MET/AKT Signaling Axis Mediates The Resistance Of Lung Cancer A549 Cell Line To Gefitinib

Objective: Lung cancer is the leading cause of cancer mortality throughout the world.Studies have found that the epidermal growth factor receptor(EGFR)is overexpressed in more than 80% of non-small cell lung cancer(NSCLC).Therefore,molecular targeting therapy for this target has achieved significant efficacy in the treatment of NSCLC,especially in patients with exon 19 deletion mutation and exon 21 point mutation(L858R).However,most patients eventually develop resistance to EGFR tyrosine kinase inhibitors(EGFR-TKIs).At present,various mechanisms of acquired resistance to EGFR-TKIs in NSCLC with sensitive EGFR mutations have been discovered.Although studies have found that patients with EGFR wild-type NSCLC can also benefit from monotherapy of EGFR-TKIs,the drug resistance mechanism of EGFR wild-type NSCLC to EGFR-TKIs is rarely reported.This study aims to study the acquired resistance mechanism of EGFR wild-type NSCLC cells to EGFR-TKI gefitinib and to provide a reference for improving tumor-targeted therapy.Methods:(1)The mechanisms of drug resistance in A549/GR cells were screened: the T790 M mutation of EGFR gene was detected by Sanger sequencing;the extrachromosomal amplification of EGFR gene was detected by fluorescence in situ hybridization,and the expression of MET and PTEN gene relative to A549 cells was detected by q PCR.(2)After A549/GR cells were treated with gefitinib and MET tyrosine kinase inhibitor PHA-665752 alone or in combination,the viability of A549/GR cells was detected by CCK8 assay,and apoptosis was detected by Hoechst staining and TUNEL assay,and the protein expression of EGFR,p-EGFR,MET,p-MET,AKT,and p-AKT was detected by Western blot.(3)After knocking down the expression of MET in A549/GR cells with MET-specific si RNA,the expression of MET,EGFR,p-EGFR,AKT,and p-AKT in A549/GR cells treated with gefitinib was detected by Western blot,and the viability of cells treated with different concentrations of gefitinib was detected by CCK8 assay.Results:(1)EGFR gene T790 M mutation and extrachromosomal amplification were not detected in A549 / GR cells.Compared with A549 cells,the expression of PTEN gene did not change significantly,but the expression of MET gene was significantly up-regulated.(2)A549/GR cells were resistant to both gefitinib alone and MET inhibitor alone,but the combined treatment of gefitinib and MET inhibitor significantly inhibited the growth of A549/GR cells and induced apoptosis of A549/GR cells.Gefitinib combined with MET inhibitor completely inhibited the phosphorylation of AKT,which could not be inhibited when they were applied to A549/GR cells alone.(3)Knockdown of MET expression in A549/GR cells by MET-specific si RNA resulted in marked inhibition of AKT phosphorylation and re-inhibition of cell growth by gefitinib alone.After infecting A549 cells with MET gene-carrying lentivirus to up-regulate the expression of MET,gefitinib could no longer inhibit the phosphorylation of AKT alone,and A549 cells became resistant to gefitinib.Conclusion: A549 cells with wild-type EGFR develop acquired resistance to gefitinib by upregulating the expression of MET to maintain sustained activation of AKT.