凝血酶調節素(thrombomodulin，TM)為一穿膜的醣蛋白，其主要功能為與凝血酶(thrombin)結合而達到其抗凝血的作用。除了其抗凝血功能之外，亦有研究指出TM的表現和腫瘤的生成以及轉移有相關。近年來許多臨床研究觀察到TM減少會造成癌症病患預後變差且腫瘤轉移情形增加，然而對於TM如何調控腫瘤轉移的機制仍不清楚。因此本篇研究主要是探討TM在癌症轉移中所扮演的角色。首先，我們利用TM為異質性分布的HeLa母細胞株作為研究的模組以及腫瘤轉移的活體實驗模式和細胞懸浮性生長二種方式分別篩選出具有惡性轉移能力 (HeLa M1, HeLa M2)及抗失巢凋亡能力的細胞亞群(HeLa AR)作為研究的模型並且進一步分析TM的表現。實驗結果得知具有惡性轉移能力及抗失巢凋亡能力(anoikis resistance)的細胞株相較於HeLa母細胞株其TM表現量較低且具有較好的懸浮性生長能力及能對抗失去細胞外間質所導致的失巢凋亡(anoikis)，即具有抗失巢凋亡的能力。除此之外，將具有惡性轉移能力的細胞株或抗失巢凋亡能力的細胞株大量表現TM，則在懸浮生長下會大量的表現失巢凋亡的死亡訊息。另外也發現TM表現量不同的子宮頸癌細胞株其懸浮生長下抗失巢凋亡能力會隨著TM的表現降低而有升高的現象。由以上結果我們首度證實TM的表現量下降會使腫瘤細胞具有抗失巢凋亡能力而抑制腫瘤轉移。

Thrombomodulin (TM), a cell surface glycoprotein, is well known as an anticoagulant which functions through binding with thrombin. Recent clinical studies indicate that the reduction of TM in cancer is associated with poor prognosis and tumor metastasis. However, the functional consequence of decreased TM expression in tumor metastasis remains unclear. To better understand the mechanism underlying the role of TM in tumor metastasis, HeLa cellswhich express heterogeneous TM were used. The sublines of HeLa cells with aggressive metastatic and anoikis-resistant properties were selected by an in vivo experimental metastasis model and an in vitro anoikis-resistant selection, respectively. The lower TM-expressing HeLa Cells exhibited resistance to apoptosis in suspension culture. Examining the phenotypes of the metastatic sublines and anoikis-resistant HeLa cells revealed less TM expression than HeLa parental cells. Loss of TM expression was sufficient to promote anchorage-independent growth, and conferred anoikis resistance to cell rounding and matrix detachment. Additionally, overexpression of TM in metastatic sublines led to increased cell death in suspension growth. We further examined that different cervical cancer cells without TM expression indeed do have anoikis resistance in suspension assay. Taken together, we first demonstrated that reduction of TM is involved in anoikis resistance that may contribute to its metastasis suppression capacity, thus providing a possible explanation for the tumor malignancy with downregulated TM.

Alberts DS (1998) Tumor heterogeneity and extreme drug resistance: meet your match! The cancer journal from Scientific American 4: 25-26

Atsushi Yawata1 MA, Hiroyuki Okuda1, Yasuyoshi Naishiro1,, Takenori Takamura1 MH, Shinichi Takayama2, John C Reed2 and Kohzoh Imai1 (1998) Prolonged cell survival enhances peritoneal dissemination of gastric cancer cells. Oncogene 16, 2681 ± 2686

Boffa MC, Burke B, Haudenschild C (1987) [Different localization of thrombomodulin]. Ann Biol Clin (Paris) 45: 191-197

Bourin MC, Boffa MC, Bjork I, Lindahl U (1986) Functional domains of rabbit thrombomodulin. Proceedings of the National Academy of Sciences of the United States of America 83: 5924-5928

Bourin MC, Lundgren-Akerlund E, Lindahl U (1990) Isolation and characterization of the glycosaminoglycan component of rabbit thrombomodulin proteoglycan. J Biol Chem 265: 15424-15431

Conway EM, Nowakowski B, Steiner-Mosonyi M (1994) Thrombomodulin lacking the cytoplasmic domain efficiently internalizes thrombin via nonclathrin-coated, pit-mediated endocytosis. Journal of cellular physiology 158: 285-298

Conway EM, Pollefeyt S, Collen D, Steiner-Mosonyi M (1997) The amino terminal lectin-like domain of thrombomodulin is required for constitutive endocytosis. Blood 89: 652-661

