CFTR基因突變或者功能缺失是否導致包括胃腸道在內的各種組織惡性腫瘤的發生風險增加目前仍然是一個充滿爭議的問題。同時，眾所周知，緊密連接分子在腫瘤發生和轉移的過程發揮了關鍵的作用。本論文首次發現了CFTR基因與一種緊密連接分子AF-6/afadin的在人類結直腸腫瘤中的表達水平呈高度相關，并研究了CFTR和AF-6/afadin之間潛在的相互作用及其在結直腸腫瘤轉移中的功能。 / 論文的第一部份首先用實時定量PCR和免疫組織化學的方法比較了CFTR在結直腸腫瘤和正常組織的表達情況，發現CFTR表達水平在腫瘤組織中有顯著的下降。令人感興趣的是，我們同時發現CFTR和AF-6/afadin在腫瘤組織中的表達呈高度正相關，并由此展開了後續的體外實驗，研究對CFTR與AF-6/afadin之間可能的相互聯繫。利用免疫螢光染色和免疫共沉澱的方法，我們發現了這兩種蛋白分子共表達在結直腸腫瘤細胞的接觸面，并存在相互作用。用CFTR突變蛋白的免疫共沉澱實驗進一步發現，這種相互作用需要CFTR分子在細胞膜表面的正確定位及其PDZ結構域結合位點。實驗還發現與CFTR的相互作用加強了AF-6/afadin與細胞骨架蛋白系統的結合。在結直腸腫瘤細胞中CFTR基因敲减导致了AF-6/afadin蛋白定位混亂，從細胞連接位點轉移到細胞漿內，并因此破壞了上皮細胞的緊密性。極性生長細胞的跨上皮電阻降低而滲透性增強的實驗結果證實了CFTR基因敲減導致的上皮細胞緊密性的破壞。同時，AF-6/afadin蛋白水平也隨著CFTR基因敲減而降低，但mRNA水平未發生明顯的改變。蛋白降解系統的抑製劑逆轉了CFTR基因敲減細胞中AF-6/afadin蛋白的減少，提示CFTR基因敲減增加了AF-6/afadin的蛋白降解。這些實驗結果揭示了通過與細胞連接分子AF-6/afadin的相互作用以及調節，CFTR可能在上皮細胞極性的調節以及腫瘤發展過程中起重要作用。 / 論文的第二部份研究了CFTR和AF-6/afadin在結直腸腫瘤細胞上皮細胞間充質化（EMT）和轉移過程中的功能及機制。我們之前的工作已經揭示抑制CFTR的功能可以誘導結直腸腫瘤LIM1863細胞的EMT過程。本研究在另外三株不同的結直腸腫瘤細胞（SW480，SW1116和HRT-18）中進一步證實了抑制CFTR誘導的EMT過程。細胞形態轉變，上皮細胞標誌物的下調，間充質細胞標誌物的上調以及受損的上皮細胞緊密性均證實了對CFTR的抑制可以在這三種細胞中成功誘導EMT的發生。我們發現在以上所有細胞EMT的過程中，AF-6/afadin的蛋白表達水平都發生了顯著的下調。在HRT-18細胞中過表達AF-6/afadin，可以逆轉由CFTR抑製劑誘導的上皮細胞標誌分子的下調和間充質標誌分子的上調，表明抑制CFTR誘導的EMT過程是由AF-6/afadin參與介導的。此外，CFTR基因敲減導致結直腸腫瘤細胞的惡性表型強化，包括減弱的細胞粘附性，增強的貼壁依賴性生長、侵襲和遷移。另外，CFTR基因敲減激活了ERK的磷酸化，過表達AF-6/afadin可以阻斷ERK途徑的激活。CFTR基因敲減而增強的細胞侵襲性也可以被外源性AF-6/afadin或者ERK途徑的抑製劑U0126完全逆轉，提示作為AF-6/afadin的下游靶信號，ERK介導了CFTR在腫瘤侵襲中的作用。更重要的是，我們分析了CFTR和AF-6/afadin的表達水平與結直腸癌病人腫瘤進展的關係，發現在嚴重TNM腫瘤分期或者有腫瘤遠處轉移的病人中CFTR的表達水平顯著低於輕型分期或未发生转移的病人中的水平，而且CFTR和/或AF-6/afadin低表達的病人的預後更差。這些實驗結果顯示CFTR的缺失可能通過抑制AF-6/afadin和激活ERK通路而與EMT和結直腸癌癥轉移的過程高度相關。 / 綜上所述，本研究揭示了以往未報道過的CFTR在結直腸腫瘤發病機理中的功能，提示CFTR可以用作一種新的腫瘤的潛在預後指標。 / The question whether mutation or dysfunction of CFTR increases the risk of malignancies in various tissues, including the gastrointestinal tract, remains highly controversial. Meanwhile, it is well-known that adherens junctions play critical roles in the process of cancer development and metastasis. In this thesis we found for the first time a highly correlation between expression levels of CFTR and an adherens junction molecule AF-6/afadin in human colorectal tumours, and investigated the potential interaction between CFTR and AF-6/afadin and their functional roles in the metastasis of colorectal cancer. / In the first section of this thesis, we started our studies with comparing the expression of CFTR between human colorectal tumours and normal colorectal tissues. Real time quantitative PCR and immunohistochemistry results revealed a dramatically reduced CFTR level in the cancer tissues. Intriguingly, we noticed a highly positive correlation between CFTR and AF-6/afadin expression in tumours, which prompted the further in vitro investigation of possible interaction between CFTR and AF-6/afadin. Using immunofluoresent staining and co-immunoprecipitation, we found that the two proteins were colocalized at cell-cell junctions and interacted with each other in colorectal cancer cell lines. Further Co-IP experiments performed with CFTR mutations revealed that this protein interaction requires the proper localization of CFTR in cell membrane and its PDZ-interacting domain. Moreover the interaction with CFTR strengthens the binding of AF-6/afadin to the cytoskeleton system. Knockdown of CFTR in colorectal cancer cells resulted in the disorganized localization of AF-6/afadin protein from junctional sites to the cytoplasm and impaired epithelial tightness, which was confirmed by significantly reduced transepithelial resistance and increased permeability of polarized cells. Meanwhile, the protein level of AF-6/afadin was down-regulated in CFTR-knockdown cells, while no significant changes were detected at the mRNA level. Protein degradation inhibitor reversed the repression of AF-6/afadin protein in CFTR knockdown