The Establishment Of Icotinib Resistant Non-small Cell Lung Cancer Cell Lines And The Study On Its Genomic Aberrations

Aims:Icotinib is the most commonly used and effective epidermal growth factor receptor(EGFR)tyrosine kinase inhibitor(TKI)for treating EGFR-mutated patients in China.Due to its unique characteristics of targeting and low toxicity,icotinib have achieved significant outcome in treatment of EGFR mutations non-small cell lung cancer(NSCLC)patients in the clinic.However,patients develop acquired resistance to icotinib within 9 to 15 months,so there is an urgent need to elucidate the underlying mechanisms of icotinib resisitance.Many studies have shown that EGFR-TKI resistance is associated with genomic aberration,including single nucleotide polymorphisms(SNP)and copy number variation(CNV).Compared to other EGFR-TKIs such as elotinib and gefitinib,icotinib has a different molecular structure and in vivo metabolism,the mechanisms of icotinib resistance may be different from other TKIs.In this study,whole exome sequencing was used to identify the genomic variations between the parent cells and icotinib-resistant cells,we hope to reveal the genomic aberrations associated with icotinib resistance and study the mechanisms of icotinib resistance.Methods:An EGFR-TKI sensitive NSCLC cell line PC-9(exon 19 deletion of EGFR)was chosen as research object.Icotinib resistance PC-9 cells was established by means of increasing concentration gradient.The tolerance level of PC-9/IR to icotinib was verified by several experiments,such as morphological observation,MTT,wound healing test,Western blot(WB)and immunofluorescence.Genomic DNA was extracted from PC-9 and PC-9/IR cells,whole exome sequencing and bioinformatic analysis were performed to characterize the mutational changes of SNPs,indels and CNVs between PC-9 and PC-9/IR cells.Several obtained SNPs and CNV were examined at the DNA,mRNA,protein and cellular biology levels.Finally,in order to verify whether the CAPN7 amplification was specific for icotinib resistance,two EGFR-TKI resistant cells A549(EGFR wild type)and H1975(EGFR T790M and exon 21 mutation of EGFR)were treated with high dose of icotinib in vitro for 40 weeks,and the CAPN7 at the DNA,mRNA and protein levels was detected.Results:The PC-9/IR cells were induced by icotinib for 40 weeks.Compared with the parental cells,the morphology of PC-9/IR cells was changed from round to fusiform,and the cell ability of PC-9/IR cells was higher than the PC-9 cells at the same drug concentration,and the migration rate and expression of p-EGFR in PC-9/IR cells were significantly higher than those in PC-9 cells.Immunofluorescence assay was used to detect the expression of cadherin and vimentin in drug-resistant cells,the results suggested that PC-9/IR did not undergo epithelial transformation.Using whole exome sequencing,we identified 76,475 and 79,150 SNPs,10,228 and 10,891 indels in PC-9 and PC-9/IR cells.Compared with the PC-9 cells,we obtained 183 single nucleotide polymorphisms(SNVs)and 10 indels in PC-9/IR cells.In addition,we also found 3 CNVs in PC-9/IR cells:the amplification of calpain-7(CAPN7),the deletion of Insulin-like growth factor 2 mRNA-binding protein 2 and KIAA0922.We confirmed the sequencing results at different levels of DNA,mRNA,protein and cytology.The results showed that T790M mutation and RAS/Raf signaling pathway activation were existed in PC-9/IR cells,and the proliferation of PC-9/IR cells was inhibited by T790M and RAS/Raf signal pathway inhibitor AZD9291 and U0126.The results of Real time PCR and WB showed that the copy number of CAPN7 in PC-9/IR cells was amplified,and the expression was higher than that of parental cells at the mRNA and protein levels.We treated EGFR-TKI resistant cells A549 and H1975 with high dose of icotinib for 40 weeks,and obtained A549/IR and H1975/IR cells.Compared with the pre-treated cells,the copy number of CAPN7 were amplified,suggesting that CAPN7 amplification may be a specific result of icotinib induction.The expression of CAPN7 in H1975/IR cells was higher than that in H1975 cells at mRNA and protein level,but not in the EGFR wild type A549/IR cells compared with A549 cells.Conclusion:This study successfully established icotinib-resistant NSCLC cells PC-9/IR.Whole exome sequencing and bioinformatic analysis were used to find the different genomic aberration between PC-9 and PC-9/IR cells.Among the obtained 183 SNVs,10 indels and 3 CNVs,which 57 aberrations may cause changes in gene function.The amplification of CAPN7 was found in PC-9/IR,A549/IR and H1975/IR cells.And the expression of CAPN7 at mRNA and protein level in the PC-9/IR and H1975/IR cells was higher than that in pre-treated cells,suggesting that the increasement of CAPN7 copy number and expression may be a specific result of icotinib induction.In conclusion,the amplification of CAPN7 may be a specific CNV of icotinib reaiatance in NSCLC cells.