The Investigation On Molecular Mechanism Of Acquired Resistance In EGFR-TKI Resistance Cell Line HCC827-TR

Objective: To explore the molecular mechanism of acquired resistance to EGFR-TKI and investigate the feasible treatment procedure for clinical drug resistant patients.To construct a EGFR-TKI resistant cell line HCC827-TR and identify genes that differ in HCC827-TR from primary cells,and to identify the role of FGF2/FGFR signal pathway mutation in the HCC827-TR resistance and explore the further molecular mechnism.Methods: EGFR sensitive mutant cells HCC827-P were continuously Erlotinib cultured to induce EGFR-TKI resistant cell line HCC827-TR.Then the first generation sequencing technology,gene chip technology,RT-PCR and Western Blot were used to screen the known mechanisms of drug resistance.The two cell lines were detected by the high throughput next generation sequencing technology.GO enrichment and KEGG pathway enrichment of differentially expressed genes were applied.All the differential genes were retrieved in the COSMIC database and GDSC database.Combined with the previous gene chip results,the frequency of the genes mutation,and the harmful prediction of each gene by SIFT,Poly Phen-2,MutationTaster and CADD,the FGF2 was finally chosen as the target gene.Then RT-PCR and Western Blot were used to verify the mRNA and protein expressions of this gene between the two cell lines.AZD4547,a high selectivity inhibitor of FGFR,was administrated to treat two cell lines.Then two drug treatment procedure combined with Erlotinib was applied in two cell lines to verify the resistance changes.Flow cytometry was used to detect the apoptosis level.Western Blot was used for the FRS protein expression changes of two cell lines after treatment and also for P-ERK and P-AKT expressions of HCC827-TR after administration.RT-PCR method was used to detect the mRNA levels changes of genes related to apoptosis and cell cycle in the downstream of the signal pathway.Results: MTS assay showed that Erlotinib significantly inhibited HCC827-P cell proliferation.Inhibition of Erlotinib on HCC827-TR cell line also increased with the increase of drug concentration,and The IC50 of HCC827-TR cell line elevated at least 100 times higher compared to primary cell.The first generation sequencing result showed that HCC827-TR and HCC827-P cell lines both appeared typical deletion mutation of E746-A750 in EGFR19 exon without T790 M mutation.Gene chip result indicated the presence of 1480 different gene expressions between the two cell lines.RT-PCR confirmed that no statistical significance was found in the comparisons of MET,EGFR,HER2 or PTEN mRNA expressions between two cell lines.Western Blot verified the similar PTEN expressions in two cell lines.NGS detection revealed that there were 581 single nucleotide mutations in HCC827-TR cells.Based on the GO enrichment and KEGG pathway enrichment screening results,the frequency of the genes mutations and the harmful prediction of each gene by softwares,and the literature reports,FGF2 was subsequently researched.Results revealed the gene loci of G101 T mutation on chromosome 4 in FGF2 exon 1 led to its synthesis of amino acid.The p.G34 V,glycine was replaced by valine.Western Blot results verified the protein level of FGF2 in resistant cells were significantly higher than the primary cells.Dectetion of the P-ERK and P-AKT,as the key positions of signal pathway,indicated that significant differences between two cell lines after drug treatment.MTS showed increased inhibition rates after combined treatment of AZD4547 and Erlotinib in two cell lines,while the inhibition rate of HCC827-TR significantly increased.Flow cytometry results confirmed that the HCC827-TR in the two-drug combination group had significantly increased levels of apoptosis.Protein levels of the key factor of FRS2 appeared significant difference in two cell lines after AZD4547 treatment.Obvious differences of P-ERK and P-AKT protein expressions were detected in the resistant cell line between single Erlotinib group and two drugs combined group.For drug-resistant strain HCC827-TR,in which the Erlotinib monotherapy group and the combined AZD4547 double drug group were applied,it was found in combined group that the BCL-2 expression decreased,BAX increased significantly,CCND1 expression decreased,and P21 did not change significantly.Conclusion: EGFR-TKI resistant cell line HCC827-TR has been successfully constructed,and no known resistance mechanism has been found in this cell line.FGF2 gene was preliminarily determined as the target gene that led to drug resistance of this cell line.It is suggested that the protein changes induced by FGF2 gene mutation can lead to the continuous activation of FGF2/FGFR signaling pathway,and induce EGFR-TKI acquired resistance.Targeted inhibition of this pathway can effectively reverse the drug resistance of cell line.