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博碩士論文 etd-0605117-225731 詳細資訊
Title page for etd-0605117-225731
論文名稱
Title
CD24通過上調Wnt信號通路誘導前列腺癌的上皮-間質轉化和骨轉移
CD24 induces epithelial-mesenchymal transition and bone metastasis in prostate cancer through the upregulation of Wnt signaling pathway
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
80
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2017-07-28
繳交日期
Date of Submission
2017-08-17
關鍵字
Keywords
P53、KRAS、CD24、前列腺癌、骨轉移、BRAF
Bone metastasis, P53, BRAF, Prostate cancer, KRAS, CD24
統計
Statistics
本論文已被瀏覽 5690 次,被下載 108
The thesis/dissertation has been browsed 5690 times, has been downloaded 108 times.
中文摘要
前列腺癌相較於其他癌症其生長速度較為緩慢,因初期無較為明顯之症狀,往往總是到中後期階段才有明顯症狀產生,且前列腺癌之預後程度主要源自於轉移是否存在而決定。在轉移上來說,骨轉移為前列腺癌中最常見的部位,而其常見之部位為椎骨、胸骨、骨盆骨、肋骨,嚴重者可能導致患者死亡。然而,於過去之研究早已證明,其腫瘤於生長及增生的過程中,必需仰賴於腫瘤內之癌症幹細胞的部分細胞,且幹細胞於惡性腫瘤的後期也扮演著很重要的角色,因此本研究將著重於探討癌症幹細胞在前列腺癌之發展與轉移上的作用。
本論文建立了兩種基因工程之小鼠模型,目的在於藉由Probasin-Cre特異性之條件於組織中剔除P53基因並且搭配KRASG12D或BRAFV600E突變體,使得小鼠產生與人類相似的前列腺癌疾病。此外,也建立了自KRASG12D以及BRAFV600E突變體的小鼠前列腺原位癌細胞,並且藉由Microarray比較分析兩者差異性,其中於前列腺癌的動物模式中,以CD24的表現較為明顯強烈進而導致腫瘤轉移的現象產生。
根據本論文研究數據所顯示,皆指出於前列腺癌的骨轉移中,CD24扮演著重要的作用分子,它促進前列腺小鼠之腫瘤的轉移,並且誘導了癌細胞之遷移 與侵襲以及我們發現藉由2-(4-Chlorobenzyl)-3-hydroxyl-7,8,9,10-tetrahydrobenzo[H] quinolone-4-carboxylic acid (PSI-697)其抑制劑,能有效減緩CD24的活性進而降低腫瘤的生成。希望藉由此項研究了解癌症幹細胞於前列腺癌中的作用,進而開發出更有效的治療藥物來對抗疾病的發生。

關鍵字: 前列腺癌,骨轉移,CD24,KRAS,P53,BRAF
Abstract
Prostate cancer is usually a very slow growing cancer, often causing no symptoms until it is in an advanced stage. The development is due to epithelial cell abnormal proliferation formation of prostatic intraepithelial neoplasia (PIN), followed by progression to invasive and metastatic cancer. Therefore, the prognosis of prostate cancer is mainly decided by the presence or absence of metastases. Bone is the most common metastatic site of prostate cancer. Metastasis to the bone most commonly involves the vertebrae, sternum, rib cage, pelvic cavity. Once tumor metastasize to bone, they are virtually incurable and result in significant disease morbidity prior to a patient's death.
In this study, we developed two genetically engineered mouse (GEM) models that crossed both conditional activate LSL / KRASG12D or mutant BRAFV600E and conditional abrogate P53 function in a prostate-specific manor to induce the development and metastatic progression of prostate adenocarcinomas, with similarities to human prostate adenocarcinoma. We also generated primary prostate cancer cell lines derived from two mouse models to characterize and compare their different molecular mechanism in vitro. Microarray studies comparing the expression profile of Pb-Cre4; P53L/L;KRASG12D and Pb-Cre4;P53L/L;BRAFV600E cell lines.