Conway EM, Pollefeyt S, Cornelissen J, DeBaere I, Steiner-Mosonyi M, Weitz JI, Weiler-Guettler H, Carmeliet P, Collen D (1999) Structure-function analyses of thrombomodulin by gene-targeting in mice: the cytoplasmic domain is not required for normal fetal development. Blood 93: 3442-3450

Conway EM, Van de Wouwer M, Pollefeyt S, Jurk K, Van Aken H, De Vriese A, Weitz JI, Weiler H, Hellings PW, Schaeffer P, Herbert JM, Collen D, Theilmeier G (2002) The lectin-like domain of thrombomodulin confers protection from neutrophil-mediated tissue damage by suppressing adhesion molecule expression via nuclear factor kappaB and mitogen-activated protein kinase pathways. The Journal of experimental medicine 196: 565-577

Derksen PW, Liu X, Saridin F, van der Gulden H, Zevenhoven J, Evers B, van Beijnum JR, Griffioen AW, Vink J, Krimpenfort P, Peterse JL, Cardiff RD, Berns A, Jonkers J (2006) Somatic inactivation of E-cadherin and p53 in mice leads to metastatic lobular mammary carcinoma through induction of anoikis resistance and angiogenesis. Cancer cell 10: 437-449

Esmon CT, Esmon NL, Harris KW (1982) Complex formation between thrombin and thrombomodulin inhibits both thrombin-catalyzed fibrin formation and factor V activation. J Biol Chem 257: 7944-7947

Fidler IJ (1978) Tumor Heterogeneity and the Biology of Cancer Invasion and Metastasis1. Cancer research: 38, 2651-2660,

Fidler IJ (2002) The pathogenesis of cancer metastasis: the 'seed and soil' hypothesis revisited. Nature reviews Cancer

Frisch SM (1994) Disruption of Epithelial Cell-MatrixInteractions Induces Apoptosis.

Frisch SM (2000) cAMP takes control. Nature cell biology 2: E167-168

Frisch SM (2001) <Anoikis mechanisms.pdf>. Current Opinion in Cell Biology 2001 13:555–562

Haenssen KK, Caldwell SA, Shahriari KS, Jackson SR, Whelan KA, Klein-Szanto AJ, Reginato MJ (2010) ErbB2 requires integrin alpha5 for anoikis resistance via Src regulation of receptor activity in human mammary epithelial cells. Journal of cell science 123: 1373-1382

Haga T, Uchide N, Tugizov S, Palefsky JM (2008) Role of E-cadherin in the induction of apoptosis of HPV16-positive CaSki cervical cancer cells during multicellular tumor spheroid formation. Apoptosis : an international journal on programmed cell death 13: 97-108

Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144: 646-674

Healy AM, Rayburn HB, Rosenberg RD, Weiler H (1995) Absence of the blood-clotting regulator thrombomodulin causes embryonic lethality in mice before development of a functional cardiovascular system. Proc Natl Acad Sci U S A 92: 850-854

Heppner GH (1984) Tumor heterogeneity. Cancer research 44: 2259-2265

Heppner GH, Kopp JS, Medina D (1976) Microcytotoxicity assay of immune responses to non-mammary tumor virus-induced, preneoplastic, and neoplastic mammary lesions in BALB/c mice. Cancer research 36: 753-758

Hsu YY, Shi GY, Kuo CH, Liu SL, Wu CM, Ma CY, Lin FY, Yang HY, Wu HL (2012) Thrombomodulin is an ezrin-interacting protein that controls epithelial morphology and promotes collective cell migration. FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Huang HC, Shi GY, Jiang SJ, Shi CS, Wu CM, Yang HY, Wu HL (2003) Thrombomodulin-mediated cell adhesion: involvement of its lectin-like domain. J Biol Chem 278: 46750-46759

Huang MT, Wei PL, Liu JJ, Liu DZ, Huey-Chun H, An J, Wu CC, Wu CH, Ho YS, Yang YY, Chang YJ (2010) Knockdown of thrombomodulin enhances HCC cell migration through increase of ZEB1 and decrease of E-cadherin gene expression. Annals of surgical oncology 17: 3379-3385

John Geisler MD, STEPHEN L. ROSE, M.D.,, HANS E. GEISLER MD, GREG A. MILLER, M.D.,, MICHAEL C. WIEMANN MD (2002) Drug resistance and tumor heterogeneity. CME Journal of Gynecologic Oncology 7:25–28

Kao YC, Wu LW, Shi CS, Chu CH, Huang CW, Kuo CP, Sheu HM, Shi GY, Wu HL (2010) Downregulation of thrombomodulin, a novel target of Snail, induces tumorigenesis through epithelial-mesenchymal transition. Mol Cell Biol 30: 4767-4785

