clin. Cancer Res. CR . 37 , 61 (2018). Nishihara, S. et al. Mechanisms of EGFR-TKI-induced apoptosis and strategies targeting apoptosis in EGFR-mutated non-small cell lung cancer. Genes 13 , 2183 (2022). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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07:10:17","extension":"html","order_by":45,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":108885,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7839640/v1/7254db66133d8da29fe8dee3.html"},{"id":95894970,"identity":"2facc19a-7cd1-4258-8717-7d6579f9cff0","added_by":"auto","created_at":"2025-11-14 07:10:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1280342,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNREP Expression and Its Prognostic Significance in Lung Adenocarcinoma (LUAD).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The expression of NREP mRNA across multiple cancer types as shown in the GEPIA2 database.\u003c/p\u003e\n\u003cp\u003e(B) Violin plots depicting NREP expression in adjacent normal tissues and different subtypes of lung adenocarcinoma (LUAD) from the GSE30219 dataset. P values were calculated by one-way ANOVA.\u003c/p\u003e\n\u003cp\u003e(C-D) Box plots showing the expression levels of NREP mRNA in tumor tissues and matched adjacent normal tissues across independent datasets. P values were determined by t-tests.\u003c/p\u003e\n\u003cp\u003e(E-F) Kaplan-Meier survival analysis of NREP expression in LUAD patients from the GSE30219 dataset. Patients were stratified based on the optimal cut-off value of 7.472. (E) Progression-free survival (PFS) and (F) overall survival (OS) curves are shown.\u003c/p\u003e\n\u003cp\u003ep values for survival analysis were calculated using the log-rank test.\u003c/p\u003e","description":"","filename":"FIG100.png","url":"https://assets-eu.researchsquare.com/files/rs-7839640/v1/cec7572a4b562783549b638a.png"},{"id":96243241,"identity":"f39a26ea-70cd-4a97-8f41-9c982a61374d","added_by":"auto","created_at":"2025-11-19 07:15:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":708849,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNREP Expression in Gefitinib-Sensitive and -Resistant Lung Adenocarcinoma Cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The relative cell viability of PC9 (gefitinib-sensitive) and PC9/GR (gefitinib-resistant) cells following 24-hour treatment with various concentrations of gefitinib, measured by the CCK-8 assay. Data are presented as the mean ± SD (n = 3) and were compared to the negative control (NC) group.\u003c/p\u003e\n\u003cp\u003e(B–C) Apoptosis rates in PC9 and PC9/GR cells 24 hours post-treatment with gefitinib (IC50-IC60) were assessed by Annexin V-FITC/PI double staining. (C) Quantification of apoptotic cells (n = 3).\u003c/p\u003e\n\u003cp\u003e(D) Relative expression of NREP mRNA in PC9 and PC9/GR cells following gefitinib treatment (IC50-IC60, 8 hours), measured by qPCR. β-actin was used as an internal reference. Data are presented as the mean ± SD (n = 3).\u003c/p\u003e\n\u003cp\u003e(E) Western blot analysis of NREP protein expression in PC9 and PC9/GR cells after 8-hour treatment with gefitinib (IC50-IC60). Quantification of NREP/β-actin protein ratio using ImageJ software (n = 3).\u003c/p\u003e","description":"","filename":"FIG200.png","url":"https://assets-eu.researchsquare.com/files/rs-7839640/v1/6444cb00155ec8cd94ef7227.png"},{"id":96242823,"identity":"c042ea0c-b872-48a9-b692-54256c906305","added_by":"auto","created_at":"2025-11-19 07:14:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":180091,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNREP modulates gefitinib sensitivity in lung adenocarcinoma cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Western blot analysis showing the protein expression of NREP in PC9 (gefitinib-sensitive) and PC9/GR (gefitinib-resistant) cells following NREP overexpression or knockdown. β-actin was used as a loading control. The relative expression of NREP (NREP/β-actin ratio) was quantified (mean ± SD, n = 3).\u003c/p\u003e\n\u003cp\u003e(B) Cell viability of PC9 and PC9/GR cells with NREP overexpression (oeNREP) or knockdown (siNREP) was measured by the CCK-8 assay after 24-hour treatment with gefitinib (IC50-IC60). Data are presented as the mean ± SD (n = 6) and compared to the negative control (NC) group.\u003c/p\u003e\n\u003cp\u003e(C–D) Apoptosis induction in PC9 (C) and PC9/GR (D) cells after 24-hour gefitinib treatment (IC50-IC60) was assessed by Annexin V-FITC/PI staining.\u003c/p\u003e\n\u003cp\u003e(E–F) Quantification of apoptosis rates in PC9 (E) and PC9/GR (F) cells, with data presented as the mean ± SD (n = 3).\u003c/p\u003e\n\u003cp\u003e(G–H) NREP expression in PC9 (G) and PC9/GR (H) cells after transfection with oeNREP or siNREP and gefitinib treatment (IC50-IC60, 8 hours), with quantitative analysis (mean ± SD, n = 6).\u003c/p\u003e","description":"","filename":"FIG300.png","url":"https://assets-eu.researchsquare.com/files/rs-7839640/v1/9b19eb7999ab0ef25e385ee9.png"},{"id":95894977,"identity":"dc514d35-f88e-454e-b3a0-e1ebfbeb6254","added_by":"auto","created_at":"2025-11-14 07:10:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1732900,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNREP promotes resistance to EGFR-TKIs in lung adenocarcinoma cells by inducing epithelial-mesenchymal transition (EMT).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Morphological comparison of PC9 (gefitinib-sensitive) and PC9/GR (gefitinib-resistant) cells after 24-hour treatment with gefitinib at IC50-IC60 concentrations. The scale bar represents 200 μm.