EJSO (2005) 31, 217220

www.ejso.com

REVIEW

Thrombomodulin: tumour biology and prognostic implications

A.M. Hanly*, A. Hayanga, D.C. Winter, D.J. Bouchier-Hayes
Department of Surgery, Research and Education Building, Beaumont Hospital, Beaumont, Dublin 9, Ireland
Accepted for publication 11 November 2004 Available online 20 January 2005

KEYWORDS
Thrombomodulin; Cancer; Manipulation

Summary Background. Thrombomodulin (TM) is an endothelial receptor that exerts anti-coagulant, anti-brinolytic, and anti-inammatory activity by inhibiting thrombin and cellular adhesion. There is growing evidence that TM plays a role in tumour behaviour. Methods. The electronic literature (19662004) was reviewed with a specic focus on tumour biology. Results. TM is expressed on both the endothelium and tumour cells in several cancers. Loss of expression denotes a more malignant prole with poorer prognosis. Loss of TM is mediated by hypoxia, endotoxin, and various cytokines, while upregulation can be achieved by pharmacological manipulation (e.g. pentoxyfylline and statins). Conclusion. Originally described as an endothelial anticoagulant, TM plays a key role in tumour biology and prognostics, and provides a potential therapeutic target in impeding cancer spread. q 2004 Elsevier Ltd. All rights reserved.

Introduction
Thrombomodulin (TM) was rst described as an integral component of the haemostatic pathway in 1981.1 Constitutively expressed on the vascular and lymphatic endothelium, TM interacts with thrombin to form a high-afnity complex that inhibits thrombin activity (brin formation) and accelerates protein C activation.2,3 In addition to this anticoagulant function, TM reduces brinolysis by

* Corresponding author. Address: Dublin 9, Ireland. Tel.: C353 86 6066 904/1 8317391; fax: C353 1 8317231. E-mail address: amhanly@indigo.ie (A.M. Hanly).

activating thrombin-activatable brinolysis inhibitor (TAFI) in plasma.4 Thus, induced TM deciency (genetic knock-out in mice) results in a hypercoagulable state with risk of arterial thrombotic disease.5 Soluble molecules of TM, released from endothelial cell surfaces, are found in plasma and urine where higher levels indicate injury and/or enhanced turnover of the endothelium.68 It is not surprising, therefore, that smokers display increased serum soluble TM concentrations that correlate with activated protein C levels, number of cigarettes smoked (per day), duration of smoking (years), degree of endothelial damage, and risk of thrombosis. 9,10

0748-7983/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.ejso.2004.11.017

218 Serum TM elevations, by neutrophil-mediated proteolytic/oxidative cleavage or hypoxiainduced shedding from the endothelium, parallel the activity of inammatory diseases (e.g. vasculidites and ulcerative colitis), ischaemiareperfusion injuries (i.e. vascular, cardiac, and transplant surgery), and atherosclerotic complications (e.g. in hypertension, smoking, and diabetes).1116 Clinical use of exogenous soluble infusions is approaching with recent report that administration of human urinary TM limited ischaemic injury, reperfusion sequelae, and systemic coagulopathy in a canine model of hepatic surgery.17 Early literature focused on TM as a placental surface protein (termed at the time fetomodulin) where it was identied as a marker of fetal endodermal differentiation essential for survival.18,19 However, since it was recognized that embryonic lethality was independent of its anticoagulant activity3 the role of TM in tumour biology has drawn clinical interest. The key points in this genesis are presented here with a focus on future directions in cancer care.

A.M. Hanly et al.

