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研究生(外文):Ting-Huan Wang
論文名稱(外文):CTHRC1 overexpression in hepatocellular carcinoma promotes tumor invasion and metastasis
指導教授(外文):Yung-Ming Jeng
口試委員(外文):Tsui-Lien MaoYen-Rong ChenJinn-Chyuan Sheu
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CTHRC1為一種分泌型醣蛋白,且能活化Wnt 訊息傳遞的細胞極化路徑。使用微陣列分析,我們發現CTHRC1基因在肝癌中會過度表現。從201位肝癌病人取得手術切除的肝臟檢體,以即時反轉錄聚合反應分析mRNA表現量,我們發現在腫瘤體積較大以及癌症晚期者,CTHRC1表現量最為顯著。此外,CTHRC1表現量是一個肝癌不良預後的因子。
我們發現在肝癌細胞中過度表現CTHRC1能促進腫瘤侵犯能力。反之,將CTHRC1表現量降低,會抑制腫瘤移行與侵犯能力。更進一步探討,我們發現CTHRC1會誘發RhoA蛋白的活化而非Rac1或Cdc42,以促進細胞移行。CTHRC1藉由活化β1-integrin / FAK / ERK訊息傳遞路徑,增加細胞貼附能力以提升腫瘤轉移及侵犯能力。CTHRC1扮演著調節者的角色,藉由增進腫瘤細胞的貼附力與移行力來強化其侵犯與轉移的機會。我們也發現CTHRC1是一個新的評估肝癌病人預後因子。

Collagen triple helix repeat containing-1 (CTHRC1) is a secreted glycoprotein that activates the planar cell polarity pathway of Wnt signaling. Using microarray analysis, we found that the CTHRC1 gene is overexpressed in hepatocellular carcinoma (HCC). The level of CTHRC1 mRNA was measured in 201 surgically resected HCCs using real time reverse transcription-polymerase chain reaction. Overexpression of CTHRC1 in HCC was associated with large tumor size and advanced tumor stage. CTHRC1 overexpression predicts poor prognosis for HCC patients. Suppression of CTHRC1 expression inhibited tumor migration and invasion whereas overexpression of CTHRC1 promoted tumor invasion. Activation of RhoA, but not Rac1 or Cdc42, was found to play a crucial role in CTHRC1-induced cell migration. CTHRC1 promoted adhesion of cancer cells to extracellular matrix through induction of integrin β1 expression and activation of focal adhesion kinase. These results suggest CTHRC1 promotes tumor invasion and metastasis by enhancing the adhesion and migratory abilities of tumor cells. It is also a promising biomarker for predicting the prognosis of patients with HCC.

口試委員會審定書 #
誌謝 I
中文摘要 II

1.1 Hepatocellular carcinoma 1
1.2 Collagen triple helix repeat containing 1 1
1.2.1 CTHRC1 is involved in Wnt pathway 2
1.2.2 CTHRC1 and disease 2
1.3 CTHRC1 in cancer 3
1.4 Regulation of cell migration 3
1.4.1 Integrin Family 4
1.4.2 FAK and focal adhesion 5
1.5 Purpose of Study 5
2.1 Liver samples 7
2.2 Histology study and tumor staging 7
2.3 Microarray analysis 8
2.4 RNA isolation and real time RT-PCR 8
2.5 Cell culture and treatment 9
2.6 Plasmid, transfection, and retroviral infection 9
2.7 Tumorigenicity assay 9
2.8 Boyden chamber assay 10
2.9 Adhesion assay 10
2.10 Immunofluorescence staining 11
2.11 Western blot 11
2.12 Detection of CTHRC1 in culture medium 12
2.13 3D collagen gel invasion assay 12
2.14 Flow cytometry 12
2.15 TOP/FOP Luciferase Reporter Assay 13
Chapter 3 RESULTS 14
3.1 CTHRC1 was overexpressed in HCC 14
3.2 CTHRC1 has no effect on cell proliferation 15
3.3 CTHRC1 overexpression increased tumor cell migration and invasion ability 15
3.4 CTHRC1 enhanced cell adhesion 16
3.5 CTHRC1 enhanced cell adhesion by enahncing β1-integrin expression 17
3.6 CTHRC1 has no effect on canonical Wnt pathway in HCC 18
Chapter 4 DISCUSSION 19
5.1 Figures 23
5.2 Tables 42

