Research On The Function And Mechanism Of SOX3 Gene Mutation In EGFR-TKI Resistance Of Lung Adenocarcinama

Objective Lung cancer is the most lethal cancer in China and even in the world.In recent years,targeted drugs targeting EGFR mutations have been shown to be effective in the treatment of specific gene subtypes of non-small cell lung cancer.But most of the patients who received the first generation of tyrosine kinase inhibitors(TKI)developed resistance after a median of 10-12 months.The mechanism of drug resistance can be divided into primary drug resistance and acquired drug resistance.Primary drug resistance mainly refers to those who are treated with TKI for the first time,but do not benefit or benefit little.Acquired drug resistance mainly refers to the fact that the tumor has known EGFR mutations related to TKI sensitivity,and has previously been treated with a continuous single drug EGFR-TKI,but the disease is still progressing.The main reasons of acquired drug resistance are the second mutation of EGFR gene(such as T790 M mutation),the channel disorder of some genes(such as c-Met amplification),etc.But about 40% of acquired drug resistance mechanism is still unknown.Based on the above research background,we collected69 tumor tissue samples and blood samples from patients with stage IIIB-IV non-small cell lung cancer(NSCLC)who were resistant to the first generation of tyrosine kinase inhibitors(TKI).Among them,21 patients underwent second biopsy and second blood sampling.After excluding the causes of T790 M mutation,amplification of c-Met gene and transformation of pathological type,we found some key mutations in the 21 patients.In the subsequent preliminary experiments,we found that the mutation of SOX3 gene had positive results in the cell function test.Therefore,the purpose of this study is to investigate the relationship between SOX3 gene mutation and EGFR-TKI resistance in PC9 and HCC827 cells with TKI sensitive exon 19 deletion mutation,and to explore its possible mechanism.Method1)Through overexpression of SOX3 gene and its mutant SOX3_Mut in PC9(EGFR19Del mutation)and HCC827 cells(EGFR 19 Del mutation),and identification by RT-q PCR(real-time fluorescence quantitative PCR)and Western blot(protein immunoblotting),a stable cell line was established.2)Through MTT and plate cloning experiments,the cell functions of the above stable cell lines were studied to evaluate whether the mutation of SOX3 gene has an effect on the cell proliferation.3)Western blot was used to detect the expression of cleaved Caspase-3,cleaved PARP and other proteins related to apoptosis in the stable cell line.The expression of Caspase-3 in the stable cell line was observed by fluorescence microscope.The level of apoptosis in the stable cell line was measured by flow cytometry.The mutation of SOX3 gene was analyzed whether it was related to apoptosis.4)Through subcutaneous tumor bearing in nude mice,we analyzed whether SOX3 gene mutation was related to TKI resistance in vivo experiments : constructing subcutaneous tumor model in nude mice,analyzing tumor weight and growth curve after TKI treatment;q PCR in tumor bearing tissue to verify whether SOX3 gene and SOX3_Mut gene were up regulated in transcriptional level;Western blot in tumor bearing tissue to verify SOX3 gene and SOX3_Mut gene in protein level.The expression of Ki-67 protein and cleaved caspase-3 protein in tumor bearing tissues was analyzed by HE staining and immunohistochemistry.5)Western blot was used to detect the phenotype related signal pathway in stable cell lines,and the mechanism of phenotype related was analyzed by RNA sequencing.Result:1)In PC9 and HCC827 cells,the cell lines stably transfected with empty vector con--trol,SOX3 and SOX3_Mut were established.MTT analysis showed that the overexpression of SOX3_Mut increased the resistance of PC9 and HCC827 cells to EGFR-TKI compared with the control group.The clonal formation of PC9 and HCC827 cells overexpression of SOX3_Mut in EGFR-TKI was significantly enhanced.2)Compared with the control cells,the level of cleared caspase-3 and cleared PARP protein in cells expressing SOX3_Mut decreased after EGFR-TKI treatment.Annexin V / PI flow cytometry also confirmed that EGFR-TKI induced apoptosis of cells expressing SOX3_Mut decreased compared with the control cells.3)After EGFR-TKI treatment,the weight of the tumor in the SOX3_Mut overexpression group was significantly greater than that in the SOX3 overexpression group and the control group;the tumor growth curve also showed that the tumor in the SOX3_Mut overexpression group grew faster than the other two groups 4 weeks after TKI treatment.Immunohistochemical analysis of the tumor tissue showed that compared with the empty vector and SOX3,the expression of Ki67 increased,while the expression of cleaved caspase-3 decreased.These in vivo results are consistent with those of in vitro cell experiments,indicating that SOX3_Mut leads to the resistance of lung adenocarcinoma cells to EGFR-TKI.4)It was found that the mechanism of SOX3_Mut leading to EGFR-TKI resistance in lung adenocarcinoma cells may be through increasing phosphorylation of Erk protein and activating MAPK signaling pathway.The transcriptome sequencing and bioinformatics analysis showed that Erk protein phosphorylation may further activate JMJD7-PLA2G4 B fusion protein,promote the proliferation of tumor cells and inhibit the apoptosis of tumor cells.Conclusion:We speculated that SOX3 gene mutation is a new mechanism of EGFR-TKI acquired drug resistance.SOX3 gene mutation may inhibit EGFR-TKI induced apoptosis by enhancing Erk protein phosphorylation,which leads to acquired esistance of some NSCLC patients with sensitive mutations to EGFR-TKI.