The endothelin-1 (ET-1)/ET A receptor (ETAR) signalling pathway is a well-established driver of epithelial ovarian cancer (EOC) progression. One key process promoted by ET-1 is tumor cell invasion, which requires the scaffolding functions of ß-arrestin-1 (ß-arr1) downstream of the receptor; however, the potential role of ET-1 in inducing invadopodia, which are crucial for cellular invasion and tumor metastasis, is completely unknown. We describe here that ET-1/ETAR, through ß-arr1, activates RhoA and RhoC GTPase and downstream ROCK (Rho-associated coiled coil-forming kinase) kinase activity, promoting actin-based dynamic remodelling and enhanced cell invasion. This is accomplished by the direct interaction of ß-arr1 with PDZ-RhoGEF (postsynaptic density protein 95/disc-large/zonula occludens-RhoGEF). Interestingly, ETAR-mediated invasive properties are related to the regulation of invadopodia, as evaluated by colocalization of actin with cortactin, as well as with TKS5 and MT1-MMP (membrane type 1-matrix metalloproteinase) with areas of matrix degradation, and activation of cofilin pathway, which is crucial for regulating invadopodia activity. Depletion of PDZ-RhoGEF, or ß-arr1, or RhoC, as well as the treatment with the dual ET-1 receptor antagonist macitentan, significantly impairs invadopodia function, MMP activity and invasion, demonstrating that ß-arr1/PDZ-RhoGEF interaction mediates ETAR-driven ROCK-LIMK-cofilin pathway through the control of RhoC activity. In vivo, macitentan is able to inhibit metastatic dissemination and cofilin phosphorylation. Collectively, our data unveil a noncanonical activation of the RhoC/ROCK pathway through the ß-arr1/PDZ-RhoGEF complex as a regulator of ETAR-induced motility and metastasis, establishing ET-1 axis as a novel regulator of invadopodia protrusions through the RhoC/ROCK/LIMK/cofilin pathway during the initial steps of EOC invasion.
Cell Movement/physiology , Ovarian Neoplasms/metabolism , Podosomes/physiology , Receptor, Endothelin A/metabolism , Rho Guanine Nucleotide Exchange Factors/metabolism , beta-Arrestins/metabolism , Actin Depolymerizing Factors/metabolism , Actins/metabolism , Adaptor Proteins, Vesicular Transport/metabolism , Animals , Cell Line, Tumor , Cell Movement/genetics , Cortactin/metabolism , Female , Humans , Immunoblotting , Lim Kinases/metabolism , Matrix Metalloproteinase 14/metabolism , Mice, Nude , Ovarian Neoplasms/genetics , Ovarian Neoplasms/pathology , Podosomes/genetics , Podosomes/metabolism , RNA Interference , Receptor, Endothelin A/genetics , Reverse Transcriptase Polymerase Chain Reaction , Rho Guanine Nucleotide Exchange Factors/genetics , Signal Transduction/genetics , Transplantation, Heterologous , beta-Arrestins/genetics , rho GTP-Binding Proteins/metabolism , rho-Associated Kinases/metabolism , rhoC GTP-Binding Protein