1. El-Serag HB, Rudolph KL (2007) Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology 132: 2557-2576.
2. Schafer DF, Sorrell MF (1999) Hepatocellular carcinoma. Lancet 353: 1253-1257.
3. Marrero CR, Marrero JA (2007) Viral hepatitis and hepatocellular carcinoma. Arch Med Res 38: 612-620.
4. Hsu H, Peng S, Lai P, Chu J, Lee P (1994) Mutations of p53 gene in hepatocellular-carcinoma (hcc) correlate with tumor progression and patient prognosis - a study of 138 patients with unifocal hcc. Int J Oncol 4: 1341-1347.
5. Hsu HC, Jeng YM, Mao TL, Chu JS, Lai PL, et al. (2000) Beta-catenin mutations are associated with a subset of low-stage hepatocellular carcinoma negative for hepatitis B virus and with favorable prognosis. Am J Pathol 157: 763-770.
6. Fujimoto A, Totoki Y, Abe T, Boroevich KA, Hosoda F, et al. (2012) Whole-genome sequencing of liver cancers identifies etiological influences on mutation patterns and recurrent mutations in chromatin regulators. Nat Genet 44: 760-764.
7. Pyagay P, Heroult M, Wang Q, Lehnert W, Belden J, et al. (2005) Collagen triple helix repeat containing 1, a novel secreted protein in injured and diseased arteries, inhibits collagen expression and promotes cell migration. Circ Res 96: 261-268.
8. Yamamoto S, Nishimura O, Misaki K, Nishita M, Minami Y, et al. (2008) Cthrc1 selectively activates the planar cell polarity pathway of Wnt signaling by stabilizing the Wnt-receptor complex. Dev Cell 15: 23-36.
9. Durmus T, LeClair RJ, Park KS, Terzic A, Yoon JK, et al. (2006) Expression analysis of the novel gene collagen triple helix repeat containing-1 (Cthrc1). Gene Expr Patterns 6: 935-940.
10. De Calisto J, Araya C, Marchant L, Riaz CF, Mayor R (2005) Essential role of non-canonical Wnt signalling in neural crest migration. Development 132: 2587-2597.
11. Kelley MW (2008) Leading Wnt down a PCP path: Cthrc1 acts as a coreceptor in the Wnt-PCP pathway. Dev Cell 15: 7-8.
12. Park EH, Kim S, Jo JY, Kim SJ, Hwang Y, et al. (2013) Collagen triple helix repeat containing-1 promotes pancreatic cancer progression by regulating migration and adhesion of tumor cells. Carcinogenesis 34: 694-702.
13. LeClair RJ, Durmus T, Wang Q, Pyagay P, Terzic A, et al. (2007) Cthrc1 is a novel inhibitor of transforming growth factor-beta signaling and neointimal lesion formation. Circ Res 100: 826-833.
14. Tang L, Dai DL, Su M, Martinka M, Li G, et al. (2006) Aberrant expression of collagen triple helix repeat containing 1 in human solid cancers. Clin Cancer Res 12: 3716-3722.
15. Kharaishvili G, Cizkova M, Bouchalova K, Mgebrishvili G, Kolar Z, et al. (2011) Collagen triple helix repeat containing 1 protein, periostin and versican in primary and metastatic breast cancer: an immunohistochemical study. J Clin Pathol 64: 977-982.
16. Kim JH, Baek TH, Yim HS, Kim KH, Jeong SH, et al. (2013) Collagen Triple Helix Repeat Containing-1 (CTHRC1) Expression in Invasive Ductal Carcinoma of the Breast: The Impact on Prognosis and Correlation to Clinicopathologic Features. Pathol Oncol Res.
17. Orloff M, Peterson C, He X, Ganapathi S, Heald B, et al. (2011) Germline mutations in MSR1, ASCC1, and CTHRC1 in patients with Barrett esophagus and esophageal adenocarcinoma. JAMA 306: 410-419.
18. Tan F, Liu F, Liu H, Hu Y, Liu D, et al. (2013) CTHRC1 is associated with peritoneal carcinomatosis in colorectal cancer: a new predictor for prognosis. Med Oncol 30: 473.
19. Liu G, Sengupta PK, Jamal B, Yang HY, Bouchie MP, et al. (2013) N-glycosylation induces collagen triple helix repeat containing 1 (CTHRC1) and drives oral cancer cell migration. J Biol Chem.
20. von der Mark K, Schober S, Goodman SL (1999) Integrins in cell migration. Methods Mol Biol 129: 219-230.
21. Cho SY, Klemke RL (2000) Extracellular-regulated kinase activation and CAS/Crk coupling regulate cell migration and suppress apoptosis during invasion of the extracellular matrix. J Cell Biol 149: 223-236.
22. Allingham MJ, van Buul JD, Burridge K (2007) ICAM-1-mediated, Src- and Pyk2-dependent vascular endothelial cadherin tyrosine phosphorylation is required for leukocyte transendothelial migration. J Immunol 179: 4053-4064.
23. Mitchison TJ, Cramer LP (1996) Actin-based cell motility and cell locomotion. Cell 84: 371-379.
24. Huveneers S, Danen EH (2009) Adhesion signaling - crosstalk between integrins, Src and Rho. J Cell Sci 122: 1059-1069.
