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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 郭明良 | |
dc.contributor.author | Pin-Ju Wu | en |
dc.contributor.author | 吳品儒 | zh_TW |
dc.date.accessioned | 2021-07-10T21:36:09Z | - |
dc.date.available | 2021-07-10T21:36:09Z | - |
dc.date.copyright | 2016-10-14 | |
dc.date.issued | 2016 | |
dc.date.submitted | 2016-07-26 | |
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The prognostic significance of amplification and overexpression of c-met and c-erb B-2 in human gastric carcinomas. Cancer 85, 1894-1902. Natali, P.G., Prat, M., Nicotra, M.R., Bigotti, A., Olivero, M., Comoglio, P.M., and Di Renzo, M.F. (1996). Overexpression of the met/HGF receptor in renal cell carcinomas. Int J Cancer 69, 212-217. Oh, Y.M., Song, Y.J., Lee, S.B., Jeong, Y., Kim, B., Kim, G.W., Kim, K.E., Lee, J.M., Cho, M.Y., Choi, J., et al. (2012). A new anti-c-Met antibody selected by a mechanism-based dual-screening method: therapeutic potential in cancer. Mol Cells 34, 523-529. Olwill, S.A., Joffroy, C., Gille, H., Vigna, E., Matschiner, G., Allersdorfer, A., Lunde, B.M., Jaworski, J., Burrows, J.F., Chiriaco, C., et al. (2013). A highly potent and specific MET therapeutic protein antagonist with both ligand-dependent and ligand-independent activity. Mol Cancer Ther 12, 2459-2471. Organ, S.L., and Tsao, M.S. (2011). An overview of the c-MET signaling pathway. Ther Adv Med Oncol 3, S7-S19. Paumelle, R., Tulasne, D., Kherrouche, Z., Plaza, S., Leroy, C., Reveneau, S., Vandenbunder, B., and Fafeur, V. (2002). Hepatocyte growth factor/scatter factor activates the ETS1 transcription factor by a RAS-RAF-MEK-ERK signaling pathway. Oncogene 21, 2309-2319. Prat, M., Crepaldi, T., Pennacchietti, S., Bussolino, F., and Comoglio, P.M. (1998). Agonistic monoclonal antibodies against the Met receptor dissect the biological responses to HGF. J Cell Sci 111 ( Pt 2), 237-247. Puri, N., and Salgia, R. (2008). Synergism of EGFR and c-Met pathways, cross-talk and inhibition, in non-small cell lung cancer. J Carcinog 7, 9. Rodrigues, G.A., and Park, M. (1994). Autophosphorylation modulates the kinase activity and oncogenic potential of the Met receptor tyrosine kinase. Oncogene 9, 2019-2027. Schmidt, L., Duh, F.M., Chen, F., Kishida, T., Glenn, G., Choyke, P., Scherer, S.W., Zhuang, Z., Lubensky, I., Dean, M., et al. (1997). Germline and somatic mutations in the tyrosine kinase domain of the MET proto-oncogene in papillary renal carcinomas. Nat Genet 16, 68-73. Siegfried, J.M., Weissfeld, L.A., Luketich, J.D., Weyant, R.J., Gubish, C.T., and Landreneau, R.J. (1998). The clinical significance of hepatocyte growth factor for non-small cell lung cancer. Ann Thorac Surg 66, 1915-1918. Surati, M., Patel, P., Peterson, A., and Salgia, R. (2011). Role of MetMAb (OA-5D5) in c-MET active lung malignancies. Expert Opin Biol Ther 11, 1655-1662. Trusolino, L., and Comoglio, P.M. (2002). Scatter-factor and semaphorin receptors: cell signalling for invasive growth. Nat Rev Cancer 2, 289-300. Wang, J., Goetsch, L., Tucker, L., Zhang, Q., Gonzalez, A., Vaidya, K.S., Oleksijew, A., Boghaert, E., Song, M., Sokolova, I., et al. (2016). Anti-c-Met