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研究生： 毛久珊
Mao, Jiou-Shan
論文名稱： 探討GAS7基因功能及其過度表現對肺癌轉移抑制作用
The Function of GAS7 Gene and Its Effects of Overexpression on Metastasis-Suppression of Lung Cancer
指導教授： 王憶卿
Wang, Yi-Ching
學位類別： 碩士
Master
系所名稱： 醫學院 - 藥理學研究所
Department of Pharmacology
論文出版年： 2010
畢業學年度： 98
語文別： 英文
論文頁數： 80
中文關鍵詞： 生長休止基因七 、肺癌 、轉移抑制
外文關鍵詞： GAS7, lung cancer, metastasis-suppression
相關次數： 點閱：86  下載：1 
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    • 摘要
    研究目的：GAS7 基因是 growth-arrest-specific gene family (GAS family) 的成員之ㄧ，本實驗室先前利用高解析染色體異質性喪失分析方法 (high-resolution genome-wide loss of heterozygosity) 發現 GAS7 基因所位於的染色體區位 17p 13.1 是一個染色體高度異質性喪失的區域。我們進一步利用 RT-PCR 及 immunohistochemistry (IHC) 方法分析肺癌病人 GAS7 mRNA 和蛋白質的表現情形，結果顯示在肺癌病人中 GAS7 mRNA 和蛋白質分別有高達 45.1% 和 57.3% 低表達的情形，促使我們深入去探討 GAS7 基因在肺癌的變異機制。
    研究材料與方法：首先我們將 GAS7C 在肺癌細胞中 overexpression 並分別用流式細胞儀 (FACS)、細胞爬行移動力實驗 (wound healing assay 及 transwell-migration assay)、細胞侵略移動力實驗 (invasion assay) 來觀察 GAS7C overexpression 對細胞週期及細胞移動能力的影響。並且在動物癌症轉移模組中，探討 GAS7C overexpression 對癌細胞轉移的影響。
    結果：GAS7C overexpression 並無明顯改變細胞週期的分佈；但 GAS7C overexpression 不僅會在細胞層次抑制肺癌細胞的移動及侵略能力，在動物模組中也同樣抑制肺癌細胞的轉移能力。深入探究其原因，我們發現 GAS7C overexpression 可能是透過減少fibronectin-integrin-FAK 路徑的相關蛋白質表現，例如：fibronectin、phosphor-FAK、phosophor-paxillin 及 Arp2/3complex 來影響 actin dynamic 而達到抑制肺癌細胞的轉移。另一方面我們想找出 GAS7C 交互作用的蛋白質 (association proteins) 並探討其影響細胞移動的作用機制，我們利用 immunoprecipitation-Western blotting 實驗確認 GAS7C 會和 fibronectin-integrin-FAK 路徑中影響 actin dynamic 的重要蛋白質 N-WASP 交互作用形成蛋白質複合體 (protein complex)；另外我們的研究結果顯示，GAS7C 也會和 β-catenin 及 ubiquitin degradation 路徑相關蛋白質，例如： hnRNP-U 形成蛋白質複合體 (protein complex)，當 GAS7C overexpression 時，使 β-catenin 的 ubiquitination 程度上升和 β-catenin 蛋白表現量下降，其中 β-catenin 是一個細胞間附著連接間隙 (adhesion junctions) 與細胞骨架 (actin cytoskeleton) 的重要調控因子，若β-catenin 蛋白表現量下降則癌細胞移動轉移能力下降。
    結論：本研究是首篇探討 GAS7 基因在肺癌癌化過程中的變異機制，並且也是首篇發現 GAS7C overexpression 可能會透過減少 fibronectin-integrin-FAK 路徑蛋白質表現及影響 actin dynamic 而達到抑制肺癌細胞的轉移，同時本篇研究也利用 immunoprecipitation-Western blotting 方法找出 GAS7 可能的交互作用蛋白，並且發現 GAS7C 除了會和影響 actin dynamic 的重要調控蛋白 N-WASP 具有交互作用外，也和 ubiquitin degradation 路徑相關蛋白質具有交互作用並使得 β-catenin 蛋白表現下降，顯示了GAS7C overexpression 可能會透過降低fibronectin-integrin-FAK訊息傳遞路徑及增加β-TrCP/β-catenin蛋白形成而達到抑制肺癌細胞移動轉移的能力。本篇研究可做為日後肺癌診斷分子標誌 (bio-markers) 的參考並且提供了日後肺癌基因標靶治療的新方向。

    ABSTRACT
    Aim: GAS7 is a member of growth-arrest-specific (GAS) gene family. Our previous study identified the chromosome 17p13.1, which harbors the GAS7 gene, as a high deletion region in lung cancer patients. In addition, the frequencies of low GAS7 mRNA and protein expression were 45.1% and 57.3%, respectively. Therefore, the present study aims to investigate the function of GAS7 gene and to study what the molecular mechanism of GAS7 alteration is in lung tumorigenesis.
