IGFBP7 Involves In Acquired Resistance To Osimertinib In Non-small Cell Lung Cancer

Background:Epidermal growth factor receptor(EGFR)mutation was the first driver mutation found to be a therapeutic target for non-small cell lung cancer(NSCLC).The identification of EGFR-sensitive mutation and the subsequent development of EGFR-TKIS targeting therapy have greatly changed the treatment approach for advanced NSCLC.The acquired resistance mechanism using the first generation of EGFR tyrosine kinase inhibitors(TKIs)is mainly T790M mutation in the EGFR 20 exon region,accounting for about 60%.To overcome first-generation TKI resistance,second-generation(such as afatinib)and third-generation(such as osimertinib)EGFR-TKIs were developed.However,afatinib has certain activity against wild-type EGFR,leading to a series of drug-related toxic and side effects,which limits its clinical application.The third generation EGFR-TKI represented by osimertinib(AZD9291)has no significant inhibitory effect on wild-type EGFR for it can selectively inhibit EGFR-sensitive mutation and EGFR T790M mutation.Although numerous evidence-based medical evidence has shown that osimertinib can benefit NSCLC patients with EGFR mutation or EGFR T790M sensitive mutation,acquired resistance after treatment is still inevitable.Although the mechanisms of acquired resistance to osimertinib have been reported,a large part of the mechanisms of resistance are still unknown.The molecular heterogeneity of NSCLC greatly influences the possible mechanism of osimertinib resistance,so for the third-generation EGFR-TKI,improving its resistance profile can help to clarify the molecular mechanism of resistance and thus provide better treatment options for subsequent generations.In our previous study,the osimertinib resistant cell line was induced by the EGFR 19 exon deletion lung adenocarcinoma cell line HCC827.In this study,the resistant monoclonal cells were screened and identified,and were named as HCC827/AZDR.Transcriptome sequencing and bioassay were used to compare the differences in transcriptional levels between parent cells and drug-resistant cells,and to screen potential drug-resistant genes.In addition,the potential drug resistance genes and the molecular mechanisms leading to osimertinib resistance were verified and explored in cell lines and tissues.Methods:1.Screening and identification of drug-resistant monoclonal cell lines:After screening the monoclonal cell lines,the cell morphology was observed under an optical microscope,and the proliferation inhibition rate of osimertinib against drug-resistant parent cells was detected by MTS method,and the IC50 and drug resistance ratio were calculated.MTS method was used to continuously detect the proliferation of cells from day 0-4 and draw the growth curve of cells.2.Exploration of potential drug-resistance mechanism:Whole exome sequencing(WES)was used to analyze the mutation status and copy number variation of 416 cancer-related genes in parent cells and drug-resistant cells;Whole transcriptome sequencing was used to compare gene expression differences between parent cells and drug-resistant cells and screen potential drug-resistant genes.The methylation difference between parent cells and drug-resistant cells was compared by 850K methylation chip;3.Verification of drug resistance mechanism:(1)Whether HCC827/AZDR cells had small cell lung cancer transformation:the expression of CD56,Syn and CgA in HCC827,HCC827/AZDR and H525 cells were detected by cellular immunochemistry assay.(2)Whether IGFBP7 is involved in the occurrence of osimertinib resistance:the expression level of IGFBP7 in NSCLC cell lines was detected by RT-PCR and Western-blot;The expression level of IGFBP7 in tumor tissues of patients with NSCLC was detected by immunohistochemical method.IGFBP7 knockdown and overexpression of NSCLC cell models were established by si-RNA interference and lentivirus infection,respectively.The proliferation inhibition rate of osimertinib cells after IGFBP7 knockdown or overexpression was detected by MTS method,and IC50 and drug resistance multiple were calculated.Colony formation assay was used to detect the changes of cell clone formation ability after knockdown or overexpression of IGFBP7.Cell apoptosis was detected by Annexin V staining.The expression and activation of EGFR,IGF1R and its downstream ERK and AKT signaling pathways as well as apoptosis-related proteins were detected by Western blot.Results:1.Osimertinib-resistant monoclonal cell strain was successfully screened and named as HCC827/AZDR.HCC827/AZDR was a cell strain with high level of osimertinib resistance,and its resistance ratio was 262 times.2.The WES sequencing results showed that the mutation status of HCC827/AZDR gene was still EGFR 19 exon deletion,and no other