Axl在肝癌中活化的机制及其作为治疗靶点应用性的研究

RTKs has been found to be a crucial molecule contributing to tumor invasiveness.The mechanisms of metastasis and recurrence of HCC are still unclear. Axl receptor are a member of TAM RTKs subfamily. Mammals have three receptors: Tyro, Axl and Mer.These receptors are activated in most contexts by Gas6 and PS ligands, Gas6 is also a protein containing four epidermal growth factor-like repeats which is likely hydroxylated by aspartyl (asparagynal)-β-hydroxylase(ASPH). Axl signaling is initiated when two Gas6 ligands interacts with two Axl transmembrane receptors expressed on an cell. This interaction triggers the tyrosine phosphorylation of the a tyrosine kinase domain at C terminus of Axl, allowing transduction of a serial of signals. Within the endochylema, , as well as to mastermind and other transcriptional regulatory proteins to form a complex, resulting in phosphorylation and coactivation of Axl downstream target proteins and thereby regulating various cellular processes, including proliferation,migration and antiapoptosis. We hypothesized that the receptor Axl may precipitate the invasion and metastasis of cacer cells of hepatocellular carcinoma cells.In order to identify this presumption, firstly,this study will investigate the over-expression and prognosis prediction of Axl in the membrane of HCC by tissue microarray (TMA) of paired HCC.Secondly,to confirm the pathway of Gas6 and ASPH involves in the Axl activation in HCC,we further assess the possible correlation of Gas6 coexpression with Axl ,the different invasiveness of HCC cell line with Gas6,Axl a high-/low-expression level,and the diverse metastasis stations of transplantation tumors in nude mice by Axl transfectants and the study of RNA interference .At the same time, to determine the upstream pathway of Axl in HCC and the the possible role of ASPH in Gas6/Axl binding,we will evaluate the variations of Gas6/Axl binding by up-/down-regulation of ASPH and antibody of Gas6.In the end,we then may find that the signal network of HCC metastasis and invasion mediated by Axl and the new therapeutic target in the cell membrane.

受体酪氨酸激酶家族成员与肿瘤侵袭转移具有明确相关性，肝癌的转移复发机制尚不清楚。我们推测，受体酪氨酸激酶Axl被其配体Gas6激活，而Gas6需接受ASPH的羟化修饰，然后Axl信号途径活化并参与促进肝癌细胞的侵袭转移。为证实此推测，本研究将首先证实肝癌细胞的ASPH、Axl与Gas6过表达状态，及与此平行的肝癌侵袭转移性增强及更差预后。其次，上调表达Axl及Gas6，观察Axl信号途径激活前后肝癌细胞Axl、Gas6和下游信号蛋白变化。第三，分别上调和下调ASPH，观察ASPH上调和ASPH表达抑制前后，Gas6表达及其与Axl结合能力、Axl下游信号蛋白活性、肝癌细胞侵袭转移能力的变化，同时进行相应裸鼠体内实验及信号蛋白检测。第四，上调ASPH并产生针对Gas6受ASPH修饰的羟化区抗体，进行肿瘤侵袭性体内外抑制试验。最终通过比较分析，初步揭示Axl信号通路介导肝癌细胞侵袭转移的机制。

本项目以分析Axl促进肝癌发展的分子机制为主要研究目标，力图为全面认识 Axl促进癌细胞转移这一重要问题提供重要线索。在具体课题研究内容上，包括了肝癌细胞增殖、迁移等的重要环节，包括Axl在肝癌表达的特征和规律，肝癌受Axl调节的分子机制，受Axl影响的分子调控机理、重要信号分子的致癌机制等。我们以实时定量RT-PCR、Western blot和免疫组化的方法，从mRNA和蛋白水平证实了Axl在肝癌组织和肝癌细胞系中的表达比正常肝组织和正常肝细胞系中的表达高，与已有的报道一致。Axl的高表达与肝癌的侵袭表型相关，即与血清AFP升高、肿瘤多发, 包膜不完整, 微血管侵犯, 较晚的BCLC或者 TNM分期相关，同时通过结合无瘤生存率的分析，发现Axl高表达和肝癌根治性切除术后无瘤生存率差相关，提示Axl高表达可能与肝癌转移复发相关。而通过实验证实， Axl敲减引起的肝癌细胞迁移能力下降； 腺病毒表达法Axl表达上调，引起肝癌细胞MAPK、PI3K表达增加。因此，Axl可能参与肝癌的MAPK/PI3K信号作用途径，来影响肝癌细胞侵袭、迁移、转移，在转移形成过程中起到了重要的作用。