We show that CD24 expression promote tumor metastasis in our prostate cancer mouse model and strongly induced cell motility and invasion. The findings here point to an important mechanism of action for CD24 in prostate cancer. Then, the 2-(4-Chlorobenzyl)-3-hydroxyl-7,8,9,10-tetrahydrobenzo[H]quinolone-4-carboxylic acid (PSI-697) inhibitor potent reduce CD24 activity, and decrease tumorigenic effects. Understanding the stem cells in prostate cancer initiation and progression, will developing of more efficacious therapeutics to fight the disease.
Keywords: Prostate cancer, Bone metastasis, CD24, KRAS, P53, BRAF
目次 Table of Contents
國立中山大學研究生學位論文審定書 i
誌謝 ii
中文摘要 iii
Abstract iv
縮寫表 v
中英對照表 vii
目錄 viii
圖次 xi
表次 xii
第一章 緒論 1
1.1 前列腺癌 1
1.2 基因工程小鼠模型 3
1.3 PB-Cre4基因基於人類癌症 4
1.4 KRAS基因基於人類癌症 4
1.5 BRAF基因基於人類癌症 5
1.6 TP53基因基於人類癌症 5
1.7 CD24基因基於人類癌症 6
1.8 PSI-697抑制劑 6
第二章 材料與方法 7
2.1 動物模式 7
2.2 DNA萃取DNA Isolation 7
2.3基因型鑑定 8
2.4 組織染色 9
2.5 免疫組織化學染色 9
2.6 即時定量聚合酶鏈鎖反應 10
2.7 細胞培養 11
2.8 MTT細胞增生試驗 12
2.9 細胞群落分析 13
2.10 傷口癒合試驗 13
2.11 腫瘤細胞侵襲實驗 13
2.12 3D懸吊式液珠實驗 14
2.13 腫瘤球體形成試驗 14
2.14 西方點墨法 14
2.15 細胞轉染 16
2.16 統計分析 17
第三章 實驗結果 18
3.1 P53缺失與突變體KRASG12D或BRAFV600E導致前列腺癌的發展 18
3.2 鑑定PKP以及PBP小鼠的前列腺癌上皮細胞來源 19
3.3 PKP小鼠模型中骨轉移情形 19
3.4 PKP的癌細胞具有tumorigenesis的能力 20
3.5 PKP的腫瘤細胞中具有癌症幹細胞CD24之高表現 20
3.6 PKP的腫瘤細胞中高表現Wnt信號通路 21
3.7 CD24促進PKP前列腺癌細胞的骨轉移 21
3.8 在人類上皮細胞中過度表現CD24導致EMT的發生 22
3.9 通過PSI-697去抑制CD24導致的骨轉移情形 23
第四章 結果與討論 25
參考文獻 57
附錄 64
附錄1 64
附錄2 65
附錄3 66
參考文獻 References
1. Society., A.C., Cancer Facts & Figures 2016. (https://www.cancer.org/research/cancer-facts-statistics/all-cancer-facts-figures/cancer-facts-figures-2016.html).
2. Yin, M., et al., Prevalence of incidental prostate cancer in the general population: a study of healthy organ donors. J Urol, 2008. 179(3): p. 892-895.
3. Edwards, C.N., E. Steinthorsson, and D. Nicholson., An autopsy study of latent prostatic cancer. Cancer 1953. 6(3): p. 531-554.
4. Halpert, B., et al., Carcinoma of the prostate. A survey of 5,000 autopsies. Cancer, 1963. 16(6): p. 737-742.
5. Liavåg, I., Atrophy and regeneration in the pathogenesis of prostatic carcinoma. APMIS, 1968. 73(3): p. 338-350.
6. Hølund, B., Latent Prostatic Cancer in a Consecutive Autopsy Series. Scandinavian Journal of Urology and Nephrology, 1980. 14(1): p. 29-35.
7. Pienta, K.J., and Peggy S. Esper., Risk factors for prostate cancer. Annals of internal medicine, 1993. 118(10): p. 793-803.
8. Knudsen, B.S. and V. Vasioukhin, Mechanisms of prostate cancer initiation and progression. Adv Cancer Res, 2010. 109: p. 1-50.
