Thrombin regulates the metastatic potential of human rhabdomyosarcoma cells: distinct role of PAR1 and PAR3 signaling

Abstract

We observed that human rhabdomyosarcoma (RMS) cells highly express a tissue factor that promotes thrombin formation, which indirectly and directly affects RMS progression. First, we found that thrombin activates platelets to generate microvesicles (PMV), which transfer to RMS cells' alpha2beta3 integrin and increase their adhesiveness to endothelial cells. Accordingly, RMS cells covered with PMVs showed higher metastatic potential after i.v. injection into immunodeficient mice. Furthermore, PMVs activate mitogen-activated protein kinase (MAPK)p42/44 and AKT to chemoattract RMS cells. We also found that RMS cells express functional protease-activated receptor-1 (PAR1) and PAR3 and respond to thrombin stimulation by MAPKp42/44 and MAPKp38 phosphorylation. To our surprise, thrombin did not affect RMS proliferation or survival; it inhibited the chemotactic and adhesive properties of RMS cells. However, when PAR1-specific agonist thrombin receptor-activating peptide 6 was used, which does not activate PAR3, selective PAR1 stimulation enhanced RMS proliferation. To learn more on the role of PAR1 and PAR3 antagonism in RMS proliferation and metastasis, we knocked down both receptors by using a short hairpin RNA strategy. We found that although thrombin does not affect growth of PAR1(-/-) cells, it stimulated the proliferation of PAR3(-/-) cells. More importantly, PAR3(-/-) cells, in contrast to PAR1(-/-) ones, formed larger tumors in immunodeficient mice. We conclude that thrombin is a novel underappreciated modulator of RMS metastasis and that we have identified a novel role for PAR3 in thrombin signaling.

Figure 1. Human RMS cells express TF and activate coagulation cascade that generates PMVs

Panel A: Human RMS cells express TF at the mRNA and protein levels. The experiment was repeated three times with similar results. A representative study is shown. Panel B: TF expressed on human RMS cell lines has the ability to initiate coagulation cascade, as shown by TF activity assay. The experiment was repeated three times with similar results. A representative study is shown. Panel C: RMS cells were incubated with PMVs, then washed and stained with anti-α2β3 (CD41)-specific Ab. As shown, PMVs transfer CD41 from platelets to RMS cells. The experiment was repeated three times with similar results. A representative study is shown. Panel D: PMVs activate AKT and MAPKp42/44 in human RMS cells in a time-dependent manner. The experiment was repeated three times with similar results. A representative study is shown.

Figure 2. PMV mediates chemotaxis and adhesion of RMS cells

Panels A and B: PMV induces chemotaxis (Panel A) and adhesion (Panel B) of RMS cells. Data from four separate experiments are pooled together * p < 0.01. CW9019 (Panel C) or RH30 (Panel D) cells were pre-incubated with PMVs or medium alone and were then injected intravenously into immunodeficient mice. Forty-eight hours later, the seeding efficiency for RMS cells in murine BM was evaluated by real-time PCR by detection of human α-satellite gene. The number of human cells present in murine BM (degree of chimerism) was calculated from the standard curve obtained by mixing different numbers of human cells with a constant number of murine cells as described (13). Data from four separate experiments are pooled together * p < 0.01.

Figure 3. RMS cells express functional thrombin receptors

Panel A: Expression of PAR1, -3, and -4 at the mRNA level in human RMS cells. The experiment was repeated two times with similar results. A representative study is shown. Panel B: Flow cytometry analysis of PAR1 on human RMS cell lines. The experiment was repeated three times with similar results. A representative study is shown. Phosphorylation of MAPK p42/44, AKT, and MAPK p38 in selected human RMS cell lines stimulated by thrombin (1U/ml) (Panel C) or PAR1-agonist TRAP6 (50uM) (Panel D) for 2, 5, 10, and 15 minutes. The experiment was repeated three times with similar results. A representative study is shown.

Figure 3. RMS cells express functional thrombin receptors

Panel A: Expression of PAR1, -3, and -4 at the mRNA level in human RMS cells. The experiment was repeated two times with similar results. A representative study is shown. Panel B: Flow cytometry analysis of PAR1 on human RMS cell lines. The experiment was repeated three times with similar results. A representative study is shown. Phosphorylation of MAPK p42/44, AKT, and MAPK p38 in selected human RMS cell lines stimulated by thrombin (1U/ml) (Panel C) or PAR1-agonist TRAP6 (50uM) (Panel D) for 2, 5, 10, and 15 minutes. The experiment was repeated three times with similar results. A representative study is shown.