\u003c/p\u003e\n\u003cp\u003e(B–C) Expression of EMT-related proteins in PC9 cells overexpressing NREP and PC9/GR cells with NREP knockdown. Protein levels were quantified, showing the impact of NREP on EMT marker expression.\u003c/p\u003e\n\u003cp\u003e(D–E) Immunofluorescence (IF) detection of CD133 (D) and α-SMA (E) in PC9 cells overexpressing NREP and PC9/GR cells with NREP knockdown, following 8-hour treatment with gefitinib (IC50-IC60). Fluorescence intensity was quantified. The scale bar represents 50 μm.\u003c/p\u003e","description":"","filename":"FIG400.png","url":"https://assets-eu.researchsquare.com/files/rs-7839640/v1/e87d5505859d7ef0385e3da7.png"},{"id":95894975,"identity":"e87a25bc-f9bd-4e5d-846e-238a911a95dd","added_by":"auto","created_at":"2025-11-14 07:10:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1726403,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNREP enhances gefitinib-induced expression of EMT markers.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Immunofluorescence (IF) analysis of PC9 cells overexpressing NREP, treated with gefitinib (IC50-IC60, 8 hours). The fluorescence intensity of CD133 (green) and α-SMA (red) was assessed, with the scale bar representing 50 μm.\u003c/p\u003e\n\u003cp\u003e(B) IF staining of PC9/GR cells with NREP knockdown, treated with gefitinib (IC50-IC60, 8 hours), showing fluorescence intensity for CD133 (green) and α-SMA (red). The scale bar represents 50 μm.\u003c/p\u003e","description":"","filename":"FIG500.png","url":"https://assets-eu.researchsquare.com/files/rs-7839640/v1/1c64d359b8ce800a96e15aa9.png"},{"id":95894980,"identity":"6188bf7a-3ef4-4979-9c5c-db864e4f919a","added_by":"auto","created_at":"2025-11-14 07:10:16","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2311413,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGPX4 enhances resistance to EGFR-TKIs in lung adenocarcinoma cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-B) Western blot analysis of NREP and GPX4 expression in PC9 (A) and PC9/GR (B) cells under various treatments. The expression levels were quantified using ImageJ software, and data are presented as the mean ± SD (n = 3).\u003c/p\u003e\n\u003cp\u003e(C-D) Western blot analysis of GPX4 expression in PC9 cells overexpressing GPX4 (C) and PC9/GR cells with GPX4 knockdown (D). β-actin was used as a loading control. Quantification of the GPX4/β-actin ratio is shown (mean ± SD, n = 3).\u003c/p\u003e\n\u003cp\u003e(E-F) Scratch assay to assess migration of PC9 cells overexpressing GPX4 (E) and PC9/GR cells with GPX4 knockdown (F) after treatment with gefitinib (IC20, 24 hours). The scale bar represents 100 μm.\u003c/p\u003e\n\u003cp\u003e(G-H) Quantitative analysis of cell migration rates in PC9 (G) and PC9/GR (H) cells. Data are expressed as the mean ± SD (n = 3).\u003c/p\u003e","description":"","filename":"FIG600.png","url":"https://assets-eu.researchsquare.com/files/rs-7839640/v1/dbabbe9d628fc1ab24b9b8b4.png"},{"id":96242612,"identity":"68234167-c81c-4ddf-8c0b-8bed008c4edc","added_by":"auto","created_at":"2025-11-19 07:13:41","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":4237784,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNREP-GPX4 mediates EMT regulation and enhances invasion in PC9 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Transwell invasion assay assessing the invasive potential of PC9 cells overexpressing NREP and GPX4, treated with or without gefitinib. The results demonstrate that NREP and GPX4 overexpression enhances invasion, and this effect is antagonized by gefitinib treatment.\u003c/p\u003e\n\u003cp\u003e(B) Western blot analysis of GPX4, CD133, and α-SMA protein expression in PC9 cells overexpressing NREP and GPX4, with or without gefitinib treatment. The corresponding quantitative analysis of protein levels was performed using ImageJ software. Data are presented as the mean ± SD (n = 3).\u003c/p\u003e\n\u003cp\u003e(C) Immunofluorescence (IF) analysis of PC9 cells overexpressing NREP and GPX4, treated with or without gefitinib. Expression of CD133 (green) and α-SMA (red) was assessed to evaluate the effects of NREP and GPX4 on EMT markers.\u003c/p\u003e","description":"","filename":"FIG700.png","url":"https://assets-eu.researchsquare.com/files/rs-7839640/v1/7ff317108eba80555d3ba067.png"},{"id":95894983,"identity":"2ddf08ac-d2b6-4963-a346-682b81cb4f55","added_by":"auto","created_at":"2025-11-14 07:10:16","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":3255051,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNREP-GPX4 synergistically activates EMT and promotes invasion in PC9/GR drug-resistant cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Transwell invasion assay assessing the invasive capacity of PC9/GR cells with NREP and GPX4 knockdown, treated with or without gefitinib. The data show that knockdown of both NREP and GPX4 significantly decreases cell invasion, and this effect is enhanced by gefitinib treatment.\u003c/p\u003e\n\u003cp\u003e(B) Immunofluorescence (IF) staining of PC9/GR cells with NREP and GPX4 knockdown, treated with or without gefitinib. CD133 (green) and α-SMA (red) expression levels were assessed to determine the impact on EMT markers.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNotes: Statistical analysis was performed using GraphPad Prism 9.0. Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons test. Statistical significance is indicated as follows: P \u0026lt; 0.05 (*), P \u0026lt; 0.01 (**), P \u0026lt; 0.001 (***), P \u0026lt; 0.0001 (****), and ns = not significant., and ns = no significant difference.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThe experimental groups include: negative control (NC), gefitinib treatment group (+Gef), NREP overexpression group (+oeNREP), NREP overexpression with gefitinib treatment group (+oeNREP+Gef), GPX4 overexpression group (+oeGPX4), GPX4 overexpression with gefitinib treatment group (+oeGPX4+Gef), NREP + GPX4 overexpression co-culture group (+oeNREP+oeGPX4), and the combined three-factor treatment group (+oeNREP+oeGPX4+Gef).