Thrombomodulin and squamous cell carcinoma

Perhaps the most striking evidence for the biological role of TM in carcinogenesis is in squamous epithelium. Initially, TM was proposed to be associated with keratinocyte differentiation as it was identied in all cell layers except the basal and upper granular levels.23 Following this, TM localisation was studied in normal and dysplastic oral epithelium and it was established that TM expression is not signicantly different in normal epithelium, lichen planus and mild dysplasia.26 However, in moderate or severe dysplasia, and well-differentiated squamous cell carcinoma (SCC) expression is markedly less than in normal epithelium.26 In addition, the proportion of TM positive cells is signicantly lower in poorly differentiated SCC and this is associated with enhanced invasiveness.27 TM expression in SCC of the lung and oesophagus is localized to the cellcell boundaries and the intratumoural cytoplasm, with a structural phenotype identical to that of the endothelium.28, 31 Lack of TM expression is an independent prognostic variable, and may be a better indicator of prognosis than traditional factors such as size, degree of invasion, and differentiation of the tumour.27,28 There is a clear association between loss of TM expression and the presence of lymphnode metastases, which have even less TM localisation than the primary tumour.27,28

The role of thrombomodulin in tumour biology

In 1987, TM was identied as a marker of invasive malignancy in vascular tumours.20 This led to further investigation that linked TM to prognosis in many tumour types including those of embryonal, epithelial and lymphatic origin.2124 Since, it is found in the placental syncytiotrophoblast of the placenta, TM was examined in chorionic diseases of the uterus and stomach.25 The nding that it is expressed in uterine choriocarcinoma but not in gastric choriocarcinoma suggests that it may be organ dependent. Nevertheless, TM is expressed in several tumours of neural (brain)21 and epithelial origin including squamous cell carcinoma (oral, oesophageal, and pulmonary)2628 and adenocarcinoma (breast and colorectal),22,24 where a consistent nding is that loss of expression correlates with advanced stage and bad prognosis. Plasma levels of soluble TM increase with progression of cancer stage in various tumour types including colorectal and pancreatic malignancy.29,30 There is considerable heterogeneity of soluble TM levels among different cancer types, perhaps due to varying degrees of tumour vascularisation, hypoxia, angiogenesis, and endothelial turnover.

Thrombomodulin and intercellular reactions

E-cadherin is an adhesion molecule that preserves epithelial structural integrity and whose loss denotes enhanced invasive potential of cancers in many tissue types.32 In an analogous fashion, loss of TM expression is associated with a poor prognosis.33 The cell-to-cell adhesion properties of TM underpin its role in limiting metastatic behaviour independent of its anticoagulant function.10 This adhesive function is reliant on the calcium-dependent, lectin-like domain of TM that inhibits neutrophil binding to the endothelium.34 The mechanism by which this occurs is through downregulation of nuclear transcription factors (NFKB) and adhesion molecules (E-selectin).35 TM is comparable to Ecadherin in adhesion and morphoregulatory activities, such that reduced tumour expression of either molecule results is a marker of invasiveness and poor prognosis in, for example, squamous cell carcinoma.31,32,34

Thrombomodulin and cancer Products of the coagulation cascade are involved in the spread of cancer, with thrombin playing a role in endothelial adhesion of tumour cells and evidence that warfarin impairs metastatic development.36 Tumour cells lacking TM may have low anticoagulant activity, facilitating adhesion to the endothelium in target tissues and thus, metastatic spread.35 For example, the presence of TM reduces intrahepatic spread, portal vein tumour thrombus, and capsular invasion in hepatocellular carcinoma.37

219

Conclusions
Thrombomodulin plays a key role in tumour biology and prognostics. The opportunities afforded by inducers of thrombomodulin expression provide potential therapies to improve tumour behaviour and impede transendothelial spread of cancer.