9. Montironi, R., et al, Workgroup 1: Origins of prostate cancer 78.2 (1996): 362-365. Cancer, 1996. 78(2): p. 362-365.
10. Bostwick, D.G., Prostatic intraepithelial neoplasia. Current urology reports, 2000. 1(1): p. 65-70.
11. DeMarzo, A.M., et al., Pathological and molecular aspects of prostate cancer. The Lancet, 2003. 361(9361): p. 955-964.
12. Bostwick, D.G. and J. Qian, High-grade prostatic intraepithelial neoplasia. Mod Pathol, 2004. 17(3): p. 360-379.
13. Tomlins, S.A., et al., Integrative molecular concept modeling of prostate cancer progression. Nat Genet, 2007. 39(1): p. 41-51.
14. Montironi, R., et al., Prostatic intraepithelial neoplasia: its morphological and molecular diagnosis and clinical significance. BJU international, 2011. 108(9): p. 1394-1404.
15. Berx, G., et al. , E-cadherin is a tumour/invasion suppressor gene mutated in human lobular breast cancers. The EMBO journal 1995. 14(24): p. 6107.
16. Li, J., PTEN, a Putative Protein Tyrosine Phosphatase Gene Mutated in Human Brain, Breast, and Prostate Cancer. Science, 1997. 275(5308): p. 1943-1947.
17. Van't Veer, L.J., et al., Gene expression profiling predicts clinical outcome of breast cancer. nature 2002. 415(6871): p. 530-536.
18. Bubendorf, L., et al., Metastatic patterns of prostate cancer: An autopsy study of 1,589 patients. Human Pathology, 2000. 31(5): p. 578-583.
19. Jacobs, S.C., Spread of prostatic cancer to bone. Urology 1983. 21(4): p. 337-344.
20. Noguchi, M., et al., Percentage of the positive area of bone metastasis is an independent predictor of disease death in advanced prostate cancer. Br J Cancer, 2003. 88(2): p. 195-201.
21. Shen, M.M. and C. Abate-Shen, Molecular genetics of prostate cancer: new prospects for old challenges. Genes Dev, 2010. 24(18): p. 1967-2000.
22. Matusik, R.J., et al., Prostate epithelial cell fate. Differentiation, 2008. 76(6): p. 682-698.
23. Wang, X., et al., A luminal epithelial stem cell that is a cell of origin for prostate cancer. Nature, 2009. 461(7263): p. 495-500.
24. Anders, C., and Lisa A. Carey., Understanding and treating triple-negative breast cancer. Oncology, 2008. 22(11): p. 1233-1243.
25. Kim, J.H., Characterization of copy number aberrations and epigenetic modifications in prostate cancer. . Diss. University of Michigan., 2010.
26. Kim, C.F.B., et al., Mouse Models of Human Non-Small-Cell Lung Cancer: Raising the Bar-1. Cold Spring Harbor Symposia on Quantitative Biology., 2005. 70.
27. Wang, X., et al., Identifying novel genes for atherosclerosis through mouse-human comparative genetics. Am J Hum Genet, 2005. 77(1): p. 1-15.
28. Brown, S.D., R.E. Hardisty-Hughes, and P. Mburu, Quiet as a mouse: dissecting the molecular and genetic basis of hearing. Nat Rev Genet, 2008. 9(4): p. 277-290.
29. Lipp, H.-P., and David P. Wolfer. , Genetically modified mice and cognition. Current opinion in neurobiology, 1998. 8(2): p. 272-280.
30. Walrath, J.C., et al., Genetically Engineered Mouse Models in Cancer Research. 2010. 106: p. 113-164.
31. Matuq, Y., et al., The androgen-dependent rat prostate protein, probasin, is a heparin-binding protein that co-purifies with heparin-binding growth factor-1. In Vitro Cellular & Developmental Biology-Plant 1989. 25(6): p. 581-584.
32. Matusik, R.J., et al, Regulation of prostatic genes: role of androgens and zinc in gene expression. Biochemistry and Cell Biology, 1986. 64(6): p. 601-607.
33. Greenberg, N.M., et al., The rat probasin gene promoter directs hormonally and developmentally regulated expression of a heterologous gene specifically to the prostate in transgenic mice. Molecular Endocrinology 1994. 8(2): p. 230-239.

