胰臟癌為非常惡性之腫瘤，其診斷與治療皆面臨各種挑戰，目前沒有任何有效治療胰臟癌的方法。胰臟癌病患檢體中可檢測出非常高比率的Kras基因突變，因此尋找針對Kras訊號傳遞路徑的下游標靶是一個解決目前胰臟癌困境的方法，其中一個非常有潛力的標靶，轉錄因子Forkhead box M1 (FoxM1)為一調控細胞週期，進而調控細胞增生、分化的重要因子，在許多癌症細胞中可觀察到FoxM1高度表現，包括胰臟癌細胞。到目前為止我們實驗室與其他研究團隊發現在細胞實驗與活體動物實驗中，Ras/MAPK訊號傳遞路徑活化FoxM1進而調控細胞週期相關基因，本實驗室近期的研究中使用SPC-rtTA/ TetO-Cre/ TetO-KrasG12D/ FoxM1fl/fl 的模式生物小鼠發現在肺臟表皮細胞中剔除FoxM1基因減少KrasG12D 突變誘導的腫瘤數量及大小，這個結果暗示FoxM1為KrasG12D誘導產生腫瘤過程中的重要因子。然而，在胰臟癌中FoxM1在突變的Kras訊號路徑中的角色尚未被確立，因此我們使用Doxycycline誘導表現系統在胰臟癌細胞株中表現FoxM1，實驗結果證實高度表現FoxM1促進胰臟癌細胞增生、遷移與上皮-間質轉化，高度表現FoxM1的胰臟癌細胞珠促進間質細胞標記物Vimentin表現量增加，以及上皮細胞標記物E-cadherin表現量下降，伴隨著明顯的細胞增生與細胞遷移特徵，反之，抑制胰臟癌細胞內本身FoxM1的表現抑制細胞增生與細胞遷移特徵。FoxM1在含有突變Kras基因的胰臟癌細胞增生、遷移與上皮-間質轉化中扮演非常重要的角色，在未來對於胰臟癌的治療與診斷上可能作為一個有潛力的胰臟癌細胞標靶。

Activating mutations in Kras gene were frequently found in human patients with pancreatic cancers and no effective chemotherapy agent is available. Hence, identifying new downstream molecular targets of KRAS signaling is critical for improving current therapeutic outcomes. The Forkhead box M1 (FoxM1), a proliferation specific transcription factor, is highly expressed in a variety of aggressive human cancers, including pancreatic cancers. We and others have demonstrated that FoxM1 is a major effector of Ras/MAPK signaling and transactivates genes required for cell cycle progression in vitro and in vivo. We recently demonstrated that depleting FoxM1 alleles in respiratory epithelial cells diminished the number and size of lung tumors induced by oncogenic KrasG12D in SPC-rtTA/ TetO-Cre/ TetO-KrasG12D/ FoxM1fl/fl mice, suggesting FoxM1 is required for gain-of-function K-Ras initiated lung tumorigenesis. However, the role of FoxM1 in Kras-mutated pancreatic cancer is still elusive. Herein , we showed that inducible overexpression of FoxM1 in pancreatic cancer cells led to an increased cell migration rate and acquisition of epithelial-mesenchymal transition(EMT) phenotypes, which were associated with an increased level of mesenchymal cell markers, vimentin, and reduction of epithelial cell marker, E-cadherin. Increased cell proliferation and cell migration can be also detected in FoxM1 gain of function pancreatic cells. In contrast, inhibition of endogenous FoxM1 expression by siRNA reduced cell proliferation and cell migration. We demonstrated that FoxM1 is critical for Kras mutated pancreatic cancer cell proliferation, migration and EMT, suggesting FoxM1 can be a potential target for pancreatic adenocarcinoma cancer diagnosis and therapies.

