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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 潘思樺(Szu-Hua Pan) | |
| dc.contributor.author | Pei-Shan Wu | en |
| dc.contributor.author | 吳佩珊 | zh_TW |
| dc.date.accessioned | 2021-07-11T14:56:26Z | - |
| dc.date.available | 2025-03-03 | |
| dc.date.copyright | 2020-03-03 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-02-20 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78427 | - |
| dc.description.abstract | 表皮生長因子接受器(Epidermal Growth Factor Receptor，EGFR)為目前癌症治療常見之標靶癌蛋白。非小細胞肺癌(Non-Small Cell Lung Cancer, NSCLC)病人當其EGFR具有活化性突變, 如L858R及deletion at exon19, 以酪胺酸激酶抑制劑(Tyrosine Kinase Inhibitor，TKI)進行標靶治療，可有效抑制癌細胞生長，但時常伴隨第二個EGFR突變點(Secondary Mutation, T790M)的產生，導致病人於投藥後一年內產生抗藥性並復發腫瘤。EGFR於細胞中扮演相當重要的角色，包括調節細胞生長、存活、移動以及分化等能力，EGFR接受表皮生長因子(EGF)刺激而活化，並藉由形成二聚體，進而使磷酸酶功能活化並促進自身之酪氨酸磷酸化，最終啟動下游訊號傳遞路徑並影響細胞生理功能。T790M突變為抗藥性之EGFR亞型，其蛋白結構因突變而受到改變，推測與其交互作用之蛋白與訊息傳遞路徑將亦受影響。為了探討藉由T790M發生所改變的細胞生理特性並了解調節機轉，如細胞增生較緩慢以及對標靶治療產生抗藥性等，本研究利用兩對具不同EGFR突變亞型之NSCLC細胞株作為蛋白交互作用實驗模型，包含對TKI敏感與抗藥性之PC9 (Del19), H3255 (L858R), CL68 (Del19-T790M)與H1975 (L858R-T790M)，並利用親和性純化策略與質譜技術(Affinity Purification Mass Spectrometry, AP-MS)，鑑定出不同EGFR突變亞型之蛋白交互作用(Protein-Protein Interactions, PPIs)。此利用質譜分析技術探討蛋白間之交互作用、生物特性與關聯性之策略，則被稱之為交互作用體(Interactome)。
接著再利用與控制組進行量化上的比較，以及與非專一性結合蛋白資料庫進行比對篩選，共可提取出189個可信度較高的EGFR交互作用蛋白。ERBB2、JAK1、SHC1、EPHA2與NOTCH3等蛋白，可同時於四個實驗細胞株中被鑑定到，這些蛋白大多皆參與EGFR調控細胞傳遞訊息之路徑；除此之外，同時也可鑑定到與特定EGFR突變亞型具專一交互作用之蛋白，如AXL、SNX12與SMURF2僅出現在PC9 (Del19)細胞株當中，以及EHD1、SHC4與MTCH1則僅可於CL68 (EGFR-T790M)細胞株中被鑑定到。進一步的生物資訊分析結果可顯示EGFR交互作用蛋白之功能與調控路徑，於TKI敏感之細胞株當中(PC9與H3255)，其交互作用蛋白多參與於PI3K-AKT signaling與adhesion junction，呼應活化之EGFR (Activated EGFR)普遍所調控之細胞生理現象；細胞內控制蛋白質運輸、位置與分解功能之相關調控路徑，如內噬作用(endocytosis)、胞內體或囊泡運輸(endosomal/ vacuolar pathway)以及自噬作用等，其參與調控路徑之蛋白多可於抗藥性肺癌細胞株中被鑑定到，此生物資訊分析結果強調，EGFR T790M突變其交互作用蛋白具有較多樣性的細胞組成與較活躍的細胞內運輸機制，同時也暗示具T790M突變之EGFR於細胞內之蛋白表現量與活性與此運輸機制有著密切關連。後續生物驗證顯示，經Gefitinib處理過後之NSCLC細胞株樣本，利用免疫螢光染色與蛋白質轉漬法進行分析，於抗藥性細胞株中可誘導自噬作用(Autophagy)活化，並進一步分解T790M突變亞型之EGFRs；反之，若以抑制劑阻斷自噬作用後，則可有效抑制自噬作用活化、減緩EGFR分解同時有效降低細胞增生能力，推測抗藥性細胞藉由自噬作用作為抵抗藥物治療之防禦機轉。而TKI敏感性之NSCLC細胞株於Gefitinib之作用下，Rab7可藉由泛素(ubiquitin)修飾調控其活性，並介導晚期包內體(late endosome)之運輸，將EGFR輸送至細胞膜周圍進行回流(recycling)作用，藉此機制降低EGFR受到TKI之抑制，同時維持EGFR於細胞內之含量。藉此研究結果可知，具T790M EGFR突變亞型之非小型肺癌細胞，可利用自噬作用維持細胞存活，並同時抵抗標靶藥物的治療，因此TKI治療合併抑制自噬作用可作為未來肺癌治療的新方向。藉由AP-MS策略分析EGFR突變亞型之交互作用蛋白，可用於了解不同突變亞型如何影響細胞內訊息傳遞與改變調控路徑，此研究結果將可提供未來藥物開發與癌症治療上新穎的方向與策略。 | zh_TW |
| dc.description.abstract | Epidermal growth factor receptor (EGFR) is one of the most popular oncoproteins for targeted cancer therapy. Activating mutations (L858R, deletion at exon 19) on EGFR are susceptible to therapy by tyrosine kinase inhibitor (TKI) in non-small cell lung cancer (NSCLC) patients, yet resistance to TKI develops within one year mainly due to secondary mutation (T790M) of EGFR. EGFR regulates cancer pathogenesis by homo-/hetero-dimerization with EGFR family members, followed by complex interactions to recruit downstream associated proteins to subsequently activate signaling cascades. Thus, it is intriguing to study whether the oncogenic T790M mutation may recruit different interacting partners and alter the downstream signaling. To study the alternated cellular features leading by T790M, such as slower cell proliferation and resistant to target therapy, affinity purification coupled mass spectrometry (AP-MS) was applied to map the protein-protein interactions (PPIs) of different mutant EGFR. Two pairs of NSCLC cell lines with primary and secondary EGFR mutations, PC9 (Del19) and CL68 (Del19-T790M), as well as H3255 (L858R) and H1975 (L858R-T790M), were used as TKI-sensitive and -resistant NSCLC models.
