基于ILK/Hippo通路的黄酮类新化合物LFG-500 抗肿瘤上皮间质转化研究

There has been no report in clinical setting about an agent with excellent abilities against tumor invasion and metastasis. According to recent studies, integrin-linked kinase (ILK) is essential for tumor metastasis, detailing its abilities to mediate the inactivation of the Hippo pathway and accelerate epithelial-mesenchymal transition (EMT) in tumor. However, few medications are reported to prevent tumor metastasis via the ILK/Hippo cascade. LFG-500 is a novel synthesized flavonoid with strong anti-metastasis activity, whose molecular mechanism however remains incompletely understood. Our previous researches demonstrated that LFG-500 could inhibit EMT, regulate the activities of key molecules involved in the Hippo pathway, and suppress the expression of ILK. These data indicate that LFG-500 may inhibit EMT in tumor via the ILK/Hippo pathway. Therefore, the present study is designed to investigate the inhibitory effects of LFG-500 on tumor EMT at molecular, cellular, and whole-animal levels. Meanwhile, we will explore the regulatory effects of LFG-500 on ILK/Hippo pathway, in order to clarify the mechanism by which the agent prevents EMT in tumor and search for possible drug targets with potent activities. This study will provide substantial theoretical supporting for the anti-tumor activities of LFG-500 and other flavonoids. Moreover, the results will contribute greatly to the research and development of more novel anti-metastatic drugs.

迄今临床上仍没有一种理想的抗肿瘤侵袭转移药物。最新研究表明，整合素连接激酶（ILK）可介导Hippo信号通路失活，促进肿瘤上皮间质转化（EMT），在肿瘤转移过程中发挥重要作用，而基于ILK/Hippo通路的抗肿瘤转移药物研究未见报道。LFG-500是自主合成的全新黄酮类化合物，前期研究发现其具有显著的抗肿瘤转移活性，但作用机制有待阐明。预实验显示LFG-500能抑制肿瘤EMT、调节Hippo通路中关键分子的活性、抑制ILK表达，提示LFG-500可能通过影响ILK/Hippo信号通路而抑制肿瘤EMT。据此，本课题拟在整体、细胞及分子水平全面研究LFG-500对肿瘤EMT的抑制作用，并以ILK/Hippo通路为切入点，探讨LFG-500对该通路的调控作用，阐明抗肿瘤EMT机制，寻找可能的药物作用靶点，为LFG-500乃至黄酮类化合物的抗肿瘤研究提供依据，并为抗肿瘤转移药物研发提供新的思路。

迄今临床上仍没有一种理想的抗肿瘤转移药物。最新研究表明，整合素连接激酶（ILK）可介导Hippo信号通路失活，促进肿瘤上皮间质转化（EMT），在肿瘤转移过程中发挥重要作用，而基于ILK/Hippo通路的抗肿瘤转移药物研究未见报道。本项目研究发现，黄酮类新化合物LFG-500可调控肿瘤EMT过程中细胞表型变化的关键指标分子E-cadherin、ZO-1、N-cadherin和vimentin的表达，抑制肿瘤EMT，继而发挥抗肿瘤转移活性。机制研究发现，LFG-500可提高Lats1/2、YAP的磷酸化水平，并减少YAP和TAZ的表达，促进Hippo通路激活。此外，LFG-500可下调Hippo通路关键转录因子TEAD 4的转录活性，并抑制EMT相关核转录因子Slug、Snail和ZEB 1的表达，继而抑制肿瘤EMT。而过表达YAPS127A使YAP处于持续活化状态，可阻断LFG-500对Hippo通路的活化作用及其抗肿瘤EMT作用，提示Hippo通路在LFG-500抗肿瘤EMT过程中发挥关键作用。进一步采用Sybyl-x2.1软件进行对接分析，结果发现LFG-500可与ILK形成稳定的相互作用，具有保守的氢键与疏水相互作用，提示LFG-500可能是ILK潜在的抑制剂。同时LFG-500可下调ILK的表达，而过表达ILK可阻断LFG-500对肿瘤细胞迁移能力的抑制作用，并逆转LFG-500对Hippo通路的激活，进而阻断LFG-500的抗肿瘤EMT作用，提示LFG-500可通过抑制ILK继而激活Hippo通路，发挥抗肿瘤EMT作用。A549细胞尾静脉接种肺转移模型结果显示，LFG-500治疗组可显著减少肺部肿瘤结节以及大转移灶数，且LFG-500治疗组小鼠肺转移结节中E-cadherin的表达水平升高，而N-cadherin、YAP和ILK的表达水平显著降低，表明LFG-500抗肿瘤转移作用与其调控ILK/Hippo通路有关。且异种移植肿瘤模型结果显示，LFG-500具有较好的抗肿瘤生长活性。本研究明确了LFG-500对肿瘤EMT的抑制作用，并以ILK/Hippo通路为切入点，阐明其抗肿瘤EMT机制。相关工作为LFG-500乃至黄酮类化合物的抗肿瘤研究提供依据，并为抗肿瘤转移药物研发提供新的思路。