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研究生:左先正
研究生(外文):Hsien-Cheng Tso
論文名稱:上皮細胞黏附因子在腫瘤生成中活化與訊息傳遞的分子機制
論文名稱(外文):The Molecular Mechanisms of EpCAM Activation and Signaling in Tumorigenesis
指導教授:吳漢忠
指導教授(外文):Han-Chung Wu
口試委員:呂仁沈家寧
口試委員(外文):Jean LuChia-Ning Shen
口試日期:2015-06-15
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:口腔生物科學研究所
學門:醫藥衛生學門
學類:牙醫學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:英文
論文頁數:66
中文關鍵詞:大腸直腸癌上皮細胞黏附因子上皮細胞黏附因子之細胞外域上皮細胞黏附因子之細胞內域肝細胞生長因子受體β-cateninFHL2
外文關鍵詞:colorectal cancerEpCAMEpEXEpICDc-Metβ-cateninFHL2
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上皮細胞黏附因子 (EpCAM),屬第一型穿膜醣蛋白,於所有的腺狀細胞癌及鱗狀上皮細胞癌都有很高的表現量。過去的研究顯示,此蛋白也出現在癌幹細胞中,並有助於惡性腫瘤的生長。其中又以大腸直腸癌的上皮細胞黏附因子表現量,在所有癌症中為最高。由我們先前的研究得知,上皮細胞黏附因子在大腸癌幹細胞中具有調節腫瘤細胞起始的能力,又與癌症的惡性程度有密切的相關性。因此在本研究中,我們透過磷酸化受體酪氨酸激酶陣列 (phosphor-receptor tyrosine kinase array) 來探討EpCAM的訊息傳遞並且發現上皮細胞黏附因子之細胞外域 (EpEX) 可以活化肝細胞生長因子受體 (Hepatocyte growth factor receptor, HGFR, c-Met)。更進一步發現EpEX可以透過活化 c-Met來促進細胞增生能力、細胞移動能力、細胞轉移能力以及細胞集落形成能力。其次,我們發現EpEX可以透過c-Met與 Erk 訊息傳遞來增加腫瘤壞死因子α轉化酶 (TACE) 以及γ-分泌酶 (γ-secretase) 的活性。為了進一步探討受到EpICD所調控的基因,我們透過融合瘤的技術成功建立了三株對抗EpICD的單株抗體。此外,EpEX會透過增加 γ-secretase 的活性來促進上皮細胞黏附因子之細胞外域 (EpICD) 以及 c-Myc 的表現量。EpICD 可以透過調節 β-catenin-FHL2 的核轉移來調控此複合體的基因調節能力。

Epithelial cell adhesion molecule (EpCAM) is a type Ⅰ transmembrane glycoprotein overexpressed in almost all adenocarcinomas and squamous cell carcinomas. It’s also a well-known cancer stem cell marker and contributes to tumor growth. EpCAM is highly expressed in colon cancer. Our previous study pointed out that EpCAM plays an important role in regulating tumorigenesis in colon cancer stem cells and in regulating tumor malignancy. In this study, we identified the signaling of EpCAM through receptor tyrosine kinase array and found that EpCAM extracellular domain (EpEX) might activate c-Met signaling, which promotes cell proliferation, migration, invasion and colony formation. Subsequent study of EpEX signaling pathway found that EpEX activated the c-Met-ERK signaling pathway and increased the activity of TACE and γ-secretase. Moreover, to investigate the genes regulated by EpICD using ChIP Assay, we successfully generated three monoclonal antibodies against EpICD by hybridoma technology. EpEX was found to upregulate EpICD and c-Myc by increasing γ-secretase activity. EpICD could regulate the nuclear translocation of β-catenin-FHL2 complex then decrease the down-stream gene expression. In conclusion, our results indicate that EpCAM can regulate tumorigenesis through production of EpEX and EpICD. EpEX can activate the c-Met-ERK signaling pathway, while EpICD can turn on gene expression after being translocated into the nucleus.

致謝 1
中文摘要 2
Abstract 3
Contents 5
List of abbreviations 7
Introduction 10
1.1 Epithelial cell adhesion molecule (EpCAM) 10
1.2 Molecular mechanism of EpCAM signaling 10
1.3 Expression of EpCAM in tumor tissue 11
1.4 EpCAM expression on cancer stem cell 12
1.5 EpCAM as a biomarker of circulating tumor cells 13
1.6 Epidemiology of colorectal cancer 14
1.7 Pathogenesis of colorectal cancer 15
1.8 Treatment of colorectal cancer 16
Materials and Methods 19
2.1 Cell lines and cell culture 19
2.2 Receptor protein tyrosine phosphatase-receptor protein tyrosine kinase array 19
2.3 Western blotting 20
2.4 Transfection with lentiviral vectors expressing shRNA 21
2.5 Cell proliferation assay (MTT assay) 22
2.6 Cell migration using the ibidi culture insert 22
2.7 Cell invasion assay 23
2.8 Colony formation assay 23
2.9 Reverse transcription/real time PCR 24
2.10 TACE activity assay 24
2.11 γ-secretase activity assay 25
2.13 Nuclear and cytoplasm lysate extraction 25
2.14 Immunofluorescence assay 25
2.15 Chromation immnoprecipitation (ChIP) 26
2.16 Flow cytometry 27
2.17 Immunoprecipitation and immunoblotting 27
2.18 Cellular ELISA 28
2.19 Immunogold labeling and transmission electron microscopy 28
2.20 Statistical analysis 29
Results 30
3.1 EpEX-Fc activates c-Met and ErbB family in colon cancer cells. 30
3.2 EpEX-Fc promotes cell proliferation, migration, invasion and colony formation through c-Met signaling in HCT116 cells. 31
3.3 Knocksown of c-Met inhibits the EpEX-induced celluar functions. 32
3.4 EpEX increases the TACE activity through c-MET/ ERK pathway. 32
3.5 EpEX increases nuclear translocation of β-catenin-FHL2-EpICD complex 34
3.6 Inhibition of EpICD cleavage attenuates the binding of β-catenin to promoters of target genes 34
3.7 Generation of EpICD monoclonal antibodies by hybridoma technology 36
Discussion 38
References 62


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