中药提取物芫花烯靶向调控Hippo信号通路逆转肝癌索拉非尼耐药的作用及机制研究

Based on the Chinese medicine theory combined with modern biological technology, exploring the molecular mechanism of hepatocellular carcinoma sorafenib resistance (HSR) and finding drug intervention is the key to reverse the drug-based treatment strategies. Hippo signaling pathway was reported to relate to the proliferation and invasion of hepatocellular carcinoma, as well as stem cell characteristics. However, whether Hippo pathway is involved in HSR process is still unknown. In our preliminary study, we found Hippo signaling pathway activity was increased in HSR cells, and predicted the patient's treatment response and prognosis. Meanwhile, the traditional Chinese medicine extract, Genkwadaphnine, was found not only can suppress Hippo signaling pathway but also reverse the HSR. However, it is need to clarify the role of Hippo signaling pathway in HSR, and the underlying mechanisms of Genkwadaphnine targeting Hippo signaling and therapeutic value of Genkwadaphnine on HSR also need to further be illustrated. Therefore, using multiple molecular biology methods, we plan to: 1)to further explore the role of Hippo pathway in HSR process, providing a clear target for reversing HSR; 2) to investigate the underlying regulation mechanisms of Genkwadaphnine on Hippo signaling pathway and clarify its characteristic of reversing HSR, which will enrich the connotation of Traditional Chinese Medicine Daphne genkwa and establish the pharmacological basis for developing Genkwadaphnine as a new drug of reversing HSR.

基于中医药理论结合现代生物学技术探索肝癌索拉非尼耐药（HSR）的分子机制及干预药物是寻找逆转耐药治疗策略的关键。Hippo信号通路被发现与肝癌的增殖、侵袭转移及干细胞特性的维持密切相关。然而，该通路是否参与HSR过程还不得而知。我们前期研究发现，Hippo信号通路在HSR细胞中活性增强，且与用药患者的反应性和预后相关，而中药芫花提取物芫花烯则可抑制Hippo信号通路，并能逆转HSR。然而，对于Hippo信号通路在HSR过程中的角色和以芫花烯分子靶向调控该通路的确切机制和治疗意义等问题还亟待阐明。为此，本课题拟采用多种分子生物学方法：1）进一步探索Hippo通路在HSR过程中的角色地位，为靶向逆转HSR策略提供明确的切入点和靶标。2）深入探究芫花烯靶向调控此信号通路的分子机制，并阐明其逆转HSR的作用特点，从而为丰富中药芫花的药用内涵，并将芫花烯开发为新型逆转HSR的药物奠定药理学基础。

近年来，从传统草药中发现生物活性天然产物已成为药物研究和开发的重要资源。本研究中，我们从瑞香属植物芫花中分离鉴定了瑞香烷型二萜类化合物芫花烯Genkwadaphnin（GD）在肝癌细胞中显示出显著的体内体外抑制肝癌细胞生长和转移的作用。索拉非尼是第一个FDA批准用于肝癌患者治疗的一线靶向药物，索拉非尼耐药是影响其临床疗效的主要原因。我们的研究结果提示索拉非尼耐药细胞表现出对索拉非尼YAP蛋白表达增加并出现核聚积，抑制YAP可增加肝癌细胞对索拉非尼的敏感性并且减少肝癌细胞的侵袭转移和上皮间质转化（EMT）特性。同时，我们发现一些瑞香烷型二萜类化合物可以抑制YAP荧光素酶转录活性，其中GD在两种肝癌细胞系中均具有显著的YAP-TEAD转录活性抑制作用。我们对GD干预的Hep3B细胞进行了基因芯片测试，通过GO分析、KEGG通路分析富集了Hippo信号通路。进一步的体外和在体动物实验研究表明GD可以通过激活Hippo信号通路有效失活YAP，减少细胞的侵袭和EMT。且GD联合索拉非尼使用可通过调控Hippo信号和逆转EMT克服肝癌细胞对索拉非尼耐药。本研究结果为丰富GD的抗肿瘤药理学作用提供了依据，为探寻肝癌索拉非尼的耐药机制及临床逆转策略提供了新的思路。