Kim R, Tanabe K, Uchida Y, Osaki A, Toge T (2002) The role of HER-2 oncoprotein in drug-sensitivity in breast cancer (review). Oncology reports 9: 3-9

Kim SJ, Shiba E, Ishii H, Inoue T, Taguchi T, Tanji Y, Kimoto Y, Izukura M, Takai S (1997) Thrombomodulin is a new biological and prognostic marker for breast cancer: an immunohistochemical study. Anticancer research 17: 2319-2323

Koeppe JR, Beach MA, Baerga-Ortiz A, Kerns SJ, Komives EA (2008) Mutations in the fourth EGF-like domain affect thrombomodulin-induced changes in the active site of thrombin. Biochemistry 47: 10933-10939

Koyama T, Parkinson JF, Aoki N, Bang NU, Muller-Berghaus G, Preissner KT (1991) Relationship between post-translational glycosylation and anticoagulant function of secretable recombinant mutants of human thrombomodulin. Br J Haematol 78: 515-522

Kuo CH, Chen PK, Chang BI, Sung MC, Shi CS, Lee JS, Chang CF, Shi GY, Wu HL (2012) The recombinant lectin-like domain of thrombomodulin inhibits angiogenesis through interaction with Lewis Y antigen. Blood 119: 1302-1313

Kurosawa S, Galvin JB, Esmon NL, Esmon CT (1987) Proteolytic formation and properties of functional domains of thrombomodulin. J Biol Chem 262: 2206-2212

Lager DJ, Callaghan EJ, Worth SF, Raife TJ, Lentz SR (1995) Cellular localization of thrombomodulin in human epithelium and squamous malignancies. Am J Pathol 146: 933-943

Lengyel E (2010) Ovarian cancer development and metastasis. The American journal of pathology 177: 1053-1064

Light DR, Glaser CB, Betts M, Blasko E, Campbell E, Clarke JH, McCaman M, McLean K, Nagashima M, Parkinson JF, Rumennik G, Young T, Morser J (1999) The interaction of thrombomodulin with Ca2+. European journal of biochemistry / FEBS 262: 522-533

Liu PL, Tsai JR, Chiu CC, Hwang JJ, Chou SH, Wang CK, Wu SJ, Chen YL, Chen WC, Chen YH, Chong IW (2010) Decreased expression of thrombomodulin is correlated with tumor cell invasiveness and poor prognosis in nonsmall cell lung cancer. Molecular carcinogenesis 49: 874-881

Maruno M, Yoshimine T, Isaka T, Kuroda R, Ishii H, Hayakawa T (1994) Expression of thrombomodulin in astrocytomas of various malignancy and in gliotic and normal brains. J Neurooncol 19: 155-160

Maruyama I (1988) [Abnormal thrombomodulin or thrombomodulin deficiency]. Tanpakushitsu Kakusan Koso 33: 1010-1012

Matsushita Y, Yoshiie K, Imamura Y, Ogawa H, Imamura H, Takao S, Yonezawa S, Aikou T, Maruyama I, Sato E (1998) A subcloned human esophageal squamous cell carcinoma cell line with low thrombomodulin expression showed increased invasiveness compared with a high thrombomodulin-expressing clone--thrombomodulin as a possible candidate for an adhesion molecule of squamous cell carcinoma. Cancer Lett 127: 195-201

McCachren SS, Diggs J, Weinberg JB, Dittman WA (1991) Thrombomodulin expression by human blood monocytes and by human synovial tissue lining macrophages. Blood 78: 3128-3132

Mehlen P, Puisieux A (2006) Metastasis: a question of life or death. Nature reviews Cancer 6: 449-458

Menschikowski M, Hagelgans A, Tiebel O, Vogel M, Eisenhofer G, Siegert G (2012) Regulation of thrombomodulin expression in prostate cancer cells. Cancer letters 322: 177-184

Nesheim M, Wang W, Boffa M, Nagashima M, Morser J, Bajzar L (1997) Thrombin, thrombomodulin and TAFI in the molecular link between coagulation and fibrinolysis. Thrombosis and haemostasis 78: 386-391

Ogawa H, Yonezawa S, Maruyama I, Matsushita Y, Tezuka Y, Toyoyama H, Yanagi M, Matsumoto H, Nishijima H, Shimotakahara T, Aikou T, Sato E (2000) Expression of thrombomodulin in squamous cell carcinoma of the lung: its relationship to lymph node metastasis and prognosis of the patients. Cancer letters 149: 95-103

