Novel Pleiotropic Effects of Thrombin in Regulation of the Metastatic Potential of Human Rhabdomyosarcoma (RMS) Cells.

Abstract

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of adolescents and children that frequently infiltrates bone marrow (BM). We identified chemokines and growth factors [i.e., stromal derived factor (SDF)-1, hepatocyte growth factor (HGF)] that play important roles in RMS metastasis (Blood 2002; 100:2597, Cancer Res. 2003; 63:7926,Cancer Res. 2007; 67:2131). However, novel evidence indicates that the metastatic process for many tumors may be modulated by components of the coagulation cascade (e.g., thrombin, activated platelets). Thus, we became interested in the role of coagulation cascade in modulating metastasis of RMS cells. We learned that RMS cells express tissue factor (TF) and thus may activate coagulation by generating thrombin. Thrombin activated in the tumor microenvironment activates platelets that release microvesicles. These interact with RMS cells and/or directly stimulate PAR 1–4 receptors that are expressed on RMS cells. Focusing on these effects, 9 human RMS cells lines were employed for our studies. We learned that platelet-derived microvesicles (PMVs) transfer several platelet integrin receptors to RMS cells (e.g., a2b3) that are important for interaction with the endothelium, thus increasing their adhesive potential to endothelial cells, fibronectin, and fibrinogen. More importantly, RMS cells covered with PMVs showed higher metastatic potential after intravenous injection into severe combined immunodeficient (SCID) mice. This could be explained by enhanced adhesiveness of PMV-covered RMS cells to endothelial cells in small vessels. On the other hand, PMVs also directly chemoattracted RMS cells and activated MAPKp42/44 and AKT. Since we found that RMS cell lines express functional PAR 1–4 receptors, we become interested in the thromin-mediated responses of these cells. In RMS, we observed cells stimulated by thrombin phosphorylation of MAPKp42/44 and p38, but not AKT. However, we did not observe any effect from thrombin on proliferation and survival of RMS cells. To our surprise, thrombin decreased RMS chemotactic responses to SDF-1, HGF, and PMVs. Thrombin also decreased adhesion of RMS cells to fibronectin and laminin. In conclusion, we demonstrate for first time that RMS-expressed TF activates prothrombin and that thrombin is a novel, underappreciated, pro-metastatic factor for these cells. Activated in tumor proximity by thrombin, platelets release PMVs that chemoattract and transfer several platelet-derived receptors/adhesion molecules to RMS cells that are crucial for adhesion/interaction with the endothelium. Conversely, by decreasing the responsiveness of RMS cells to local chemoattractants and decreasing adhesiveness of RMS cells, thrombin promotes their release from the primary tumor into circulation. Consequently, RMS cells that are covered by PMVs release into circulation and respond to chemoattractants in distant organs for metastasis.