缺氧调控肝癌YAP信号通路介导巨噬细胞M2型极化的机制研究

Macrophages are educated by tumor cells to be M2-like phenotype, which is critical for hypoxia drived metastasis of heptocarcinoma. How the heptocarcinoma cells obtain stronger ability of education in hypoxia remains exclusive, which is a pressing problem to be solved. Our previous studies have found for the first time that the aberrant activation of YAP by hypoxia plays a key role in hypoxia triggered M2 polarization of macrophages. NEDD8 is involved in hypoxia drived activation of YAP. Based on these previous studies, we will further clarify the relationship between neddylation and YAP activation in hypoxia, discover the key regulators during the modification, find the downstream secretory protein that participate in M2 polarization stimulated by hypoxia. We will also discuss the feasibility of anti-metastasis by targeting M2 polarization through interfere neddylation of YAP in heptocarcinoma. Our study will elucidate the underlying mechanisms responsible for YAP activation in hypoxia, discover the new biological functions of YAP in macrophages education, and enrich the molecular mechanisms of hypoxia drived metastasis, in the hope to provide new targets for the design and discovery of novel anti-metastasis drugs and potential therapeutic targets for anti-metastasis therapy.

缺氧环境下，肝癌细胞“驯化”巨噬细胞成为M2型是缺氧促肝癌转移的关键因素之一。但肝癌细胞如何在缺氧环境中获得更强的“驯化”能力尚不清楚，是该领域亟需解决的问题。申请人前期研究发现肝癌细胞中YAP通路的缺氧异常激活可能是其促巨噬细胞M2型极化的关键，而缺氧下YAP蛋白Neddylation修饰水平的变化是导致YAP通路异常激活的关键因素。本课题将在此全新发现的基础上，进一步研究Neddylation修饰如何调控YAP通路的异常激活，发现调控该修饰的关键分子，探索缺氧YAP激活后肝癌细胞“驯化”巨噬细胞的关键分泌蛋白，探讨通过调控YAP蛋白Neddylation修饰从而干预巨噬细胞M2型极化成为抗肝癌转移新策略的可能性。本课题将阐明YAP缺氧异常激活的全新机制，发现YAP蛋白“驯化”巨噬细胞的新生物学功能，揭示缺氧促肿瘤转移的新分子机制，为抗肝癌转移的药物研究和临床治疗提供新靶点和新策略。

肝癌细胞“驯化”肿瘤相关巨噬细胞在肝癌发生发展过程中发挥关键作用。但肝癌细胞如何获得更强的“驯化”能力尚不明确，是该领域的研究热点。本项目研究发现，缺氧微环境促进肝癌细胞的YAP蛋白显著入核，发挥启动下游靶基因转录的作用；肝癌细胞高表达YAP可显著增加巨噬细胞的募集和迁移作用；敲低YAP后，HCC募集巨噬细胞的能力则显著减少；提示YAP的表达水平对于HCC细胞募集巨噬细胞能力至关重要。进一步研究发现NEDD8过表达显著抑制YAP蛋白的水平及下游靶基因的表达，而Neddylation抑制剂MLN4924则明显促进YAP蛋白的表达和下游靶基因激活，说明Neddylation修饰是调控YAP通路活性的关键因素。深入研究发现YAP蛋白的E3泛素连接酶SCFβ-Trcp复合物中的Cullin-1蛋白的Neddylation修饰改变是该过程的关键因素。我们发现，当肿瘤细胞处于缺氧、代谢酸刺激等异常微环境中，Cullin-1蛋白的Neddylation修饰显著减少，从而妨碍SCFβ-Trcp复合物的组装，导致该E3泛素连接酶复合物与YAP蛋白的结合大大减少，使得YAP蛋白稳定性增加而持续激活。Neddylation造成的类似调控在YAP的旁系同源物（Paralog）TAZ蛋白也观察到，我们推测，主要原因可能是由于调控TAZ的E3泛素连接酶也是E3泛素连接酶SCFβ-Trcp复合物。进一步我们采用细胞因子抗体芯片发现YAP过度激活的肝癌细胞主要通过分泌IL-6，导致肿瘤相关巨噬细胞的募集。而可阻断YAP核转位的他汀类化合物可以通过抑制YAP激活，减少肝癌细胞对巨噬细胞的募集作用，提示靶向该通路可能是干预肝癌恶性演进的潜在策略。