Contents

Abstract ----------------------------------------------------------------------------------------- 4
Introduction ------------------------------------------------------------------------------------ 6
Materials and Methods-----------------------------------------------------------------------12
Cell culture --------------------------------------------------------------------------------------12
Transient transfections-------------------------------------------------------------------------12
Generation of lentiviral vectors---------------------------------------------------------------13
Western blot-------------------------------------------------------------------------------------13
MTT and WST-1 assay------------------------------------------------------------------------14
Cell migration assay----------------------------------------------------------------------------14
Real-time reverse transcription-PCR analysis ---------------------------------------------15
Statistical methods------------------------------------------------------------------------------16
Results ------------------------------------------------------------------------------------------17
Depleting FoxM1 expression by siRNA diminished PC cell growth -------------------17
Depleting FoxM1 expression in pancreatic cancer cells inhibited cell migration in vitro-----------------------------------------------------------------------------------------------18
pINDUCER20 lentivirus elicits inducible expression of GFP tagged FoxM1 and N-terminal deletion mutant--------------------------------------------------------------------18
Effect of FoxM1 overexpression on pancreatic cancer cells migration in vitro--------19
Overexpression of FoxM1-FL increased cell proliferation in PANC-1 cells-----------19
Overexpression of FoxM1 induced epithelial to mesenchymal transition in PANC-1 cells ----------------------------------------------------------------------------------------------20
Discussion --------------------------------------------------------------------------------------21
Appendix----------------------------------------------------------------------------------------23
Tables -------------------------------------------------------------------------------------------23
Table 1. Chemicals of acrylamide gels------------------------------------------------------23
Table 2. Chemicals of Running/Transfer buffer(10X) ------------------------------------23
Table 3. Chemicals of Loading dyes(5X) ---------------------------------------------------23
Table 4. Chemicals of Protein IP Buffer-----------------------------------------------------24
Table 5. Chemicals of Stripping buffer------------------------------------------------------24
Table 6. Chemicals of TBST(10X) ----------------------------------------------------------24
Experimental procedures--------------------------------------------------------------------25
Western blot protocol--------------------------------------------------------------------------25
References--------------------------------------------------------------------------------------28

1. Torre, L.A., et al., Global cancer statistics, 2012. CA Cancer J Clin, 2015. 65(2): p. 87-108.
2. Siegel, R., et al., Cancer statistics, 2014. CA Cancer J Clin, 2014. 64(1): p. 9-29.
3. Ryan, D.P., T.S. Hong, and N. Bardeesy, Pancreatic adenocarcinoma. N Engl J Med, 2014. 371(22): p. 2140-1.
4. Pliarchopoulou, K. and D. Pectasides, Pancreatic cancer: Current and future treatment strategies. Cancer Treatment Reviews, 2009. 35(5): p. 431-436.
5. Jemal, A., et al., Cancer Statistics, 2010. Ca-a Cancer Journal for Clinicians, 2010. 60(5): p. 277-300.
6. Limani, P., et al., [Pancreatic cancer- a curable disease]. Praxis (Bern 1994), 2015. 104(9): p. 453-60.
7. La Rosa, S., F. Sessa, and C. Capella, Acinar Cell Carcinoma of the Pancreas: Overview of Clinicopathologic Features and Insights into the Molecular Pathology. Front Med (Lausanne), 2015. 2: p. 41.
8. Klimstra, D.S. and D.S. Longnecker, K-ras mutations in pancreatic ductal proliferative lesions. Am J Pathol, 1994. 145(6): p. 1547-50.
9. Almoguera, C., et al., Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes. Cell, 1988. 53(4): p. 549-54.
10. Almoguera, C., et al., Most Human Carcinomas of the Exocrine Pancreas Contain Mutant C-K-Ras Genes. Cell, 1988. 53(4): p. 549-554.
11. Schubbert, S., K. Shannon, and G. Bollag, Hyperactive Ras in developmental disorders and cancer. Nat Rev Cancer, 2007. 7(4): p. 295-308.