References
1. Esmon CT, Owen WG. Identication of an endothelial cell cofactor for thrombin-catalyzed activation of protein C. Proc Natl Acad Sci USA 1981;78:224952. 2. Esmon CT. The roles of protein C and thrombomodulin in the regulation of blood coagulation. J Biol Chem 1987;264: 47436. 3. Imada M, Yamaguchi H, Nagumo N, Katayanagi S, Iwasaki H, Imada M. Identication of fetomodulin, a surface marker protein of fetal development, as thrombomodulin by gene cloning and functional assays. Dev Biol 1990;140:11322. 4. De Munk GAW, Groeneveld E, Rijken DC. Acceleration of the thrombin inactivation of single chain urokinase type plasminogen activator, by thrombomodulin. J Clin Invest 1991;88: 16804. 5. Dorfer-Melly J, De kruif M, Schwarte LA, Franco RF, Florquin S, Spek CA, et al. Functional thrombomodulin deciency causes enhanced thrombus growth in a murine model of carotid artery thrombosis. Basic Res Cardiol 2003; 98(6):34752. 6. Ishii H, Majerus PW. Thrombomodulin is present in human plasma and urine. J Clin Invest 1985;76:217881. 7. Ishii H, Uchiyama H, Kazama M. Soluble thrombomodulin antigen in conditioned medium is increased by damage of endothelial cells. Thromb Haemost 1991;65(5):61823. 8. Nadar S, Blann AD, Lip GY. Endothelial dysfunction methods of assessment and application to hypertension. Curr Pharm Des 2004;10(29):3591605. 9. Fernandez JA, Gruber A, Heeb MJ, Grifn JH. Protein C pathway impairment in non-symptomatic cigarette smokers. Blood Cells Mol Dis 2002;29(1):7382. 10. Markulujak I, Ivankova J, Kubisz P. Thrombomodulin and von Willebrand factor in smokers and during smoking. Nouv Rev Fr Hematol 1995;37(2):1379. 11. Boehme MWJ, Raeth U, Scherbaum WA, Galle PR, Stremmel W. Interaction of endothelial cells and neutrophils in vitro. Kinetics of thrombomodulin, intercellular adhesion molecule-1 (ICAM-1), E-selectin, and vascular cell adhesion molecule-1 (VCAM-1): implications for the relevance as serological disease activity markers in vasculitides. Clin Exp Immunol 2000;119:2504. 12. Boehme MWJ, Autschbach F, Zuna I, Scherbaum WA, Stange E, Raeth U, et al. Elevate serum levels and reduced immunohistochemical expression of thrombomodulin in active ulcerative colitis. Gastroenterology 1997;113: 10717. 13. Nishida K, Miyazawa Y, Hatano M, Suzuki K, Hirose A, Fukushima R, Okinaga K. Reperfusion induces sublethal endothelial injury. J Surg Res 1998;79:8590. 14. Ileri M, Hisar I, Yetkin E, Kosar F, Cehreli S, Korkmaz S, et al. Increased levels of plasma thrombomodulin in patients with acute myocardial infarction who had thrombolytic therapy and achieved successful reperfusion. Clin Cardiol 2001;24: 3779.

Manipulating thrombomodulin expression in cancer

The regulation of TM is complex and vascular endothelial growth factor (VEGF), which may be tumour-derived or released following major surgery, induces endothelial TM and protein C during angiogenesis.38 Perhaps of more interest to surgeons, because of the potential to reverse negative characteristics of primary tumours and inhibit circulating cancer cell-endothelial interactions, two pharmacological substances upregulate membrane expression of TM. Pentoxifylline increases TM expression in endothelial cells under hypoxic conditions (tumours commonly display areas of diminished oxygenation) at a clinically-achievable dose.39 Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (statins) induce TM expression thereby, contributing to the benecial effects on endothelial function independent of cholesterol synthesis.40 However, the potential impact of these manipulations on tumour progression has yet to be tested in the clinical setting. TM expression is downregulated by a variety of inammatory mediators, cytokines, and bacterial products such as interleukin-1, transforming growth factor beta (TGF-b), and lipopolysaccharide (endotoxin).4143 Such post-operative stress responses and noxious stimuli enhance tumour cell-endothelial adhesion, facilitate the genesis of metastases, and contribute to thromboembolism.44,45 The potential to impede perioperative tumour cell-endothelial interactions exists by administering hypertonic saline.46 Similarly, exogenous administration of TM limits endothelial interactions,17 and infusion of recombinant TM may be of clinical benet to cancer patients in various scenarios that include: inhibiting adhesion of shed tumour cells at operation; limiting hepatic dysfunction following oncologic liver resection with vascular inow occlusion; and reduced risk of venous thromboembolism with minimal haemorrhagic complications.