The confident EGFR interactors were filtered by quantitative comparison with IgG control and non-specific binding proteins were systematically eliminated using contaminant frequency extracted from databases. The results reveals total of 189 EGFR interacting proteins, including ERBB2, JAK1, SHC1, EPHA2 and NOTCH3 known to involve in EGFR signaling pathway and commonly identified in four interactomes. Comparison between the NSCLC cell lines harboring primary- or secondary-mutation revealed EGFR subtype-specific interacting protein, such as AXL/ SNX12/ SMURF2 in PC9 (Del19) and EHD1/ SHC4/ MTCH1 in CL68 (Del19-T790M). Pathway enrichment analysis of EGFR interactors showed that PI3K-AKT signaling and adhesion junction were mapped in TKI-sensitive models (PC9 and H3255), presenting general signal regulation of activated EGFR. Functional annotation and pathway mapping reveals significant enrichment in endocytosis, endosomal/ vacuolar pathway and autophagy, in TKI-resistant EGFR interactomes, suggesting the more dynamic intracellular trafficking of protein. These results implied EGFR interactors of T790M-containing subtype may mediate mutant EGFR level and activity via endocytic trafficking. Biological validation by immunofluorescence and immunoblotting revealed that gefitinib induced autophagic activation and mediated EGFR degradation in T790M acquired TKI-resistant cells. We further evaluate synergetic effect by combined treatment of gefitinib and autophagy inhibitors. The inhibitors of autophagy dramatically suppressed the activation of autophagy, degradation of EGFR and cell proliferation in TKI-resistant cells. In addition, gefitinib treatment induced ubiquitination of Rab7 and regulated late endosome to translocate EGFRs to the cell periphery in TKI-sensitive cells, suggesting that endosomal recycling of activated EGFRs through ubiquitylated Rab7 was utilized by TKI-sensitive NSCLC cells to protect EGFRs from TKI blockage and sustain EGFR level. These finding suggested that autophagy may provide survival advantage to induce resistance to TKI inhibition in NSCLC cells bearing EGFR-T790M, and the combination of gefitinib with autophagy inhibitors may be a promising strategy to restore the TKI sensitivity in lung cancer treatment. In summary, the characterization of PPI of mutant EGFR provide insight on its association with EGFR internalization and trafficking and suggest novel therapeutic strategy to manage drug resistance in NSCLC. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-11T14:56:26Z (GMT). No. of bitstreams: 1 ntu-109-D04b48002-1.pdf: 10182786 bytes, checksum: 35f27f981effe88970cf3686fe303dbd (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | 口試委員會審定書………………………………………………….…….……….…… i
中文摘要…………………………………………………………….…………………. ii Abstract …………………………………………………………..……………….….... iv 目錄...….…………………………………………………………….……….......……. vi 圖目錄…………………………………………………………….………….............. viii 表目錄……………………………………………………………….………....…….... xi 1. Introduction ……………………………………………….……………..………... 1 1.1 Lung Cancer and Epidermal Growth Factor Receptor (EGFR) ...……….…..... 1 1.2 EGFR Mutation and TKI-Resistance………………………………………...... 2 1.3 Affinity Purification Coupled Mass Spectrometry (AP-MS) and Protein-protein Interaction Study …………………………………….……………………………. 5 1.4 Thesis Objective ……………………………...……………...……..…………. 7 2. Materials and Methods ……………………….………………….………………. 8 2. 1 Cell Culture ………………….………..………………..…………………... 8 2.2 Reagents and Antibodies .……………………………………………………... 