HGF/c-MET信号通路与EML4-ALK阳性非小细胞肺癌获得性耐药的相互关系及其机制研究

EML4-ALK was defined as new subtype of NSCLC with distinct clinical and pathological characteristics. Crizotinib, a tyrosine kinase inhibitor of dual-targeting ALK and c-MET genes, has been increasingly applicated in clinics. But the subsequent resistance seems to be inevitable and the potential mechanisms need to be elucidated. It has been well-knowed that HGF/c-MET signaling pathway has play an important role in carcinogenesis.However, the relationship between this pathway and crizotinib resistence remains no reported. In this study, the full and intermediate resistence cell lines are to be induced by increasing concentrations of crizotinib and TEA684 (a potent tyrosine kinase inhibitor of c-MET ) in H3122, BA/F cell lines and lung cancer cells which were obtained from previous screenning. EML4-ALK and c-MET gene are observed in parent and resistent cell lines with the purpose to draw mutation profiles in drug resistenc condition. Relationship bewteen HGF/c-MET signaling pathway and changes of ALK and c-MET genes need to be verified. Cross-talk mechanisms with other core signaling pathways are also to be detected for selecting key proteins and aim to provide molecular biological basis for anti-resistence for further investigation. If the hyperactivities of HGF/c-MET signaling pathway were observed in our study, the small interference RNA will attempt to reverse drug-resistence though interfering c-MET genes. This strategy to overcome resistance can be mirrored by other micromolecular TKI drugs.

EML4-ALK融合基因阳性的非小细胞肺癌作为新的肺癌亚型，针对ALK/c-MET双靶点的酪氨酸激酶抑制剂crizotinib正逐渐应用于临床，但随之而来的耐药难以避免，其机制有待阐明。已知HGF/c-MET信号通路在肿瘤演进中发挥重要调控作用，但该通路与耐药的关系迄今未有报道。本研究拟对H3122、BA/F细胞株及前期筛获的EML4-ALK肺癌细胞采用浓度递增法诱导crizotinib和TAE684完全和不完全耐药株，观察亲本和耐药株EML4-ALK和c-MET基因扩增和突变情况，绘制耐药突变谱，并求证ALK和c-MET基因的变化与HGF/c-MET通路活性的相关性，同时检测其它关健信号通路，了解HGF/c-MET与其它通路的交互机制，遴选关健节点蛋白，为将来耐药拮抗提供分子生物学依据和参考，最后采用siRNA干扰c-MET基因尝试耐药逆转，为类似小分子TKI药物的耐药克服提供新的思路。

EML4-ALK非小细胞肺癌作为新的肺癌亚型，针对ALK/c-MET双靶点抑制剂crizotinib正逐渐应用于临床，但耐药难以避免，本课题旨在探寻HGF/c-MET信号通路在肿瘤耐药中的作用机制，为克服耐药提供新的思路。研究结果有：(1) Crizotinib 对EML4-ALK阳性细胞株H2228、H3122和c-MET基因扩增的H1993细胞株有促凋亡作用, 均呈时间和剂量依赖性, 在H2228中，发现细胞增殖被阻滞在G1 期；(2)采用crizotinib浓度递增法获得耐药指数为10的H2228CR；(3)对耐药前后亲本和子代细胞在多条信号通路上关健蛋白进行检测(mTOR, BIM, Bid,Bcl-2,Bcl-xL, stat3, survivin, ALK、Met、EGFR, p70S6K,PI3K, AKT, MET, ERK以及磷酸化的p-mTOR, p-stat3, p-survivin, p-ALK、p-Met、p-EGFR, p-p70S6K, p-PI3K, p- AKT, p-MET, p-ERK), 遴选出的核心调控蛋白包括：Bim, c-Met、Stat3和survivin； (4)证实低氧处理时氯化钴可下调 H2228细胞HIF-1α的mRNA表达,氯化钴浓度为20μmol/L时下调最明显；(5)证实HGF、EGF、TGF-α为强旁路激活因子, 而IGF-1为弱旁路激活因子，二甲双胍逆转耐药作用弱且持效短暂；(6)对耐药株EML4-ALK基因进行测序, 首次发现EML4-ALK位点2067G->A和2182G->C的点突变,它们参与BIM高表达和survivin低表达, 后者与克唑替尼的耐药有关; (7)较早采用siRNA 技术“沉默” BIM 基因的表达可成功逆转耐药；(8) 动物实验证实50mg/kg/day克唑替尼可完全抑制了c-MET基因扩增型肿瘤的c-MET磷酸化物p-c-MET，提示p-c-MET水平可作为c-MET抑制剂疗效的标志物。完成了课题拟定检测和研究的全部内容，并增加尝试了多种方法进行耐药逆转，为今后分子靶向药物的研究开发和药物治疗提供理论依据和重要的参考。本课题培养了6位硕士研究生，共在国内外专业期刊上发表论文8篇,成果获得2015年度广西医药卫生适宜技术推广奖二等奖和2016年 度广西科学技术奖三等奖。