肝星状细胞在细胞外基质高硬度微环境中活化并分泌COMP促进肝细胞癌进展的机制研究

Extracellular matrix stiffness-related microenvironment, resulted from liver cirrhosis, is the main reason for the initiation and progression of hepatocellular carcinoma (HCC). However, the underlying mechanisms remain largely unknown. Our published data proved that hepatic stellate cells (HSCs) play an important role in the liver tumor microenvironment (Hepatology, 2015). Our previous studies found that matrix stiffness induced HSCs activation and COMP secretion, and P300 exerted an critical regulatory role in this process. High level of serum COMP indicated a poor prognosis of HCC patients. In addition, recombinant human COMP promoted the activation of PI3K/AKT and MEK/ERK pathways, and facilitated HCC cell growth and metastasis. Thus, clinical and biological experiments will be performed to verify the following scientific hypothesis that P300-RhoA positive feedback loop maintains HSCs activation and promotes COMP secretion in extracellular matrix stiffness-related microenvironment. HSCs-sourced COMP binds to membrane receptor CD36 and activates PI3K/AKT and MEK/ERK pathways, and subsequently promotes HCC cell growth and metastasis. Furthermore, high level of serum COMP is an independent prognostic factor for predicting poor prognosis of HCC patients. Meanwhile, the anti-HCC effect of the specific COMP monoclonal antibody will be investigated in a mouse model. Altogether, our project is aimed to provide the basis for the development of new HCC targeted therapy drugs.

肝硬化所致的细胞外基质高硬度微环境是导致肝细胞癌（HCC）发生发展的关键因素，但具体机制目前仍很不清楚。我们已发表成果证实肝星状细胞（HSCs）在肝脏肿瘤微环境中发挥重要作用（Hepatology,2015）。我们前期发现①高基质硬度诱导HSCs活化并分泌COMP蛋白，P300在此过程中发挥关键调控作用；②血清COMP高表达提示HCC患者预后不良；③重组人COMP激活PI3K/AKT和MEK/ERK通路并促进HCC细胞生长和侵袭转移。因此，我们拟通过临床和功能学实验证实如下科学假说：细胞外基质高硬度微环境中P300与RhoA形成正反馈环路促进HSCs活化并分泌COMP蛋白，HSCs源性COMP与膜受体CD36结合并激活PI3K/AKT和MEK/ERK通路，从而促进HCC细胞生长和侵袭转移。同时，本项目拟研究特异性COMP单抗在动物体内抗HCC作用，为临床上开发新HCC靶向治疗药物提供依据。

2020年全球肝癌新发病例达91万，其中45%来源于我国。手术切除仍然是肝癌治疗的最佳选择，但大部分患者初诊即为晚期，丧失了手术机会。靶向治疗和免疫治疗的应用给部分晚期肝癌患者带来了曙光，但对于整体预后改善不显著。在所有肝癌中，80%-90%发生在纤维化或肝硬化中。肝纤维化是一个动态过程，其特征是由任何病因的慢性肝损伤引起的细胞外基质（ECM）的净积累。在细胞水平上，激活的肝星状细胞（HSCs）是产生ECM的主要效应细胞。ECM重组和过度产生导致肝脏硬度增加，临床上肝脏硬度已作为预测肝癌发生、进展和预后的指标。既往研究证实基质硬度通过机械信号通路（如YAP/TAZ通路或SRF通路）促进肝癌细胞增殖、侵袭、化疗抵抗和有氧糖酵解等。综上所述，基质硬度微环境是肝癌的重要特征，也是肝癌发生发展的关键驱动因素，但是基质硬度通过何种分子机制发挥促肝癌效应，目前仍有待进一步深入研究。本项目发现HSCs分泌的COMP蛋白与肝癌细胞表面CD36受体结合，激活MEK/ERK和PI3K/AKT信号通路，从而上调EMT调节因子Slug和Twist以及MMP-2/9，进而促进肝癌细胞增殖、迁移和侵袭。证实内源性抗炎脂质介质RvD1可以阻断HSCs诱导的肝癌细胞生长和转移，RvD1通过结合FPR2减少ROS和FOXM1表达，导致COMP启动子区域募集的FOXM1减少，最终导致HSCs分泌的COMP蛋白降低及其促肝癌效应减弱。明确HSCs塑造的细胞外基质硬度通过诱导CXCR4表达促进肝癌细胞增殖、EMT及干性。基质硬度通过CXCR4降低UBTD1水平，破坏了UBTD1/UbcH5c/β-TrCp复合体介导的YAP泛素化蛋白酶解，导致YAP靶基因及其下游信号通路激活。本研究揭示了HSCs与肝癌细胞相互作用新模式及分子机制，有利于发现抑制HSCs活化和阻断促肿瘤肝脏微环境的新靶点。同时，阐明了基质硬度对肝癌细胞生物学行为的影响及新调控机制，有助于从新角度探索肝癌治疗的新策略。