Supplementary File PDF 277K, Supplementary Methods: Includes additional details of reagents and methodology, and references. Supplementary Figure 1: Effect of FVIIa and thrombin on cell proliferation of wild-type REN, TF- or PAR1- knocked-down REN MPM cells. Supplementary Figure 2: TF- or PAR1 knock-down in REN MPM cells suppresses in vivo tumor cell proliferation. Supplementary Figure 3: TF- or PAR1 knock-down in REN MPM cells increases tumor cell apoptosis in vivo. Supplementary Figure 4: Over expression EPCR did not alter in vitro growth potential of REN MPM cells. Supplementary Figure 5: Tissue factor pathway inhibitor (TFPI) and thrombomodulin (TM) protein levels in parental MS-1 and M9K MPM cells and their variants as analyzed by western blot analysis. Supplementary Figure 6: EPCR knock-down did not alter cell proliferation of MS-1 or M9K MPM cells in vitro
ARTICLE ABSTRACT
The procoagulant protein tissue factor (F3) is a powerful growth promoter in many tumors, but its mechanism of action is not well understood. More generally, it is unknown whether hemostatic factors expressed on tumor cells influence tissue factor-mediated effects on cancer progression. In this study, we investigated the influence of tissue factor, endothelial cell protein C receptor (EPCR, PROCR), and protease activated receptor-1 (PAR1, F2R) on the growth of malignant pleural mesothelioma (MPM), using human MPM cells that lack or express tissue factor, EPCR or PAR1, and an orthotopic nude mouse model of MPM. Intrapleural administration of MPM cells expressing tissue factor and PAR1 but lacking EPCR and PAR2 (F2RL1) generated large tumors in the pleural cavity. Suppression of tissue factor or PAR1 expression in these cells markedly reduced tumor growth. In contrast, tissue factor overexpression in nonaggressive MPM cells that expressed EPCR and PAR1 with minimal levels of tissue factor did not increase their limited tumorigenicity. More importantly, ectopic expression of EPCR in aggressive MPM cells attenuated their growth potential, whereas EPCR silencing in nonaggressive MPM cells engineered to overexpress tissue factor increased their tumorigenicity. Immunohistochemical analyses revealed that EPCR expression in tumor cells reduced tumor cell proliferation and enhanced apoptosis. Overall, our results enlighten the mechanism by which tissue factor promotes tumor growth through PAR1, and they show how EPCR can attenuate the growth of tissue factor-expressing tumor cells. Cancer Res; 73(13); 3963–73. ©2013 AACR.
