Identification And Functional Study Of Acquired Resistance Markers Of Third-generation EGFR-TKI Osimertinib

Objective EGFR-TKIs significantly prolonged the survival period of lung cancer patients with EGFR mutations and achieved remarkable therapeutic effect.However,most patients developed acquired resistance after continuous usage of EGFR-TKIs.Exploring resistance markers of EGFR-TKIs is helpful to adjust the treatment strategy in time.The object of the study is to establish third-generation EGFR-TKI osimertinib resiatant non-small cell lung cancer cell line resiatance signature and investigate the potential acquired resistance mechanism.Methods 1.The third generation of EGFR-TKI resistant cell line was screened and established by the gradient increment of osimertinib concentration.Exon PCR and Sanger sequencing were used to detect resistance mutations such as EGFR exon 18,EGFR exon 19,EGFR exon 20,EGFR exon 21,Kras exon2,Kras exon3,Kras exon4,Braf exon15,Pik3 ca exon9,Nras exon3.2.Bioinformatics mining of gene expression profile of drug-resistant cells from Geo database and screening differentially expressed genes were done to build drug resistance signature and evaluate its diagnostic efficacy.3.Epithelial mesenchymal transformation markers were detected by fluorescence quantitative PCR and Western blot.The expression of CTGF and N-cadherin were interfered by plasmid transfection to investigate the effect of N-cadherin on osimertinib sensitivity and apoptosis.Results 1.Five amino acids were absent from exon 19 In PC9 cells(?ELREA).The IC50 of p C9 to osimertinib was 0.0079?M(95% CI: 0.006 ?M-0.009?M)while the IC50 of PC9-OR was 21.24?M(95%CI: 9.39?M-48.03?M)and the resistance index was proved to be almost 2688.From the sight of morphology,PC9 was similar to epithelial cells while PC9-OR was round,like stromal cells,phalloidine staining showed that PC9-OR exhibit pseudopodium.Common resistance mechanisms such as EGFR exon 19 C797S/G,L798 I,792F/H,G796S/R,L718 Q,Kras exon2 G12 S,G12A,G12 D,Q61H,exon3 Q64 H,exon4 A146 T,Nras exon3 E63 K,Braf exon15 V600 E and Pik3 ca exon9 E545 K were not found.The IC50 of p C9 and PC9-OR to cisplatin was 2.403?M(95% CI: 2.138?M-2.701?M),2.818?M(95% CI: 2.485?M-3.196?M)respectively.The IC50 of p C9 and PC9-OR to Gefitinib was 0.056?M(95%CI: 0.027?M-0.118?M),44.29?M(95%CI: 8.355?M-234.8?M)respectively.There was no statistical significance of the difference in cell proliferation between PC9 and PC9-OR.2.Based on the analysis of GEO database(GSE106765,GSE75602 and GSE103350),a number of AZD9291 resistance related differentially expressed genes were identified,including 47,372 and 45 up-regulated genes respectively,among which MRAS,CTGF and ANKRD1 were concurrent up-regulated genes.The screened relatively high expression genes were verified by logistic regression in an independent dataset GSE35228.The results showed that MRAS,MET,ANKRD1,MCAM and FGFR1 had better diagnostic efficacy with the area under the ROC curve 0.9822.3.The expression lecvel of vimentin in PC9-OR was up-regulated at RNA level,but there was no significant difference in E-cadherin expression.The expression level of N-cadherin in PC9-OR was up-regulated at protein level,but there was no significant difference between E-cadherin and vimentin.The IC50 of PC9-OR was 7.493?M(95%CI: 5.751?M-9.764?M)after the expression level of CTGF was down-regulated with CTGFsh1 plasmid transfection while 8.696?M(95%CI: 4.645?M-16.28 ?M)with CTGFsh2 and the IC50 of the cells in the control group was 7.958?M(95%CI: 6.640?M-9.537?M).The IC50 of PC9-OR was 4.043?M(2.850?M-5.735?M)after the expression level of N-cadherin was down-regulated with Ncadsh1 plasmid transfection while 3.811?M(2.862?M-5.074?M)with Ncadsh2 and the IC50 of control group was 7.958?M(6.640?M-9.537?M).After interfering the expression of Ncadherin in PC9-OR,the proportion of apoptosis cells in N-cadherin sh1 and Ncadherin Sh2 group were significantly higher than that in control group.Conclusions 1.The third-generation EGFR-TKI osimertinib resistant cell line PC9-OR was successfully established and the IC50 of it to osimertinib was 21.24?M(95% CI: 9.39?M-48.03?M).The resistance index proved to be almost 2688.PC9-OR is resistant to the first-generation EGFR-TKI gefitinib as well,with a resistance index up to 791.However,it was sensitive to cisplatin,a traditional chemotherapy drug,as well as its parent p C9 cell line.The resistance mechanism of this cell is independent of common acquired resistance mechanism such as EGFR exon 19 C797S/G,L798 I,792F/H,G796S/R,L718 Q,Kras exon2 G12 S,G12A,G12 D,Q61H,exon3 Q64 H,exon4 A146 T,Nras exon3 E63 K,Braf exon15 V600 E and Pik3 ca exon9 E545 K.2.It was found that ANKRD1,Met,MRAS,MCAM,ANKRD1 had a good predictive efficacy of resistance to third generations EGFR-TKI,and we successfully construct the resistance signature to predict the sensitivity to osimertinib.It may provide a basis for the clinical to identify EGFR-TKIs resistance timely.3.The expression of mesenchymal cell markers such as N-cadherin and vemintin in PC9-OR increased.Overexpression of N-cadherin can promote cell apoptosis and inhibiting the expression of N-cadherin in pc9-or cells can decrease the sensitivity of PC9-OR to osimertinib.Interfering the expression of N-cadherin may increase cell sensitivity to osimertinib in PC9-OR.Moreover,it was found that N-cadherin may be involved in osimertinib resistance by inducing cell phenotype transformation to mesenchymal state or inducing cell apoptosis,making it a potential therapeutic target.