12. Mccormick, F., Ras Gtpase Activating Protein - Signal Transmitter and Signal Terminator. Cell, 1989. 56(1): p. 5-8.
13. Hruban, R.H., et al., K-ras oncogene activation in adenocarcinoma of the human pancreas. A study of 82 carcinomas using a combination of mutant-enriched polymerase chain reaction analysis and allele-specific oligonucleotide hybridization. Am J Pathol, 1993. 143(2): p. 545-54.
14. Grunewald, K., et al., High frequency of Ki-ras codon 12 mutations in pancreatic adenocarcinomas. Int J Cancer, 1989. 43(6): p. 1037-41.
15. Behren, A., et al., Phenotype-assisted transcriptome analysis identifies FOXM1 downstream from Ras-MKK3-p38 to regulate in vitro cellular invasion. Oncogene, 2010. 29(10): p. 1519-30.
16. Major, M.L., R. Lepe, and R.H. Costa, Forkhead box M1B transcriptional activity requires binding of Cdk-cyclin complexes for phosphorylation-dependent recruitment of p300/CBP coactivators. Mol Cell Biol, 2004. 24(7): p. 2649-61.
17. Ma, R.Y., et al., Raf/MEK/MAPK signaling stimulates the nuclear translocation and transactivating activity of FOXM1. Methods Mol Biol, 2010. 647: p. 113-23.
18. Zhong, H., et al., Synergistic effects of concurrent blockade of PI3K and MEK pathways in pancreatic cancer preclinical models. PLoS One, 2013. 8(10): p. e77243.
19. Clark, K.L., et al., Co-crystal structure of the HNF-3/fork head DNA-recognition motif resembles histone H5. Nature, 1993. 364(6436): p. 412-20.
20. Halasi, M. and A.L. Gartel, A novel mode of FoxM1 regulation: positive auto-regulatory loop. Cell Cycle, 2009. 8(12): p. 1966-7.
21. Korver, W., J. Roose, and H. Clevers, The winged-helix transcription factor Trident is expressed in cycling cells. Nucleic Acids Res, 1997. 25(9): p. 1715-9.
22. Wang, I.C., et al., Forkhead Box M1 Regulates the Transcriptional Network of Genes Essential for Mitotic Progression and Genes Encoding the SCF (Skp2-Cks1) Ubiquitin Ligase. Mol Cell Biol, 2005. 25(24): p. 10875-94.
23. Wang, I.C., et al., FoxM1 regulates transcription of JNK1 to promote the G1/S transition and tumor cell invasiveness. J Biol Chem, 2008. 283(30): p. 20770-8.
24. Costa, R.H., et al., New and unexpected: forkhead meets ARF. Curr Opin Genet Dev, 2005. 15(1): p. 42-8.
25. Laoukili, J., et al., FoxM1 is required for execution of the mitotic programme and chromosome stability. Nat Cell Biol, 2005. 7(2): p. 126-36.
26. Kim, I.M., et al., The Forkhead Box m1 transcription factor stimulates the proliferation of tumor cells during development of lung cancer. Cancer Res, 2006. 66(4): p. 2153-61.
27. Chan, D.W., et al., Over-expression of FOXM1 transcription factor is associated with cervical cancer progression and pathogenesis. J Pathol, 2008. 215(3): p. 245-52.
28. Li, Q., et al., Critical role and regulation of transcription factor FoxM1 in human gastric cancer angiogenesis and progression. Cancer Res, 2009. 69(8): p. 3501-9.
29. Kalin, T.V., et al., Increased levels of the FoxM1 transcription factor accelerate development and progression of prostate carcinomas in both TRAMP and LADY transgenic mice. Cancer Res, 2006. 66(3): p. 1712-20.
30. Spirin, K.S., et al., p27/Kip1 mutation found in breast cancer. Cancer Res, 1996. 56(10): p. 2400-4.
31. Singh, B., et al., Molecular cytogenetic characterization of head and neck squamous cell carcinoma and refinement of 3q amplification. Cancer Res, 2001. 61(11): p. 4506-13.
