胰腺導管腺癌（PDAC）是全球主要的惡性腫瘤之一。不論是中期或末期的胰臟導管腺癌，其五年生存率均低於 7 %。主要由於癌症初期形成及轉移初期都沒有明顯的症狀。許多研究表明，癌症轉移與癌細胞間質上皮細胞轉化（EMT）及癌細胞代謝中皆有重要關聯。先前許多研究指出Forkhead Box M1（FOXM1）轉錄因子可以調節細胞週期基因網絡並在許多器官的腫瘤發生中扮演關鍵作用。我們和其他學者過去在基因工程小鼠的研究發現，剔除細胞的Foxm1基因會導致抑制許多不同癌症的腫瘤發生。在這項研究中，我們使用致癌KrasG12D基因小鼠來研究Foxm1在胰腺癌起始過程中的作用，但是我們並未觀察到剔除Foxm1可以防止PanIN病變形成，但可以減少PanIN 細胞分裂生長。在細胞培養實驗中，我們發現抑制人類胰腺癌細胞中FOXM1基因表現，可造成細胞生長速度變慢以及降低細胞球體(sphere)形成的能力。同時，我們發現剔除FOXM1的胰臟癌細胞粒腺體動態轉變為延長型態(elongated) ，增加Mfn2和Drp1表現量並且增加粒腺體的膜電位，以及降低ATP的生成。我們使用OROBOROS測量，發現抑制FOXM1無法改變PDAC細胞株的粒腺體耗氧率（OCR）。因此我們認為剔除FOXM1會導致PDAC細胞生長速度降低及影響球體形成(sphere-forming)能力，並促使代償性地增加細胞內能量產生，以及改變粒線體型態轉變成延長態。

Pancreatic ductal adenocarcinoma (PDAC) is one of the malignant tumors worldwide. The five-year survival rate is less than 7 % for all stages of PDAC combined. This is because of no obvious symptoms in the early stages and early metastases. Many research indicated that cancer cell metastasis is associated with mesenchymal-epithelial transition (EMT) and their metabolism. The Forkhead Box M1 (FOXM1) transcription factor has been shown to regulate cell cycle gene and play a critical role in tumorigenesis of many organs. Genetically depletion of Foxm1 gene resulted in a decreased of tumorigenesis in many cancer mouse models. However, the role of FOXM1 in pancreatic cancer initiation, progression remain unclear. In this study, we used a genetic engineered mouse model to clarify the role of Foxm1 during initiation of pancreatic cancer by oncogenic KrasG12D. Although we did not observe the deletion of Foxm1 alleles in pancreatic acinar cells caused inhibition of PanIN lesion formation as anticipated, we found depleting FOXM1 decreased PanIN cell proliferation. Additionally, in vitro cell culture experiment showed that diminished expression of FOXM1 in human pancreatic cancer cell lines caused a decrease of cell proliferation on plates and sphere-forming ability. This phenotype is associated with elongated mitochondrial morphology increased mitochondrial membrane potential, and decreased ATP production. Interestingly, diminished expression of FOXM1 decreased mitochondrial oxygen consumption rate (OCR) as determined by OROBOROS, which was associated with increase mRNA levels of Mfn2 and Drp1in Kras mutated PDAC. We thus conclude that diminished FOXM1 levels causes a significantly decrease of PDAC cell proliferation, sphere-forming ability which correlates elevation of cellular ATP level via mitochondrial fusion.