cells, suggesting the protein degradation of AF-6/afadin was increased by CFTR knockdown. These data revealed that CFTR interacts with and regulates the cell adhesion molecular AF-6/afadin in colorectal cells, which may be important in the regulation of epithelial cell polarity and cancer development. / In the second section of this thesis, we studied the functional roles and mechanisms of CFTR and AF-6/afadin in the epithelial-mesenchymal transition (EMT) and metastasis of human colorectal cancer cells. Our previous work has revealed inhibition of CFTR can induce EMT in a colorectal cancer cell line, LIM1863. This study further confirmed the induction of EMT by inhibiting CFTR in several other colorectal cancer cell lines (SW480, SW1116 and HRT-18), which was evaluated by morphological changes, down-regulation of epithelial markers or up-regulation of mesenchymal markers, and impaired epithelial cell tightness. In all these cell lines, we found that the protein levels of AF-6/afadin were significantly reduced. Over-expression of AF-6/afadin in HRT-18 cells reversed the down-regulated epithelial markers and up-regulated mesenchymal markers induced by CFTR inhibition, indicating that the CFTR inhibition-induced EMT is mediated by AF-6/afadin. Moreover, knockdown of CFTR in HRT-18 or RKO cells resulted in enhanced malignant phenotypes, including decreased cell adhesion, increased anchorage-independent cell growth, invasion, and migration. In addition, extracellular signal-regulated kinase (ERK) phosphorylation was activated by CFTR knockdown, which was abolished by over-expression of AF-6/afadin. The enhanced invasiveness of CFTR knockdown cells was also completely inhibited by either exogenous AF-6/afadin or ERK inhibitor, U0126, suggesting that ERK, the downstream target of AF-6/afadin, is involved in mediating the effect of CFTR in cancer invasion. More importantly, we analyzed the association of CFTR and AF-6/afadin expression levels with tumour progression of patients with colorectal cancer, and revealed that CFTR expression was significantly lower in patients with more severe TNM stage or with metastasis to distant organs than those with milder stage or with no metastasis. The prognosis was poorer in patients with lower expression of CFTR and/or AF-6/afadin than those with higher expressions. These data showed that dysfunction of CFTR is highly associated with EMT and colorectal cancer metastasis, probably via repression of AF-6/afadin and activation of ERK pathways. / In summary, the present study has revealed a previously undefined role of CFTR in the pathogenesis of colorectal cancer and indicated its potential as a new prognostic indicator. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Sun, Tingting. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 113-127). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i / 中文摘要 --- p.iv / Publications --- p.vi / Conference Abstract --- p.vii / Declaration --- p.viii / Acknowledgements --- p.x / List of Figures --- p.xi / List of Tables --- p.xiii / List of Abbreviations --- p.xiv / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1. --- Colorectal Cancer --- p.1 / Chapter 1.1.1. --- Structure of Human Normal Colon and Rectum Epithelium --- p.1 / Chapter 1.1.2. --- Staging of Colorectal Cancer --- p.3 / Chapter 1.1.3. --- Metastasis of Colorectal Cancer --- p.3 / Chapter 1.1.4. --- K-Ras mutation and It Downstream Pathways in Colorectal Cancer Metastasis --- p.11 / Chapter 1.1.5. --- Prognosis of Colorectal Cancer --- p.14 / Chapter 1.2. --- Epithelial Cell Junctional Complexes --- p.14 / Chapter 1.2.1. --- Junctional Complexes and Epithelial Cell Polarity --- p.15 / Chapter 1.2.2. --- Classic Cadherin-catenin Complex --- p.17 / Chapter 1.2.3. --- Novel Nectin-afadin Complex --- p.19 / Chapter 