    Materials and Methods: We overexpressed GAS7C in lung cancer cells lines to evaluate the changes of cell cycle distribution and cell migration ability by FACS, wound healing assay, transwell-migration assay, and invasion assay. In addition, tail-vein experimental metastasis assay using GAS7C overexpression cells was examined in animal model. Results: The results indicated that GAS7C overexpression did not alter the cell cycle distribution, but decreased the lung cancer cells migration and invasion abilities both in vitro and in vivo. Immunoprecipitation-Western blotting was performed to further identify GAS7 association proteins. The results showed that GAS7C interacted with N-WASP, which is a key regulator in actin dynamic via fibronectin-integrin-FAK pathway. In addition, GAS7C interacted with β-catenin and ubiquitin-degradation pathway proteins such as hnRNP-U. Overexpression of GAS7C increased β-catenin ubiquitination level, thus decreased the β-catenin protein level. Collectively, these data suggested that GAS7C overexpression decreases the lung cancer cells migration may partly through its association proteins via fibronectin/integrin/FAK/N-WASP/actin dynamic pathway and hnRNP-U/β-TrCP/β-catenin ubiquitin-degradation pathway.
    Conclusion: This is the first study to investigate the function of GAS7 gene and its alteration mechanisms in lung tumorigenesis. We demonstrated that GAS7C overexpression decreases lung cancer cells migration ability both in vivo and in vitro which may reduce the fibronectin-integrin-FAK pathway protein expression and increase protein complex formation of β-TrCP/β-catenin. This study provides new evidence that GAS7C may be used as a prediction biomarker or the target for gene therapy of lung cancer.

    CONTENTS Introduction--------------------------------------------1 I.Clinical significance of lung cancer------------------1 II.Study basis on the role of growth-arrest specific 7 (GAS7)in lung cancer------------------------------------2 III.Overview of GAS family and GAS7 gene----------------3 (A)GAS family-------------------------------------------4 (B)The GAS7 gene and GAS7 protein isoforms--------------5 IV.Overview of GAS7 WW domain association proteins and their cellular functions--------------------------------6 (A)Overview of GAS7 WW domain association protein N-WASP in cell migration-----------------------7 (B)Overview of GAS7 WW domain association protein hnRNP-U in protein ubiquitination---------------------8 V.Overview of GAS7 in neuron differentiation------------9 Study Basis and Purposes-------------------------------11 Materials and Methods----------------------------------12 I.Cell culture-----------------------------------------12 II.Plasmids transfection-------------------------------12 III.Western blotting-----------------------------------12 IV.Immunofluorescent staining--------------------------13 V.Analysis of cell cycle distribution------------------14 VI.Wound healing assay---------------------------------14 VII.Cell transwell-migration and cell invasion assays--15 VIII.Experimental metastasis animal model--------------15 IX.NNK treatment for GAS7 knockout mice----------------16 X.Immunoprecipitation and Western blotting-------------17 XI.Statistical analysis--------------------------------17 Results------------------------------------------------19 I.The expression of GAS7 isoforms in human lung cancer cell lines---------------------------------------------19 II.The effects of UV treatment after GAS7C overexpression on cell cycle progression---------------19 III.GAS7C overexpression decreases lung cancer cells migration and invasion abilities-----------------------20 IV.GAS7C overexpression decreases the expression level of fibronectin-integrin-FAK pathway and alters actin arrangement--------------------------------------------22 V.GAS7C overexpression decreases human lung cancer metastasis in experimental metastasis animal model-----23 VI.The effect of NNK treatment with GAS7 knockout