Ebp1介导的Akt/HDM2/p53信号通路在ErbB3异常激活引起的肝癌细胞EMT中的作用

The over activation of ErbB3 signaling pathway can enhance the migration and invasion of HCC cells, and its mechanism is related to epithelial mesenchymal transition (EMT). Ebpl is a newly discovered intracellular binding protein of ErbB-3, which can regulate the phosphorylation of Akt. We found that the expression of Ebp1 in HCC tissues was positively correlated with the expression of p53 and lymphatic metastasis, Stable transfection of Ebp1 promotes invasion and metastasis of hepatocellular carcinoma cells and activate the phosphorylation of Akt. Akt/p53 signaling pathway can induce tumor cell EMT. Therefore, we hypothesized that the Akt/HDM2/p53 signaling pathway regulated by Ebp1 may be involved in the occurrence of EMT in hepatocellular carcinoma cells induced by abnormal activation of ErbB3. This project intends to use the clinical liver cancer specimens, animal models, hepatocellular carcinoma cell lines, molecular and cellular biology techniques, (1) To clarify the possibility of Ebp1 mediated EMT in hepatocellular carcinoma cells, (2)To elucidate the molecular mechanism of hepatoma cells EMT induced by Ebp1 through the AKT/HDM2/p53 signaling pathway, (3) To investigate the dependence of molecular signaling network regulated by Ebp1 in hepatocellular carcinoma cell EMT induced by abnormal activation of ErbB3. To provide a new target and experimental basis for the mechanism of invasion and metastasis of HCC.

ErbB3信号通路的过度激活可提高肝癌细胞的迁移和侵袭性，其作用机制与上皮间质转化（EMT）有关。Ebp1是新发现的ErbB-3胞内结合蛋白，可调控Akt的磷酸化。我们前期研究发现，Ebp1在肝癌组织中的表达量与p53的表达和淋巴转移正相关，且稳定转染Ebp1促进肝癌细胞侵袭转移，并激活Akt的磷酸化。Akt/p53信号通路能够诱导肿瘤细胞发生EMT，因此我们推测Ebp1调控的Akt/ HDM2/p53信号通路可能参与ErbB3异常激活引起的肝癌细胞EMT的发生。本项目拟利用临床肝癌标本、动物模型、肝癌细胞系、分子生物学技术，（1）明确Ebp1参与肝癌细胞发生EMT的可能性；（2）靶向于AKT/HDM2/p53信号通路阐明Ebp1在肝癌细胞发生EMT中的分子机制；（3）探讨ErbB3异常激活引起的肝癌细胞EMT对Ebp1调控的信号网络的依赖性。旨在为肝癌侵袭转移机制提供新的靶点和理论依据。

肝细胞肝癌（HCC）是危害我国居民健康的主要恶性肿瘤之一，其恶性程度高，治疗效果不理想。ErbB3信号通路的过度激活可提高肝癌细胞的迁移和侵袭性，其作用机制与EMT有关。Ebp1是新发现的ErbB-3胞内结合蛋白，其长片段p48 Ebp1主要位于细胞质和核内，过度表达抑制细胞凋亡。癌基因HDM2是活化的Akt下游信号分子，能够通过与p53结合抑制p53的抑癌功能，并导致p53的泛素化降解，从而抑制细胞凋亡。近年来p53在肝癌细胞侵袭转移中EMT发生相关性研究逐渐增多，本研究1）选取长片段p48 Ebp1来检测ErbB3、p53、EMT标志物的表达，分析各基因之间的相关性和肝癌临床病理参数之间的关系。结果表明，Ebp1在肝癌组织中高表达，其表达量与p53的表达、肿瘤大小及转移正相关，与EMT相关蛋白均有相关，表明Ebp1可能参与肝癌侵袭转移过程中EMT的发生。2）选取ErbB3高表达的HepG2细胞进行划痕实验检测了细胞生长速度和细胞迁移，并Transwell实验观察沉默Ebp1对肝癌细胞侵袭转移的影响，结果沉默Ebp1组细胞增殖迁移能力明显低于对照组；Transwell侵袭实验观察结果显示沉默Ebp1组迁移到小室的细胞数明显少于对照组。3）p53 Ser15和20位被磷酸化修饰，阻断了MDM2与p53之间的相互作用，使p53蛋白水平升高。本研究发现Ebp1的沉默导致p53Ser15和Ser46激活；敲低Ebp1改变了HepG2和Sk-hep1细胞中EMT相关标记物的蛋白表达。主要表现为敲低Ebp1的HepG2和Sk-hep1细胞中Vimentin、N-cadherin、Snail和Slug的表达明显下降，而E-cadherin的表达显著升高。提示，Ebp1参与调控肝癌细胞的EMT，且其作用是通过p53下调介导的。4）Ebp1的沉默抑制Huh7细胞增殖，p38α表达下降，SB203580剂量依赖性地降低了p38的磷酸化和HIF-1α的表达，表明Ebp1可能是p38MAPK和Hif1α的上游，而p38激活可能在HIF-1α表达的上游。总之，以上结果提示Ebp1通过调控Akt/HDM2/p53和p38 MAPK/HIF-1α信号通路参与ErbB3异常激活的肝癌细胞生长增殖及EMT的发生。