肺癌在世界和臺灣中，都位居癌症死亡率之首，而發生突變的EGFR是肺癌發生與惡化的原兇。臨床上，針對EGFR突變的酪胺酸酶抑制劑 (EGFR-TKI)，例如：Gefitinib、Erlotinib和Afatinib。雖然EGFR-TKI藥物能夠延長帶有EGFR突變的肺癌患者的其無惡化存活期，但於一年之內肺癌即會產生抗藥性。通常腫瘤的惡化會伴隨著幹細胞的訊號，例如於轉移和抗藥性產生之時，因此我們研究SOX2一個對肺幹細胞的自我更新極為重要的轉錄因子，與對TKI藥物產生抗性的關聯。首先，我們選用帶有EGFR突變的肺癌細胞株HCC827將其先培養在逐漸增高Gefitinib濃度的培養液中，篩選出對EGFR-TKI藥物產生抗性的細胞株，並將其命名為HCC827GR。由實驗發現HCC827GR有EMT的發生並增加移動能力，然而其SOX2的表達量低於原本的母株細胞。再者，我們從HCC827中篩選出帶有不同內生性SOX2表達量的細胞株，發現其SOX2的表達量與對EGFR-TKI藥物的敏感性高低有所關聯。此外，若於HCC827GR中大量表達SOX2則能會抑制其移動能力。我們的研究顯示SOX2能夠調控肺癌細胞的塑性，並且具有潛力成為EGFR-TKI治療上的生物標記。

Lung cancer is the leading cause of cancer mortality in the world as well as in Taiwan. Abnormal expression of epidermal growth factor receptor (EGFR) has been attributed to lung cancer initiation and progression. EGFR mutations occur in lung adenocarcinoma, contributing to clinical efficacy of EGFR-tyrosine kinase inhibitors (TKIs), such as Gefitinib, Erlotinib and Afatinib. EGFR-TKI treatments prolong progression-free survival in EGFR-mutated lung cancer patients. Nonetheless, EGFR-TKI resistance often occurs in one year. Because stem cell signaling is associated with tumor malignancy, such as drug resistance and metastasis, we further investigate the role of SOX2, a transcriptional factor essential for self-renewal in lung stem cells, in development of TKI-resistance. With sequentially increasing concentration of Gefitinib in HCC827, an EGFR-mutated lung adenocarcinoma cell line, we established EGFR-TKI resistant cells, named as HCC827GR. Intriguingly, HCC827GR had increased migration ability and underwent epithelial-to-mesenchymal transition (EMT). Moreover, we observed that SOX2 expression was diminished in HCC827GR compared to parental cells. By selecting HCC827 single clones with different intrinsic SOX2 expression, we found that SOX2 expression was correlated with EGFR-TKI sensitivity. Ectopic expression of SOX2 skewed HCC827GR trans-differentiation from EMT to a reverse process, mesenchymal-to- epithelial transition (MET). In addition, SOX2 expression attenuated migration of HCC827GR. Thus, our data support that SOX2 regulates cancer plasticity with a potential as a biomarker for EGFR-TKI treatment in lung cancer.

中文摘要……………………………………………………………………………………………1
英文摘要…………………………………………………………………………………………2
背景介紹
1. 肺癌…………………………………………………………………………………………...…3
2. 上皮細胞生長因子受體及酪胺酸酶抑制劑…………………………..………………………..3
3. 表皮間質轉化………………………………………………………………..…………………..4
4. 癌症幹細胞……………………………………………………………….………….…………5
5. 轉錄因子SOX2………………………………………………………………………………….6
目標…………………………………………………………………………………………………7
材料與方法
1. 細胞培養………………………………………………………………………………………….8
2. 聚落形成分析…………………………………………………………………………………....8
3. Alamar blue 細胞存活分析…………………………………………………………………..8
4. 反轉錄聚合酶鏈反應……………………………………………………………………………9
5. 即時聚合酶鏈鎖反應…………………………………………………………………………..9
6. 移動能力測試……………………………………………………………………………………9
7. Lentivirus 轉殖基因系統…………………………………………………………………….9
8. 蛋白質萃取和西方墨點法……………………………………………………………………10
結果
1. HCC827GR具有抗EGFR-TKI之特性……………………………………………………….11
2. HCC827GR的型態改變及EMT之產生…………………………………………………..11
3. HCC827GR的SOX2表達量下降可能跟EMT有關 ………………………………………12
4. 增加HCC827的SOX2表達量導致MET之產生與對TKI藥物更加敏感……………….....12
5. 增加HCC827GR的SOX2表達量，致其型態改變及MET之產生………..…………13
討論…………………………………………………………………………………………….…..14
圖表及圖說……………………………………………………………………………….17
補充的圖表及圖說………………………………………………………………….31
補充的表格………………………………………………………………………….39
參考文獻……………………………………………………………………………………….…41

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