220
15. Sido B, Datsis K, Mehrabi A, Kraus T, Klar E, Otto G, Nawroth PP. Soluble thrombomodulina marker of reperfusion injury after orthotpic liver transplantation. Transplantation 1995;60:4626. 16. Funiak S, Kubisz P, Kvetensky J, Cronberg S. Von Willebrand factor and endothelial damage in essential hypertension. J Hum Hypertens 1995;36:4835. 17. Kaneko H, Joubara N, Toshino M, Yamazaki K, Mitumaru A, Miki Y, et al. Protective effect of human urinary thrombomodulin on ischemia-reperfusion injury in the canine liver. Eur Surg Res 2000;32(2):8793. 18. Imada M, Imada H, Iwasaki A, Kume H, Yamaguchi H, Moore EE. Fetomodulin: marker surface protein of fetal development which is modulatalble by cyclic AMP. Dev Biol 1987;122:48391. 19. Imada M, Yamaguchi H, Imada M. Differential expression of fetomodulin and tissue plasminogen activator to characterize parietal endoderm differentiation of F9 embryonal carcinoma cells. Dev Biol 1990;144:42630. 20. Yonezawa S, Maruyama I, Sakae K, Igata A, Majerus PW, Sato E. Thrombomodulin as a marker for vascular tumours. Comparative study with factor VIII and Ulex europaeus I lectin. Am J Clin Pathol 1987;88(4):40511. 21. Appelton MA, Attanoos RL, Jasani B. Thrombomodulin as a marker of vascular and lymphatic tumours. Histopathology 1996;29(2):1537. 22. Kim SJ, Shiba E, Ishii H, Inoue T, Taguchi T, Tanji Y, et al. Thrombomodulin is a new biological and prognostic marker for breast cancer: an immunohistochemical study. Anticancer Res; 17: 231923. 23. Jackson DE, Mitchell CA, Bird P, Salem HH, Hayman JA. Immunohistochemical localisation of thrombomodulin in normal human skin and skin tumours. J Pathol 1995; 175(4):42132. 24. Takebayashi Y, Yamada K, Maruiyama E, Fujii R, Akiyama S, Aikou T. The expression of thymidine phosphorylase and thrombomodulin in human colorectal carcinoma. Cancer Lett 1995;92(1):17. 25. Yonezawa S, Maruyama I, Tanaka S, Nakamura T, Sato E. Immunohistochemical localisation of thrombomodulin in chorionic diseases of the uterus and choriocarcinoma of the stomach. A comparitive study with the distribution of human chorionic gonadotropin. Cancer 1988;62(3):56976. 26. Tabata M, Yonezawa S, Sugihara K, Yamashita S, Maruyama I. The use of thrombomodulin to study epithelial cell differentiation in neoplastic and non-neoplastic oral lesions. J Oral Pathol Med 1995;24(10):4439. 27. Matsushita Y, Ushiie K, Imamura Y, Ogawa H, Imamura H, Takao S, et al. A subcloned human oesophageal cell carcinoma cell line with low thrombomodulin expression showed increased invasiveness compared with a high thrombomodulin expressing clone thromobmodulin as a possible candidate for an adhesion molecule of squamous cell carcinoma. Cancer Lett 1998;127(12):195201. 28. Ogawa H, Yanizawa S, Maruyama I, Matsushita Y, Tezuka Y, Toyyama H, et al. Expression of thrombomodulin in squamous cell carcinoma of the lung: its relationship to lymph node metastasis and prognosis of the patients. Cancer Lett 2000;149:95103. 29. Lindahl AK, Boffa MC, Abilgaard U. Increased plasma thrombomodulin in cancer patients. Thromb Haemost 1993;69(2):1124. 30. Boffa MC, Lapeyrere C, Berard M, Lindahl AK, Flageul B, Chemaly P, et al. Plasma thrombomodulin level in malignancy varies according to the tumour type. Nouv Rev Fr Hematol 1994;36:S87S8. 31. Tezuka Y, Yonezawa S, Maruyama Y, Matsushita T,

A.M. Hanly et al.

