CD44调控肝癌细胞侵袭伪足形成及其机制的研究

Hepatocellular carcinoma (HCC) is a disease with high morbidity. There are about 300,000 people die of HCC each year, in China. Metastasis of HCC is the main reason responsible for high mortality of HCC, which can not be effectively controlled by regular treatment. Therefore, it is of critical importance to understand the mechanisms of invasion and metastasis of HCC, and to explore a more effective therapeutic target for HCC treatment. CD44, a single transmembrane protein, mediate the interaction between cells and extracellular matrix. Recent studies show that CD44 is involved in HCC progression, and is associated with metastasis and prognosis after local ablation therapy. In this project, we will explore the role and the potential mechanism of CD44 alternative splice variants (CD44s or CD44v) in invadopodia formation of HCC cells, and determine which CD44 isoform plays the main role in invadopodia formation. Furthermore, we will define the interaction between CD44 and Integrin β1, CD44 and Annexin II, and try to block the above interaction to analyze effects of CD44 on invadopodia formation and on transition between focal adhesions (FAs) and invadopodia formation. This study will focus on Integrin β1/FAK/Src and Annexin II/PIP2/Tks5 signal pathway. We will try to block Integrin β1/FAK/Src signal pathway by knockdown Integrin β1 or overexpression focal adhesion kinase (FAK) mutant,to determine whether CD44 regultes FAs-invadopodia transition through Integrin β1/FAK/Src signal pathway. We will also block Annexin II/PIP2/Tks5 signal pathway by knockdown Annexin II or overexpression CD44 mutant which was abolished of Annexin II-binding ability, to analyze the mechanism of PI(3,4)P2 recruitment which promotes initiation of invadopodia formation.

肝癌发病率高，侵袭转移是肝癌致死的主要原因，现有治疗措施尚不能有效控制肝癌侵袭转移，因此深入探讨其机制，寻找有效的治疗靶点是肝癌治疗研究领域的热点。近期研究表明，CD44分子参与肝癌的发生发展过程，与肝癌细胞侵袭转移和肝癌手术预后密切相关。本项目拟对CD44选择性剪切变异体(CD44s和CD44v)在肝癌细胞侵袭伪足形成中的作用及其机制进行系列研究，明确参与侵袭伪足形成的CD44剪切变异体类型；并以CD44与Integrin β1和Annexin II 相互作用为切入点，通过阻断Integrin β1信号通路及CD44和Annexin II的相互作用，分析CD44在侵袭伪足始发过程中黏着斑和侵袭伪足相互转换中的作用；围绕Integrin β1/FAK/Src和AnnexinII/PIP2/Tks5两条信号通路解析CD44促进肝癌细胞侵袭伪足形成的分子机制，为寻找有效的肝癌治疗靶点奠定基础。

肝癌是严重影响人类健康的恶性疾病，居世界肿瘤死因第三位。尽管以外科手术切除为基础的 综合治疗在原发性肝癌的治疗上已经取得了长足进展。但肝癌患者的总体5年生存率仍较低。侵袭和转移是肝癌致死的重要原因。因此，揭示肝癌细胞侵袭转移的细胞和分子机制，寻找更为有效的治疗手段是肝癌研究领域的热点内容之一。近期研究表明，CD44 分子参与肝癌的发生发展过程，与肝癌细胞侵袭转移和肝癌手术预后密切相关。. 本项目对CD44 选择性剪切变异体(CD44s 和CD44v)在肝癌细胞侵袭伪足形成中的作用及其机制进行系列研究，明确了CD44s是参与肝癌细胞侵袭伪足形成的主要CD44剪切变异体类型；下调CD44分子能抑制细胞PTPRB的表达，影响Src 527位酪氨酸磷酸化，进而促进Src的活化。然而CD44分子表达下调后会通过Integrin β1/FAK信号通路，上调FAK 397酪氨酸磷酸化。而磷酸化后的FAK则在空间上束缚了活化的Src，因此，下调CD44表达后，虽然活化的Src分子增多，但由于活化的Src均被磷酸化的FAK束缚于黏着斑部位，从而无法发挥其促进侵袭伪足的功能。由此可见，当下调CD44表达时，黏着斑无法解聚释放出活性Src，进而抑制了侵袭伪足的形成过程。 . 本实验还采用免疫共沉淀的方法证实了在CD44分子能与钙离子依赖的膜磷脂结合蛋白Annexin II分子发生相互作用。而AnnexinII分子为PI(4,5)P2的结合蛋白，且PI(4,5)P2的结合蛋白Dynamin 2能通过加速Integrin β1内化，而促进黏着斑的解聚。因此我们推测下调CD44分子后Annexin II分子不能与CD44分子相结合，因此间接降低了PI(4,5)P2及其结合蛋白Dynamin2在黏着斑周围的聚集，进而降低了黏着斑的解聚速度，使黏着斑更为稳定。此外沉默CD44能显著抑制Annexin II分子酪氨酸磷酸化。通过免疫共沉淀实验发现，下调CD44分子表达后，与pY397-FAK结合的Annexin II蛋白显著减少，提示CD44分子通过与Annexin II结合，促进并保护AnnexinII的磷酸化，进而募集PI(4,5)P2及其结合蛋白Dynamin2到达黏着斑部位，从而加速黏着斑的解聚。