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig800.png","url":"https://assets-eu.researchsquare.com/files/rs-7839640/v1/b1ba341e284fd44a9d220d89.png"},{"id":101643710,"identity":"bfd30876-6077-4297-b456-72c8bef9ada9","added_by":"auto","created_at":"2026-02-02 08:11:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":15763027,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7839640/v1/c93057a5-37cb-4155-a956-ae0243f08b99.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The NREP-GPX4 Axis Regulates Epithelial-Mesenchymal Transition and Enhances EGFR-TKI Resistance in Lung Adenocarcinoma Cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLung adenocarcinoma (LUAD) is the most prevalent form of non-small cell lung cancer (NSCLC), and despite advancements in targeted therapies such as epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), acquired resistance remains a major challenge. EGFR-TKIs, which target mutations in the EGFR gene, initially offer substantial clinical benefits for patients with EGFR mutations\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. However, the emergence of resistance, particularly through secondary mutations like T790M, presents a significant barrier to long-term efficacy\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. In addition to genetic mutations, cellular processes such as epithelial-to-mesenchymal transition (EMT) have been implicated in mediating resistance to EGFR-TKIs\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. EMT enables tumor cells to acquire migratory and invasive properties, facilitating metastasis and reducing sensitivity to targeted therapies\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. As such, understanding the molecular mechanisms underlying EMT and EGFR-TKI resistance is critical for the development of effective therapeutic strategies.\u003c/p\u003e\u003cp\u003eNeuronal Regeneration-Related Protein (NREP), also known as P311, is a highly conserved 8 kDa intracellular multifunctional protein\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. In cancer research, it has been extensively documented to participate in cellular processes including tumor cell migration, invasion, and EMT\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. NREP has been found to be upregulated in several cancers, where it is associated with poor prognosis and resistance to treatment\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. In LUAD, NREP expression has been linked to tumor progression and metastasis, suggesting its potential role in mediating resistance to EGFR-TKIs\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Additionally, NREP\u0026rsquo;s interaction with other proteins such as GPX4, a key regulator of oxidative stress, may contribute to resistance by modulating survival pathways and influencing the cellular response to EGFR inhibition.\u003c/p\u003e\u003cp\u003eRecent studies have highlighted the role of GPX4 in promoting EGFR-TKI resistance by maintaining redox balance and preventing ferroptosis, a regulated form of cell death\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Overexpression of GPX4 has been correlated with both intrinsic and acquired resistance to EGFR-TKIs in LUAD\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Furthermore, the NREP-GPX4 axis has been shown to synergistically enhance cancer cell survival under therapeutic stress, contributing to drug resistance\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. The cooperative role of NREP and GPX4 in regulating cell invasion, migration, and resistance highlights the importance of these molecules in LUAD progression. This study aims to further elucidate the molecular mechanisms by which the NREP-GPX4 axis influences EMT, invasion, and EGFR-TKI resistance, providing potential targets for overcoming therapeutic resistance in LUAD.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eBioinformatic Analysis\u003c/h2\u003e\u003cp\u003eThe GEPIA database(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gepia.cancer-pku.cn/\u003c/span\u003e\u003cspan address=\"http://gepia.cancer-pku.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was utilized to analyze the differential expression of NREP between various cancer tissues and normal tissues. Based on the publicly available GEO database, datasets GSE30219, GSE31210, and GSE68465 were selected to examine the differential expression of NREP in lung cancer tissues. Details of the datasets are provided in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The optimal cutoff value was determined using the \"surv_cutpoint\" package, and Kaplan-Meier survival analysis was performed with the \"survival\" package to assess the impact of different NREP expression levels on overall survival(OS) and progression-free survival (PFS).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eGEO Dataset Information.