Figure 4. Thrombin regulates the pro-metastatic potential of RMS cells

Thrombin (Panel A) and TRAP6 (Panel B) decrease the chemotactic response of human RMS cells to BM-CM. Data from four separate experiments are pooled together * p < 0.001. Panel C: Hirudin reverses an inhibitory effect of thrombin but not TRAP6 on the chemotactic response of RMS cells to BM-CM. Data from three separate experiments are pooled together * p < 0.01. Panel D: Thrombin and TRAP6, but not PAR2- and PAR4-agonist, inhibit the chemotactic response of human RMS cells to BM-CM. Data from four separate experiments are pooled together * p < 0.01.

Figure 5. Effect of thrombin on the SDF-1-induced motility of RMS cell lines

The composite trajectories of RD, CW9019, RH28, RH5, and RH30 cells migrating in the absence (left panel) or presence (middle panel) of SDF-1 and (right panel) SDF-1 + thrombin in the culture media are shown in circular diagrams drawn with the initial point of each trajectory at the origin of the plot. Data from 4 separate experiments are pooled together.

Figure 6. Thrombin regulates the adhesive potential of RMS cells

Panel A: Thrombin decreases the BM-CM induced adhesive response of human RMS cells. Data from four separate experiments are pooled together * p < 0.001. Panel B: Thrombin inhibits phosphorylation of AKT, but not MAPKp42/44 induced by BM-CM in human RMS cells (RH5). The experiment was repeated three times with similar results. A representative study is shown.

Figure 7. Effect of thrombin and thrombin receptors on proliferation of RMS cells in vitro and in vivo

Panel A: RMS cells were cultured in the presence or absence of thrombin (1u/mL). After 72 hours, the number of cells was evaluated by hematocytometer. Panel B: RMS cells were cultured in the presence or absence of TRAP6 (100uM). After 72 hours, the number of cells was evaluated by hematocytometer. Panels C and D: CW9019, CW9019 PAR1^−/−, and -PAR3^−/− cells were cultured in the presence of thrombin (Panel C) or TRAP6 (Panel D). Numbers of cells were evaluated 72 hours after stimulation. Data from four separate experiments are pooled together * p < 0.001. Panel E: CW9019, CW9019 scrambled, or CW9019 PAR1^−/− cells were inoculated into the hind limb muscles of SCID-Beige inbred mice. Tumors were measured after 6 weeks. Panel F: CW9019, CW9019 scrambled, or CW9019 PAR3^−/− cells were inoculated into the hind limb muscles of SCID-Beige inbred mice. Tumors were measured after 3 weeks. Data from four separate experiments (6 mice/group each) are pooled together * p < 0.01.

Figure 7. Effect of thrombin and thrombin receptors on proliferation of RMS cells in vitro and in vivo

Panel A: RMS cells were cultured in the presence or absence of thrombin (1u/mL). After 72 hours, the number of cells was evaluated by hematocytometer. Panel B: RMS cells were cultured in the presence or absence of TRAP6 (100uM). After 72 hours, the number of cells was evaluated by hematocytometer. Panels C and D: CW9019, CW9019 PAR1^−/−, and -PAR3^−/− cells were cultured in the presence of thrombin (Panel C) or TRAP6 (Panel D). Numbers of cells were evaluated 72 hours after stimulation. Data from four separate experiments are pooled together * p < 0.001. Panel E: CW9019, CW9019 scrambled, or CW9019 PAR1^−/− cells were inoculated into the hind limb muscles of SCID-Beige inbred mice. Tumors were measured after 6 weeks. Panel F: CW9019, CW9019 scrambled, or CW9019 PAR3^−/− cells were inoculated into the hind limb muscles of SCID-Beige inbred mice. Tumors were measured after 3 weeks. Data from four separate experiments (6 mice/group each) are pooled together * p < 0.01.

Figure 8. Pro-metastatic consequences of TF expression by RMS cells

TF expressed on surface of RMS cells activates conversion of prothrombin to thrombin, which: i) activates platelets that release pro-metastatic PMVs; and ii) directly interacts with PAR1 and PAR3 on RMS cells. A negative regulatory effect of PAR3 on PAR1 is indicated.