鈣粘蛋白是一類通過影響細胞粘附和細胞信號通路在腫瘤發生中起重要作用的細胞間粘附分子。鈣粘蛋白超家族包括經典鈣粘蛋白和非經典鈣粘蛋白，其中非經典鈣粘蛋白包含了原鈣粘蛋白。啟動子CpG甲基化調控下的基因沉默或表達下調是腫瘤發生中一個關鍵事件的觀點現已得到廣泛認可。一些鈣粘蛋白家族成員，如鈣粘蛋白-1/4/13（CDH1，CDH4，CDH13）被已有研究報導是受擬遺傳學調控沉默的功能性腫瘤抑制基因。本研究主要針對兩個鈣粘蛋白家族成員鈣粘蛋白-11（CDH11）和原鈣粘蛋白-10（PCDH10）進行腫瘤發生相關功能和機制的研究。 / CDH11位於雜合性缺失（LOH）經常發生而預示可能存在抑癌基因的染色體16q21-22區域，我們實驗室先前通過基因組芯片雜交技術（aCGH）對腫瘤細胞系的研究已發現它是該區域一個可能的抑癌基因。我們通過進一步的半定量反轉錄聚合酶鏈反應（RT-PCR）發現CDH11在正常組織和永生化正常上皮細胞中廣泛表達，但在各腫瘤細胞系中表達下降。甲基化特異性聚合酶鏈反應（MSP）和亞硫酸氫鹽處理的基因組測序（BGS）檢測到CDH11啟動子甲基化常發生于腫瘤細胞和腫瘤組織中。在CDH11表達缺失的腫瘤細胞中重新導入該基因的表達可顯著減少細胞克隆的形成，誘導細胞凋亡并抑制腫瘤細胞的遷移。通過更深入的機制研究，我們發現CDH11通過抑制Wnt/β-catenin信號通路發揮功能。 / 本研究的另一個鈣粘蛋白家族成員是PCDH10。之前我們實驗室的工作已經證實了PCDH10是一個在鼻咽癌和食管癌中受啟動子甲基化調控的抑癌基因,這裡我們主要研究它在大腸癌發病中的功能和機制。我們發現在PCDH10表達缺失的大腸癌細胞中重新導入PCDH10表達可顯著抑制腫瘤細胞的克隆形成，細胞遷移和幹細胞性。機制研究顯示PCDH10抑制Wnt/β-catenin和RhoA信號轉導通路，并進一步抑制腫瘤上皮細胞-間充質轉化（EMT）的過程，誘導幹細胞標記的下調。 / 綜上所述，本研究顯示CDH11和PCDH10兩個鈣粘蛋白家族成員在多種腫瘤中廣泛受甲基化調控失活，它們是重要的Wnt/β-catenin信號通路拮抗因素，可抑制腫瘤細胞的克隆形成和細胞遷移 / Cadherins are an important group of cell-cell adhesion molecules, which play crucial roles in tumorigenesis by affecting cell adhesion and cell signaling. Cadherin superfamily consists of classical cadherins and non-classical cadherins including protocadherins. It has been well recognized that silencing or downregulation of tumor suppressor genes (TSGs) by promoter CpG methylation is a critical event in human tumorigenesis. Some cadherin family members, such as CDH1, CDH4, CDH13, have been reported as functional TSGs silenced through epigenetic regulation. In this study, I mainly focus on the function and mechanism studies of two cadherin members-Cadherin 11(CDH11) and Protocadherin 10 (PCDH10). / CDH11 is located in 16q21-22, a region with frequent loss of heterozygosity (LOH), indicating the presence of candidate TSG. Previously, our lab also identified CDH11 as a candidate TSG through array-CGH of tumor cell lines. I further found by semi-quantitative RT-PCR that CDH11 was broadly expressed in normal tissues while frequently downregulated in multiple tumor cell lines, but not in immortalized normal epithelial cells. Methylation-specific PCR (MSP) and bisulfite genomic sequencing (BGS) detected frequent promoter methylation of CDH11 in tumor cell lines and primary tumor samples. Ectopic expression of CDH11 dramatically reduced tumor cell clonogenecity, induced tumor cell apoptosis and inhibited tumor cell migration. By further mechanism study, I found that CDH11 is a negative inhibitor of Wnt/β-catenin signaling pathway. / Another cadherin family protein which I chose to study is PCDH10. Previously our lab identified PCDH10 as a TSG by promoter methylation in nasopharyngeal and esophageal carcinomas. I studied the function and mechanism of PCDH10 in the pathogenesis of colon cancer. I found ectopic expression of PCDH10 strongly suppressed colon tumor cell clonogenecity, migration and stemness. Moreover, I found that PCDH10 repressed Wnt/β-catenin and RhoA signaling, thus further inhibited the epithelial-to-mesenchymal transition (EMT) of tumor cells and downregulated stem cell markers. / In summary, this study demonstrates two cadherin family members CDH11 and PCDH10, as important antagonists to Wnt/β-catenin signaling pathway, suppress tumor cell clonogenecity, migration, and are also frequently inactivated by epigenetic mechanism in multiple tumors. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhang, Yanjiao. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 84-95). / Abstracts also in Chinese. / Abstract --- p.i / Acknowledgements --- p.iii / Table of Contents --- p.vi / List of Tables --- p.x / List of Figures --- p.xi / List of Abbreviations --- p.xiii / List of Publications --- p.xvi / Chapter Chapter 1 --- Introduction and Literature Review --- p.1 / Chapter 1.1 --- Cancer --- p.1 / Chapter 1.1.1 --- General introduction about cancer --- p.1 / Chapter 1.1.2 --- Oncogenes and TSGs --- p.3 / Chapter 1.1.3 --- Cancer mechanism models --- p.3 / Chapter 1.2 --- Cancer Epigenetics --- p.5 / Chapter 1.2.1 --- DNA methylation --- p.6 / Chapter 1.2.2 --- DNA methylation and gene silencing --- p.7 / Chapter 1.2.3 --- DNA methylation and cancer --- p.7 / Chapter 1.2.4 --- Clinical implications of DNA methylation --- p.8 / Chapter 1.3 --- Cadherins --- p.10 / Chapter 1.3.1 --- General introduction of cadherin superfamily --- p.10 / Chapter 1.3.2 --- Cadherin classification --- p.10 / Chapter 1.3.3 --- Cadherin and cancers --- p.12 / Chapter 1.3.4 --- Cadherin switch and EMT in cancer --- p.16 / Chapter 1.4 --- Wnt/β-catenin signaling pathway and cancer --- p.16 / Chapter 1.4.1 --- Wnt/β-catenin signaling pathway --- p.16 / Chapter 1.4.2 --- Wnt/β-catenin signaling pathway in cancer --- p.18 / Chapter 1.4.3 --- Epigenetic silencing of Wnt/β-catenin signaling --- p.20 / Chapter 1.4.4 --- Wnt/β-catenin signaling pathway in CRC --- p.21 / Chapter Chapter 2 --- Aims of the Study --- p.22 / Chapter Chapter 3 --- Materials and Methods --- p.25 / Chapter 3.1 --- Cell lines and tissue samples --- p.25 / Chapter 3.1.1 --- Cell lines --- p.25 / Chapter 3.1.2 --- Maintenance of cell lines --- p.25 / Chapter 3.1.3 --- Drug treatment of cell lines --- p.26 / Chapter 3.1.4 --- Normal and primary tissues --- p.26 / Chapter 3.1.5 --- Total RNA extraction --- p.27 / Chapter 3.1.6 --- Genomic DNA extraction --- p.28 / Chapter 3.2 --- Gene expression analysis --- p.29 / Chapter 3.2.1 --- Reverse transcription (RT) --- p.29 / Chapter 3.2.2 --- Semi-quantitative RT-PCR --- p.30 / Chapter 3.3 --- Methylation Analysis --- p.32 / Chapter 3.3.1 --- CpG island prediction --- p.32 / Chapter 3.3.2 --- Sodium bisulfite treatment of genomic DNA --- p.33 / Chapter 3.3.3 --- Methylation-specific PCR (MSP) --- p.33 / Chapter 3.3.4 --- Bisulfite Genomic Sequencing (BGS) --- p.34 / Chapter 3.4 --- Construction of expression plasmids --- p.36 / Chapter 3.4.1 --- Construction of CDH11 expression vector --- p.36 / Chapter 3.4.2 --- Construction of PCDH10 expression plasmid --- p.38 / Chapter 3.4.3 --- Plasmid extraction --- p.39 / Chapter 3.5 --- Plasmid transfection --- p.41 / Chapter 3.6 --- Subcellular localization --- p.42 / Chapter 3.7 --- Function analyses --- p.43 / Chapter 3.7.1 --- Colony formation assay --- p.43 / Chapter 3.7.2 --- Wound healing assay --- p.44 / Chapter 3.8 --- Mechanism exploration --- p.44 / Chapter 3.8.1 --- Protein extraction and western-blot --- p.44 / Chapter 3.8.2 --- Dual-luciferase reporter assay --- p.47 / Chapter 3.9 --- Statistical analysis --- p.48 / Chapter Chapter 4 --- CDH11 functions as a tumor suppressor via modulating Wnt/β-catenin signaling and is frequently downregulated by promoter methylation --- p.49 / Chapter 4.1 --- The CpG island of CDH11 gene promoter --- p.50 / Chapter 4.2 --- CDH11 expression profile in normal tissues --- p.50 / Chapter 4.3 --- Frequent silencing of CDH11 by promoter methylation in multiple tumors --- p.51 / Chapter 4.4 --- Restoration of CDH11 expression by pharmacologic demethylation --- p.53 / Chapter 4.5 --- Frequent CDH11 methylation in primary tumors --- p.54 / Chapter 4.6 --- Function studies --- p.56 / Chapter 4.6.1 --- Ectopic expression of CDH11 inhibited tumor cell clonogenecity --- p.57 / Chapter 4.6.2 --- CDH11 induced tumor cell apoptosis --- p.57 / Chapter 4.6.3 --- CDH11 inhibited tumor cell migration --- p.58 / Chapter 4.7 --- CDH11 antagonized Wnt/β-catenin signaling --- p.59 / Chapter 4.8 --- Discussion --- p.60 / Chapter Chapter 5 --- Epigenetic inactivated tumor suppressor PCDH10 exerts tumor suppressive functions through modulating Wnt/β-catenin signaling and cell stemness in colon cancer --- p.66 / Chapter 5.1 --- PCDH10 was broadly expressed in normal tissues and frequently silenced in CRC cell lines --- p.67 / Chapter 5.2 --- Promoter methylation mediated PCDH10 silencing in CRC cell lines --- p.68 / Chapter 5.3 --- Frequent PCDH10 methylation in CRC primary tumors --- p.69 / Chapter 5.4 --- PCDH10 was located in cytoplasm and membrane --- p.70 / Chapter 5.5 --- Function Studies --- p.71 / Chapter 5.5.1 --- PCDH10 inhibited clonogenicity of tumor cells --- p.71 / Chapter 5.5.2 --- PCDH10 suppressed tumor cell mobility --- p.72 / Chapter 5.6 --- Mechanism exploration of PCDH10 in CRC --- p.72 / Chapter 5.6.1 --- PCDH10 antagonized Wnt/β-catenin signaling --- p.72 / Chapter 5.6.2 --- PCDH10 negatively regulated EMT and stemness of tumor cells --- p.74 / Chapter 5.6.3 --- PCDH10 inhibited RhoA signaling --- p.75 / Chapter 5.7 --- Discussion --- p.75 / Chapter Chapter 6 --- Conclusions and Future studies --- p.80 / Chapter 6.1 --- Conclusions --- p.80 / Chapter 6.2 --- Future studies --- p.82 / Reference List --- p.84
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328714 |
| Date | January 2012 |
| Contributors | Zhang, Yanjiao., Chinese University of Hong Kong Graduate School. Division of Medical Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | electronic resource, electronic resource, remote, 1 online resource (xvi, 95 leaves) : ill. (some col.) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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