monoclonal antibody ABT-700 breaks oncogene addiction in tumors with MET amplification. BMC Cancer 16, 105. Zeng, Z.S., Weiser, M.R., Kuntz, E., Chen, C.T., Khan, S.A., Forslund, A., Nash, G.M., Gimbel, M., Yamaguchi, Y., Culliford, A.T.t., et al. (2008). c-Met gene amplification is associated with advanced stage colorectal cancer and liver metastases. Cancer Lett 265, 258-269. Zhang, W., Chu, Y.Q., Ye, Z.Y., Zhao, Z.S., and Tao, H.Q. (2009). Expression of hepatocyte growth factor and basic fibroblast growth factor as prognostic indicators in gastric cancer. Anat Rec (Hoboken) 292, 1114-1121. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76746 | - |
dc.description.abstract | 肝細胞生長因子(Hepatocyte growth factor, HGF)為細胞表面上的酪胺酸激酶受體MET之受質。HGF/MET 的訊息傳遞路徑調節了許多細胞生理過程,包括了胚胎發育及組織修復等。然而不正常的MET訊號活化透過促進癌細胞增生、增加癌細胞移動與侵犯的能力及產生癌細胞對藥物的抗性等,增進了癌症進程的惡化。因此以抑制MET訊息路徑為標的,是對於多種癌症的治療之良好方針。在我們實驗室先前的研究當中發現肝臟分泌的白血球趨化因子2 (Leukocyte cell-derived chemotoxin 2, LECT2) 能內生性和MET受體結合,降低MET訊息路徑活化並抑制肝癌的血管侵犯。因此我們以MET對LECT2結合的片段作為抗原,生產了新型的MET單株抗體#5-9,評估其對於MET專一性的結合及抑制MET訊息路徑活化之效益。我們評估MET抗體#5-9的結合型態,發現此抗體能在不與HGF競爭下和MET受體結合。接著我們評估MET抗體#5-9 抑制MET訊號活化的效果,無論在是否經由HGF所誘導的MET活化模式當中,MET 5-9抗體皆能抑制癌細胞增生、移動與侵犯的能力。此外MET 抗體#5-9 抑制腦癌細胞球體形成 (sphere formation),可能與MET抗體降低癌細胞產生對藥物的抗性有關。接著進一步去探討MET抗體#5-9 抑制MET訊息路徑的分子機制,我們發現MET 抗體#5-9 能防止MET形成活化的二聚體(dimerization)、促進MET受體內吞作用(internalization)而降低表面MET的表現,進而在無論是否由HGF所誘導的模式中都能降低MET磷酸化及達到下游訊息的抑制。最後在癌症異種移植(xenograft)的動物模式中驗證了MET抗#5-9拮抗MET訊息傳遞及抑制腫瘤生長的效果。總結來說,新型的MET單株抗體 #5-9能以抑制癌症MET訊息路徑為標的,未來若能應用於臨床可能是對於多種癌症的治療之良好方針。 | zh_TW |
dc.description.abstract | The MET tyrosine kinase is a cell surface receptor for hepatocyte growth factor (HGF). The HGF/MET signaling regulates many cellular processes, such as embryogenesis and tissue regeneration. However, aberrant activation of MET pathway contributes to cancer malignancy by promoting proliferation, migration, and invasion and resistance. Thus, targeting the MET oncogenic pathway is considered to be a promising therapeutic strategy in multiple cancers. In our previous study, we have identified Leukocyte cell-derived chemotaxin 2 (LECT2) as a novel endogenous MET antagonist to suppress vascular invasion without competing HGF binding in hepatocellular carcinoma (HCC). By using the LECT2 binding site as an antigen, we have generated a monoclonal antibody, anti-MET mAb #5-9 against the specific binding site of MET protein. The in vitro binding assay and competitive ELISA analysis demonstrates that this neutralizing antibody specifically binds to extracellular domain of MET without competing HGF binding. We investigate the effect of the anti-MET mAb #5-9 to inhibit both HGF-dependent and HGF-independent MET activation. The antagonistic effect of anti-MET mAb #5-9 was evaluated by the suppression of MET phosphorylation, cell proliferation, cell migration, sphere formation, and the tumor growth in xenograft model. Moreover, we characterized the molecular mechanism of antibody-mediated MET inactivation, including phosphatases recruitment, receptor internalization and degradation, as well as the ability to prevent MET receptor from dimerization. Collectively, this novel MET antibody may provide a new therapeutic strategy for blocking MET-dependent signaling and eliminating the cancer progression. | en |