32. Liu, M., et al., FoxM1B is overexpressed in human glioblastomas and critically regulates the tumorigenicity of glioma cells. Cancer Res, 2006. 66(7): p. 3593-602.
33. Priller, M., et al., Expression of FoxM1 is required for the proliferation of medulloblastoma cells and indicates worse survival of patients. Clin Cancer Res, 2011. 17(21): p. 6791-801.
34. Xia, J.-T., et al., Overexpression of FOXM1 Is Associated With Poor Prognosis and Clinicopathologic Stage of Pancreatic Ductal Adenocarcinoma. Pancreas, 2012. 41(4): p. 629-635.
35. Wang, Z., et al., Down-regulation of Forkhead Box M1 transcription factor leads to the inhibition of invasion and angiogenesis of pancreatic cancer cells. Cancer Res, 2007. 67(17): p. 8293-300.
36. Bao, B., et al., Over-expression of FoxM1 leads to epithelial-mesenchymal transition and cancer stem cell phenotype in pancreatic cancer cells. J Cell Biochem, 2011. 112(9): p. 2296-306.
37. Huang, C., et al., A novel FoxM1-caveolin signaling pathway promotes pancreatic cancer invasion and metastasis. Cancer Res, 2012. 72(3): p. 655-65.
38. Kalluri, R. and R.A. Weinberg, The basics of epithelial-mesenchymal transition. J Clin Invest, 2009. 119(6): p. 1420-8.
39. Thiery, J.P., Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer, 2002. 2(6): p. 442-54.
40. Fodde, R. and T. Brabletz, Wnt/beta-catenin signaling in cancer stemness and malignant behavior. Curr Opin Cell Biol, 2007. 19(2): p. 150-8.
41. Yang, K., et al., Short hairpin RNA- mediated gene knockdown of FOXM1 inhibits the proliferation and metastasis of human colon cancer cells through reversal of epithelial-to-mesenchymal transformation. J Exp Clin Cancer Res, 2015. 34: p. 40.
42. Yu, C., et al., Targeting FoxM1 inhibits proliferation, invasion and migration of nasopharyngeal carcinoma through the epithelialto-mesenchymal transition pathway. Oncol Rep, 2015. 33(5): p. 2402-10.
43. Kong, F.F., et al., FOXM1 regulated by ERK pathway mediates TGF-beta1-induced EMT in NSCLC. Oncol Res, 2014. 22(1): p. 29-37.
44. Meng, F.D., et al., FoxM1 overexpression promotes epithelial-mesenchymal transition and metastasis of hepatocellular carcinoma. World J Gastroenterol, 2015. 21(1): p. 196-213.
45. Chiu, W.T., et al., FOXM1 confers to epithelial-mesenchymal transition, stemness and chemoresistance in epithelial ovarian carcinoma cells. Oncotarget, 2015. 6(4): p. 2349-65.
46. Wang, I.C., et al., Increased expression of FoxM1 transcription factor in respiratory epithelium inhibits lung sacculation and causes Clara cell hyperplasia. Dev Biol, 2010. 347(2): p. 301-14.
47. Wang, I.C., et al., Foxm1 transcription factor is required for the initiation of lung tumorigenesis by oncogenic Kras(G12D.). Oncogene, 2014. 33(46): p. 5391-6.
48. Yang, C., et al., FOXM1 promotes the epithelial to mesenchymal transition by stimulating the transcription of Slug in human breast cancer. Cancer Lett, 2013. 340(1): p. 104-12.
49. Wei, P., et al., FOXM1 promotes lung adenocarcinoma invasion and metastasis by upregulating SNAIL. Int J Biol Sci, 2015. 11(2): p. 186-98.
50. Qian, J., et al., Twist1 promotes gastric cancer cell proliferation through up-regulation of FoxM1. PLoS One, 2013. 8(10): p. e77625.
51. Yang, J. and R.A. Weinberg, Epithelial-mesenchymal transition: at the crossroads of development and tumor metastasis. Dev Cell, 2008. 14(6): p. 818-29.