NRP1 overexpression potentially enhances osimertinib resistance in NSCLC via activation of the PI3K/AKT signaling pathway

Resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) is the main cause of mortality in lung cancer. This study aimed to investigate the roles of neuropilin 1 (NRP1) in non-small cell lung cancer (NSCLC). NRP1 expression was assessed in tumor tissues from patients with osimertinib-resistant (OR) NSCLC and osimertinib-responsive NSCLC as well as in patients with paracancerous NSCLC tissues who did not undergo radiotherapy or chemotherapy. In vitro experiments were conducted using five cell lines: BEAS-2B, HCC827, and PC9 cells, and the constructed OR cell lines, HCC827-OR and PC9-OR. HCC827-OR cells showing significant differences in osimertinib IC50 were selected for further study. After investigating the effects of altering NRP1 expression on cell sensitivity to osimertinib, NRP1 expression was inhibited to further investigate changes in cell viability, proliferation, migration, invasion, and apoptosis in OR cells. Additionally, bioinformatics techniques were used to detect targets (Integrin β3) and signaling pathways (PI3K/AKT) downstream of NRP1; subsequent cell experiments verified their interactivity. Finally, an orthotopic mouse tumor model was constructed using HCC827-OR cells treated with a PI3K/AKT signaling pathway activator (740Y-P), allowing exploration of the role played by the PI3K/AKT signaling pathway via NRP1 regulation on NSCLC resistance both in vivo and in vitro. Results showed that NRP1 expression was significantly increased in the cells of patients with NSCLC-OR, and increased NRP1 expression reduced HCC827 cell sensitivity to osimertinib. Both in vitro and in vivo experiments showed that NRP1 deficiency mediated by NRP1 inhibitors inhibited HCC827-OR cell proliferation, migration, and invasion, promoted tumor cell apoptosis, and enhanced osimertinib efficacy. In contrast, 740Y-P partially inhibited the effects of NRP1 inhibitors combined with osimertinib on the PI3K/AKT signaling pathway and on tumor growth in vivo and in vitro. Cellular experimental results showed that NRP1 positively regulates the Integrin β3 expression and activation of the PI3K/AKT signaling pathway. Bioinformatics analysis showed that both NRP1 and Integrin β3 may jointly participate in regulating the PI3K/AKT signaling pathway. In conclusion, our findings suggest that elevated NRP1 expression in NSCLC tumor tissues may promote NSCLC resistance to osimertinib by activating the PI3K/AKT signaling pathway, and integrin β3 potentially being involved in this process. These insights may provide a novel strategy for combination therapy for OR NSCLC.