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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳炳宏(Ping-Hung Chen) | |
| dc.contributor.author | Yu-Chi Su | en |
| dc.contributor.author | 蘇宇琦 | zh_TW |
| dc.date.accessioned | 2023-03-19T22:19:40Z | - |
| dc.date.copyright | 2022-10-07 | |
| dc.date.issued | 2022 | |
| dc.date.submitted | 2022-09-13 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/84665 | - |
| dc.description.abstract | 表皮生長因子受體 (EGFR) 屬於受體酪氨酸激酶 (RTK) ErbB 家族，對調節細胞功能至關重要。野生型EGFR過度表達或發生突變時，會導致細胞增加轉化能力。 在東亞地區，EGFR是非小細胞肺癌 (NSCLC) 中最常見的突變腫瘤驅動基因。酪氨酸激酶抑製劑 (TKI) (Scharping et al.) 被開發用於靶向 NSCLC 中的突變 EGFR。然而，獲得抗藥性限制了酪氨酸激酶抑製劑TKI 的療效。越來越多的研究指出，在具TKI抗藥性的細胞中，EGFR 的運輸發生了改變。癌細胞會採”適應性內吞運輸”來改變訊號傳導，以增加腫瘤癌化能力。但是，詳細的機制仍有待建立。我的研究發現具TKI抗藥性的PC9細胞中，神經元富集的同功型 Dynamin1 (Dyn1) 蛋白質的量增加，並與 EGFR 的表現增加相關，進而增加細胞遷移能力。此外，與親代 PC9 細胞相比，在具TKI抗藥性的PC9 細胞中，細胞表面的EGFR 增加，顯示 EGFR 的運輸改變。因此，我們開發了識別 EGFR 細胞外結構域的抗 EGFR 納米抗體，作為進一步研究改變的突變型 EGFR 運輸機制的新工具。總之，這項工作為理解改變的 EGFR 運輸如何加劇 TKI 抗性及影響NSCLC 細胞的遷移能力提供了新的視角。 | zh_TW |
| dc.description.abstract | Epidermal growth factor receptor (EGFR) belongs to the receptor tyrosine kinases (RTKs) ErbB family and is crucial in regulating cellular functions. Overexpression of wild-type or mutations of EGFR leads to increased cell transforming abilities. EGFR is the most frequently mutant tumor-driver gene in non-small cell lung cancer (NSCLC) in East Asians. Tyrosine kinase inhibitors (TKIs) are developed to target mutant EGFRs in NSCLC. However, acquired resistance limited the TKIs efficacy. Increasing studies reveal altered trafficking of EGFRs in TKIs-resistant cells, and cancer cells adopt “adaptive endocytic trafficking” to bias signaling to augment malignancy. However, the detailed mechanisms remain to establish. This study reveals that up-regulated neuronal enriched isoform dynamin1 (Dyn1) correlates with increased EGFR to augment migration in gefitinib-resistant PC9 cells. Furthermore, increased surface EGFR is observed in gefitinib-resistant PC9 cells compared to parental PC9 cells indicating the altered trafficking of EGFR. Therefore, we develop the anti-EGFR nanobodies recognizing the EGFR extracellular domain as a new tool to study the mechanism of altered mutant-EGFR trafficking. In summary, the work provides a new perspective for understanding how altered EGFR trafficking intensifies migration in TKI-resistant NSCLC cells. | en |
| dc.description.provenance | Made available in DSpace on 2023-03-19T22:19:40Z (GMT). No. of bitstreams: 1 U0001-1209202216285200.pdf: 3124849 bytes, checksum: 5e070d488c142ca6eca425d644f2a665 (MD5) Previous issue date: 2022 | en |
| dc.description.tableofcontents | 誌謝 i 中文摘要 ii Abstract iii 縮寫 iv Table of contents v Chapter 1: Introduction 1 1.1 The role of EGFR in non-small cell lung cancer 1 1.2 Treatment of NSCLC harboring mutant-EGFR 2 1.3 The crosstalk between endocytosis and signaling in cancer 2 1.4 Contribution of Dyn1 in regulating trafficking of NSCLC cells 3 1.5 A new tool for monitoring mutant-EGFR trafficking 5 1.6 The purpose of this study 6 Chapter 2: Materials and Methods 7 2.1 Materials 7 2.1.1 Cell lines 7 2.1.2 Competent cells 8 2.1.3 Plasmids 8 2.1.4 Chemicals and reagents 10 2.1.5 Commercial Kits 14 2.1.6 Antibody 15 2.1.7 Enzymes 16 2.2 Methods 16 2.2.1 Cell culture 16 2.2.2 Western blotting 17 2.2.3 EGFR half-life assay 17 2.2.4 Cell viability assay 18 2.2.5 Lentivirus production 18 2.2.6 Overexpression of EGFR (Ex19Del) in PC9 cells 19 2.2.7 siRNA transfection 19 2.2.8 Wound healing assay 20 2.2.9 Expression of anti-EGFR nanobody G10 20 2.2.10 Purification of anti-EGFR nanobody G10 21 2.2.11 Flow cytometry 22 2.2.12 Establishment of EGFR ECD-Fc expressing cells 22 2.2.13 Protein expression of 6xHis tagged EGFR ECD 23 2.2.14 Preparation of EGFR ECD/magnetic beads 23 2.2.15 Nanobody screen 24 2.2.16 Yeast colony PCR 25 2.2.17 Construction of nanobody expressing plasmids 26 2.2.18 Expression of nanobody 28 Chapter 3: Results 29 3.1 Expressions of EGFR and Akt are up-regulated in gefitinib-resistant PC9 cells. 