中文摘要
肺癌為全球死亡率極高的癌症。上皮生長因子受體(epidermal growth factor receptor; EGFR)的異常表現已被歸因於肺癌發生及惡化的原因之一。EGFR的突變好發於肺腺癌中，而EGFR的突變也使得肺腺癌對酪胺酸激酶抑制劑 (tyrosine kinsae inhibitor; TKI)具有高度的敏感性。肺癌病患若有EGFR突變其使用EGFR-TKI 的療效較傳統鉑金類及紫杉醇類的化療，可使病患有更佳的無惡化存活期(Progression-free survival)。但不幸的是，此類病患常於EGFR-TKI治療的一年內會有抗藥性的產生。為了瞭解在EGFR變異的肺腺癌細胞中產生EGFR-TKI抗藥性的分子機制，我們藉由含EGFR (del19) 突變且對EGFR-TKI敏感的HCC827肺腺癌細胞來進行EGFR-TKI抗藥株的篩選。我們將於gefitinib處理過60天後存活的HCC827細胞放大增生後命名為HCC827GR。有趣的是HCC827GR不只產生對EGFR-TKI的抗藥性，同時也發生了上皮─間質細胞轉化(epithelial- mesenchymal transition)的現象，且具有增強的移動和侵襲的能力。透過基因表現之微陣列 (gene expression microarray) 分析，我們發現神經內分泌蛋白VGF高量表現於HCC827GR，也藉由即時定量PCR 與免疫墨點法分析HCC827GR 確實有高量VGF表現。在HCC827GR細胞中剃除VGF 會導致細胞死亡且對gefitinib 藥物敏感性增加。進一步發現，在HCC827細胞內表現VGF會對gefitinib、 erlatinib 和 afatinib 等EGFR-TKI藥物具有抗藥性。此外，在HCC827 高量表現VGF會造成上皮─間質細胞轉化(epithelial-to-mesenchymal transition; EMT)的現象且增加細胞移動和侵襲的能力。 研究結果提供有利證據支持VGF的表現能造成肺癌細胞對EGFR-TKI 的抗藥性且會誘導細胞EMT及增加移動和侵襲的能力。VGF有潛力可以作為肺癌細胞的生物標記，以利於在治療上預後和診斷的方針。此外VGF基因也可發展成為新的肺癌藥物治療標的。

Lung cancer is the leading cause of cancer death in the world. Abnormal expression of epidermal growth factor receptor (EGFR) has been attributed to lung cancer initiation and progression. Mutations in EGFR often occur in lung adenocarcinoma and render cancer cells sensitive to EGFR tyrosine kinase inhibitors (EGFR-TKIs). Lung cancer patients harboring EGFR mutations once treated with EGFR-TKIs have better progression-free survival than those under platinum/taxol based chemotherapy. Nonetheless, resistance often develops in one year during EGFR-TKI treatment. To understand the molecular mechanism of EGFR-TKI resistance in EGFR mutated lung adenocarcinoma, we adopted HCC827, a lung adenocarcinoma cell line with del19 mutation and sensitive to EGFR-TKIs, to screen EGFR-TKI resistant cells. Through gefitinib treatment for 60 days, survived HCC827 cells were propagated and named as HCC827GR. Intriguingly, HCC827GR not only developed EGFR-TKI resistance but also acquired epithelial-to-mesenchymal transition (EMT) with increased migration and invasion abilities. Through gene expression microarray analyses followed by Q-PCR and immunoblotting, we identified VGF, encoding a neuroendocrine protein, as one of the genes differentially expressed in HCC827GR but not in HCC827. Knockdown of VGF in HCC827GR resensitized cells to gefitinib treatment with sever cell death. Forced expression of VGF in HCC827 rendered cells resistant to the treatment of EGFR-TKIs, such as gefitinib, erlatinib and afatinib. Moreover, ectopic expression of VGF in HCC827 caused EMT with increased migration and invasion. Our findings provide compelling evidence that VGF encourages EGFR-TKI resistance, EMT and invasion with potential as a novel biomarker for prognosis and diagnosis and a therapeutic target for lung cancer treatment.

文獻回顧
1. 肺癌……………………………………………………………………………………1
2. 上皮細胞生長因子受體和癌症………………………………………………………2
3. 上皮細胞生長因子受體-酪胺酸酶抑制劑 …………………………………………3
4. 上皮─間質細胞轉化和癌症 ………………………………………………………4
5. VGF 神經生長因子(nerve growth factor)……………………………………………6
研究動機和目的 …………………………………………………………………………… 7
材料和方法
1. 細胞培養 ……………………………………………………………………… …8
2. Alamar blue細胞存活試驗 ………………………………………………………8
3. 加藥處理和聚落形成析 ……………………………………………………………8
4. lentivirus 轉殖基因系統 ……………………………………………………………9
5. 基因分析試驗法………………………………………………………………………9
6. 蛋白質萃取和定量分析……………………………………………………………… 10
7. 西方墨法 ……………………………………………………………………………10
8. 創傷癒合實驗(wound healing assay) …………………………………………………11
9. 細胞轉移和侵襲驗 …………………………………………………………………11
10. 海馬XF生物能量測定儀分析細胞耗氧量 …………………………………………12
11. 細胞免疫螢光染色法…………………………………………………………………13
結果
1. 篩選EGFR-TKI抗藥性細胞株和測量抗藥性細胞株存活之差異 …………………14
2. 抗藥性產生非MET amplification和T790M 的改變 ……………………………15
3. 細胞株獲得EGFR-TKI抗藥性導致細胞型態和EMT改變 ………………………15
4. 細胞株獲得EGFR-TKI抗藥性促使細胞轉移能力增加 …………………………16
5. 細胞株獲得抗藥性調控細胞生長和影響細胞耗氧…………………………………17
6. 細胞株獲得抗藥性影響細胞存活訊息…………………………………………17
7. VGF在HCC827GR 中大量表現 ……………………………………………………18
8. 剔除VGF 促使抗藥性細胞株死亡和抗藥性下降………………………………19
9. 大量表現VGF1伴隨細胞抗藥性增加 ……………………………………………19
10. VGF大量表現會伴隨細胞型態和EMT改變 ……………………………………20
11. VGF高量表現促使細胞轉移能力增加 …………………………………………21
12. VGF表現調控細胞生長和細胞耗氧………………………………………………21
13. VGF表現調控細胞生長和存活訊號………………………………………………22
14. 惡性肺腺癌組織有高量VGF 表現 ………………………………………………22
討論 ……………………………………………………………………………………24
參考文獻 ………………………………………………………………………………26
圖表 ……………………………………………………………………………………30
附錄
中英文對照表 ………………………………………………………………………………63

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