Petersen TE (1988) The amino-terminal domain of thrombomodulin and pancreatic stone protein are homologous with lectins. FEBS letters 231: 51-53

Raife TJ, Lager DJ, Madison KC, Piette WW, Howard EJ, Sturm MT, Chen Y, Lentz SR (1994) Thrombomodulin expression by human keratinocytes. Induction of cofactor activity during epidermal differentiation. J Clin Invest 93: 1846-1851

Sadler JE (1997) Thrombomodulin structure and function. Thrombosis and haemostasis 78: 392-395

Sakamoto S, Kyprianou N (2010) Targeting anoikis resistance in prostate cancer metastasis. Molecular aspects of medicine 31: 205-214

Sakuma Y, Takeuchi T, Nakamura Y, Yoshihara M, Matsukuma S, Nakayama H, Ohgane N, Yokose T, Kameda Y, Tsuchiya E, Miyagi Y (2010) Lung adenocarcinoma cells floating in lymphatic vessels resist anoikis by expressing phosphorylated Src. The Journal of pathology 220: 574-585

Schackmann RC, van Amersfoort M, Haarhuis JH, Vlug EJ, Halim VA, Roodhart JM, Vermaat JS, Voest EE, van der Groep P, van Diest PJ, Jonkers J, Derksen PW (2011) Cytosolic p120-catenin regulates growth of metastatic lobular carcinoma through Rock1-mediated anoikis resistance. The Journal of clinical investigation 121: 3176-3188

Schenk-Braat EA, Morser J, Rijken DC (2001) Identification of the epidermal growth factor-like domains of thrombomodulin essential for the acceleration of thrombin-mediated inactivation of single-chain urokinase-type plasminogen activator. European journal of biochemistry / FEBS 268: 5562-5569

Shanmugathasan M, Jothy S (2000) Apoptosis, anoikis and their relevance to the pathobiology of colon cancer. Pathology international 50: 273-279

Shi CS, Shi GY, Chang YS, Han HS, Kuo CH, Liu C, Huang HC, Chang YJ, Chen PS, Wu HL (2005) Evidence of human thrombomodulin domain as a novel angiogenic factor. Circulation 111: 1627-1636

Shi CS, Shi GY, Hsiao SM, Kao YC, Kuo KL, Ma CY, Kuo CH, Chang BI, Chang CF, Lin CH, Wong CH, Wu HL (2008) Lectin-like domain of thrombomodulin binds to its specific ligand Lewis Y antigen and neutralizes lipopolysaccharide-induced inflammatory response. Blood 112: 3661-3670

Shirai T, Shiojiri S, Ito H, Yamamoto S, Kusumoto H, Deyashiki Y, Maruyama I, Suzuki K (1988) Gene structure of human thrombomodulin, a cofactor for thrombin-catalyzed activation of protein C. Journal of biochemistry 103: 281-285

Simpson CD, Anyiwe K, Schimmer AD (2008) Anoikis resistance and tumor metastasis. Cancer letters 272: 177-185

Singh AB, Sharma A, Smith JJ, Krishnan M, Chen X, Eschrich S, Washington MK, Yeatman TJ, Beauchamp RD, Dhawan P (2011) Claudin-1 up-regulates the repressor ZEB-1 to inhibit E-cadherin expression in colon cancer cells. Gastroenterology 141: 2140-2153

Smit MA, Peeper DS (2011) Zeb1 is required for TrkB-induced epithelial-mesenchymal transition, anoikis resistance and metastasis. Oncogene 30: 3735-3744

Sosic D, Richardson JA, Yu K, Ornitz DM, Olson EN (2003) Twist regulates cytokine gene expression through a negative feedback loop that represses NF-kappaB activity. Cell 112: 169-180

Spencer SL, Gerety RA, Pienta KJ, Forrest S (2006) Modeling somatic evolution in tumorigenesis. PLoS computational biology 2: e108

Steven M. Frisch KV, * Erkki Ruoslahti,* and Po-Ying Chan-Hui* (1996) Control of Adhesion-dependent Cell Survival by Focal Adhesion Kinase. The Journal of cell biology 134

Streuli CH, Gilmore AP (1999) Adhesion-mediated signaling in the regulation of mammary epithelial cell survival. Journal of mammary gland biology and neoplasia 4: 183-191

Su Y, Luo X, He BC, Wang Y, Chen L, Zuo GW, Liu B, Bi Y, Huang J, Zhu GH, He Y, Kang Q, Luo J, Shen J, Chen J, Jin X, Haydon RC, He TC, Luu HH (2009) Establishment and characterization of a new highly metastatic human osteosarcoma cell line. Clinical & experimental metastasis 26: 599-610