34. Wu, X., et al., Generation of a prostate epithelial cell-specific Cre transgenic mouse model for tissue-specific gene ablation. Mechanisms of development 2001. 101(1): p. 61-69.
35. Trotman, L.C., et al., Pten dose dictates cancer progression in the prostate. PLoS Biol, 2003. 1(3): p. E59.
36. Wang, S., et al., Prostate-specific deletion of the murine Pten tumor suppressor gene leads to metastatic prostate cancer. Cancer cell, 2003. 4(3): p. 209-221.
37. Maddison, L.A., et al., Conditional deletion of Rb causes early stage prostate cancer. Cancer research, 2004. 64(17): p. 6018-6025.
38. Zhou, Z., et al., Synergy of p53 and Rb deficiency in a conditional mouse model for metastatic prostate cancer. Cancer Res, 2006. 66(16): p. 7889-7898.
39. Scheidig, A.J., C. Burmester, and R.S. Goody, The pre-hydrolysis state of p21ras in complex with GTP: new insights into the role of water molecules in the GTP hydrolysis reaction of ras-like proteins. Structure, 1999. 7(11): p. 1311-S2.
40. Henis, Y.I., J.F. Hancock, and I.A. Prior, Ras acylation, compartmentalization and signaling nanoclusters (Review). Mol Membr Biol, 2009. 26(1): p. 80-92.
41. Pylayeva-Gupta, Y., E. Grabocka, and D. Bar-Sagi, RAS oncogenes: weaving a tumorigenic web. Nat Rev Cancer, 2011. 11(11): p. 761-774.
42. Prior, I.A., P.D. Lewis, and C. Mattos, A comprehensive survey of Ras mutations in cancer. Cancer Res, 2012. 72(10): p. 2457-2467.
43. Kreeger, P.K., et al., Integration of multiple signaling pathway activities resolves K-RAS/N-RAS mutation paradox in colon epithelial cell response to inflammatory cytokine stimulation. Integr Biol (Camb), 2010. 2(4): p. 202-208.
44. Stephen, A.G., et al., Dragging ras back in the ring. Cancer Cell, 2014. 25(3): p. 272-281.
45. Wellbrock, C., M. Karasarides, and R. Marais, The RAF proteins take centre stage. Nat Rev Mol Cell Biol, 2004. 5(11): p. 875-885.
46. Davies, H., et al. , Mutations of the BRAF gene in human cancer. Nature 2002. 417(6892): p. 949-954.
47. Emuss, V., et al., Mutations of C-RAF are rare in human cancer because C-RAF has a low basal kinase activity compared with B-RAF. Cancer Res, 2005. 65(21): p. 9719-9726.
48. Mercer, K., et al., Expression of endogenous oncogenic V600EB-raf induces proliferation and developmental defects in mice and transformation of primary fibroblasts. Cancer Res, 2005. 65(24): p. 11493-11500.
49. Villanueva, J., et al., Acquired resistance to BRAF inhibitors mediated by a RAF kinase switch in melanoma can be overcome by cotargeting MEK and IGF-1R/PI3K. Cancer Cell, 2010. 18(6): p. 683-695.
50. Chapman, P.B., et al. , Improved survival with vemurafenib in melanoma with BRAF V600E mutation. New England Journal of Medicine, 2011. 364(26): p. 2507-2516.
51. Levine, A.J., Jamil Momand, and Cathy A. Finlay., The p53 tumour supressor gene. Nature, 1991. 351(6326): p. 453.
52. Mulligan, L.M., et al. , Mechanisms of p53 loss in human sarcomas. Proceedings of the National Academy of Sciences 1990. 87(15): p. 5863-5867.
53. Wahl, A.F., et al. , Loss of normal p53 function confers sensitization to Taxol by increasing G2/M arrest and apoptosis. Nature medicine, 1996. 2(1): p. 72-79.
54. Petitjean, A., et al., Impact of mutant p53 functional properties on TP53 mutation patterns and tumor phenotype: lessons from recent developments in the IARC TP53 database. Hum Mutat, 2007. 28(6): p. 622-629.
