RESUMEN
Extracellular vesicles, such as microvesicles (MVs), were identified as important players in tumor progression and acquisition of an aggressive phenotype. Tissue factor (TF) is a transmembrane protein that initiates the blood coagulation cascade. In tumor cells, TF has been associated with aggressiveness and cancer progression. Previous studies demonstrate that TF is incorporated into MVs secreted by tumor cells; however, it is unknown whether TF is actively involved in the release of MVs. Here, we investigated the influence of TF expression on the release of MVs. TF silencing was achieved through CRISPR/Cas9 approaches in the human breast cancer cell line, MDA-MB-231. TF knockout in MDA-MB-231â¯cells efficiently reduced TF-dependent signaling and procoagulant activity. Remarkably, silencing of TF caused a significant decrease in the number of MVs released by MDA-MB-231â¯cells. We also observed an increase in actin-positive membrane projections in TF knockout cells and a reduction in RhoA expression when compared to TF-expressing cells. Treatment of MDA-MB-231â¯cells with the RhoA-ROCK signaling pathway inhibitor, fasudil, significantly reduced the release of MVs. Taken together, our results suggest a novel and relevant role for TF in tumor biology by playing an active role in the MVs secretion.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/patología , Tromboplastina/metabolismo , Neoplasias de la Mama/genética , Línea Celular Tumoral , Vesículas Extracelulares/genética , Factor VIIa/análisis , Factor VIIa/metabolismo , Femenino , Silenciador del Gen , Humanos , Transducción de Señal , Tromboplastina/genética , Quinasas Asociadas a rho/análisis , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/análisis , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Activated Protein C (APC) is a serine-protease that displays antithrombotic and anti-inflammatory properties. In addition, cleavage of protease-activated receptor 1 (PAR1) by APC exerts endothelial cytoprotective actions. The effects of APC on endothelial cells may be reproduced by TR47, a PAR1-based peptide that mimics the novel N-terminus of PAR1 generated upon cleavage at Arg-46 by APC. In this study we demonstrate that wild-type APC and its signaling-proficient mutant, APC-2Cys (which has dramatically reduced anticoagulant activity), display similar inhibitory effects towards the transendothelial migration of A375 human melanoma cells. Consistent with this observation, APC and APC-2Cys significantly reduced the in vivo metastatic potential of the B16F10 murine melanoma cells. TR47 recapitulated the in vitro and in vivo protective profiles of APC and APC-2Cys. Treatment of EA.hy926 endothelial cells with TR47 (20 µM) significantly decreased the A375 cell migration. In addition, treatment of C57/BL6 mice with a single TR47 dose (125 µg/animal) strongly reduced the metastatic burden of B16F10 cells. Together, our results suggest that protection of the endothelial barrier by APC/TR47-mediated signaling pathways might be a valuable therapeutic approach to prevent metastasis.