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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 沈湯龍(Tang-Long Shen) | |
| dc.contributor.author | Pei-Yu Chu | en |
| dc.contributor.author | 朱珮瑜 | zh_TW |
| dc.date.accessioned | 2021-06-16T23:12:35Z | - |
| dc.date.available | 2016-08-09 | |
| dc.date.copyright | 2012-08-09 | |
| dc.date.issued | 2012 | |
| dc.date.submitted | 2012-08-03 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/64986 | - |
| dc.description.abstract | Growth factor receptor bound protein-7 (Grb7) 是一個有數個功能區域 (domain) 的銜接蛋白 (adaptor protein)，能與許多訊息分子產生交互作用並藉此調控細胞功能。我們首先確認了上游激酶 focal adhesion kinase (FAK) 磷酸化Grb7的位置 (Tyr188 和 Tyr 338)。當這些位置被突變，Grb7便無法被磷酸化也不能影響下游如 paxillin 及 ERK1/2 的磷酸化。此外，在 integrin 的訊息傳遞鏈中，FAK和Grb7會結合形成複合物，進而有效影響癌細胞的移動，複製，與不須附著的生長能力。我們進一步發現在 erbB2 及 Grb7 共同大量表現的乳癌細胞株中，當受到表皮生長因子 (EGF) 的刺激，Grb7 會被磷酸化，接著與 Ras-GTPase 產生結合後促進其活性，並影響下游ERK1/2之磷酸化。這條 EGFR-Grb7-Ras- ERK1/2 訊息傳導鏈能提高乳癌細胞的生長能力，進而促進癌化現象。另外，我們在非小細胞肺癌細胞中同樣發現 Grb7會受到 EGF 刺激而磷酸化。在此系統中，Grb7 能透過調控轉錄因子 STAT3 的活性來影響 EphA4 基因表現。EphA4 能和 EGFR 交互作用，產生互相磷酸化與活化的現象，並因此增強 EGFR 的下游訊息傳遞。因此，在 EGFR-Grb7-STAT3-EphA4 的訊息傳遞下，細胞的生長，移動，侵染 (invasion) 與腫瘤進程 (tumor progression) 都受到調控。我們的研究提供了一個對 Grb7參與之訊息傳遞有更多了解的機會。基於眾多證據直指 Grb7 在許多癌症中皆扮演著重要角色，我們的實驗結果也突顯出未來針對 Grb7 發展疾病偵測標誌及標靶治療的可能性。 | zh_TW |
| dc.description.abstract | Growth factor receptor bound protein-7 (Grb7) is a multi-domain adaptor protein which is regulated cell functions by interacting with signaling molecules to transmit signal transduction. We first demonstrate that FAK was capable of phosphorylating the Tyr-188 and Tyr-338 within Grb7, and the tyrosine phosphorylation-deficient mutants evidently altered the phosphorylation of paxillin and ERK1/2 but less on AKT, implying their involvement in the FAK•Grb7-mediated cellular functions. Additionally, the formation of FAK-Grb7 complexes and Grb7 phosphorylation by FAK in an integrin-dependent manner were essential for cell migration, proliferation and anchorage-independent growth in A431 cells, indicating the importance of FAK-Grb7 complexes in tumorigenesis. We further show that Grb7 promotes tumorigenesis through the formation of a novel EGFR-Grb7-Ras signaling complex. Grb7-mediated cell proliferation and growth are essential for the tumorigenesis in erbB2-Grb7-overexpressing SK-Br3 breast cancer cells. EGF-induced de novo Grb7 tyrosine phosphorylation recruits and activates Ras-GTPases and subsequently promotes the phosphorylation of ERK1/2, thereby stimulating tumor growth. We also reveal a novel mechanism by which Grb7 regulates cell migration, proliferation, invasion and tumorigenesis of A549 NSCLC cells through the EGF-triggered EGFR-Grb7-STAT3-EphA4 signaling pathway. Our data provide a better understanding on the signal transduction event for Grb7-mediated cellular functions. Grb7 might have the potential to develop as a prognostic biomarker model and improve the overall sensitivity and specificity of cancer therapies. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T23:12:35Z (GMT). No. of bitstreams: 1 ntu-101-D95633004-1.pdf: 4437724 bytes, checksum: ef650cd970ec28edeb99bad61fd31116 (MD5) Previous issue date: 2012 | en |
| dc.description.tableofcontents | 口試委員會審定書 ……………………………………………………...…..…….. i