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGSE30219\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGSE68465\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eGSE31210\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePlatform\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGPL570\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eGPL96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eGPL570\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSpecies\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ehomo\u0026nbsp;sapiens\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ehomo\u0026nbsp;sapiens\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ehomo\u0026nbsp;sapiens\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTissue\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLungs\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLungs\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eLungs\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNormal\u0026nbsp;group\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTumor group\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e443\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e226\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCell Lines and Culture Conditions\u003c/h3\u003e\n\u003cp\u003eThis study utilized PC9 (EGFR-sensitive) and PC9/GR (EGFR-resistant) cell lines to investigate the roles of NREP and GPX4 in cell invasion and resistance to gefitinib, an EGFR-TKI. PC9 cells were obtained from the cell bank of the Procell, and PC9/GR cells were generated by long-term exposure to gefitinib. Cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, maintained in a humidified incubator at 37\u0026deg;C with 5% CO₂.\u003c/p\u003e\n\u003ch3\u003eOverexpression and Knockdown of NREP and GPX4\u003c/h3\u003e\n\u003cp\u003eTo study the effects of NREP and GPX4 on gefitinib resistance, overexpression and knockdown models of NREP and GPX4 were generated in both PC9 and PC9/GR cells. Overexpression of NREP and GPX4 was achieved by viral transfection, while gene knockdown was performed using siRNA targeting NREP and GPX4. Overexpression and knockdown efficiency were confirmed by Western blotting and qPCR.\u003c/p\u003e\n\u003ch3\u003eCell Invasion and Migration Assays\u003c/h3\u003e\n\u003cp\u003eCell invasion was assessed using Transwell invasion assays. Cells were seeded in the upper chamber of the Transwell inserts pre-coated with Matrigel. After 24 hours, invading cells on the lower surface were stained and counted. Scratch assays (wound healing assays) were performed to evaluate cell migration. Cells were seeded to form a monolayer, and a scratch was made in the middle of the well. Migration was assessed by imaging the wound area at various time points after treatment.\u003c/p\u003e\n\u003ch3\u003eProtein Expression and EMT Marker Analysis\u003c/h3\u003e\n\u003cp\u003eWestern blotting was used to evaluate the expression of NREP, GPX4, and EMT markers such as CD133 and α-SMA. For protein extraction, cells were lysed in RIPA buffer, and protein concentrations were determined using a BCA protein assay. The samples were then analyzed by SDS-PAGE, transferred to PVDF membranes, and incubated with specific primary antibodies. Immunofluorescence staining was used to visualize CD133 and α-SMA expression in cells under various conditions. Cells were fixed, incubated with primary antibodies, and stained with appropriate secondary antibodies. Fluorescent images were captured using a fluorescence microscope.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eCell Viability and Apoptosis Assays\u003c/h2\u003e\u003cp\u003eCell viability was assessed using the CCK-8 assay following treatment with various concentrations of gefitinib (IC50-IC60). Cells were plated in 96-well plates and treated with gefitinib for 24 hours. Apoptosis was analyzed by Annexin V-FITC/PI staining followed by flow cytometry. The apoptosis rate was quantified by analyzing the percentage of Annexin V-positive cells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eStatistical Analysis\u003c/h2\u003e\u003cp\u003eAll data were analyzed using GraphPad Prism 9.0 (GraphPad Software, USA). Statistical comparisons between multiple groups were performed using one-way ANOVA, followed by Tukey\u0026rsquo;s multiple comparisons test. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Kaplan-Meier survival analysis was used to analyze survival data, and statistical significance statistical significance is indicated as follows: P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (*), P\u0026thinsp;\u0026lt;\u0026thinsp;0.01 (**), P\u0026thinsp;\u0026lt;\u0026thinsp;0.001 (***), P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 (****), and ns\u0026thinsp;=\u0026thinsp;not significant., and ns\u0026thinsp;=\u0026thinsp;no significant difference.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eElevated Expression of NREP in Lung Adenocarcinoma and Its Association with Poor Prognosis\u003c/h2\u003e\u003cp\u003eTo investigate the role of NREP in lung cancer, its expression across multiple cancer types was first assessed using the GEPIA2 database. The results revealed that NREP mRNA is aberrantly upregulated in a variety of malignancies, including lung adenocarcinoma (LUAD) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). To further evaluate its expression pattern within lung cancer subtypes, the GSE30219 dataset was analyzed, as detailed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Violin plots demonstrated that NREP expression was significantly higher in LUAD tissues compared to adjacent normal tissues and showed differential expression among various histological subtypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Consistently, box plots from independent datasets confirmed that NREP mRNA expression was markedly elevated in tumor tissues relative to matched normal counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eC\u0026ndash;D).