Establishment Of Third Generation EGFR-TKI Osimertinib Acquired Resistant NSCLC Cell Line And Preliminary Study On Resistance Mechanism

Objective:The third generation of egfr-tki showed more surprising data in clinical trials than the first and second generation of TKI.As the only approved third generation of TKI,oxitinib gradually gained wide recognition in clinical practice and successfully won the throne of the four guidelines combined recommended first-line treatment.However,as a new generation of star targeted drugs,oxitinib will eventually face the problem of drug resistance.It has become an important research direction to clarify the mechanism of drug resistance and explore the potential therapeutic strategies after drug resistance.In this study,the third generation EGFR-TKI Osimertinib resistant cell line H1975/OR was established in vitro,and the key genes which may induce to Osimertinib acquired resistance were detected by Next-Generation Sequencing(NGS),so as to explore the molecular mechanism of Osimertinib acquired resistance.In order to provide more experimental basis for acquired resistance mechanism of EGFR-TKI,and providing potential therapeutic strategies for patients with clinical drug resistance.Methods:NCI-H1975 cell line containing EGFR L858R/T790 M mutation was induced to establish the third generation EGFR-TKI Osimertinib resistant cell line H1975/OR by low-concentration addition method in vitro.Comparision of parental cells and resistant cells in morphology and biology characteristic differences,including proliferation level differences by CCK8 and clone formation,apoptosis and cell cycle differences by flowcytometry,migration,invasion ability differences by Scratching and transwell experiment.To explore the EMT role in resistance,the expression levels of EMT markers were detected in both parental cells and resistant cells.Then we used RNA interference technology to knowkdown the key regulator Snail in resistant cells.After the interference efficiency was confirmed,changes in EMT-related molecules of Snail were explicitly down-regulated,and changes in sensitivity and migration and invasion ability were also examined.Finally,the activation levels of EMT-related key pathways in parental cells and resistant cell lines were preliminarily detected to preliminarily explore the signal transduction pathway that may lead to TKI resistance by inducing EMT.A big panel of genes mutation was detected by NGS,COSMIC,ICGC,and CCLE databases were used to find the mutation information analysis.In addition,SIFT,Polyphen2,Mutation Assessor online software were used to predict the potential toxicity of new single nucleotide mutations in resistant cells.Finally,differential genes were analyzed by KEGG pathway enrichment.Results:In the first part,this study first verified that both the drug and the cell lines used in the experiment met the requirements of the study.The drug Osimertinib low-concentration gradient method was used for 5 months to successfully established the Osimertinib resistant cell line H1975/OR,with an IC50 about 4.5 ?mol/L and drug resistance index of 391.3.In terms of morphology,the resistant cell line H1975/OR showed irregular cell characteristics,losing the morphology of epithelioid cells,and most of them were of spindle type.In addition,the cytoplasm was mostly filled with round vacuoles and the cell space was enlarged.In terms of biological characteristics,resistant cell line H1975/OR was characterized as slow proliferation,prolonged multiplication time,reduced apoptosis,G2/M cell cycle arrest,high migration and high invasion compared with the parental cells.According to the results of the previous part,we supposed that EMT may plays an important role in the process of acquired resistance to Osimertinib.Therefore,in the third part,the mRNA level and protein level of H1975 and H1975/OR EMT-related markers and analyzed to confirm that the expression of E-cadherin,an epithelial phenotype,was down-regulated,while the expression of N-cadherin and Vimentin,an interstitial phenotype,was increased,which promoted the expression of Snail and TWIST1,key transcription factors of EMT,and MMP-2/9,which was related to the migration and invasion ability,was highly expressed in the resistant cells.When we lowered Snail,we found that EMT was reversed.At the same time,the sensitivity of resistant cell line H1975/OR to Osimertinib was significantly restored after EMT reversal,and the migration ability was decreased.In addition,we preliminarily screened the activation of some star pathways related to EMT,and found that IGF1 R,Wnt/?-catenin,and NF-?B signaling pathways were abnormally activated in resistant cells H1975/OR,and may be involved in the signal transduction of TKI acquired resistance.The role of EMT in the resistance of Osimertinib was fully confirmed.In the third part,we used the next-generation sequencing technology to carry out a large Panel gene mutation detection on the gene level of parent cell H1975 and drug-resistant cell H1975/OR,and then analyzed the sequencing results after the machine quality control was qualified.A total of 54 gene mutation sites were detected in H1975,and 61 mutation sites were detected in H1975/OR cell line,among which single nucleotide variation was the most common type of mutation.After comparing the two cell lines,it was found that there were 4 sites for increasing mutation frequency,3 sites for decreasing mutation frequency,13 new mutation sites and 6 mutation vanishing sites.The sequencing results showed that all mutation site information had not been recorded or reported through database and literature search,and no currently known mechanism of Osimertinib acquired resistance had been found,such as EGFR C797 S mutation,MET amplification and HER2 amplification.After comparing the differentially expressed genes,13 new gene mutation sites of drug-resistant strains were found,among which 10 belonged to single nucleotide variation,1 belonged to insertion mutation,and 2 belonged to deletion mutation.Furthermore,multiple databases were used to predict and evaluate the sequence,the harmfulness of encoded proteins and the enrichment of pathways of the corresponding mutation sites.Several mutated genes have been reported to be involved in the EMT process.Conclusion:Osimertinib acquired resistance cell line H1975/OR was successfully established in vitro,no clear known resistance mechanism and relevant gene site mutations were found by next generation sequencing,suggesting that epithelial mesenchymal transformation may involved in the process of Osimertinib acquired resistance.