中文摘要 ………………………………………………………….………………. ii ABSTRACT ………………………………………………………………………. iii TABLE OF CONTENTS …………………………,……………………………… v LIST OF TABLE …………………………………,………………………………. viii LIST OF FIGURES ………………………………………,……………………… ix LIST OF ABBREVIATIONS ………………………,……………………………. x CHAPTER I: INTRODUCTION ……………………………………………..... 1 A. Grb7 …………………………………………………………………………... 2 B. EGFR family ………………………………………………………………… 38 C. EphA4 ……………………………………………………………………….. 44 D. References …………………………………………………………………… 49 CHAPTER II: MATERIALS AND METHODS ……………………………… 78 A. Reagents………………………………………………………………………… 79 B. Cell Culture…………………………………………………………………….. 80 C. Lentiviral Production and Infection…………………………………………… 81 D. Plasmid Construction …………………………………….…………..……….. 83 D.1 Construction of Expression Vectors ……………………………..……….. 83 D.2. Construction of Luciferase Reporter Gene Constructs ………………………85 E. Immunoprecipitation, Co-immunoprecipitation and Western Blotting Analyses . 85 F. Cell Motility Assay ……………………….…………………………………... 86 G. Cell Migration Assay …………………….….………………………………... 87 G.1. Wound Healing Assay …………………………………………………… 87 G.2. Boyden Chamber Assay ………………….……………………………… 88 G.3. Scattering Onto Immobilized Ligands (SOIL) assay ……………………. 88 H. BrdU Incorporation Assay …………………….……………………………... 89 I. Cell Survival Assay ……………………………….…………………………... 90 I.1. Starvation ……………………………………….……….………………... 90 I.2. Anti-tumor Assays by Herceptin …………………………………………. 90 J. Tumor Growth in SCID Mice ……………………...…………………………... 91 K. Soft Agar Assay …………………………….…………….…………………... 91 L. Matrigel Invasion Assay ………………………….…………………………… 92 M. Tissue Microarray ……………………………………………………………. 93 N. GST-(Raf)-RBD Pulldown Assay ……………………………………………..95 O. Quantitative Real-Time PCR and RT-PCR ……………………………………95 P. Northern Blotting ………………………………………………………………96 Q. Nuclear and Cytosolic Fractionation ………………………………………… 97 R. Immunofluorescence Assay (IFA) ………………………………………….. 98 S. Promoter Luciferase Assay …………………………………………………... 99 T. Chromatin Immunoprecipitation (ChIP) Assay ……………………………… 100 CHAPTER III: RESULTS …………………………………….………………. 102 A. Tyrosine Phosphorylation of Growth Factor Receptor-bound Protein-7 by Focal Adhesion Kinase in the Regulation of Cell Migration, Proliferation, and Tumorigenesis .……………………………………………….……………….. 103 B. EGF-induced Grb7 Recruits and Promotes Ras Activity Essential for the Tumorigenicity of Sk-Br3 Breast Cancer Cells ……………….…………….… 115 C. Grb7-Mediated Transcriptional Regulation of EphA4 Gene Expression in Cancer Progression …………………………………………………………….……… 121 D. Figures …..…………………………………….…………….………………… 136 E. Tables ………………………………………………………………………….. 213 F. References ……………………………………………………………………... 215 CHAPTER IV: DISCUSSION ………………………………………………….. 218 CHAPTER V: CONCLUSION AND PROSPECTS ………………………… 239 APPENDIX: Curriculum Vitae ……………………………………………….. 244 | |
| dc.language.iso | en | |
| dc.subject | FAK | zh_TW |
| dc.subject | Grb7 | zh_TW |
| dc.subject | 癌症治療藥物 | zh_TW |
| dc.subject | 非小細胞肺癌 | zh_TW |
| dc.subject | 乳癌 | zh_TW |
| dc.subject | 表皮生長因子 | zh_TW |
| dc.subject | cancer therapy | en |
| dc.subject | FAK | en |
| dc.subject | EGF | en |
| dc.subject | breast cancer | en |
| dc.subject | non-small-cell-lung-cancer | en |
| dc.subject | Grb7 | en |
| dc.title | Grb7參與在細胞移動與腫瘤進程之訊息調控 | zh_TW |
| dc.title | Signal Transduction of Grb7 in Cell Migration and
Tumor Progression | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 100-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 吳國瑞(Kou-Juey Wu),孟子青(Tzu-Ching Meng),劉俊揚(Jun-Yang Liou),王萬波(Won-Bo Wang),李財坤(Tsai-Kun Li) | |
| dc.subject.keyword | Grb7,FAK,表皮生長因子,乳癌,非小細胞肺癌,癌症治療藥物, | zh_TW |
| dc.subject.keyword | Grb7,FAK,EGF,breast cancer,non-small-cell-lung-cancer,cancer therapy, | en |
| dc.relation.page | 246 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2012-08-03 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 植物病理與微生物學研究所 | zh_TW |
| 顯示於系所單位： | 植物病理與微生物學系 | |
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