【作者】 于涛；

【导师】 钟殿胜；

【作者基本信息】 天津医科大学 ， 内科学 呼吸系病（专业学位）， 2018， 博士

【摘要】 目的:研究EGFR-TKI获得性耐药的分子机制,探索临床耐药患者可行的治疗方向。构建EGFR-TKI耐药细胞株HCC827-TR,研究该细胞株发生差异改变的基因,筛选可能导致其耐药发生的位点,进而明确FGF2/FGFR信号通路活性改变对于该细胞株耐药的作用并探索其机制。方法:对EGFR敏感突变细胞株HCC827-P进行持续药物培养诱导EGFR-TKI耐药细胞株HCC827-TR,利用MTS法检测Erlotinib对于两株细胞抑制率的影响,采用一代测序、基因芯片技术、RT-PCR和Western Blot的方法对目前已经明确的耐药机制进行筛查。对HCC827-P和HCC827-TR进行高通量二代测序,检测出的全部差异基因进行GO富集和KEGG通路富集,并在COSMIC数据库以及GDSC数据库分别检索全部差异基因,结合前期基因芯片的结果,以及各个基因突变的频率,在SIFT,Poly Phen-2,MutationTaster和CADD对所有差异基因进行有害性预测,最终选取FGF2作为目的基因,采用RT-PCR和Western Blot的方法检测所选取的突变基因及其下游信号通路分子表达量上的差异。应用FGFR高选择性抑制剂AZD4547处理两株细胞,明确其单药抑制率,随后联合Erlotinib双药处理两株细胞,验证其耐药性能变化情况;流式细胞仪分别检测Erlotinib单药和联合AZD4547处理后的两株细胞凋亡水平的变化情况;Western Blot验证AZD4547处理后的两株细胞FRS的蛋白水平是否存在差异,并对耐药细胞株检测其在Erlotinib单药和和联合AZD4547作用下信号通路关键激酶P-ERK和P-AKT的变化情况,最后应用RT-PCR的方法检测该信号通路下游相关基因mRNA水平变化。结果:MTS法显示Erlotinib明显抑制HCC827-P细胞株的增殖,呈现明显的量效关系,其IC50约为0.1μmol/L;Erlotinib对于HCC827-TR细胞株的抑制作用随着浓度的升高也有所增加,但其量效关系不如原代细胞显著,其IC50与原代细胞相比至少升高100倍;一代测序结果表明,HCC827-TR和HCC827-P细胞株均存在典型的EGFR19外显子E746-A750缺失突变,但无T790M突变;基因芯片结果提示HCC827-TR和HCC827-P之间存在1480个差异基因,后续的RT-PCR证实HCC827-TR和HCC827-P二者的MET,EGFR,HER2和PTEN的mRNA表达水平没有统计学差异,Western Blot验证两株细胞的PTEN蛋白表达水平一致,耐药株中不存在PTEN的缺失。NGS检测发现HCC827-TR细胞株中存在581个单核苷酸突变,全部为杂合突变,未发现已知的可以造成EGFR-TKI耐药的点突变;HCC827-TR细胞株中存在38处基因的插入缺失,基因组拷贝数目变异区域为10个,未发现已知可以导致耐药的机制,利用GO富集和KEGG通路富集对全部差异基因进行筛选,根据其突变频率和四大软件预测的有害性评分以及既往文献报道选取FGF2这一基因位点进行后续研究,位于4号染色体的FGF2外显子1区的点突变G101T,导致其合成的氨基酸发生了改变p.G34V,34位的甘氨酸被缬氨酸取代;RT-PCR证实在耐药细胞株中FGF2和其受体FGFR2的mRNA水平与原代细胞相比无统计学差异,Western Blot印证FGF2的蛋白水平与原代细胞相比明显增高,而其受体FGFR2的蛋白水平在两株细胞间表达无明显差异,P-ERK和P-AKT这两个下游信号通路关键位置的蛋白激酶在两株细胞分别接受Erlotinib处理后的表达存在显著性差异。MTS法检测AZD4547与Erlotinib联合处理后两株细胞抑制率均有所增加,但HCC827-TR的抑制率增加更为显著并具有统计学差异;流式细胞仪检测证实HCC827-P的两药联合处理组凋亡指数与Erlotinib单药相比没有统计学差异,而HCC827-TR在两药联合处理组与Erlotinib单药组相比发生了凋亡水平的显著增加;两株细胞在单药AZD4547处理后信号通路关键因子FRS2的蛋白水平存在显著性差异,耐药细胞株中Erlotinib单药组与联合AZD4547双药组相比其P-ERK和P-AKT表达水平存在显著性差异;对于耐药株HCC827-TR应用Erlotinib单药组与联合AZD4547双药组,后者信号通路下游BCL-2表达减低,BAX明显增加,CCND1表达减低,P21变化不显著。结论:成功构建EGFR-TKI耐药细胞株HCC827-TR,在该细胞株中未发现已知的耐药机制,初步确定导致该细胞株耐药的靶向基因为FGF2基因,其分子机制为由于FGF2基因点突变导致的蛋白改变造成FGF2/FGFR信号通路的持续活化,从而诱导产生EGFR-TKI获得性耐药,通过靶向抑制该通路可有效逆转细胞株耐药性。更多还原

【Abstract】 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.更多还原