P27kip1/CDK通路介导肺癌EGFR-TKI耐药新机制的研究

Acquired resistance to EGFR-TKI is the main problem for targeted therapy in non small cell lung cancer, therefore it is critical to explore the mechanism of drug resistance and develop the approach to reverse it. Our preliminary works indicated that P27kip1 dysfunction was the key molecular event and P27kip1/CDK pathway was a potential target to reverse EGFR-TKI acquired resistance. However little is known about the mechanism of P27kip1/CDK in EGFR-TKI resistance. Hence we plan to study the mechanism of P27kip1/CDK guided EGFR-TKI resistance and precise therapy. Firstly, we propose to demonstrate the role and mechanism of P27kip1 dysfunction in EGFR-TKI resistance. Subsequently, we will analyze the expression heterogeneity of P27kip1/CDK pathway and the mechanism of its inherent regulation in EGFR-TKI resistance, and validate the targets of CDK inhibitors in vitro. Finally, we plan to determine the role of P27kip1/CDK pathway in EGFR-TKI resistance in clinical samples by immunohistochemistry and verify the feasibility of targeting CDK for reversing acquired resistance to EGFR-TKI in vivo. Our study will help to demonstrate the mechanisms of acquired resistance to EGFR-TKI and provide the basis of a possible therapeutic strategy for lung cancer.

EGFR-TKI耐药制约了晚期NSCLC的有效治疗，探索耐药分子机制、寻求对抗耐药措施成为该研究领域的重要课题。前期工作提示P27kip1功能失调是多种肺癌EGFR-TKI耐药细胞共同的分子事件，而其发生机制及异质性表达对下游CDK亚型的调控作用仍需进一步探究。结合前期基础，我们提出科学假说：P27kip1功能失调异质性调控CDK参与肺癌EGFR-TKI耐药并指导靶向CDK精准干预。为验证该假说，本项目拟首先在分子水平阐明P27kip1功能失调在EGFR-TKI耐药中的发生机制；随后体外实验分析P27kip1/CDK通路内部调控异质性，明确CDK抑制剂有效干预靶点；最终分别在临床样本及动物体内验证P27kip1/CDK通路在EGFR-TKI耐药中的作用及基于该通路异质性为指导的CDK干预有效性评估。本研究有助于进一步阐明肺癌EGFR-TKI耐药的分子机制，为寻求耐药后治疗策略提供新思路。

表皮生长因子受体（epidermal growth factor receptor, EGFR）酪氨酸激酶抑制剂（Tyrosine kinase inhibitor, TKI）是存在EGFR敏感性突变晚期非小细胞肺癌（Non-small cell lung cancer，NSCLC）的主要治疗药物。但靶向药物的耐药不可避免，因此探索EGFR-TKIs获得性耐药的分子机制，寻求对抗耐药措施是NSCLC治疗领域的重要研究课题。我们的前期研究发现P27功能失调是多种EGFR-TKIs耐药机制共同的下游分子特征，但其发生机制及对下游CDK亚型的调控作用仍不明确，有必要深入研究。为此本项目通过体外实验明确P27/CDK通路信号分子在EGFR-TKIs耐药细胞中的异质性表达情况，通过临床样本研究分析了上述因子与EGFR-TKIs耐药的相关性。体外研究针对P27下游CDK/Cyclins异质性表达的细胞应用针对性的CDK抑制剂对磷酸化Rb具有明显抑制作用，并产生不同程度的细胞生长抑制作用。我们进一步从分子水平阐明细胞核PKCδ表达是导致P27功能失调的关键因素。在该课题的支持下，项目组进一步开展逆转EGFR-TKIs耐药的药物筛选工作。文献挖掘及初期细胞毒性实验提示一系列姜黄素二芳基庚烷衍生物能明显抑制原发及获得性EGFR-TKIs耐药细胞生长，体内动物实验证实了该化合物的有效性和安全性。进一步体外实验提示该类化合物能够明显抑制EGFR-TKIs耐药细胞PKCδ及EGFR总蛋白表达。本项目研究从P27/CDK参与EGFR-TKIs耐药机制出发，结合肿瘤细胞异质性的特点，通过将细胞核PKCδ与P27/CDK通路调控相结合，最终筛选能够逆转EGFR-TKIs耐药的潜在药物。研究成果有助于进一步揭示EGFR-TKIs耐药分子机制，为肺癌EGFR-TKIs耐药治疗提供理论基础，具有重要的转化医学意义。