\u003c/p\u003e\u003cp\u003eTo assess the clinical relevance of NREP expression, LUAD patients in the GSE30219 cohort were stratified into high and low expression groups based on the optimal cut-off value (7.472). Kaplan\u0026ndash;Meier survival analyses indicated that high NREP expression was significantly associated with reduced progression-free survival (PFS) and overall survival (OS) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eE\u0026ndash;F). These findings suggest that NREP may serve not only as a potential biomarker for lung adenocarcinoma but also as a prognostic indicator of poor clinical outcomes.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eExpression of NREP in Gefitinib-Sensitive and -Resistant Lung Adenocarcinoma Cells\u003c/h2\u003e\u003cp\u003eThe expression of NREP in gefitinib-sensitive (PC9) and gefitinib-resistant (PC9/GR) lung adenocarcinoma cells was assessed under various experimental conditions. The relative cell viability of both cell lines was measured using the CCK-8 assay following 24-hour treatment with different concentrations of gefitinib. The results showed a significant difference in cell survival between the PC9 and PC9/GR cell lines, with the resistant PC9/GR cells exhibiting higher viability compared to the sensitive PC9 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003eApoptosis rates were also evaluated by Annexin V-FITC/PI staining following gefitinib treatment (IC50-IC60 concentrations). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and quantified in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, a higher apoptosis rate was observed in the sensitive PC9 cells compared to the resistant PC9/GR cells, further confirming the differential response to gefitinib treatment. In addition, the expression of NREP mRNA and protein was measured by qPCR and Western blot, respectively, after gefitinib treatment (IC50-IC60, 8 hours). Both mRNA and protein levels of NREP were significantly higher in the resistant PC9/GR cells compared to the sensitive PC9 cells, suggesting that NREP may be involved in the gefitinib resistance mechanism (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eD\u0026ndash;E). These findings highlight the potential role of NREP in regulating the sensitivity of lung adenocarcinoma cells to gefitinib and its potential as a therapeutic target in overcoming drug resistance.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eNREP Modulates Gefitinib Sensitivity in Lung Adenocarcinoma Cells\u003c/h2\u003e\u003cp\u003eThe role of NREP in regulating gefitinib sensitivity in lung adenocarcinoma cells was evaluated in PC9 (gefitinib-sensitive) and PC9/GR (gefitinib-resistant) cell lines. Western blot analysis showed that overexpression or knockdown of NREP resulted in corresponding changes in NREP protein levels in both cell lines, with PC9/GR cells showing higher baseline NREP expression compared to PC9 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The CCK-8 assay revealed that PC9/GR cells with NREP knockdown exhibited significantly reduced cell viability after gefitinib treatment, whereas NREP overexpression in PC9 cells conferred increased resistance to gefitinib, compared to the negative control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eAnnexin V-FITC/PI staining showed that gefitinib treatment induced higher apoptosis in PC9 cells compared to PC9/GR cells, and apoptosis was further enhanced in PC9/GR cells with NREP knockdown (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u0026ndash;E). In contrast, NREP overexpression in PC9 cells reduced gefitinib-induced apoptosis (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eD\u0026ndash;F). Furthermore, the impact of NREP overexpression and knockdown on NREP expression following gefitinib treatment was assessed by qPCR and western blot. Both PC9 and PC9/GR cells showed significant alterations in NREP expression after treatment, suggesting that NREP modulates gefitinib resistance by regulating apoptosis pathways (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eG\u0026ndash;H). These results indicate that NREP plays a crucial role in modulating the gefitinib sensitivity of lung adenocarcinoma cells, potentially serving as a therapeutic target for overcoming drug resistance.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eNREP Induces EMT and Promotes Resistance to EGFR-TKIs in Lung Adenocarcinoma Cells\u003c/h2\u003e\u003cp\u003eTo investigate the role of NREP in regulating gefitinib resistance, morphological differences between PC9 (gefitinib-sensitive) and PC9/GR (gefitinib-resistant) cells were observed after 24-hour treatment with gefitinib (IC50-IC60). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, PC9/GR cells exhibited a more mesenchymal-like morphology compared to PC9 cells, suggesting a potential role of NREP in inducing epithelial-mesenchymal transition (EMT).\u003c/p\u003e\u003cp\u003eFurther, the expression of EMT-related proteins was assessed in PC9 cells overexpressing NREP and PC9/GR cells with NREP knockdown. The results in Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eB\u0026ndash;C indicated that NREP overexpression in PC9 cells and NREP knockdown in PC9/GR cells significantly altered the levels of key EMT markers. Immunofluorescence analysis showed increased fluorescence intensity for CD133 and α-SMA in PC9 cells overexpressing NREP and reduced expression in PC9/GR cells with NREP knockdown following gefitinib treatment (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eD\u0026ndash;E). These findings suggest that NREP induces EMT, which contributes to the acquisition of resistance to EGFR-TKIs in lung adenocarcinoma cells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eNREP Enhances Gefitinib-Induced Expression of EMT Markers in Lung Adenocarcinoma Cells\u003c/h2\u003e\u003cp\u003eTo examine the effect of NREP on gefitinib-induced EMT, PC9 cells overexpressing NREP and PC9/GR cells with NREP knockdown were treated with gefitinib (IC50-IC60) for 