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Aims: In epithelial ovarian cancer (EOC), activation of endothelin-1 (ET-1)/endothelin A receptor (ETAR) and ET-1/ETBR signaling is linked to many tumor promoting effects, such as proliferation, angiogenesis, invasion, metastasis and chemoresistance. Understanding how to hamper the distinct mechanisms that facilitate epithelial plasticity and propagation is therefore central for improving the clinical outcome for EOC patients., Main Methods: The phosphorylation status of Akt and MAPK was evaluated by immunoblotting in A2780 and 2008 EOC cell lines and their cisplatinum-resistant variants. Vasculogenic mimicry was analyzed by vascular tubules formation assay. Tumor growth and metastases inhibition was performed in chemoresistant EOC xenografts., Key Findings: We found that the dual ETAR/ETBR antagonist macitentan was able to inhibit the ET-1-induced activation of Akt and MAPK signaling pathways in chemoresistant EOC cells. Moreover, chemoresistant EOC cells displayed higher capability to engage vasculogenic mimicry compared to sensitive cells that was inhibited after treatment with macitentan. Finally, the specific ETAR antagonist zibotentan was less efficacious compared to macitentan to suppress tumor growth in chemoresistant EOC xenografts and the co-treatment of macitentan and cisplatinum reduced the metastatic progression., Significance: Our findings better clarify the ET-1-induced molecular mechanisms underlying the aggressive behavior of chemoresistant EOC cells. These results also support the use of macitentan in combination with chemotherapy as a rational therapeutic strategy for circumventing drug resistance in EOC., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Aims: The endothelin-1 (ET-1)/ET A receptor (ETAR) signaling pathway is critical driver of epithelial ovarian cancer (EOC) progression. Emerging evidences demonstrate that the scaffolding protein β-arrestin-1 (β-arr1) downstream of ETAR guides cell motility, although the signaling pathways by which ETAR activation controls these process are not well understood. Here, we set out to molecularly dissect whether RhoA GTPase activation is a mediator of ET-1 signaling controlling EOC cell migration., Main Methods: We cultured EOC cell lines (HEY, SKOV3, OVCAR, A2780 and 2008) with ET-1 and the ET-1R antagonist macitentan. RhoA expression was evaluated by RT-PCR. Activation of RhoA and ROCK1 was evaluated by pull down and kinase assays, respectively. Cell motility was evaluated by chemotaxis and wound healing assays, in untrasfected cells by using ROCK chemical inhibitors, Y-27632 or Fasudil, or in cells after transfection with dominant negative RhoA construct. The phosphorylation of myosin light chain 2 (MLC2) was evaluated by immunoblotting. Pseudopodia formation was evaluated by a pseudopodia kit assay., Key Findings: In EOC cells, ET-1 activates RhoA and downstream ROCK1 and MLC2. These effects were inhibited by β-arr1 silencing, suggesting that ET-1/ETAR regulate RhoA signaling through β-arr1. At functional level, the activation of RhoA/ROCK signaling led to enhanced cell migration and pseudopodia formation. The suppressive effect of the ROCK inhibitors, as well as of macitentan, demonstrates that RhoA is involved in ET-1/ETAR-induced cell migration., Significance: Altogether these findings reveal a new pathway that depends on β-arr1 to sustain RhoA/ROCK signaling in response to ETAR activation in EOC., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Aims: In epithelial ovarian cancer (EOC), activation of endothelin-1 (ET-1)/endothelin A receptor (ETAR) signalling is linked to many tumor promoting effects, such as proliferation, angiogenesis, invasion and metastasis. These effects are dependent by the activation of critical signalling pathways, such as MAPK, Akt, and β-catenin, through specific cytosolic and nuclear scaffolding functions of β-arrestin 1 (β-arr1). Here, we have assessed the potential role of ET-1/ETAR in promoting NF-κB signalling in EOC cells through β-arr-1 recruitment., Main Methods: We used cultured HEY EOC cells cultured in the presence or absence of ET-1 and the ETAR antagonist BQ123. The phosphorylation of p65 and Iκ-Bα was evaluated by immunoblotting analysis. The interaction between p65 and β-arr1 was evaluated by immunoprecipitation experiments in nuclear extracts. NF-κB promoter activity was evaluated by transfection with NF-κB-driven luciferase reporter construct. Assessment of the function of β-arr1 was achieved by β-arr1 silencing with shRNA and expression of β-arr1-FLAG expression vector., Key Findings: In EOC cells, ET-1 promotes the phosphorylation of p65 subunit and the cytoplasmic inhibitor IκB that in turn led to increased NF-κB transcriptional activity. These effects were inhibited by the use of BQ123, as well as by β-arr-1 silencing, suggesting that ET-1 through ETAR promotes the recruitment of β-arr1 to regulate NF-κB signalling. Moreover, the nuclear physical interaction between p65 and β-arr1 indicates a nuclear function of β-arr-1 in ETAR-driven NF-κB transcriptional activity., Significance: Altogether these findings reveal a previously unrecognized pathway that depends on β-arr1 to sustain NF-κB signalling in response to ETAR activation in ovarian cancer., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Aims: Endothelin A receptor (ET(A)R) and epidermal growth factor receptor (EGFR) cross-talk enhances the metastatic potential of epithelial ovarian cancer (EOC) cells activating different pathways, including β-catenin signalling. Here, we evaluated β-catenin as one of ET(A)R/EGFR downstream pathway in the invasive behaviour of EOC cells and their therapeutic potential to co-target ET(A)R and EGFR., Main Methods: The phosphorylation status and interactions of different proteins were analysed by immunoblotting and immunoprecipitation. Reporter activity and RT-PCR was used for evaluation of β-catenin transcriptional activity and gene expression. Functional effects were evaluated by gelatin zymography and cell invasion assays. An orthotopic model of metastatic human EOC in mice was used for in vivo studies., Key Findings: In EOC cell lines, ET-1 induced Src-dependent EGFR transactivation, causing tyrosine (Y) phosphorylation of β-catenin at the residue Y654, its dissociation from E-cadherin complexes and the accumulation as an active form. This pool of Tyr-β-catenin relocalised to the nucleus promoting its transcriptional activity, and the expression of its target genes, such as MMP-2. At functional level, ET-1 and EGFR circuits enhanced protease activity and cell invasion. All these effects were significantly inhibited by the ET(A)R antagonist, zibotentan, or EGFR inhibitor, gefitinib, and are completely blocked by co-addition of both drugs. In vivo, zibotentan treatment significantly inhibited metastases, associated with reduced expression and activation of MMPs and active β-catenin, especially when combined with gefitinib., Significance: Altogether these findings provide additional support to the potential use of ET(A)R and EGFR blockade as a new therapeutic opportunity for EOC treatment., (Copyright © 2012 Elsevier Inc. All rights reserved.)