AAH羟化酶活性介导肝内胆管癌的经淋巴途径转移及分子机制

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.

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。

肝内胆管癌（ICC）是第二常见的原发性肝癌，疗效极差，肝切除术是相对早期ICC 的主要治疗手段。ICC表现高度的周围肝组织侵犯特性和经淋巴转移特征，其淋巴结转移率远远超过肝细胞癌，区域淋巴结转移是最重要的预后影响因素。既往发现天冬氨酰-(天冬酰胺)-β羟化酶(ASPH)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。本研究在106例ICC患者的肿瘤组织中证实高表达ASPH者更易出现肿瘤的淋巴途径转移，且预后不良。我们在体外实验中发现ASPH与ICC的侵袭和迁移能力呈现正相关，通过对上皮细胞-间充质转化标记物的检测，明确了过表达ASPH对ICC细胞的上皮细胞-间充质细胞转化（EMT）有促进作用。同时ASPH对ICC细胞系的GSK-3β磷酸化有抑制作用，但可促进Sonic Hedgehog信号通路的激活。进一步的机制探讨发现，在裸鼠肿瘤转移模型中，验证了过表达ASPH通过下调ICC细胞GSK-3β的磷酸化，激活Sonic Hedgehog信号通路，促进ICC细胞的EMT过程，最终促进肿瘤的转移。在对双中心的296例接受了根治性肝切除和淋巴结清扫的ICC患者分析，通过多因素Logistic回归分析将CT所示肿瘤位置、直径、血管侵犯和肝周肿大淋巴结的大小纳入临床-影像模型。采用经过机器学习和mRMR方法从四期CT图像中筛选出的6、8、5和5个代表性特征，使用Logistic回归分析构建融合模型。进一步基于Logistic回归，联合临床-影像模型中的变量和CT影像组特征融合模型，构建联合列线图预测模型。Hosmer-仿真验证检验结果表明，联合列线图预测所得到的淋巴结转移概率与实际结果之间的一致性优于Fujian模型、临床-影像模型、CT影像组特征融合模型等模型。DeLong检验与决策曲线分析显示显示，联合列线图的表现最好。本研究明确了ASPH对ICC转移的促进作用，阐释了其调控GSK/Shh信号通路的机制，建立了基于影像学组数据的ICC淋巴结转移的术前预测列线图模型，该模型结合了传统的临床放射学特征和新的放射学特征，为ICC患者提供了准确的LN转移预测，并可能有助于治疗决策。