8 hours. Immunofluorescence analysis in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eA showed that NREP overexpression in PC9 cells led to an increased fluorescence intensity of the EMT markers CD133 (green) and α-SMA (red) following gefitinib treatment. In contrast, PC9/GR cells with NREP knockdown exhibited significantly reduced fluorescence intensity for CD133 and α-SMA (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). These findings suggest that NREP enhances gefitinib-induced EMT marker expression, which may contribute to the development of resistance to EGFR-TKIs in lung adenocarcinoma cells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eGPX4 Enhances Gefitinib Resistance in Lung Adenocarcinoma Cells\u003c/h2\u003e\u003cp\u003eThe role of GPX4 in regulating gefitinib resistance was examined in PC9 and PC9/GR cells. Western blot analysis revealed that the expression of GPX4 was significantly altered in both cell lines under different treatment conditions. Specifically, PC9 cells overexpressing GPX4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eC) showed increased GPX4 expression, while PC9/GR cells with GPX4 knockdown exhibited reduced GPX4 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Additionally, the expression of NREP and GPX4 was also quantified, with significant changes observed upon treatment (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-B).\u003c/p\u003e\u003cp\u003eIn functional assays, PC9 cells overexpressing GPX4 exhibited accelerated migration in the scratch assay after gefitinib treatment (IC20, 24 hours) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eE), whereas PC9/GR cells with GPX4 knockdown showed impaired migration (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). Quantitative analysis of migration rates further supported these observations, with PC9 cells overexpressing GPX4 displaying enhanced migration, while PC9/GR cells with GPX4 knockdown showed significantly reduced migration (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eG-H). These findings suggest that GPX4 plays a key role in promoting migration and resistance to gefitinib in lung adenocarcinoma cells, potentially contributing to the development of EGFR-TKI resistance.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eNREP-GPX4 Mediates EMT Regulation and Enhances Invasion in PC9 Cells\u003c/h2\u003e\u003cp\u003eTo investigate the combined effect of NREP and GPX4 on lung adenocarcinoma cell invasion, Transwell assays showed that overexpression of both NREP and GPX4 significantly enhanced the invasion of PC9 cells, and this effect was counteracted by gefitinib treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Western blot analysis revealed that GPX4, CD133, and α-SMA protein levels were upregulated in PC9 cells overexpressing NREP and GPX4, and these markers were further modulated by gefitinib treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Immunofluorescence staining confirmed the increased expression of CD133 and α-SMA in these cells, indicating EMT activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). These results suggest that the combined overexpression of NREP and GPX4 promotes invasion and EMT, contributing to gefitinib resistance in PC9 cells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eNREP-GPX4 Synergistically Activates EMT and Enhances Invasion in PC9/GR Drug-Resistant Cells\u003c/h2\u003e\u003cp\u003eTo assess the role of NREP and GPX4 in PC9/GR drug-resistant cells, Transwell invasion assays revealed that knockdown of NREP and GPX4 significantly reduced the invasive potential of these cells. Additionally, gefitinib treatment further enhanced this effect, indicating that the combined knockdown of NREP and GPX4 sensitizes the cells to gefitinib (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Immunofluorescence analysis confirmed these findings, showing decreased expression of the EMT markers CD133 and α-SMA in PC9/GR cells upon knockdown of NREP and GPX4, particularly after gefitinib treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). These results suggest that the NREP-GPX4 axis contributes to EMT activation and invasion in gefitinib-resistant cells, and its modulation may reverse drug resistance in PC9/GR cells.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we explored the role of NREP and GPX4 in mediating resistance to EGFR-TKIs in lung adenocarcinoma (LUAD) cells. Our results demonstrate that overexpression of NREP and GPX4 in PC9 (gefitinib-sensitive) cells significantly enhances cell invasion and resistance to gefitinib, as evidenced by increased expression of EMT markers and enhanced migration capabilities. In contrast, knockdown of NREP and GPX4 in PC9/GR (gefitinib-resistant) cells sensitized them to gefitinib treatment, reducing invasion and modulating the expression of key EMT markers. The NREP-GPX4 axis appears to play a crucial role in promoting EMT and survival in LUAD cells, contributing to both intrinsic and acquired resistance to EGFR-TKIs. These findings highlight the potential of targeting the NREP-GPX4 pathway as a therapeutic strategy to overcome drug resistance and improve treatment outcomes in LUAD patients.