肺癌是一種全球性的致死率相當高的疾病，而癌症轉移是其中一項主要的死亡原因。目前雖然有很多關於癌症轉移的研究與發表，但癌症轉移相關之調控路徑以及機制仍不清楚。Forkhead Box M1（FOXM1）轉錄因子在先前研究中已經被證明可調節細胞週期相關基因，而在許多癌症相關研究中也證明這個特性對腫瘤的起始發展和轉移至關重要。在本研究中，我們增加FOXM1表現量於帶有KrasG12D / p53 +/-突變的肺癌小鼠模式，研究結果中發現FOXM1促使肺腺癌轉移發生且增加肺癌的嚴重程度進而降低小鼠的存活時間。然而這些腫瘤大多轉移至小鼠的骨頭組織(肋骨)。而在細胞實驗中，我們透過細胞球體培養實驗發現FOXM1促進了細胞球體的形成且與N-cadherin、CXCR4和SOX9的mRNA表現量上升有關。因此我們推論FOXM1促進了肺癌的骨轉移且可能與活化CXCR4 / CXCL12訊號軸線有關。

Lung cancer is one of the leading cause of death worldwide, and metastasis of lung cancer is associated with high mortality. Although the studies of cancer metastasis are accumulating, the mechanisms of cancer metastasis are still unclear. The Forkhead Box M1 (FOXM1) transcription factor has already been proved to regulate cell cycle-related genes, showing that FOXM1 is crucial in tumor initiation, progression and metastasis. In this study, we established a lung cancer mouse model with metastatic phenotype by conditional overexpressing FOXM1 in lung adenocarcinoma harboring Kras*G12Dtg/+/p53+/-. Overexpression of FOXM1 in adenocarcinoma caused a decreasing of overall survival, which was associated with the severity of lung cancer in mice. Majority of FOXM1tg/+/Kras*G12Dtg/+/p53+/- metastatic tumors were detected in the ribs in mice with overexpressing FOXM1. Furthermore, an increased expression level of FOXM1 promoted the formation of cell sphere in culture, which was associated with up-regulated mRNA levels of N-cadherin, CXCR4 and SOX9. Our results demonstrate that the FOXM1 promotes bone metastasis in lung adenocarcinoma development via CXCR4/CXCL12 axis.

Abstract 1
中文摘要 2
Contents 3
Introduction 7
Lung cancer 7
Lung adenocarcinoma (LUAD) 7
Kras mutant and p53 loss genetically engineering mouse model 8
Cancer metastasis 9
Forkhead box M1 (FOXM1) 11
FOXM1 and cancer metastasis 12
CXCR4/CXCL12 axis 13
Hypothesis 15
Materials and methods 16
Transgenic mouse model 16
Micro-CT image and PET image taking 17
Mouse harvest 17
Bone decalcification 18
Immunohistochemistry staining 18
Generation of lentiviral vectors and stable cell lines 20
Cell culture 21
Produce the slides of cell 22
Western blot 22
Sphere culture 24
Promoter analysis 25
Total RNA isolation and Real-time reverse transcription-PCR analysis 25
Results 27
Overexpression of FOXM1 in KrasG12D/p53+/- mouse lung adenocarcinoma promoted tumor progression, bone metastasis and poor survival. 27
Identify bone tumors originated from lung adenocarcinoma harboring transgenic FOXM1 and oncogenic KrasG12D 30
Overexpression of FOXM1 promoted sphere formation of Beas2B-KrasG12D-GSE56 cell line. 32
Overexpression of FOXM1 promotes increasing expression level of mesenchymal-related gene and CXCR4. 34
Conclusion and discussion 36
Figure 1. Overexpress FOXM1 in KrasG12D/p53+/- transgenic mouse model promoted tumor progression, bone tumor formation and made mice had poor survive. 45
Figure 2. Overexpress FOXM1 in KrasG12D/p53+/- transgenic mouse model promoted lung adenocarcinoma metastasis occurrence. 50
Figure 3. Overexpression of FOXM1 promoted sphere formation of Beas2B-KrasG12D-GSE56 cell line. 54
Figure 4. Overexpression of FOXM1 promotes increasing expression level of mesenchymal-related gene and CXCR4. 58
Appendix 59
Table 1. Antibody list 59
Table 2. Mouse list of survival curve 60
Table 3. Primer list 66
Table 4. FOXM1 binding motifs on CXCR4 and Cxcr4 promoters list 67
Table 5. Mouse list of lung tumor formation, bone tumor formation and metastatic-phenotype ratio 68
Supplementary Fugure1. Statistic method of ki67 positive cells in tumor region 69
Reference 71

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