新的候选癌基因EphB1在食管癌转移中的作用机制及临床意义研究

EphB1 gene locates at a frequently amplified region 3q22.2 in esophageal squamous cell carcinoma (ESCC). Previous study showed that EphB1 overexpressed in paired human ESCC tissue samples at both mRNA and protein levels. Frequent overexpression of EphB1 was significantly associated with poor survival of ESCC patients and was an independent prognostic factor in ESCC. To study the functional roles of EphB1 in ESCC, we initially transfected EphB1 gene into human normal epithelial cell line LO-2 and selected EphB1 stable expressing clones for in vitro studies including wound healing, migration assay and invasion assay. Our results revealed that overexpression of EphB1 in LO-2 cells causes increased migration and invasion potency. Moreover, immunofluorescence assay showed that epithelial-mesenchymal transition (EMT) was induced in EphB1-transfected LO-2 cells. GST pull down assay further demonstrated expression of active RhoA (RhoA-GTP) and total RhoA were upregulated in EphB-transfected LO-2 cells. In this proposal, we will identify the amplification of EphB1 gene by FISH and detect EphB1 mutations by direct sequencing in ESCC. The roles of EphB1 in ESCC metastasis will be demonstrated in EphB1-overexpressed and -depleted ESCC cell lines. We will use RT-PCR, western blot and immunofluorescence to test the expression of EMT markers, EMT regulating transcription factors, and matrix metalloproteinases (MMPs) in these cells. GST pull down assay and western blot will be used to test total and active RhoGTPase (RhoA/Rac1/Cdc42). We will also detect the microvessels by CD31/CD34 staining in mouse xerographs induced by EphB1-overexpressed or -depleted ESCC cell lines. Proteomic (2D protein electrophoresis) and genomic (cDNA microarray) approaches will be used to screen the key molecules involved in EphB1-related signaling pathways. Tissue microarray and IHC will be used to detect these candidate molecules. Bioinformatics platform will be used to analyze these data. We will use support vector machines (SVM)-based methods to develop immunomarker-SVM-based prognostic classifiers for ESCC and build new models to predict the overall survival of ESCC.

酪氨酸激酶受体EphB1基因位于食管癌中常常发生扩增的区域3q22.2。 前期实验发现EphB1的mRNA及蛋白水平在肿瘤组织中增高，且与预后不良密切相关。将EphB1基因导入上皮来源的人永生化细胞系LO-2，发现EphB1高表达增强细胞在体内外的侵袭和转移能力，且诱导细胞发生EMT，活化RhoA GTPase。在本项目中，我们拟采用FISH进一步确定EphB1在食管癌中的扩增，并测序其有无突变。通过调高或阻断EphB1基因的表达，从EMT、RhoGTPase活化、基质金属蛋白酶及血管生成调节四方面，在体内外实验中研究其对食管癌细胞侵袭和转移的影响。进一步通过cDNA芯片及双向蛋白电泳筛选可能与EphB1密切相关的信号通路关键分子以阐明其分子机制。应用组织芯片检测相关分子的表达，结合患者临床病理资料，利用生物信息学平台进行数据挖掘，建立预测模型用于判断预后，以指导临床治疗。

课题组在前期的CGH array中发现食管癌的3q22.2区域存在扩增，且与肿瘤的进展和浸润相关，而EphB1正位于此区域。本研究的目的是证明EphB1在食管癌转移中的作用，并探讨其分子机制。.我们通过FISH检测发现食管癌组织中存在着EphB1基因的扩增。 测序未发现肿瘤组织中EphB1基因存在错义突变、缺失或插入。RT-PCR检测100例，免疫组化检测251例食管癌及癌旁相对正常组织样本，发现食管癌组织中EphB1基因表达增加与预后不良相关。.在LO-2及EC109及EC9706中分别用脂质体及慢病毒转染入EphB1基因，构建Knock-in细胞株。用shRNA敲除KYSE180中EphB1基因的表达，建立knock-out细胞株。高表达EphB1基因的细胞较低表达EphB1的细胞，在体外（划痕实验、matrigel侵袭实验）具有较强的迁移和侵袭能力。在裸鼠皮下移植瘤形成实验中显示具有更强的体内成瘤能力。在SCID鼠和裸鼠尾静脉注射肿瘤细胞的肝及肺转移模型显示，高表达EphB1 基因的细胞具有更强的肝及肺转移瘤形成能力。.通过质谱分析及生物信息学分析，发现转染EphB1基因的细胞较转染空载体的细胞，干细胞相关通路表达上调。荧光定量PCR及western blot证实高表达EphB1基因的食管癌细胞HIF1表达增加，干细胞标志蛋白CD133、SOX2、CD44表达增加，多药耐药蛋白ABCB1表达上调。肿瘤球形成实验表明高表达EphB1基因的细胞肿瘤球形成能力及对化疗药物的抵抗能力增强。免疫荧光、western blot、免疫组化证明高表达EphB1基因的上皮细胞发生了上皮基质转化，肌动蛋白紧张度增加， E-cadherin的表达降低，β-catenin从细胞膜转位入核， N-cadherin和Vimentin表达升高。亲和沉淀／免疫印迹实验表明总RhoA及活化RhoA表达增加。MMP9，MMP10表达增加。. 本研究证明在ESCC存在着EphB1基因的扩增及高表达，且与病人的预后相关。初步证明EphB1基因在ESCC中的高表达，通过上调HIF1表达，促进干细胞性，通过激活 RhoA 信号，促进肿瘤的上皮基质转化，增强基质金属蛋白酶的表达，从而促进食管癌的转移。本研究结果对于指导食管癌的临床治疗及预后判断具有积极作用 。