Shimizu H, Obama M, et al. Expression of thrombomodulin in esophageal squamous cell cancer and its relationship to lymph node metastasis. Cancer Res 1995;55(18):4196200. Conacci-Sorrell M, Zhurinsky J, Ben-Zeev A. The cadherin catenin adhesion system in signalling and cancer. J Clin Invest 2002;109:98791. Hosaka Y, Higuchi T, Tsumagari M, Ishii H. Inhibition of invasion and experimental metastasis of murine melanoma cells by human soluble thrombomodulin. Cancer Lett 2000; 161(2):23140. Huang HC, Shi GY, Jiang SJ, Shi CS, Wu CM, Yang HY, et al. Thrombomodulin-mediated cell adhesion: involvement of its lectin-like domain. J Biol Chem 2003;278(47):467509. Conway EM, Van de Wauwer M, Pollefey S, Jurk K, Van Achen H. The lectin-like domain of thrombomodulin confers protection from neutrophil mediated tissue damage by suppressing adhesion molecule expression via nuclear factor ?B and mitogen activated protein kinase pathways J Exp Med 2002;196:56577. Brown JM. A study of the mechanism by which anticoagulation with warfarin inhibits blood borne metastasis. Cancer Res 1973;33(6):121724. Sueihro T, Shimada M, Matsumata T, Taketomi A, Yamamoto K, Sugimachi K. Thrombomodulin inhibits intrahepatic spread in human hepatocellular carcinoma. Hepatology 1995;21(5):128590. Calnek DS, Grinnell BW. Thrombomoduliin dependent anticoagulant activity is regulated by vascular endothelial growth factor. Exp Cell Res 1998;238(1):2948. Seigneur M, Dufourcq P, Belloc F, Lenoble M, Renard M, Boisseau MR. Inuence of pentoxifylline on membrane thrombomodulin levels in endothelial cells submitted to hypoxic conditions. J Cardiovasc Pharmacol 1995;25(Suppl 2):S85S7. Morikawa S, Takabe W, Mataki C, Kanke T, Itoh T, Wada Y, et al. The effect of statins on mRNA levels of genes related to inammation, coagulation and vascular constriction in HUVEC Human umbilical vein endothelial cells. J Atheroscler Thromb 2002;9(4):17883. Moore KL, Andreoli SP, Esmon N, Esmon CT, Bang NU. Endotoxin enhances tissue factor and suppresses thrombomodulin expression of human vascular endothelium in vitro. J Clin Invest 1987;79:12430. Ohji T, Urano H, Shirahata A, Yamagishi M, Higashi K, Gotoh S, et al. Transforming growth factor beta 1 and beta 2 induce down modulation of thrombomodulin in human umbilical endothelial cells. Thromb Haemost 1995;73: 8128. Archipoff G, Beretz A, Fretssinet JM, Brison C, Cazenave JP. Role of cylic AMP in promoting the thromboresistence of human endothelial cells by enhancing thrombomodulin and decreasing tissue factor activities. Br J Pharmacol 1993;109: 1828. Andrews E, Wang JH, Winter DC, Laug WE, Redmond HP. Tumour cell adhesion to endothelial cells is increased by endotoxin via an upregulation of beta-1 integrin expression. J Surg Res 2001;97(1):1419. Dowdall JF, Winter DC, Andrews E, Laug WE, Wang JH, Redmond HP. Soluble interleukin 6 receptor (sIL-6R) mediates colonic tumour cell adherence to the vascular endothelium: a mechanism for metastatic initiation? J Surg Res 2002;107(1):15. Shields CJ, Winter DC, Wang JH, Andrews E, Laug WE, Redmond HP. Hypertonic saline impedes tumour cellendothelial cell interaction by reducing adhesion molecule and laminin expression. Surgery 2004;136(1):7683.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.