55. Akashi, T., Takuji Shirasawa, and Katsuiku Hirokawa., Gene expression of CD24 core polypeptide molecule in normal rat tissues and human tumor cell lines. Virchows Archiv 1994. 425(4): p. 399-406.
56. Senner, V., et al. , CD24 promotes invasion of glioma cells in vivo. Journal of neuropathology and experimental neurology 1999. 58(8): p. 795-802.
57. Welsh, J.B., et al. , Analysis of gene expression profiles in normal and neoplastic ovarian tissue samples identifies candidate molecular markers of epithelial ovarian cancer. Proceedings of the National Academy of Sciences, 2001. 98(3): p. 1176-7781.
58. Kristiansen, G., et al. , CD24 expression is a new prognostic marker in breast cancer. Clinical cancer research., 2003. 9(13): p. 4906-4913.
59. Kristiansen, G., et al., CD24 is an independent prognostic marker of survival in nonsmall cell lung cancer patients. Br J Cancer, 2003. 88(2): p. 231-236.
60. Sagiv, E., D. Kazanov, and N. Arber, CD24 plays an important role in the carcinogenesis process of the pancreas. Biomed Pharmacother, 2006. 60(6): p. 280-284.
61. Sagiv, E., et al., CD24 is a new oncogene, early at the multistep process of colorectal cancer carcinogenesis. Gastroenterology, 2006. 131(2): p. 630-639.
62. Aigner, S., et al. , CD24, a mucin-type glycoprotein, is a ligand for P-selectin on human tumor cells. Blood 1997. 89(9): p. 3385-3395.
63. Sano, A., et al., CD24 expression is a novel prognostic factor in esophageal squamous cell carcinoma. Ann Surg Oncol, 2009. 16(2): p. 506-514.
64. Robinson, S.D., et al. , Multiple, targeted deficiencies in selectins reveal a predominant role for P-selectin in leukocyte recruitment. Proceedings of the National Academy of Sciences, 1999. 96(20): p. 11452-11457.

65. Wagner, D.D. and P.C. Burger, Platelets in inflammation and thrombosis. Arterioscler Thromb Vasc Biol, 2003. 23(12): p. 2131-2137.
66. Bedard, P.W., et al., Characterization of the novel P-selectin inhibitor PSI-697 [2-(4-chlorobenzyl)-3-hydroxy-7,8,9,10-tetrahydrobenzo[h] quinoline-4-carboxylic acid] in vitro and in rodent models of vascular inflammation and thrombosis. J Pharmacol Exp Ther, 2008. 324(2): p. 497-506.
67. Wakefield, T.W., et al. , Venous thrombosis prophylaxis by inflammatory inhibition without anticoagulation therapy. Journal of vascular surgery, 2000. 31(2): p. 309-324.
68. Myers, D.D., Jr., et al., Treatment with an oral small molecule inhibitor of P selectin (PSI-697) decreases vein wall injury in a rat stenosis model of venous thrombosis. J Vasc Surg, 2006. 44(3): p. 625-632.
69. Risbridger, G.P. and R.A. Taylor, The complexities of identifying a cell of origin for human prostate cancer. Asian J Androl, 2011. 13(1): p. 118-119.
70. Ohori, M., Thomas M. Wheeler, and Peter T. Scardino. , The new American joint committee on cancer and international union against cancer TNM classification of prostate cancer. Cancer 1994. 74: p. 104-114.
71. Sobin, L.H., and Irvin D. Fleming., TNM classification of malignant tumors, (1997). Cancer, 1997. 80(9): p. 1803-1804.
72. Edge, S.B. and C.C. Compton, The American Joint Committee on Cancer: the 7th edition of the AJCC cancer staging manual and the future of TNM. Ann Surg Oncol, 2010. 17(6): p. 1471-1474.
73. McMenamin, M.E., et al. , Loss of PTEN expression in paraffin-embedded primary prostate cancer correlates with high Gleason score and advanced stage. Cancer research, 1999. 59(17): p. 4291-4296.
74. Chan, T.Y., et al. , Prognostic significance of Gleason score 3+ 4 versus Gleason score 4+ 3 tumor at radical prostatectomy. Urology, 2000. 56(5): p. 823-827.