mutation sites of EGFR were found.At the same time,the copy number of new RBI gene in HCC827/AZDR cells decreased.Further detection of small-cell lung cancer marker CD56,Syn and CgA showed negative staining of HCC827/AZDR cells,suggesting that drug-resistant cells did not undergo small cell transformation.3.Total transcription sequencing showed that IGFBP7 was one of the differentially expressed genes(DEGs)of HCC827 and HCC827/AZDR cells,and its expression level was significantly increased in HCC827/AZDR cells.Further verification in HCC827/AZDR cells showed that both the protein and mRNA levels of IGFBP7 were higher than those of HCC827.4.The results of 850K methylation microarray showed that the IGFBP7 methylation region was significantly different between HCC827 and HCC827/AZDR cells.The IGFBP7 gene in HCC827 cells was hypermethylated,while the IGFBP7 gene in HCC827/AZDR cells was hypomethylated.5.In NSCLC patients with EGFR mutations who progressed after first-or second-line treatment with osimertinib,the level of IGFBP7 staining in tumor tissues was significantly increased after acquired resistance to osimertinib(P=0.046).6.The effect of IGFBP7 on the sensitivity of cells to osimertinib.(1)Inhibition of IGFBP7 expression could increase the proliferation inhibition rate of osimertinib to HCC827/AZDR cells,and the IC50 decreased from 8.514±0.295 ?M to 4.684±0.253 ?M(si-RNA 1#)and 5.574±0.177 ?M(si-RNA 2#).(2)Overexpression of IGFBP7 could reduce the proliferation inhibition rate of osimertinib to three NSCLC cell lines,and was IC50 increased:HCC827-OE-IGFBP7 0.247±0.018 ?M vs.HCC827-OE-NC 0.037±0.005 ?M.PC9-OE-IGFBP7 0.250±0.038 ?M vs.PC9-OE-NC 0.051±0.009 ±M;And H1975-OE-IGFBP7 0.221±0.033 ?M vs.H1975-OE-NC 0.057±0.003 ?M,respectively.7.The effect of IGFBP7 on cell proliferation.(1)Inhibition of IGFBP7 expression can inhibit the proliferation of HCC827/AZDR cells;Under the treatment of 1?M concentration of osimertinib,the above inhibition effect was further strengthened.(2)Overexpression of IGFBP7 could promote the proliferation of HCC827 and H1975 cells,but could not promote the proliferation of PC9 cells;Compared with the control group with IGFBP7 overexpression but no treatment,the above three cells could maintain active proliferative state at 0.05 ?M of osimertinib.8.Effects of IGFBP7 on the ability of cell colony formation.(1)After knockdown of IGFBP7,the clone formation ability of HCC827/AZDR was significantly decreased(p<0.05).(2)After the overexpression of IGFBP7,the clone formation ability of HCC827 and H1975 cells was significantly increased(p<0.05).There was no significant difference in the clone formation ability of PC9-OE-IGFBP7 cells.9.The effect of IGFBP7 on apoptosis level.(1)In HCC827/AZDR cells with IGFBP7 expression inhibited,the early apoptosis rate of cells was significantly increased(p<0.05),and the activation levels of apoptosis-related proteins BIM,caspase3 and PARP were increased in HCC827/AZDR cells with 1 ?M osimertinib.The average rate of early apoptosis in HCC827/AZDR-SI-IGFBP7-1 cells increased from 4.80%to 17.23%.The average early apoptosis rate of HCC827/AZDR-SI-IGFBP7-2 cells increased from 4.02%to 14.62%.There was no significant difference in the proportion of early apoptosis between HCC827/AZDR cells and HCC827/AZDR-si-NC cells before and after drug exposure.(2)The early apoptosis rate of HCC827,PC9 and H1975 cells overexpressed with IGFBP7 was significantly decreased under the treatment of 0.05 ?M osimertinib(p<0.05).10.IGFBP7 is involved in regulating the activation of IGF1R and its downstream AKT pathway.(1)IGF1R signaling pathway was abnormally activated in HCC827/AZDR cells.Inhibition of IGFBP7 inhibited the phosphorylation level of IGF1R and its downstream AKT pathway.The phosphorylation level of IGF1R and its downstream AKT pathway was further reduced by ?M osimertinib(2)Overexpression of IGFBP7 can enhance the phosphorylation level of IGF1R and its downstream AKT pathway in HCC827,H1975 and PC9 cells.Conclusions:1.Osimertinib-resistant monoclonal cell strain HCC827/AZDR was a high-level laboratory model with high levels of drug resistance.2.IGFBP7 expression was significantly up-regulated in osimertinib-resistant NSCLC cell lines,which was caused by demethylation of IGFBP7;3.IGFBP7 is highly expressed in NSCLC tumor tissues with disease progression after first-line or second-line osimertinib treatment;4.IGFBP7 can promote the resistance of NSCLC cells to osimertinib;5.IGFBP7 can promote the proliferation of cells,however,the survival of drug-resistant cells under the treatment of osimertinib is not completely dependent on this,IGFBP7 can also help cells survival by inhibiting apoptosis.6.IGFBP7 promoted osimertinib resistance by mediating the activation of IGF1R and its downstream AKT pathway.