29 3.2 Degradation dynamics of EGFR are similar in PC9 and PC9/GR cells. 29 3.3 Increased surface EGFR in PC9/GR cells compared to PC9 cells. 30 3.4 Dyn1 expression is up-regulated in gefitinib-resistant PC9 cells. 32 3.5 Overexpression of mutant EGFR in PC9 cells increases Dyn1 levels. 32 3.6 Depletion of Dyn1 suppresses the migration ability of PC9/GR cells. 33 3.7 Anti-EGFR nanobody (G10) slightly increases Akt activation. 34 3.8 Expression and purification of EGFR ECD-Fc fusion protein 35 3.9 Screening of anti-EGFR ECD nanobodies 36 3.10 Characterization of the candidate anti-EGFR nanobodies 36 3.11 Construction, expression, and purification of EGFR ECD-6xHis proteins 37 3.12 Identification and characterization of EGFR-interacting Nb 12 and Nb 16 37 3.13 Purified EGFR-interacting Nb 16 does not interfere Akt activation 38 3.14 Flow cytometry assay of dose-dependent binding of Nb 16 for EGFR 39 Chapter 4: Discussion 39 Figures 43 Figure 1. Expressions of EGFR and Akt are up-regulated in gefitinib-resistant PC9 cells. 43 Figure 2. Degradation dynamics of EGFR in PC9 and PC9/GR cells are similar. 46 Figure 3. Increased surface EGFR is detected by anti-EGFR nanobody (G10) in PC9/GR cells compared to PC9 cells. 47 Figure 4. Dyn1 expression is up-regulated in gefitinib-resistant PC9 cells. 49 Figure 5. Overexpression of mutant EGFR in PC9 cells increases Dyn1 levels. 50 Figure 6. Depletion of Dyn1 suppresses the migration ability of PC9/GR cells. 54 Figure 7. Anti-EGFR nanobody (G10) slightly increases Akt activation. 55 Figure 8. Expression and purification of EGFR ECD-Fc fusion proteins. 56 Figure 9. Screening of anti-EGFR ECD-Fc nanobodies. 57 Figure 10. Characterization of the candidate anti-EGFR nanobodies. 58 Figure 11. Expression and purification of EGFR ECD-6xHis proteins. 59 Figure 12. Characterization of ani-EGFR Nbs, Nb12, and Nb16. 60 Figure 13. Purified EGFR-interacting Nb 16 does not interfere Akt activation 61 Figure 14. Flow cytometry assay of dose-dependent binding of Nb 16. 63 Supplementary Figures 64 Supplementary Figure 1. Genes expression analysis of DNM genes in tumor and normal lung tissues. 64 Supplementary Figure 2. DNM1 is predicted as a high-risk factor in NSCLC. 65 Supplementary Figure 3. Dyn1 proteins are significantly up-regulated in gefitinib-resistant HCC827 cells. 66 References 67 | |
| dc.language.iso | en | |
| dc.subject | gefitinib 抗藥性 | zh_TW |
| dc.subject | 表皮生長因子受體 | zh_TW |
| dc.subject | 第一型縊斷蛋白 | zh_TW |
| dc.subject | 內吞運輸 | zh_TW |
| dc.subject | 細胞遷移 | zh_TW |
| dc.subject | 奈米抗體 | zh_TW |
| dc.subject | 非小細胞肺癌 | zh_TW |
| dc.subject | EGFR | en |
| dc.subject | gefitinib resistance | en |
| dc.subject | NSCLC | en |
| dc.subject | nanobody | en |
| dc.subject | cell migration | en |
| dc.subject | endocytic trafficking | en |
| dc.subject | Dynamin 1 | en |
| dc.title | 研究縊斷蛋白(Dynamin)在有突變表皮生長因子受體的非小細胞肺癌中調節受體運輸及細胞遷移能力的作用 | zh_TW |
| dc.title | To study the role of dynamins in regulating EGFR trafficking and migration of gefitinib-resistant NSCLC cells | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 110-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 李明學(Ming-Shyue Lee),余永倫(Yung-Luen Yu) | |
| dc.subject.keyword | 表皮生長因子受體,第一型縊斷蛋白,內吞運輸,細胞遷移,奈米抗體,非小細胞肺癌,gefitinib 抗藥性, | zh_TW |
| dc.subject.keyword | EGFR,Dynamin 1,endocytic trafficking,cell migration,nanobody,NSCLC,gefitinib resistance, | en |
| dc.relation.page | 70 | |
| dc.identifier.doi | 10.6342/NTU202203309 | |
| dc.rights.note | 同意授權(限校園內公開) | |
| dc.date.accepted | 2022-09-14 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
| dc.date.embargo-lift | 2024-09-12 | - |
| 顯示於系所單位： | 生物化學暨分子生物學科研究所 | |
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