9 2.3 EGFR Complexes Pull-down by Immuno-precipitation (IP) ….……..........… 10 2.4 Protein Digestion and Peptide Processing ………….……………….......….... 11 2.5 Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) ……..… 12 2.6 Database search and quantification …......…………………………..……..… 12 2.7 Non-specific Binding Exclusion ………..……………………………..…..… 13 2.8 EGFR Interactome Data Analysis ………………..………………………..… 13 2.9 Western Blotting Analysis ……..…………………………………………...... 14 2.10 Co-immunoprecipitation ……………………………………………..…..… 15 2.11 Immunofluorescence and Confocal Microscopy ………………….………... 15 2.12 Subcellular Fractionation by High-speed Centrifugation …………………... 17 2.13 MTS Cell Proliferation Assay ……………………………………………… 17 2.14 Statistical Analysis …………………………………………………………. 18 3. Result and discussion …………………………………….……………………...… 19 3.1 The AP-MS of EGFR interactome in TKI-sensitive and -resistant NSCLC cells …………………………………………………………….…………...…… 19 3.2 Elimination of non-specific interactors in AP-MS-derived EGFR interactome ………………………………………………..………………..……. 20 3.3 Network construction and bioinformatics analysis of EGFR interactomes indicated vigorous protein translocation in T790M-acquired resistant NSCLC cells ………………………………………………………….……………...…… 24 3.4 Characterization of EGFR localization in TKI-sensitive and -resistant NSCLC cells ………………………………………….…………………………………... 28 3.5 Mutant EGFR trafficking induced by therapeutic molecule - tyrosine kinase inhibitor ……………………………………………..………………………..….. 30 3.6 Autophagy activation induced by gefitinib in T790M-acquired resistant NSCLC cells ………………………………………………….…………………………... 33 3.7 Inhibition of autophagy recover the TKI sensitivity in T790M-acquired resistant NSCLC ……………………………………………..……………………….…… 35 3.8 Effect of gefitinib on Rab7-mediated endosomal transportation in TKI-sensitive NSCLC ………………………………………………………..………….……… 38 3.9 NEDD4L indicates a regulatory role in autophagy-mediated EGFR degradation …………………………………………..……………..……………. 40 4. Conclusion ……………………………………...…..……………………………… 43 Reference …………………………………………………….………………….…….. 49 Appendix …………………………………………………….…………………...….... 99 | |
| dc.language.iso | en | |
| dc.subject | 交互作用體 | zh_TW |
| dc.subject | 自噬作用 | zh_TW |
| dc.subject | 內吞作用 | zh_TW |
| dc.subject | 酪胺酸?抑制劑抗藥性 | zh_TW |
| dc.subject | 體細胞突變 | zh_TW |
| dc.subject | 親和性純化策略與質譜 | zh_TW |
| dc.subject | 表皮生長因子接受器 | zh_TW |
| dc.subject | Endocytic trafficking | en |
| dc.subject | TKI resistance | en |
| dc.subject | Autophagy | en |
| dc.subject | EGFR Interactome | en |
| dc.subject | EGFR mutation | en |
| dc.subject | Affinity purification coupled mass spectrometry | en |
| dc.title | 從表皮生長因子接受器蛋白交互作用體研究肺腺癌抗藥性機轉與調控路徑 | zh_TW |
| dc.title | EGFR Interactome Reveals Multiple Pathways and Mechanism of Drug Resistance in Non-Small Cell Lung Cancer | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-1 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 陳玉如(Yu-Ju Chen),游佳融(Chia-Jung Yu),俞松良(Sung-Liang Yu),施金元(Jin-Yuan Shih) | |
| dc.subject.keyword | 表皮生長因子接受器,交互作用體,親和性純化策略與質譜,體細胞突變,酪胺酸?抑制劑抗藥性,內吞作用,自噬作用, | zh_TW |
| dc.subject.keyword | EGFR Interactome,Affinity purification coupled mass spectrometry,EGFR mutation,TKI resistance,Endocytic trafficking,Autophagy, | en |
| dc.relation.page | 132 | |
| dc.identifier.doi | 10.6342/NTU202000272 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2020-02-21 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 基因體與系統生物學學位學程 | zh_TW |
| dc.date.embargo-lift | 2025-03-03 | - |
| 顯示於系所單位： | 基因體與系統生物學學位學程 | |
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