Suehiro T, Shimada M, Matsumata T, Taketomi A, Yamamoto K, Sugimachi K (1995) Thrombomodulin inhibits intrahepatic spread in human hepatocellular carcinoma. Hepatology 21: 1285-1290

Suzuki K, Nishioka J, Hayashi T, Kosaka Y (1988) Functionally active thrombomodulin is present in human platelets. J Biochem 104: 628-632

Swan EA, Jasser SA, Holsinger FC, Doan D, Bucana C, Myers JN (2003) Acquisition of anoikis resistance is a critical step in the progression of oral tongue cancer. Oral oncology 39: 648-655

Tabata M, Sugihara K, Yonezawa S, Yamashita S, Maruyama I (1997) An immunohistochemical study of thrombomodulin in oral squamous cell carcinoma and its association with invasive and metastatic potential. Journal of oral pathology & medicine : official publication of the International Association of Oral Pathologists and the American Academy of Oral Pathology 26: 258-264

Tabata M, Yonezawa S, Sugihara K, Yamashita S, Maruyama I (1995) The use of thrombomodulin to study epithelial cell differentiation in neoplastic and non-neoplastic oral lesions. Journal of oral pathology & medicine : official publication of the International Association of Oral Pathologists and the American Academy of Oral Pathology 24: 443-449

Taddei ML, Giannoni E, Fiaschi T, Chiarugi P (2012) Anoikis: an emerging hallmark in health and diseases. The Journal of pathology 226: 380-393

Takebayashi Y, Yamada K, Maruyama I, Fujii R, Akiyama S, Aikou T (1995) The expression of thymidine phosphorylase and thrombomodulin in human colorectal carcinomas. Cancer letters 92: 1-7

Tezuka Y, Yonezawa S, Maruyama I, Matsushita Y, Shimizu T, Obama H, Sagara M, Shirao K, Kusano C, Natsugoe S, et al. (1995) Expression of thrombomodulin in esophageal squamous cell carcinoma and its relationship to lymph node metastasis. Cancer Res 55: 4196-4200

Tiwari N, Gheldof A, Tatari M, Christofori G (2012) EMT as the ultimate survival mechanism of cancer cells. Seminars in cancer biology 22: 194-207

Valentijn AJ. (2002) Anoikis. Biochemical Society Transactions, Vol. 32.

Vega S, Morales AV, Ocana OH, Valdes F, Fabregat I, Nieto MA (2004) Snail blocks the cell cycle and confers resistance to cell death. Genes & development 18: 1131-1143

Watt FM (2002) Role of integrins in regulating epidermal adhesion, growth and differentiation. The EMBO journal 21: 3919-3926

Weiler-Guettler H, Aird WC, Rayburn H, Husain M, Rosenberg RD (1996) Developmentally regulated gene expression of thrombomodulin in postimplantation mouse embryos. Development 122: 2271-2281

Weiler H, Isermann BH (2003) Thrombomodulin. J Thromb Haemost 1: 1515-1524

Wen DZ, Dittman WA, Ye RD, Deaven LL, Majerus PW, Sadler JE (1987) Human thrombomodulin: complete cDNA sequence and chromosome localization of the gene. Biochemistry 26: 4350-4357

Yonezawa S, Maruyama I, Sakae K, Igata A, Majerus PW, Sato E (1987) Thrombomodulin as a marker for vascular tumors. Comparative study with factor VIII and Ulex europaeus I lectin. American journal of clinical pathology 88: 405-411

Yu Q, Toole BP, Stamenkovic I (1997) Induction of apoptosis of metastatic mammary carcinoma cells in vivo by disruption of tumor cell surface CD44 function. The Journal of experimental medicine 186: 1985-1996

Zhang Y, Weiler-Guettler H, Chen J, Wilhelm O, Deng Y, Qiu F, Nakagawa K, Klevesath M, Wilhelm S, Bohrer H, Nakagawa M, Graeff H, Martin E, Stern DM, Rosenberg RD, Ziegler R, Nawroth PP (1998) Thrombomodulin modulates growth of tumor cells independent of its anticoagulant activity. The Journal of clinical investigation 101: 1301-1309

曾舜鴻 (2011) Thrombomodulin mediates contact inhibition to suppress cell growth. 成功大學碩士論文

黃衍筠 (2008) Thrombomodulin suppresses tumor cell proliferation by the inhibition of protein kinase A. 成功大學碩士論文