CDK2AP1在EGFR-TKI诱导非小细胞肺癌获得性耐药中作用及其分子机制研究

Resently, it is well accepted that tyrosine receptor inhibitor (TKI) whose major target is epidermal growth factor receptor (EGFR) can improve the overall survival of patients with non-small cell lung cancer. However, it becomes a great challenge that there will be acquired drug resistance observed in patients treated with EGFR-TKI. During our clinical practice, we found that there is a relationship between the expression level of CDK2AP1 and the acquired drug resistance triggered by EGFR-TKI. We proposed that CDK2AP1 could play an important role in EGFR-TKI induced acquired drug resistance by modulation of cell cycle. In our previous study, we have already demonstrated that CDK2AP2 functions as a tumor suppressor gene in non-small cell lung cancer and its downregulation is necessary for EGFR-induced acquired drug resistance. We plan to elucidate the the relationship between the expression level of CDK2AP1 and acquired drug resistance on the samples from patients suffered with non-small cell lung cancer via tissure array. Furthermore, we hope to find potential drug targets and the methods which can overcome acquied drug resistance by investigation of the downstream signaling pathway induced by EGFR-TKI treatment.

近年来，分子靶向治疗药物EGFR-TKI因其在非小细胞肺癌中的显著疗效而备受关注。然而，接受EGFR-TKI治疗过程中大部分患者出现获得性耐药，成为其在临床应用中的瓶颈。我们在临床工作中发现CDK2ap1与EGFR-TKI产生获得性耐药关系密切，结合文献分析，推测该基因通过细胞周期调控，在EGFR-TKI的获得性耐药机制中发挥着重要作用。前期工作中，我们已明确CDK2AP1基因在肺癌中的抑癌基因功能表型，同时，其表达缺失对于EGFR-TKI诱导非小细胞肺癌获得性耐药具有关键影响。本项目拟在前期工作基础上应用组织芯片对产生EGFR-TKI获得性耐药的肺癌患者病理标本回顾性分析CDK2AP1表达与耐药的相关性，并观察EGFR-TKI处理前后CDK2ap1及其下游信号的变化，深入探讨CDK2AP1作用的分子机制，从而为寻找新的药物作用靶点和耐药逆转机制提供可靠的理论依据。

CDK2ap1基因是细胞内广泛存在并在进化中高度保守的基因，与周期素依赖性蛋白激酶CDK2密切相关。研究表明CDK2ap1的过量表达可以抑制CDK2相关激酶的活性，影响细胞周期，藉此抑制肿瘤细胞的生长。在人类不同恶性肿瘤细胞中CDK2ap1对其增殖具有显著的抑制作用。在本课题中我们克隆了CDK2AP1的全长cDNA，制备了过表达和RNAi的慢病毒载体，通过MTT实验发现在A549细胞中上调CDK2AP1后，细胞增殖显著减慢，而抑制CDK2AP1后，功能相反，同时我们应用流式细胞仪技术，发现其在肺癌细胞的周期调控中具有重要功能。通过肿瘤细胞耐药实验，我们发现CDK2AP1过表达通过阻滞G1/S期细胞周期转换，部分逆转肿瘤细胞的耐药机制。为了进一步探求CDK2ap1在肺癌细胞中的功能及其下游信号通路，并从中找出新的药物作用靶点，我们在本课题中在体内、体外水平观察CDK2ap1基因表达对恶性表型的影响并深入探讨了相关的分子机制；同时，应用组织芯片回顾性分析了CDK2ap1与肺癌患者临床相关性，为改善非小细胞肺癌的临床治疗提供了可靠的理论依据。