75. Kweldam, C.F., et al., Cribriform growth is highly predictive for postoperative metastasis and disease-specific death in Gleason score 7 prostate cancer. Mod Pathol, 2015. 28(3): p. 457-464.
76. Rodan, G.A., The development and function of the skeleton and bone metastases. Cancer, 2003. 97(3 Suppl): p. 726-732.
77. Janckila, A.J., et al. , Stable expression of human tartrate-resistant acid phosphatase isoforms by CHO cells. Clinica chimica acta 2002. 326(1): p. 113-122.
78. Salminen, E., et al., Serum tartrate-resistant acid phosphatase 5b (TRACP 5b) as a marker of skeletal changes in prostate cancer. Acta Oncol, 2005. 44(7): p. 742-747.
79. Schubbert, S., K. Shannon, and G. Bollag, Hyperactive Ras in developmental disorders and cancer. Nat Rev Cancer, 2007. 7(4): p. 295-308.
80. Courtney, K.D., R.B. Corcoran, and J.A. Engelman, The PI3K pathway as drug target in human cancer. J Clin Oncol, 2010. 28(6): p. 1075-1083.
81. Katz, M., I. Amit, and Y. Yarden, Regulation of MAPKs by growth factors and receptor tyrosine kinases. Biochim Biophys Acta, 2007. 1773(8): p. 1161-1176.
82. De Luca, A., et al. , The RAS/RAF/MEK/ERK and the PI3K/AKT signalling pathways: role in cancer pathogenesis and implications for therapeutic approaches. Expert opinion on therapeutic targets, 2012. 16(sup2): p. S17-S27.
83. Smalley, M. and A. Ashworth, Stem cells and breast cancer: A field in transit. Nat Rev Cancer, 2003. 3(11): p. 832-844.
84. Sleeman, K.E., et al. , CD24 staining of mouse mammary gland cells defines luminal epithelial, myoepithelial/basal and non-epithelial cells." Breast cancer research, 2005. 8(1): p. R7.
85. Kristiansen, G., et al., CD24 Is Expressed in Ovarian Cancer and Is a New Independent Prognostic Marker of Patient Survival. The American Journal of Pathology, 2002. 161(4): p. 1215-1221.
86. Kristiansen, G., M. Sammar, and P. Altevogt. , Tumour biological aspects of CD24, a mucin-like adhesion molecule. Journal of molecular histology 2004. 35(3): p. 255-262.
87. Borah, A., et al., Targeting self-renewal pathways in cancer stem cells: clinical implications for cancer therapy. Oncogenesis, 2015. 4(11).
88. Lau, E.Y., N.P. Ho, and T.K. Lee, Cancer Stem Cells and Their Microenvironment: Biology and Therapeutic Implications. Stem Cells Int, 2017. 2017.
89. Nusse, R., and Harold E. Varmus., Many tumors induced by the mouse mammary tumor virus contain a provirus integrated in the same region of the host genome. Cell, 1982. 31(1): p. 99-109.
90. Cadigan, K.M., and Roel Nusse. , Wnt signaling: a common theme in animal development. Genes & development, 1997. 11(24): p. 3286-3305.
91. Gat, U., et al. , De novo hair follicle morphogenesis and hair tumors in mice expressing a truncated β-catenin in skin. Cell, 1998. 95(5): p. 605-614.
92. Wend, P., et al., Wnt signaling in stem and cancer stem cells. Semin Cell Dev Biol, 2010. 21(8): p. 855-863.
93. Milla, L.A., Claudia N. González-Ramírez, and Verónica Palma. , Sonic Hedgehog in cancer stem cells: a novel link with autophagy. Biological research 2012. 45(3): p. 223-230.

94. Zhou, X.L. and J.C. Liu, Role of Notch signaling in the mammalian heart. Braz J Med Biol Res, 2014. 47(1): p. 1-10.
95. Yilmaz, M., G. Christofori, and F. Lehembre, Distinct mechanisms of tumor invasion and metastasis. Trends Mol Med, 2007. 13(12): p. 535-541.
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