Abstract Aberrant activation of the endothelin-1 receptor (ET-1R) elicits pleiotropic effects relevant for serous ovarian cancer (SOC) cell invasion. The network activated by this receptor might be finely, spatially and temporary orchestrated by β-arrestin1 (β-arr1)-driven interactome. Recently, we uncovered a novel role for ET-1R/β-arr1 as regulator of cytoskeletal remodelling and invasive protrusions, invadopodia. Emerging evidence demonstrated that hMENA protein, an Ena/Vasp family member, is a key invadopodia component. In this study, we set out to molecularly dissect whether hMENA might represent a novel interacting partner of β-arr1 necessary for invadopodial function downstream of ET-1R in SOC cells. ENAH mRNA is significantly upregulated in OC tissues, and in particular in high-grade (HG)-SOC tumors, compared with normal tissues. In a panel of SOC cells, the expression hMENA, along with the spliced isoform hMENAΔv6, is upregulated by ET-1, at mRNA and protein levels, through β-arr1, restricted to mesenchymal phenotype. This effect is inhibited by treatment with the dual ETAR/ETBR antagonist macitentan. As shown by biochemical and imaging assays, ET-1 promotes a physical association between hMENA and β-arr1 as well PDZ-RhoGEF, which in turn activate RhoC. Most importantly, ET-1 induces localization of hMENA in F-actin-containing puncta, which co-localize with cortactin, and extracellular matrix degradation sites, thus promoting invadopodia maturation. Silencing of hMENA, as well as of β-arr1, or treatment with macitentan, impairs ET-1-dependent invadopodia activity, MMP secretion and activation, invasion, transendothelial migration and cell plasticity. In vivo, macitentan is able to inhibit SOC metastatic dissemination and hMENA expression along with other invadopodia markers. Finally, high ETAR/ARRB1/ENAH gene expression is associated with a poor prognosis in SOC patients. Collectively, these data define a pivotal function of hMENA/ hMENAΔv6, which is required for ET-1/β-arr1-induced invadopodial function and metastatic spreading of SOC. Citation Format: Valentina Caprara, Francesca Di Modugno, Lidia Chellini, Piera Tocci, Francesca Spadaro, Andrea Sacconi, Giovanni Blandino, Paola Nisticò, Anna Bagnato, Laura Rosanò. Endothelin-1 receptor/beta-arrestin-1 pathway promotes invadopodia and metastatic process by integration with hMENA in human serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3162.