肺癌是一種非常嚴重及惡性的疾病。其中，肺腺癌是肺癌中最主要的一種，每一年造成全球大約五十萬人死亡。KRAS基因的突變是肺腺癌的主要成因之一，然而截至目前為止臨床上仍然沒有有效的藥物可以直接抑制突變的KRAS蛋白。因此，找出新的KRAS訊號下游分子標的並可藉由抑制KRAS訊號下游標的來幫助治療帶有KRAS突變的肺癌病人非常重要。KRAS 經由RAF/MEK/ERK 訊號傳遞路徑活化轉錄因子Forkhead box M1 (FOXM1) 進而調控細胞週期相關基因並且促進細胞增生。除此之外，FOXM1也被發現會調控血管新生、上皮間質轉化、轉移等癌惡化等特徵。根據先前的研究，我們發現在SPC-rtTA/TetO-Cre/TetO-KrasG12D/Foxm1fl/fl 基因轉殖鼠中，表現致癌基因KrasG12D並同時剔除Foxm1基因可抑制因KrasG12D所誘導的腫瘤數量及體積。這結果顯示出Foxm1 對於KrasG12D 所誘導的腫瘤之起始是重要且必須的。在此，我們進一步探討Foxm1 對於KrasG12D 所誘導的腫瘤之維持是否也扮演不可或缺的角色。我們給予CCSP-rtTA/TetO-KrasG12D/Foxm1fl/fl 基因轉殖鼠含有Doxycycline的食物來誘導肺腫瘤的產生，之後再將帶有CRE 基因的線病毒利用氣管注射感染肺腫瘤並且剔除Foxm1。 我們使用micro CT 檢測同一隻小鼠給予病毒前後的腫瘤大小，發現將Foxm1 去除後造成腫瘤的縮小。同時，細胞實驗也證明抑制FOXM1 可以抑制肺癌細胞以及帶有KrasG12D 的肺支氣管上皮細胞之細胞增生和細胞非貼附生長等癌化特性。我們的研究顯示Foxm1對於維持KrasG12D所誘導的細胞癌化特性和腫瘤大小的必要性。因此在臨床上，抑制FOXM1 的方式將具有潛力來治療帶有KRAS基因突變的肺癌病人。

Lung adenocarcinoma, the leading subtype of non-small cell lung cancer (NSCLC), is responsible for 500,000 deaths per year worldwide. Gain-of-function mutation of Kras gene is one of the major causes of NSCLC and no effective chemotherapy agent is available for cancers carry KRAS mutation. Hence, identifying new downstream molecular targets of KRAS signaling is critical for improving current therapeutic outcomes. Many reports indicated KRAS signaling activates downstream Forkhead box M1 (Foxm1) transcription factor that regulates transcription network of cell cycle regulatory genes, angiogenesis, genes related to epithelial to mesenchymal transition (EMT), cell migration and cancer stemness. We have previously generated SPC-rtTA/TetO-Cre/TetO-KrasG12D/Foxm1fl/fl mice and demonstrated that deleting Foxm1 alleles in respiratory epithelial cells diminished the number and size of lung tumors induced by oncogenic Kras. This result shows that Foxm1 is required for tumor initiation and proliferation of Kras gain-of-function mutant cells. Herein, we further investigate the role of Foxm1 in tumor maintenance and progression. After feeding doxycycline food for 5.5 or 7.5 months, lung tumors bearing CCSP-rtTA/TetO-KrasG12D/Foxm1fl/fl mice were intratracheal injected with CRE recombinase expressing viruses to cause genetically deletion of Foxm1 gene alleles in lungs. Micro Computed tomography (micro CT) images demonstrated that Foxm1 knockout caused lung tumor regression in vivo. Moreover, shRNA mediated knock down of FOXM1 expression inhibits cell proliferation, anchorage independent growth of KRAS mutated lung cancer cell lines and oncogenic KRAS expressed lung immortalized cell line with p53 inhibiton in vitro. This result suggested FOXM1 could be a potential therapeutic target to improve the outcome of KRAS mutant lung cancer treatment.

Acknowledgement 3
中文摘要 4
Abstract 5
Introduction 6
Results 12
AD-CRE-IRES-GFP virus is functional to delete Foxm1 gene alleles in vitro 12
Cre adenovirus mediated knockout of Foxm1 gene alleles in preexist lung tumors of CCSP-rtTA/TetO-KrasG12D/Foxm1fl/fl mouse model caused tumor regression. 13
pINDUCER-shFOXM1 construct is functional and knockdown Foxm1 inhibit H23 and A549 cell proliferation and anchorage independent properties 14
EGFP-KRAS fusion proteins localized to plasma membrane are functional to activate KRAS downstream signaling pathway 15
Generation of stable cell lines expressing EGFP-KRAS fusion protein by lentivirus infection 16
EGFP-KRAS can increase cell proliferation but anchorage independent property 16
EGFP-KRAS activation and P53 inhibition synergistically promote anchorage independent property. 17
Conclusions and Discussion 19
EGFP-KRAS fusion protein promoted cell proliferation of BEAS-2B and induced anchorage independent property synergistically with p53 inhibition. 19
Knockdown of Foxm1 in BEAS2B-p53DN-GSE56 and lung cancer cell lung (H23 and A549) decrease cell proliferation and anchorage independent growth property 19
Knockout of Foxm1 gene alleles in preexisted KRAS mutated lung tumors caused tumor regression 20
Prospective 24
Materials and Methods 25
Plasmids constructions 25
Cell culture 27
Transient transfection 28
Lentivirus generation and cell line generation 28
Fluorescent microscopy 29
Western blotting 29
MTT assay 30
Soft agar assay 31
Real-time reverse transcription-PCR analysis for gene expression studies 31
Adenovirus generation, purification and titer determination 32
intratracheal injection of adenovirus, microCT and image analysis 32
Genotyping 33
Statistics 34
References 35
Figures 40
Tables 69
Supplementary figures 78

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