High-level secretion of tissue factor-rich extracellular vesicles from ovarian cancer cells mediated by filamin-A and protease-activated receptors

Summary

Thromboembolic events occur frequently in ovarian cancer patients. Tissue factor (TF) is often overexpressed in tumours, including ovarian clear-cell carcinoma (CCC), a subtype with a generally poor prognosis. TF-coagulation factor VII (fVII) complexes on the cell surface activate downstream coagulation mechanisms. Moreover, cancer cells secrete extracellular vesicles (EVs), which act as vehicles for TF. We therefore examined the characteristics of EVs produced by ovarian cancer cells of various histological subtypes. CCC cells secreted high levels of TF within EVs, while the high-TF expressing breast cancer cell line MDA-MB-231 shed fewer TF-positive EVs. We also found that CCC tumours with hypoxic tissue areas synthesised TF and fVII in vivo, rendering the blood of xenograft mice bearing these tumours hypercoagulable compared with mice bearing MDA-MB-231 tumours. Incorporation of TF into EVs and secretion of EVs from CCC cells exposed to hypoxia were both dependent on the actin-binding protein, filamin-A (filA). Furthermore, production of these EVs was dependent on different protease-activated receptors (PARs) on the cell surface. These results show that CCC cells could produce large numbers of TF-positive EVs dependent upon filA and PARs. This phenomenon may be the mechanism underlying the increased incidence of venous thromboembolism in ovarian cancer patients.

Supplementary Material to this article is available online at www.thrombosis-online.com.

Keywords

Thrombosis - ovarian cancer - tissue factor - microvesicles - coagulation factor VII