25. Rottner K, Stradal TE (2011) Actin dynamics and turnover in cell motility. Curr Opin Cell Biol 23: 569-578.
26. Bhadriraju K, Yang M, Alom Ruiz S, Pirone D, Tan J, et al. (2007) Activation of ROCK by RhoA is regulated by cell adhesion, shape, and cytoskeletal tension. Exp Cell Res 313: 3616-3623.
27. Humphries MJ (2000) Integrin structure. Biochem Soc Trans 28: 311-339.
28. Hynes RO (2002) Integrins: bidirectional, allosteric signaling machines. Cell 110: 673-687.
29. Guan JL, Trevithick JE, Hynes RO (1991) Fibronectin/integrin interaction induces tyrosine phosphorylation of a 120-kDa protein. Cell Regul 2: 951-964.
30. Parsons JT (2003) Focal adhesion kinase: the first ten years. J Cell Sci 116: 1409-1416.
31. Klingbeil CK, Hauck CR, Hsia DA, Jones KC, Reider SR, et al. (2001) Targeting Pyk2 to beta 1-integrin-containing focal contacts rescues fibronectin-stimulated signaling and haptotactic motility defects of focal adhesion kinase-null cells. J Cell Biol 152: 97-110.
32. Nabeshima K, Inoue T, Shimao Y, Okada Y, Itoh Y, et al. (2000) Front-cell-specific expression of membrane-type 1 matrix metalloproteinase and gelatinase A during cohort migration of colon carcinoma cells induced by hepatocyte growth factor/scatter factor. Cancer Res 60: 3364-3369.
33. Webb DJ, Parsons JT, Horwitz AF (2002) Adhesion assembly, disassembly and turnover in migrating cells -- over and over and over again. Nat Cell Biol 4: E97-100.
34. Garcia AJ, Schwarzbauer JE, Boettiger D (2002) Distinct activation states of alpha5beta1 integrin show differential binding to RGD and synergy domains of fibronectin. Biochemistry 41: 9063-9069.
35. Miranti CK, Brugge JS (2002) Sensing the environment: a historical perspective on integrin signal transduction. Nat Cell Biol 4: E83-90.
36. Poullet P, Gautreau A, Kadare G, Girault JA, Louvard D, et al. (2001) Ezrin interacts with focal adhesion kinase and induces its activation independently of cell-matrix adhesion. J Biol Chem 276: 37686-37691.
37. Zhai J, Lin H, Nie Z, Wu J, Canete-Soler R, et al. (2003) Direct interaction of focal adhesion kinase with p190RhoGEF. J Biol Chem 278: 24865-24873.
38. Zhao J, Bian ZC, Yee K, Chen BP, Chien S, et al. (2003) Identification of transcription factor KLF8 as a downstream target of focal adhesion kinase in its regulation of cyclin D1 and cell cycle progression. Mol Cell 11: 1503-1515.
39. Hayashi I, Vuori K, Liddington RC (2002) The focal adhesion targeting (FAT) region of focal adhesion kinase is a four-helix bundle that binds paxillin. Nat Struct Biol 9: 101-106.
40. Turner CE (2000) Paxillin and focal adhesion signalling. Nat Cell Biol 2: E231-236.
41. Schaller MD (2001) Paxillin: a focal adhesion-associated adaptor protein. Oncogene 20: 6459-6472.
42. Mitra SK, Schlaepfer DD (2006) Integrin-regulated FAK-Src signaling in normal and cancer cells. Curr Opin Cell Biol 18: 516-523.
43. Ruest PJ, Roy S, Shi E, Mernaugh RL, Hanks SK (2000) Phosphospecific antibodies reveal focal adhesion kinase activation loop phosphorylation in nascent and mature focal adhesions and requirement for the autophosphorylation site. Cell Growth Differ 11: 41-48.
44. Edmondson HA, Steiner PE (1954) Primary carcinoma of the liver: a study of 100 cases among 48,900 necropsies. Cancer 7: 462-503.
45. Edge SB, Compton CC (2010) The American Joint Committee on Cancer: the 7th edition of the AJCC cancer staging manual and the future of TNM. Ann Surg Oncol 17: 1471-1474.
46. Wessel D, Flugge UI (1984) A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem 138: 141-143.
47. Leclair RJ, Wang Q, Benson MA, Prudovsky I, Lindner V (2008) Intracellular localization of Cthrc1 characterizes differentiated smooth muscle. Arterioscler Thromb Vasc Biol 28: 1332-1338.
48. Thannickal VJ, Lee DY, White ES, Cui Z, Larios JM, et al. (2003) Myofibroblast differentiation by transforming growth factor-beta1 is dependent on cell adhesion and integrin signaling via focal adhesion kinase. J Biol Chem 278: 12384-12389.
49. Webb DJ, Donais K, Whitmore LA, Thomas SM, Turner CE, et al. (2004) FAK-Src signalling through paxillin, ERK and MLCK regulates adhesion disassembly. Nat Cell Biol 6: 154-161.
50. Logan CY, Nusse R (2004) The Wnt signaling pathway in development and disease. Annu Rev Cell Dev Biol 20: 781-810.
51. Schlessinger K, Hall A, Tolwinski N (2009) Wnt signaling pathways meet Rho GTPases. Genes Dev 23: 265-277.

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