臺灣博碩士論文加值系統

在占85%肺癌比例的非小細胞肺癌(non small cell lung cancer, NSCLC)患者中，肺腺癌(adenocarcinoma)屬於最大宗且在亞洲被指出與特定基因如ERBB家族的突變有關。其中，human epidermal growth factor receptor 2 (HER2)被指出過度表現或其酪氨酸激酶區域(tyrosine kinase domain)的突變在人類許多癌症的致癌進程中扮演重要的角色。在非小細胞肺癌患者中約有2-4%具有HER2突變的情形，且被指出往往與其他基因突變的發生無關。近來，由臺大醫院楊志新教授團隊所鑑定出一位非小細胞肺癌病患帶有的HER2 755PL插入性突變(可簡稱為HER2PL)，同時帶有野生型(wild-type)的EGFR (epidermal growth factor receptor),臨床治療上發現使用第一代酪氨酸激酶抑制劑(tyrosine kinase inhibitor, TKI) Gefitinib的治療成效不佳，而對第二代中的Afatinib則有較好的治療效果。目前對於此插入性突變在肺癌進程中扮演的角色與EGFR-TKI的相關性尚未清楚。在本篇論文中，我們先建立帶有此病患HER2PL突變位點序列的質體並轉染至小鼠胚胎成纖維細胞(NIH/3T3)和表現野生型(wild-type)EGFR的人支氣管肺泡癌細胞(NCI-H358)。根據研究結果顯示，過度表現HER2PL可活化HER信號傳遞路徑且相較於HER2WT有更高的p-HER2和p-AKT表現量，進一步發現afatinib和gefitinib相比可更顯著降低HER2PL所活化的信號路徑。此外，也利用免疫螢光染色觀察在穩定表現HER2WT或HER2PL的NCI-H358中，HER2WT主要分布在細胞膜，而HER2PL則除細胞膜外在細胞質也有明顯分布，且在afatinib處理下和gefitinib相比HER2PL其p-HER2有顯著下降的情形。而在細胞毒殺性實驗中，可觀察到HER2PL其對於afatinib相對於gefitinib是更為敏感有效。HER2PL其對細胞生長、藥物靈敏度與致癌進程等仍須在未來進一步探討，本次的研究結果希望能進一步了解HER2PL在肺癌進程中所扮演的角色，以及應用於未來對於HER2PL的相關治療方針。

Adenocarcinoma is the most common form of non-small cell lung cancer (NSCLC) that accounts for about 85 percent of lung cancers. Lung adenocarcinoma in Asian are usually accompanied with frequent mutations in oncogenic drivers. Human epidermal growth factor receptor 2 (HER2) is frequently overexpressed in various human cancers, and many preclinical studies have shown that overexpression of HER2 or mutations of the HER2 kinase domain plays an important role in oncogenic transformation and tumorigenesis. HER2 mutations were identified in approximately 2–4% of NSCLCs and were usually mutually exclusive with other driver mutations. Recently, a novel HER2 755PL in-frame (also called HER2PL) mutation in a lung adenocarcinoma patient carrying wild type EGFR gene has been identified from Dr. Chih-Hsin Yang in National Taiwan University Hospital and this patient was sensitive to a second generation of EGFR tyrosine kinase inhibitor (TKI), afatinib. However, the role of HER2PL mutation in lung tumorigenesis and its response to EGFR TKIs have never been addressed before. In this study, we established a plasmid construct carrying a HER2 gene with HER2PL and transfected into normal murine fibroblasts, NIH/3T3 and human lung adenocarcinoma, NCI-H358 which expressing wild-type EGFR. We found that overexpression of HER2PL mutation can activate HER signaling pathways in both NIH/3T3 and NCI-H358. HER2PL mutation induces much higher phosphorylation of HER2 and downstream AKT signaling pathway compared to wide-type HER2. In addition, we found that afatinib can significantly decrease HER2PL mutation-induced HER2 signaling pathway compared to a first generation of EGFR TKI, gefitinib. Furthermore, we found that the distribution of HER2PL mutation is in cytosol as well as on the membrane and the expression of p-HER2 (Tyr1221/1222) can be effectively attenuated with afatinib treatment in NCI-H358 stable lines using immunofluorescence assay. In addition, we found that HER2PL mutation is more sensitive to afatinib than gefitinib in NCI-H358 stable lines using SRB assay. The cell growth, drug sensitivity and tumorigencity of HER2PL mutation in these NCI-H358 stable lines will be investigated in the nearly future. This research may bring us new insights to understand the oncogenic significance of HER2PL mutation and be applied to relevant therapeutics.