• References

• 1 Khorana AA, Francis CW, Culakova E. et al. Frequency, risk factors, and trends for venous thromboembolism among hospitalized cancer patients. Cancer 2007; 110: 2339-2345.
• 2 Iodice S, Gandini S, Löhr M. et al. Venous thromboembolic events and organ-specific occult cancers: a review and meta-analysis. J Thromb Haemost 2008; 06: 781-788.
• 3 Timp JF, Braekkan SK, Versteeg HH. et al. Epidemiology of cancer-associated venous thrombosis. Blood 2013; 122: 1712-1722.
• 4 Arora M, Wun T. Adverse impact of venous thromboembolism on patients with cancer. Semin Thromb Hemost 2014; 40: 313-318.
• 5 Bakhru A. Effect of ovarian tumor characteristics on venous thromboembolic risk. J Gynecol Oncol 2012; 24: 52-58.
• 6 Yokota N, Koizume S, Miyagi E. et al. Self-production of tissue factor-coagulation factor VII complex by ovarian cancer cells. Br J Cancer 2009; 101: 2023-2029.
• 7 Uno K, Homma S, Satoh T. et al. Tissue factor expression as a possible determinant of thromboembolism in ovarian cancer. Br J Cancer 2007; 96: 290-295.
• 8 Furie B, Furie BC. The molecular basis of blood coagulation. Cell 1988; 53: 505-518.
• 9 Semenza GL. Hypoxia Inducible factors in physiology and medicine. Cell 2012; 148: 399-408.
• 10 Koizume S, Jin M-S, Miyagi E. et al. Activation of cancer cell migration and invasion by ectopic synthesis of coagulation factor VII. Cancer Res 2006; 66: 9453-9460.
• 11 Koizume S, Ito S, Miyagi E. et al. HIF2a-Sp1 interaction mediates a deacety-lation-dependent FVII-gene activation under hypoxic conditions in ovarian cancer cells. Nucleic Acids Res 2012; 40: 5389-5401.
• 12 Koizume S, Miyagi Y. Breast cancer phenotypes regulated by tissue factor-factor VII pathway: possible therapeutic targets. World J Clin Oncol 2014; 05: 908-920.
• 13 D’Souza-Schorey C, Clancy JW. Tumor-derived microvesicles: shedding light on novel microenvironment modulators and prospective cancer biomarkers. Genes Dev 2012; 26: 1287-1299.
• 14 Giusti I, D’Ascenzo S, Dolo V. Microvesicles as potential ovarian cancer biom-arkers. Biomed Res Int 2013; 2013: 1-12.
• 15 Lopez JA, del Conde I, Shrimpton CN. Receptors, rafts, and microvesicles in thrombosis and inflammation. J Thromb Haemost 2005; 03: 1737-1744.
• 16 Davila M, Amirkhosravi A, Coll E. et al. Tissue factor-bearing microvesicles derived from tumor cells: impact on coagulation activation. J Thromb Haemost 2008; 61: 517-1524.
• 17 Zwicker JI, Liebman HA, Neuberg D. et al. Tumor-derived tissue factor-bearing microparticles are associated with venous thromboembolic events in malignancy. Clin Cancer Res 209 15: 6830-6840.
• 18 Wang J-G, Geddings JE, Aleman MM. et al. Tumor-derived tissue factor activates coagulation and enhances thrombosis in a mouse xenograft model of human pancreatic cancer. Blood 2012; 119: 5543-5551.
• 19 Thomas GM, Panicot-Dubois L, Lacroix R. et al. Cancer-cell derived micropar-ticles bearing P-selectin glycoprotein ligand 1 accelerate thrombus formation in vivo. J Exp Med 2009; 206: 1913-1927.
• 20 Lima LG, Oliveira AS, Campos LC. et al. Malignant transformation in melan-ocytes is associated with increased production of procoagulant microvesicles. Thromb Haemost 2011; 106: 712-723.
• 21 van der Berg YW, van den Hengel LG, Myers HR. et al. Alternatively spliced tissue factor induces angiogenesis through integrin ligation. Proc Natl Acad Sci USA 2009; 106: 19497-19502.
• 22 Kirwan CC, McDowell G, McCollum CN. et al. Early changes in the haemostatic and procoagulant systems after chemotherapy for breast cancer. Br J Cancer 2008; 2008; 99: 1000-1006.
• 23 Collier MEW, Maraveyas A, Ettelaie C. Filamin-A is required for the incorporation of tissue factor into cell-derived microvesicles. Thromb Haemost 2014; 111: 647-655.
• 24 Koizume S, Yokota N, Miyagi E. et al. Hepatocyte nuclear factor-4-independent synthesis of coagulation factor VII in breast cancer cells and its inhibition by targeting selective histone acetyltransferase. Mol Cancer Res 2009; 07: 1928-1936.
• 25 Rebello SS, Blank HS, Rote WE. et al. Antithrombotic efficacy of a recombinant nematode anticoagulant peptide (rNAP5) in canine models of thrombosis after single subcutaneous administration. J Pharmacol Exp Ther 1997; 283: 91-99.
• 26 Antonyak MA, Li B, Boroughs LK. et al. Cancer-cell derived microvesicles induce transformation by transferring tissue transglutaminase and fibronectin to recipient cells. Proc Natl Acad Sci USA 2011; 108: 4852-4857.
• 27 Lee TH, D’Asti E, Magnus N. et al. Microvesicles as mediators of intercellular communication in cancer-the emerging science of cellular debris. Semin Im-munopathol 2011; 33: 455-467.
• 28 Versteeg HH, Schaffner F, Kerver M. et al. Inhibition of tissue factor signaling suppresses tumor growth. Blood 2008; 111: 190-199.
• 29 Wang T, Gilkes DM, Takano N. et al. Hypoxia-inducible factors and RAB22A mediate formation of microvesicles that stimulate breast cancer invasion and metastasis. Proc Natl Acad Sci USA 2014; E3234-E3242.
• 30 Ott I, Fischer EG, Miyagi Y. et al. A role for tissue factor in cell adhesion and migration mediated by interaction with actin-binding protein 280. J Cell Biol 1998; 140: 1241-1253.
• 31 Fox JEB, Austin CD, Boyles JK. et al. Role of the membrane skeleton in preventing the shedding of procoagulant-rich microvesicles from the platelet plasma membrane. J Cell Biol 1990; 111: 483-493.
• 32 Rothmeier AS, Marchese P, Petrich BG. et al. Caspase-1-mediated pathway promotes generation of thromboinflammatory microparticles. J Clin Invest 2015; 125: 1471-1484.
• 33 Zheng X, Zhou A-X, Rouhi P. et al. Hypoxia-induced and calpain-dependent cleavage of filamin A regulates the hypoxic response. Proc Natl Acad Sci USA 2014; 111: 2560-2565.
• 34 Collier MEW, Ettelaie C. Regulation of the incorporation of tissue factor nito microparticles by serine phosphorylation of the cytoplasmic domain of tissue factor. J Biol Chem 2011; 86: 11977-11984.
• 35 Svensson KJ, Kucharzewska P, Christianson HC. et al. Hypoxia triggers a proan-giogenic pathway involving cancer cell microvessicles and PAR-2 mediated he-parin-binding EGF signaling in endotherial cells. Proc Natl Acad Sci USA 2011; 108: 13147-13152.
• 36 Riewald M, Ruf W. Mechanistic coupling of protease signalling and initiation of coagulation by tissue factor. Proc Natl Acad Sci USA 2001; 98: 7742-7747.
• 37 Stany MP, Vathipadiekal V, Ozbun L. et al. Identification of novel therapeutic targets in microdissected clear cell ovarian cancers. PLoS ONE 2011; 06: e21121.
• 38 Spowart JE, Townsend KN, Huwait H. et al. The autophagy protein LC3A correlates with hypoxia and is a prognostic marker of patient survival in clear cell ovarian cancer. J Pathol 2012; 228: 437-447.
• 39 van der Berg YW, Osanto S, Reitsma PH. et al. The relationship between tissue factor and cancer progression: insights from bench and bedside. Blood 2012; 119: 924-932.
• 40 Falet H, Pollitt AY, Begonja AJ. et al. A novel interaction between FlnA and Syk regulates platelet ITAM-mediated receptor signaling and function. J Exp Med 2010; 207: 1967-1979.
• 41 Zhou A-X, Toylu A, Nallapalli RK. et al. Filamin A mediates HGF/c-MET signaling in tumor cell migration. Int J Cancer 2011; 128: 839-846.
• 42 Begonia AJ, Hoffmeister KM, Hartwig JH. et al. FlnA-null megakaryocytes prematurely release large and fragile platelets that circulate poorly. Blood 2011; 118: 2285-2295.
• 43 Berrou E, Adam F, Lebret M. et al. Heterogeneity of platelet functional alterations in patients with filamin A mutations. Arterioscler Thromb Vasc Biol 2013; 33: e11-e18.