\u003c/p\u003e\u003cp\u003eOur findings are consistent with previous studies that have explored the role of NREP and GPX4 in promoting drug resistance in various cancers, particularly in lung adenocarcinoma. Similar to Liu et al. (2021)\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, who demonstrated that NREP overexpression enhances migration and invasion in gastric cancer through EMT activation, we found that NREP overexpression in LUAD cells significantly increased cell invasion and resistance to gefitinib. This supports the idea that NREP plays a crucial role in facilitating cancer cell migration and resistance across different cancer types. In line with the work by Zhang et al. (2022)\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, who identified GPX4 as a key mediator of resistance to EGFR-TKIs in LUAD, our study further confirmed that GPX4 overexpression contributes to the maintenance of drug resistance by modulating redox homeostasis. However, while Zhang et al. (2022) focused on the role of GPX4 in ferroptosis and oxidative stress, our study provides new insight into how NREP and GPX4 cooperate to enhance resistance by regulating both EMT and apoptosis resistance. This synergistic interaction between NREP and GPX4 was not previously explored, making our findings an important extension of existing knowledge. Additionally, our results differ from the study by Pan et al. (2022)\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, which demonstrated that cholesterol accumulation mediates resistance to EGFR-TKIs in NSCLC through a different signaling axis. In contrast, our study emphasizes the combined effect of NREP and GPX4 in regulating EMT and invasion, indicating that these two proteins may contribute to resistance through complementary mechanisms rather than through cholesterol-related pathways alone. This highlights the complex nature of resistance mechanisms in LUAD and suggests that targeting both NREP and GPX4 could be a promising strategy for overcoming drug resistance.\u003c/p\u003e\u003cp\u003eA unique aspect of this study lies in the combination of multiple experimental approaches to assess the role of NREP and GPX4 in both drug resistance and cell migration in LUAD. By employing both overexpression and knockdown models of NREP and GPX4 in PC9 and PC9/GR cells, we were able to directly evaluate the impact of these proteins on gefitinib resistance and invasion. The use of both Western blotting and immunofluorescence to assess the expression of EMT markers (CD133 and α-SMA) was particularly effective in confirming the involvement of NREP and GPX4 in regulating EMT. This approach builds on the work of Song et al. (2022)\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, who used similar methods to evaluate drug resistance pathways in LUAD, but with a specific focus on methyltransferases. Furthermore, our application of the Transwell invasion assay to assess cell migration under gefitinib treatment provided strong evidence for the role of NREP and GPX4 in modulating invasive potential, a finding that supports the work of Ruan et al. (2024)\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e in breast cancer, where NREP\u0026rsquo;s role in promoting invasion was also demonstrated. Another strength of our study is the incorporation of apoptosis assays using Annexin V-FITC/PI staining, which allowed us to demonstrate the protective effect of NREP and GPX4 overexpression against gefitinib-induced apoptosis. This methodology is consistent with that used in previous studies\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, but here it highlights the combination of NREP and GPX4 as key modulators of cellular survival in response to EGFR-TKI treatment. These methodologies and their findings contribute to a more comprehensive understanding of the molecular mechanisms underlying drug resistance in LUAD.\u003c/p\u003e\u003cp\u003eWhile our study provides valuable insights into the role of the NREP-GPX4 axis in EGFR-TKI resistance in LUAD, there are several limitations that should be addressed in future research. First, the use of cell line models, although a powerful tool for understanding resistance mechanisms, does not fully replicate the complexity of tumor microenvironments and the heterogeneity found in patient-derived samples. Additionally, while we demonstrated the synergistic role of NREP and GPX4 in promoting invasion and resistance, the precise molecular mechanisms underlying their interaction remain unclear. Moreover, we did not evaluate the in vivo efficacy of targeting NREP and GPX4 in LUAD, which limits the translational relevance of our findings. Future studies should aim to validate these findings in patient-derived xenografts (PDX) or organoid models, which more closely resemble human tumors and their response to treatment\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Furthermore, we did not explore the potential role of other resistance mechanisms, such as alterations in DNA repair or immune evasion, which may also contribute to EGFR-TKI resistance\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eFuture studies should focus on several key aspects to further investigate and overcome EGFR-TKI resistance in LUAD. In vivo validation using patient-derived xenografts (PDX) or organoid models would be crucial for confirming the role of NREP and GPX4 in a clinically relevant context, as these models more closely mimic the human tumor microenvironment. Furthermore, a deeper exploration of the downstream signaling pathways through which NREP and GPX4 interact is needed to elucidate their precise roles in drug resistance. Investigating potential cross-talk with other resistance pathways, including those involved in oxidative stress and apoptosis, could offer new insights into the molecular mechanisms of resistance\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Additionally, combination therapies targeting both NREP and GPX4, along with EGFR-TKIs, should be explored to improve therapeutic efficacy and overcome resistance in LUAD patients. Combining targeted therapies may provide a more comprehensive approach to tackling drug resistance\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Lastly, considering the role of the immune system in modulating resistance mechanisms could provide valuable information for overcoming EGFR-TKI resistance. Immune evasion plays a significant role in cancer progression and drug resistance, and future studies should explore the potential benefits of combining immune-modulating therapies with EGFR-TKIs\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. These efforts could lead to the development of more effective treatment strategies and improved outcomes for LUAD patients facing EGFR-TKI resistance.