肝内胆管癌(ICC)是恶性程度极高的原发性肝癌，淋巴结转移是最重要的恶性表现之一，机制未知。既往发现天冬氨酰-(天冬酰胺)-β羟化酶(AAH)在ICC中高表达，但是否参与ICC淋巴结转移及相关机制尚无研究。基于之前对AAH羟化酶活性的工作，我们推测该酶可能介导ICC的淋巴道转移。本项目将首先分析AAH羟化酶活性与ICC侵袭性病理特征(尤其是淋巴结转移)及临床预后的关系；然后对AAH进行不同基因操作(过表达、突变和RNA干扰)，观察人ICC细胞促淋巴管新生和淋巴结转移能力的变化，及AAH酶活区域的封闭抗体和酶活抑制剂对该能力的抑制作用，以证实推测。其次，通过过表达、敲减、稳定激活分子导入和免疫共沉淀等方法，明确AAH结合GSK-3β，使后者持续激活，并作用于下游分子p16和VEGF-C/D，促进ICC细胞的淋巴道转移。本项目将部分揭示ICC淋巴道转移的重要机制，为靶向干预提供靶点。

Intrahepatic cholangiocarcinoma (ICC) is a primary liver cancer with aggressive invasiveness which results in a high rate of lymphatic spread, contributing to poor clinical prognoses. The mechanism of ICC lymphatic spread remains largely unknown. Previous studies have demonstrated that aspartyl-(asparaginyl)-β-hydroxylase (AAH) is specifically and highly expressed in ICC cells. However, whether its hydroxylase activity is required for ICC lymphatic metastasis is still unclear. Based on our previous studies on the oncological function of AAH, we hypothesize that the activity of AAH might be critical for the lymphatic metastasis of this malignancy. To verify this, we will firstly observe the association between hydroxylase activity of AAH and aggressive clinicopathological features (particularly lymph node metastasis) and surgical prognosis in ICC patients. Then, we will transfect the human ICC cell lines with wild-type, mutant AAH and AAH RNAi vectors and compare their capability in invasion, lymphangiogenesis and lymphatic metastasis in vitro and in vivo. In addition, we will also analyze the inhibitory effect on invasion, lymphangiogenesis and lymphatic metastasis by the antibodies specific for AAH catalytic domain associated with its hydroxylase activity and the specific hydroxylase inhibitors. Secondly, we will adopt the approaches including gene knock in/out, overexpresssion of constitutively active molecule and co-immunoprecipitation to identify the downstream signal pathways of AAH which mediate ICC lymphatic metastasis. The possible pathway is that AAH overexpression is followed by binding to glycogen synthase kinase 3β and keeps it constitutive active, which leads ICC lymphangiogenesis and lymphatic metastasis by promoting degradation of p16 and upregulation of vascular endothelial growth factor-C/D. This study will reveal the mechanism that involved in the ICC lymphatic spread and develop a new therapeutic target.