1.2.4. --- Cell Polarity and Cancer Progression --- p.23 / Chapter 1.3. --- Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) --- p.24 / Chapter 1.3.1. --- Structure of CFTR --- p.24 / Chapter 1.3.2. --- Mutations of CFTR --- p.24 / Chapter 1.3.3. --- Functions of CFTR --- p.26 / Chapter 1.3.4. --- Cancer Risk of CF Patients --- p.33 / Chapter 1.4. --- Hypothesis and Aims --- p.34 / Chapter Chapter 2 --- Materials and Methods --- p.35 / Chapter 2.1. --- Materials --- p.35 / Chapter 2.1.1. --- Reagents and Chemicals --- p.35 / Chapter 2.1.2. --- Antibodies --- p.35 / Chapter 2.1.3. --- Primers --- p.35 / Chapter 2.1.4. --- Solutions and Buffers --- p.35 / Chapter 2.1.5. --- Human Specimens --- p.36 / Chapter 2.2. --- Methods --- p.36 / Chapter 2.2.1. --- Cell Culture --- p.36 / Chapter 2.2.2. --- Transfection --- p.36 / Chapter 2.2.3. --- Selection of Stable Clones --- p.40 / Chapter 2.2.4. --- RNA Extraction and RT-PCR --- p.40 / Chapter 2.2.5. --- Quantitative Real Time PCR --- p.41 / Chapter 2.2.6. --- Protein Extraction and Western Blotting --- p.42 / Chapter 2.2.7. --- Immunostaining --- p.45 / Chapter 2.2.8. --- In vitro Cell Functional Assays --- p.46 / Chapter 2.2.9. --- Epithelial Tightness Measurement --- p.48 / Chapter 2.2.10. --- Statistical Analysis --- p.49 / Chapter Chapter 3 --- Interaction of CFTR with AF-6/afadin and Its Importance in Maintaining Colorectal Epithelial Cell Polarity --- p.50 / Chapter 3.1. --- Introduction --- p.50 / Chapter 3.2. --- Objectives --- p.53 / Chapter 3.3. --- Experimental plan --- p.54 / Chapter 3.4. --- Results --- p.55 / Chapter 3.4.1. --- The expression of CFTR and AF-6/afadin is decreased and positively correlated in human colorectal cancer --- p.55 / Chapter 3.4.2. --- CFTR colocalizes and interacts with AF-6/afadin in human colorectal cancer cells --- p.58 / Chapter 3.4.3. --- PDZ binding motif and membrane localization of CFTR are necessary for the interaction between CFTR and AF-6/afadin --- p.64 / Chapter 3.4.4. --- Knockdown of CFTR interferes with cell junction formation in colorectal cancer cells --- p.66 / Chapter 3.5. --- Discussion --- p.71 / Chapter Chapter 4 --- CFTR as a Suppressor and Prognosis Indicator of Metastasis in Human Colorectal Cancer --- p.77 / Chapter 4.1. --- Introduction --- p.77 / Chapter 4.2. --- Objectives --- p.80 / Chapter 4.3. --- Experimental plan --- p.81 / Chapter 4.4. --- Results --- p.82 / Chapter 4.4.1. --- CFTR inhibition-induced EMT in colorectal cancer cells involves AF-6/afadin --- p.82 / Chapter 4.4.2. --- Knockdown of CFTR aggravates malignant phenotype of colorectal cancer cells --- p.86 / Chapter 4.4.3. --- AF-6/afadin mediates the effect of CFTR on cell invasion in colon cancer through ERK --- p.91 / Chapter 4.4.4. --- CFTR and AF-6/afadin expression is correlated with the prognosis of colorectal cancer --- p.97 / Chapter 4.5. --- Discussion --- p.100 / Chapter Chapter 5 --- General Discussion and Conclusion --- p.105 / Chapter 5.1. --- The diversified roles of CFTR in epithelial cells --- p.105 / Chapter 5.2. --- The unfolding relationship between CFTR and cancer development --- p.107 / Chapter 5.3. --- Future studies --- p.109 / Chapter 5.4. --- Conclusions --- p.112 / Reference List --- p.113 / Chapter Appendix A --- Reagents and Chemicals --- p.128 / Chapter Appendix B --- Antibody List --- p.131 / Chapter Appendix C --- Primer List --- p.132 / Chapter Appendix D --- Solution Recipe --- p.133
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328118 |
| Date | January 2012 |
| Contributors | Sun, Tingting., Chinese University of Hong Kong Graduate School. Division of Biomedical 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 (xv, 136 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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