Abstract 2
中文摘要 3
Contents 4
Introduction 7
Pancreatic ductal adenocarcinoma (PDAC) 7
Forkhead box M1 (FOXM1) 7
Metabolic reprogramming 7
Glycolysis metabolism 7
Pentose phosphate pathway (PPP) metabolism 7
Mitochondrial metabolism 7
Pancreatic ductal adenocarcinoma and metabolic reprogramming 7
FOXM1 and metabolic reprogramming 7
Mitochondrial morphology and the regulation of cellular processes 7
Hypothesis 7
Materials and methods 7
Cell culture 7
Transient transfection 7
Generation of lentiviral vectors 7
MTT assay 7
Western blot 7
Sphere culture 7
Real-time reverse transcription-PCR analysis 7
Measurement of ATP levels 7
Measurement of lactate levels 7
Measurement of mitochondrial respiration by OROBOROS 7
Measurement of mitochondrial membrane potential 7
Measurement of reductive oxidative species (ROS) 7
Cell cycle analysis 7
Immunofluorescence staining 7
Mouse model 7
Tail DNA extraction and genotyping 7
Harvest mouse 7
Immunohistochemistry staining 7
Results 7
Diminished expression of FOXM1 by RNAi caused a decrease of cell proliferation in pancreatic cancer cell lines 7
Knockdown FOXM1 inhibited sphere-forming ability in PDAC cell lines 7
Knockdown FOXM1 caused a decrease of ATP levels 7
Knockdown FOXM1 caused decrease of lactate levels 7
Diminished expression of FOXM1 by RNAi did not caused mitochondrial respiration change 7
Knockdown FOXM1 caused mitochondrial dynamics 7
Knockdown FOXM1 affected the mRNA expression levels of mitochondrial fusion and fission protein 7
Knockdown FOXM1 increased mitochondrial membrane potential 7
Knockdown FOXM1 caused-increase levels of reductive oxidative species (ROS) 7
Knockout Foxm1 in transgenic mouse model did not reduce the formation of PanIN 7
Conclusion and discussion 7
Diminished expression of FOXM1 caused decrease of ATP levels via affected glycolysis but not oxidative phosphorylation 7
Knockdown FOXM1 affected mitochondrial dynamics 7
Diminish expression of FOXM1 changed cellular energy level in Kras gain-of-function mutated PDAC cells rather than in KrasWT BxPC-3 cells 7
Knockout Foxm1 reduced the inflammation of pancreas in mouse model 7
Knockout Foxm1 did not reduce the formation of PanIN lesion in mouse model 7
Knockout Foxm1 decreased the numbers of mitochondria 7
Perspectives 7
Reference 7
Figure 7
Figure 1. Diminished expression of FOXM1 by RNAi caused decrease of cell proliferation in PDAC cell lines 7
Figure 2. Knockdown FOXM1 inhibited sphere-forming ability in PDAC cell lines. 7
Figure 3. Knockdown FOXM1 caused decrease of ATP levels in PDAC cell lines 7
Figure 4. Knockdown FOXM1 caused increase of lactate levels in PDAC cell lines 7
Figure 5. Diminished expression of FOXM1 in human pancreatic cancer cell lines by RNAi did not cause mitochondrial oxidative phosphorylation change 7
Figure 6. Knockdown FOXM1 expression caused mitochondrial morphology change in PDAC cell lines 7
Figure 7. Knockdown FOXM1 caused G1 arrest 7
Figure 8. The FOXM1 mRNA expression levels of knockdown FOXM1 by siRNA in PDAC cell lines 7
Figure 9. The Opa1 mRNA expression levels of knockdown FOXM1 by siRNA in PDAC cell lines 7
Figure 10. The Mfn2 mRNA expression levels of knockdown FOXM1 by siRNA in PDAC cell lines 7
Figure 11. Increase of Drp1 mRNA expression levels were detected in siFOXM1 transfected PDAC cell lines 7
Figure 12. Knockdown FOXM1 expression increased mitochondrial membrane potential in PDAC cell lines 7
Figure 13. Knockdown FOXM1 expression caused increase of reactive oxygen species (ROS) levels in PDAC human cell lines 7
Figure 14. Schemes shows generation of las-creER / Kras+/LSLG12D / Foxm1fl/fl mouse model for pancreatic cancer study 7
Figure 15. Conditional knockout of Foxm1 alleles in acinar cells did not affect the formation of PanIN 7
Figure 16. Body weight decreased in mice with oncogenic Kras-induced PanIN 7
Figure 17. Knockout Foxm1 did not inhibit PanIN initiation in thhe Elas-Kras pancreatic cancer model 7
Table 7
Table 1. qPCR primer list 7
Table 2. Antibody list 7
Table 3. NTES Lysis Buffer 7
Table 4. Mouse genotyping PCR primer and PCR program list 7

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