誌謝 ........................................i
中文摘要 ........................................ii
Abstract ................................iv
縮寫檢索表 ................................vi
目錄 ......................................viii
目錄圖次 ...................................xii
附錄次 ....................................xiii
第一章 研究背景 ................................1
1 肺癌 ................................1
1-1 肺癌流行病學 ........................1
1-2 肺癌成因 ................................1
1-3 肺癌症狀 ................................2
1-4 肺癌診斷方式 ........................3
1-5 肺癌治療方式 ........................3
2 HER家族 (Human epidermal growth factor receptor) ................................................5
2-1 HER1/ErbB1/EGFR ........................5
2-2 HER2/ErbB2/Neu ........................6
2-3 HER3/ErbB3 ........................6
2-4 HER4/ErbB4 ........................6
3 HER 信號傳遞路徑 ........................7
3-1 PI3K/AKT signaling pathway ........7
3-2 Ras-Raf-MEK-ERK signaling pathway 7
4 EGFR與HER2突變 ........................8
5 Tyrosine kinase inhibitors (TKIs) 9
5-1 第一代EGFR-TKIs: Gefitinib ........9
5-2 第二代EGFR-TKIs: Afatinib ........9
5-3 第三代EGFR-TKIs ........................10
6 EGFR-TKIs之抗藥性 ................10
6-1 先天性 (intrinsic resistance) ........10
6-2 後天性 (acquired resistance) ........10
第二章 研究目的 ................................12
第三章 研究材料與方法 ........................13
1 實驗材料 ................................13
1-1 藥品與試劑 ........................13
1-2 抗體 ................................14
1-3 實驗儀器 ................................15
1-4 cDNA序列 ........................17
2 實驗方法 ................................18
2-1 細胞培養 ................................18
2-2 Construction of plasmid with HER2PL site ................................................18
2-3 轉染 (Transfection) ................19
2-4 細胞生長測定 (Sulforhodamine B assay; SRB assay) ................................................20
2-5 RNA 萃取 (RNA extraction) ........21
2-6 反轉錄-聚合酵素連鎖反應 (RT-PCR assay) 21
2-7 西方點墨法 (Western blot analysis) 22
2-8 免疫螢光染色 (Immunofluorescence staining analysis) ................................23
2-9 數據統計分析 (Statistics analysis) ........24
第四章 研究結果.......................... 25
1-1 帶有人類非小細胞肺癌病患HER2PL突變位點序列的質體建構 ................................................25
1-2 過度表現HER2PL 對NIH/3T3和NCI-H358兩種細胞株的影響 ................................................26
1-3 探討HER2PL 的活化與EGFR的關聯性 ........27
1-4 Gefitinib和afatinib對於過度表現HER2PL 的NIH/3T3和NCI-H358兩種細胞株的影響 ........................28
1-5 Gefitinib和afatinib對穩定表現HER2PL NCI-H358之HER2分布與表現的影響 ................................29
1-6 Gefitinib和afatinib對穩定表現HER2PL NCI-H358之細胞毒殺性 ........................................29
第五章 討論 ................................31
第六章 結論 ................................36
第七章 參考文獻 ................................37
圖示 ........................................42
附錄 ........................................50

目錄圖次
Fig 1. Construction of plasmids with HER2PL and sequencing results.. .........................42
Fig 2. Overexpression of HER2PL induces over-activation of HER2 signaling pathways.. .................44
Fig 3. Overexpression of HER2PL mutation can activate HER signaling pathways.. .........................45
Fig 4. Afatinib attenuates HER2PL-induced over-activation of HER2 signaling.. .........................46
Fig 5. Afatinib decreases the expression of phosphorylation of HER2 in NCI-H358 stable lines.. .................................................47
Fig 6. Cytotoxicity of gefitinib and afatinib in NCI-H358 stable lines with overexpression of HER2WT or HER2PL.. .................................................49


附錄次
Fig S1. HER signaling pathway .................50
Fig S2. PI3K/AKT signaling pathway .........51
Fig S3. Ras/Raf/MAPK signaling pathway .........52
Table S1. 聚丙烯醯胺膠體 (SDS-PAGE) 的配製濃度與比例 .................................................53
Table S2. 西方點墨法中buffer的配製濃度與比例 .53

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