肺腺癌是最主要的非小細胞肺癌。許多導致肺腺癌的基因已被確認。上皮生長因子接受器(EGFR)的突變盛行於東南亞國家包含臺灣的不抽菸肺腺癌病患。EGFRL858R 突變，其最主要的突變是858位點的白胺酸(Leucine)被置換突變成精胺酸(Arginine)。
Forkhead box protein M1 (FOXM1)是一個轉錄因子，在調控細胞週期、細胞增生、細胞衰老以及癌症發展等扮演重要的角色。多篇研究指出FOXM1在許多種類的癌症中過量表現，並且與惡性程度呈正向相關。我們的實驗室先前發現刪除老鼠Foxm1基因可以避免小鼠模式中的肺癌發生。然而，在腫瘤裡Foxm1對腫瘤的維持與進展角色為何，依然是不清楚的。在這篇論文，我們在四個基因的轉殖小鼠模式CCSP-CreER/CCSP-rtTA/tetO-EGFRL858R/Foxm1fl/fl中，條件性地在有EGFR突變的肺癌中刪除Foxm1等位基因。在動物實驗，以微米級電腦斷層掃描造影系統(microCT)及病理學分析呈現的實驗結果，我們發現剔除Foxm1的肺腫瘤顯著地降低了腫瘤的體積。這個結果指出Foxm1 對於腫瘤生長與維持是非常重要的因子。此外，我們發現用酪胺酸激酶抑制劑(TKI)抑制EGFR的活性後，導致人類肺腺癌細胞H3255與H1975的FOXM1蛋白質表現量下降。根據我們的實驗結果，我們提出FOXM1是EGFR訊息傳導的重要下游，並且可以做為肺腺癌標靶治療的標的之一。

Lung adenocarcinoma is a major type of non-small cell lung cancer. Several mutations on driver genes have been identified in lung adenocarcinoma. The p.L858R substitution mutation in epidermal growth factor receptor (EGFR) is predominantly found in non-smoker lung adenocarcinoma patients in East Asia, including in Taiwan.
Forkhead box protein M1 (FOXM1) is a transcription factor that plays a crucial role in cell cycle and proliferation, cell senescence, and cancer progression. Numerous studies reported that FOXM1 is upregulated in various types of cancer and positive correlated with malignancy status. Previously, we found deletion of Foxm1 gene prevented the initiation of lung cancer in mouse models. However, the role of Foxm1 in tumor maintenance and progression is still not clear. In this study, we conditionally knockout Foxm1 alleles in the lung cancer harboring EGFR mutation using the CCSP-CreER/CCSP-rtTA/tetO-EGFRL858R/Foxm1fl/fl transgenic mouse model, and we found that Foxm1 deletion significantly decreased the volumes of lung tumor in vivo as shown by micro-Computed Tomography (microCT). Using H&E stain to examine pathological change, we found Foxm1 is important for tumor growth and tumor maintenance. In addition, we found that inhibition of EGFR activity by tyrosine kinase inhibitor (EGFR-TKI) treatment caused a reduction of FOXM1 protein levels in EGFR mutated human lung cell H3255 and H1975. Accordingly, our results suggest that FOXM1 is an important downstream molecule of EGFR signaling and could be a therapeutic target for lung adenocarcinoma treatment.

摘要 I
Abstract II
Table of contents III
Chapter 1 Introduction - 1 -
1-1 Lung cancer prevalence and subtypes - 1 -
1-2 Epidermal growth factor receptor - 2 -
1-3 Tyrosine kinase inhibitors - 3 -
1-4 Forkhead box protein M1 (FOXM1) - 4 -
1-5 Transcription factor FOXM1 is required for mouse lung tumorigenesis. - 5 -
Aim of this study - 8 -
Chapter 2 Materials and Methods - 9 -
2-1 Quadruple transgene mouse breeding - 9 -
2-2 MicroCT scan imaging analysis - 10 -
2-3 Mouse lung sample processing - 11 -
2-4 Process of tissue paraffin embedding - 11 -
2-5 Hematoxylin and Eosin staining (H&E stain) - 12 -
2-6 Immunohistochemistry (IHC) staining - 12 -
2-7 Cell culture - 13 -
2-8 MTT assay for IC50 - 14 -
2-9 Western Blots - 14 -
2-10 Real-time reverse transcription quantitative PCR (RT-qPCR) analysis - 15 -
2-11 siRNA delivery - 16 -
2-12 EGFR-overexpressed cell line generation - 16 -
Chapter 3 Results - 18 -
3-1 Conditional knockout of Foxm1 caused a decrease of soft tissue volume in the lung with EGFR p.L858R tumor - 18 -
3-2 The knockout of Foxm1 decreased the pre-existed lung tumor in the EGFRL858R mouse model. - 20 -
3-3 Inhibition of EGFR activity reduced the FOXM1 expression in human EGFRL858R mutant cells. - 20 -
3-4 The knockdown of EGFR decreased FOXM1 in H3255 and H1975 in the context of human EGFRL858R mutant. - 22 -
3-5 Overexpression of EGFR did not change the FOXM1 levels and cell growth in human normal airway epithelial cells NL-20 - 23 -
Chapter 4 Discussion - 25 -
MicroCT image analysis of Foxm1 knockout mice - 25 -
Inhibition of EGFR activity by tyrosine kinase inhibitors and knockdown of EGFR in H3255 and H1975 - 26 -
Conclusion - 28 -
Figures - 29 -
Figure 1. Knockout of Foxm1 prevent lung tumor initiation in EGFR lung cancer mouse model - 30 -
Figure 2. Foxm1 knockdown showed the reduced volume of soft tissue in EGFRL858R lung cancer mouse model in micro CT image - 30 -
Figure 3. Knockdown of Foxm1 by Cre-ER system reduced the volume of soft tissue in EGFRL858R lung cancer mouse model - 32 -
Figure 4. The mouse lung tissue analyzed by IHC and H&E staining - 34 -
Figure 5. The half-maximal inhibitory concentrations (IC50) of tyrosine kinase inhibitors for EGFR mutant NSCLC cell line H3255 and H1975 - 35 -
Figure 6. EGFR mutant NSCLC cell lines are sensitive to TKI - 37 -
Figure 7. Inhibition of EGFR activity by Gefitinib decreases expression of FOXM1 in H3255 - 39 -
Figure 8. Osimertinib treated H3255 reduced the FOXM1 levels - 41 -
Figure 9. Knockdown of EGFR by siRNA reduced expression of FOXM1 in H3255 and H1975 - 42 -
Figure 10. Growth curve of EGFR overexpression in NL-20 cell by MTT assay - 43 -
Tables - 44 -
Table 1. The mice information of the Foxm1 knockout experiment in detail - 44 -
Table 2. Full list of analyzed mice by microCT - 45 -
Supplementary data - 46 -
Figure S1. Mouse genotyping results of mouse Foxm1 knockout experiment checked by PCR. - 47 -
Figure S2. Foxm1 knockdown showed the reduced volume of soft tissue in EGFRL858R lung cancer mouse model in microCT imaging - 52 -
Figure S3. Inhibition of EGFR activity by Gefitinib decreases expression of FOXM1 level and FOXM1 transcription level in H3255 - 54 -
Figure S4. FOXM1 level decreased after Osimertinib treatment in H1975 - 55 -
References - 56 -

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