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, our study highlights the critical role of the NREP-GPX4 axis in mediating resistance to EGFR-TKIs in lung adenocarcinoma, offering potential new therapeutic targets to overcome this resistance. The complex interplay between NREP and GPX4 in regulating epithelial-to-mesenchymal transition (EMT), invasion, and cell survival provides valuable insights into the mechanisms driving both intrinsic and acquired resistance to EGFR inhibitors. While the in vitro data presented here lay the groundwork for targeting this pathway, future studies are needed to validate these findings in more clinically relevant models, such as patient-derived xenografts and organoid systems, to better understand the translation of these results into therapeutic strategies. Additionally, exploring the downstream signaling pathways of NREP and GPX4, as well as their potential interactions with other key resistance mechanisms, could provide a deeper understanding of resistance biology. Further investigation into combination therapies targeting both NREP and GPX4, along with EGFR-TKIs, may enhance therapeutic efficacy and provide new avenues for treatment. Overall, the NREP-GPX4 axis presents a promising target for overcoming EGFR-TKI resistance in LUAD, and future research should continue to refine and expand these therapeutic possibilities.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics Approval Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data utilized in this study were obtained from publicly available databases. As the data are de-identified and publicly accessible, this study did not require additional ethical approval or patient consent.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe public datasets used in this study are openly accessible from the GEO database (https://www.ncbi.nlm.nih.gov/geo/). The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWX.W., QL.L. and H.Y. designed the experiment and write the draft, T.Z. and LJ.M. provided technical guidance in experiments, MJ.C., WJ.D., HY.W. and YJ.Z. accomplished data collection and analysis, W.Y., and LJ.M. provided methodology and validation. All authors contributed to the design and interpretation of the study and further drafts.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research is funded by the Provincial Natural Science Foundation of Sichuan, No. 2017SZ0066 and 2024NSFSC1292. The Scientific Research Project of General Hospital of Western Theater Command, No. 2024-YGJC-A13.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHowlader, N. et al. The effect of advances in lung-cancer treatment on population mortality. \u003cem\u003eN Engl. J. Med.\u003c/em\u003e \u003cb\u003e383\u003c/b\u003e, 640\u0026ndash;649 (2020).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSantoni-Rugiu, E. et al. Intrinsic resistance to EGFR-tyrosine kinase inhibitors in EGFR-mutant non-small cell lung cancer: Differences and similarities with acquired resistance. \u003cem\u003eCancers\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e, 923 (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNishino, M. et al. 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Mechanisms of EGFR-TKI-induced apoptosis and strategies targeting apoptosis in EGFR-mutated non-small cell lung cancer. \u003cem\u003eGenes\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 2183 (2022).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Lung adenocarcinoma, EGFR-TKI resistance, NREP, GPX4, epithelial-mesenchymal transition (EMT), drug resistance, invasion, gefitinib.","lastPublishedDoi":"10.21203/rs.3.rs-7839640/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7839640/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground:\u003c/h2\u003e\u003cp\u003eLung 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.\u003c/p\u003e\u003ch2\u003eMethods:\u003c/h2\u003e\u003cp\u003eIn 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.\u003c/p\u003e\u003ch2\u003eResults:\u003c/h2\u003e\u003cp\u003eOverexpression 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.\u003c/p\u003e\u003ch2\u003eConclusions:\u003c/h2\u003e\u003cp\u003eThe 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.\u003c/p\u003e","manuscriptTitle":"The NREP-GPX4 Axis Regulates Epithelial-Mesenchymal Transition and Enhances EGFR-TKI Resistance in Lung Adenocarcinoma Cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-14 07:10:11","doi":"10.21203/rs.3.rs-7839640/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"def05f67-f029-426f-beb8-7d0b74ae16b0","owner":[],"postedDate":"November 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":57747510,"name":"Biological sciences/Cancer"},{"id":57747511,"name":"Biological sciences/Cell biology"},{"id":57747512,"name":"Biological sciences/Molecular biology"},{"id":57747513,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2026-02-02T08:10:08+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-14 07:10:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7839640","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7839640","identity":"rs-7839640","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}