dc.description.provenance | Made available in DSpace on 2021-07-10T21:36:09Z (GMT). No. of bitstreams: 1 ntu-105-R03b46018-1.pdf: 3791800 bytes, checksum: a18604122723e299519b6f8673fe26fa (MD5) Previous issue date: 2016 | en |
dc.description.tableofcontents | 口試委員審定書 …………………………………………… Ⅰ
誌謝………………………………………………………… Ⅱ 中文摘要 …………………………………………… Ⅲ Abstract ………………………………………………………………………….... Ⅴ Chapter 1. Introduction ………………………… 1 1.1 Hepatocyte growth factor (HGF)/MET axis 1.2 HGF/MET axis in cancer 1.3 Current developmental status of MET inhibitors 1.4 Design non-competitive anti-MET antibody by LECT2 binding epitope 1.5 Motivation and purpose Chapter 2. Materials and Methods …………………12 2.1 Anti-MET antibody generation and purification 2.2 Cell culture and cell transfection 2.3 Enzyme-linked immunosorbent assay (ELISA) 2.4 Endogenous immunoprecipitation and in vitro binding assay 2.5 Western blotting analysis 2.6 Flow cytometry 2.7 Cell proliferation, migration and invasion assay 2.8 Sphere formation assay 2.9 RNA extraction and quantitative reverse transcription-polymerase chain reaction (RT-qPCR) 2.10 Immunofluorescence staining 2.11 Mouse xenograft model 2.12 Immunohistochemistry (IHC) 2.13 Statistical analysis Chapter 3. Results ………………………………22 3.1 Generation and characterization of the anti-MET mAb #5-9 3.2 The anti-MET mAb #5-9 binds to MET in the presence of HGF 3.3 The anti-MET mAb #5-9 antagonizes MET-mediated cell functions 3.4 The molecular mechanism of antibody-mediated MET inactivation 3.5 The anti-MET mAb #5-9 suppresses MET phosphorylation and downstream signaling 3.6 The anti-MET mAb #5-9 suppresses tumor growth in mouse xenograft model Chapter 4. Discussion ..................................31 Chapter 5. Figures and figure legends.......................................40 Figure 1.Generation of the anti-MET mAb #5-9 Figure 2.Specific binding of the anti-MET mAb #5-9 Figure 3.Non-competitive binding between HGF and anti-MET mAb #5-9 to MET Figure 4.Functional assay Figure 5.Sphere formation Figure 6.Mechanism of antibody-mediated MET inactivation, Figure 7.Inhibition of MET phosphorylation and downstream signaling Figure 8.Xenograft model Chapter 6. Tables ............................................65 Table 1.The primer sequences for RT-qPCR used in the current study Chapter 7. References.........................................67 | |
dc.language.iso | en | |
dc.title | 探討新型MET抗體抑制MET受體活化之療效與機轉 | zh_TW |
dc.title | A novel MET antibody antagonizes MET receptor activation | en |
dc.type | Thesis | |
dc.date.schoolyear | 104-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 洪文俊,張震東,華國泰,蕭宏昇 | |
dc.subject.keyword | 白血球趨化因子2,肝細胞生長因子,MET受體,MET抗體,癌症進程, | zh_TW |
dc.subject.keyword | leukocyte cell-derived chemotaxin 2 (LECT2),hepatocyte growth factor (HGF),MET receptor,MET antibody,cancer progression, | en |
dc.relation.page | 73 | |
dc.identifier.doi | 10.6342/NTU201601327 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2016-07-27 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科學研究所 | zh_TW |
顯示於系所單位: | 生化科學研究所 |
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