mice-------------------24 VII.Identify GAS7C association proteins by immunoprecipitation - Western blotting (IP-WB)---------25 Discussion---------------------------------------------27 References---------------------------------------------32 Tables-------------------------------------------------40 Figures------------------------------------------------43 Appendix-----------------------------------------------65 TABLE CONTENTS Table 1 The antibodies and their reaction conditions used in the present study------------------------------41 Table 2 Pathologic findings from wild type (WT) and GAS7 (GAS7-/-) knockout Mice---------------------------42 FIGURE CONTENTS Figure 1 The plasmids and their characteristics used in the current study--------------------------------------44 Figure 2 Western blot analyses of expression of GAS7 isoforms in a panel of human lung cancer cell lines and normal lung cells lines--------------------------------46 Figure 3 The GAS7C transfection efficiency and GAS7C protein expression after transfection were determined by immunofluorescent staining and Western blot---------47 Figure 4 The effects of GAS7C overexpression and UV treatment on cell proliferation and cell cycle distribution-------------------------------------------49 Figure 5 Wound healing assay showed that GAS7C overexpression decreased cell motility without affecting cell proliferation---------------------------51 Figure 6 Transwell-migration assay showed that GAS7C overexpression decreased cell migration ability--------53 Figure 7 Cell invasion assay showed that GAS7C overexpression decreased cell invasion ability---------55 Figure 8 GAS7C overexpression decreased the fibronectin-integrin-FAK pathway and altered actin arrangement--------------------------------------------57 Figure 9 The animal model showed that GAS7C overexpression effectively reduced A549 lung cancer cells metastasis ability-------------------------------58 Figure 10 Tail vein injection with GAS7C overexpression cells did not influence the body weight and biochemistry/hematology evaluations of tested animals------60 Figure 11 The presence of a lung adenocarcinoma and a liver Ito cell tumor were observed in one GAS7 knockout mouse--------------------------------------------------62 Figure 12 GAS7C protein is associated with N-WASP, which is a key regulator in actin dynamic--------------63 Figure 13 GAS7C protein is associated with β-catenin and altered the hnRNP-U/β-TrCP ubiquitin degradation pathway------------------------------------------------64 APPENDIX Appendix Table 1 Alterations of GAS7C or GAS7B mRNA expression in relation to clinicopathological parameters of resected primary lung tumors-------------66 Appendix Table 2 Summary of GAS gene functions---------67 Appendix Figure 1 Representative figures and correlation analyses of immunohistochemistry and Western blot analysis of GAS7 protein in lung tumor tissues------------------------------------------------68 Appendix Figure 2 GAS7B and GAS7C mRNA expression of lung cancer patients in relation to cancer stage-------70 Appendix Figure 3 The functions of GAS family in controlling cell cycle---------------------------------71 Appendix Figure 4 Human GAS7 gene DNA composition and transcript isoforms------------------------------------72 Appendix Figure 5 Schematic presentation of predicted protein functional domain of GAS7C, GAS7B,and GAS7A----74 Appendix Figure 6 LC-MS/MS analysis of interacting proteins for known proline-based WW domain-binding motifs including GAS WW motif--------------------------75 Appendix Figure 7 GAS7 WW domain interacts with WAS family protein, N-WASP, a crucial regulator of actin polymerization-----------------------------------------77 Appendix Figure 8 Schema of fibronectin/integrin/FAK/N-WASP/actin polymerization pathway--------------------79 Appendix Figure 9 Schematic presentation of the role of hnRNP-U in protein degradation pathway--------------80

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