Bim在介导非小细胞肺癌ALK抑制剂获得性耐药中的机制研究

Anaplastic lymphoma kinase (ALK) inhibito palys an important role in the therapy of advanced NSCLC patients with EML4-ALK genes arrangement, however, resistance has become its treatment bottleneck and resistance mechanisms has not been elucidated. ALK gene is activated primarily through Akt, ERK and STAT3 pathways and ALK inhibitor induces apoptosis in the senstive cells through the expression of Bim . Our previous study found that Crizotinib (a kind of ALK inhibitor) could induced the expression of Bim in the sensitive cell lines while not in the drug-resistant cell lines. Which suggest that Bim participate in the acquired resistance of ALK inhibitor, however, the detailed regulated mechanism is still not so clear. This program used the methods of gene chips、genes transfection、siRNA、RT-PCR and western bolt to investigate the accociation of Bim and PI3K/Akt, MEK/ERK, JAK/STAT3 signal transduction pathways in cell and animal levels, trying to indentify the role and function of Bim in the resistant mechanism of ALK inhibtor. The results got from the above studies will be further verified in the clinical specimens. As a result,this program try to clarify the molocular mechanism of ALK inhibtors and might provide the potiential target and stategy to reversed the resistance of ALK inhibtor.

间变淋巴瘤激酶（ALK）抑制剂在具有EML4-ALK基因融合的晚期非小细胞肺癌患者中发挥重要作用，然而耐药已成为其治疗瓶颈，耐药机制尚未阐明。ALK基因活化主要通过Akt、ERK和STAT3通路发挥作用, ALK抑制剂则通过诱导敏感细胞Bim的表达来发生凋亡。我们前期研究发现Crizotinib（ALK抑制剂）诱导敏感细胞株产生Bim的表达，而在耐药珠中并未出现。 我们推测Bim参与了ALK抑制剂使用后的获得性耐药，但具体调控机制有待阐明。因此本课题拟采用基因芯片、基因转染、siRNA、RT-PCR、western bolt等方法在细胞和动物水平探讨Bim与PI3K/Akt、MEK/ERK、JAK/STAT3信号传导通路的关系，明确Bim在ALK抑制剂获得性耐药中的作用和机制,并通过临床标本加以验证，从而进一步阐明ALK抑制剂获得性耐药的分子调控机制，并为逆转耐药提供可能的候选靶点和思路。

本研究通过CRIZOTINIB 诱导含EML-ALK融合基因的NSCLC细胞株H2228和H3122， 并检测BIM在敏感株和耐药株中表达的区别， 发现在耐药株中存在BIM的低表达， 并沉默和转染BIM后发现细胞对于CRIZOTINIB的敏感性发生变化。同时， 采用Western blot 检测Crizotinib 敏感株与耐药株对于信号转导通路的影响， 发现PI3K/AK时影响BIM表达的主要通路。 随后在临床标本上检测了BIM的SNP缺失，发现其SNP缺失为原发性耐药的主要原因，在检测BIM的SNP的同时， 发现并报道了一例非典型ALK融合基因阳性的患者， 提示ALK检测的金标准FISH方法可能存在漏诊ALK融合基因检测的可能。 随后在临床上255例患者中对比了这2中检测方法的敏感性， 发现RT-PCR的方法能够更敏感的检测出ALK融合基因。获得性耐药机制在临床上的探索应用首先需要确定患者是否存在ALK融合基因， 本研究临床部分内容也进一步的采用RT-PCR的方法来检测ALK融合， 发现细胞学的方法也是一种可行的方法用于ALK融合基因的检测。