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The majority of ovarian cancer (OC) patients recur with a platinum-resistant disease. OC cells activate adaptive resistance mechanisms that are only partially described. Here we show that OC cells can adapt to chemotherapy through a positive-feedback loop that favors chemoresistance. In platinum-resistant OC cells we document that the endothelin-1 (ET-1)/endothelin A receptor axis intercepts the YAP pathway. This cross-talk occurs through the LATS/RhoA/actin-dependent pathway and contributes to prevent the chemotherapy-induced apoptosis. Mechanistically, β-arrestin1 (β-arr1) and YAP form a complex shaping TEAD-dependent transcriptional activity on the promoters of YAP target genes, including EDN1, which fuels a feed-forward signaling circuit that sustains a platinum-tolerant state. The FDA approved dual ET-1 receptor antagonist macitentan in co-therapy with cisplatin sensitizes resistant cells to the platinum-based therapy, reducing their metastatic potential. Furthermore, high ET A R/YAP gene expression signature is associated with a poor platinum-response in OC patients. Collectively, our findings identify in the networking between ET-1 and YAP pathways an escape strategy from chemotherapy. ET-1 receptor blockade interferes with such adaptive network and enhances platinum-induced apoptosis, representing a promising therapeutic opportunity to restore drug sensitivity in OC patients., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Hypoxia-inducible factor-1α (HIF-1α) mediates the response to hypoxia or other stimuli, such as growth factors, including endothelin-1 (ET-1), to promote malignant progression in numerous tumors. The importance of cofactors that regulate HIF-1α signalling within tumor is not well understood. Here we elucidate that ET-1/ET(A) receptor (ET(A)R)-induced pathway physically and functionally couples the scaffold protein β-arrestin1 (β-arr1) to HIF-1α signalling. In epithelial ovarian cancer (EOC) cells, ET-1/ET(A)R axis induced vascular-endothelial growth factor (VEGF) expression through HIF-1α nuclear accumulation. In these cells, activation of ET(A)R by ET-1, by mimicking hypoxia, promoted the nuclear interaction between β-arr1 and HIF-1α and the recruitment of p300 acetyltransferase to hypoxia response elements on the target gene promoters, resulting in enhanced histone acetylation, and HIF-1α target gene transcription. Indeed, β-arr1-HIF-1α interaction regulated the enhanced expression and release of downstream targets, such as ET-1 and VEGF, required for tumor cell invasion and pro-angiogenic effects in endothelial cells. These effects were abrogated by β-arr1 or HIF-1α silencing or by pharmacological treatment with the dual ET-1 receptor antagonist macitentan. Interestingly, ET(A)R/β-arr1 promoted the self-amplifying HIF-1α-mediated transcription of ET-1 that sustained a regulatory circuit involved in invasive and angiogenic behaviors. In a murine orthotopic model of metastatic human EOC, treatment with macitentan, or silencing of β-arr1, inhibits intravasation and metastasis formation. Collectively, these findings reveal the interplay of β-arr1 with HIF-1α in the complexity of ET-1/ET(A)R signalling, mediating epigenetic modifications directly involved in the metastatic process, and suggest that targeting ET-1-dependent β-arr1/HIF-1α pathway by using macitentan may impair EOC progression.

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.)

Reciprocal growth factor exchanges between endothelial and malignant cells within the hypoxic microenvironment determine tumor progression. However, the nature of these exchanges has not yet been fully explored. We studied the mutual regulation between endothelial cells (EC), melanoma cells and hypoxia that dictate tumor aggressiveness and angiogenic activity. Here, we investigated the presence of bidirectional autocrine/paracrine endothelin (ET)-1/ET receptor (ETBR) signaling in melanoma cells, blood and lymphatic EC. In all these cells, hypoxia enhanced ET-1 expression, which in turn induced vascular endothelial growth factor (VEGF)-A and VEGF-C secretion, through the hypoxia-inducible growth factor (HIF)-1α and HIF-2α. Autocrine/paracrine exchanges of ET-1, VEGF-A and VEGF-C promoted tumor aggressiveness and morphological changes in blood and lymphatic EC. Furthermore, conditioned media from EC enhanced melanoma cell migration and vessel-like channel formation. This regulation was inhibited by ETBR blockade, by using the selective ETBR antagonist, or ETBR small interfering RNA (siRNA), and by VEGFR-2/-3 antibodies, indicating that ET-1, VEGF-A/VEGF-C, produced by melanoma cells or EC mediated inter-regulation between these cells. Interestingly, HIF-1α/HIF-2α siRNA, impaired this reciprocal regulation, demonstrating the key role of these transcriptional factors in signaling exchanges. In melanoma xenografts, the ETBR antagonist reduced tumor growth and the number of blood and lymphatic vessels. These results reveal an interplay between melanoma cells and EC mediated by ET-1 and VEGF-A/-C and coordinated by the hypoxic microenvironment through HIF-1α/2α transcriptional programs. Thus, targeting ETBR may improve melanoma treatment for tumor and EC, by inhibiting autocrine/paracrine signaling that sustains melanoma progression.

Despite the fundamental pathophysiological importance of β-catenin in tumor progression, the mechanism underlying its final transcriptional output has been partially elucidated. Here, we report that β-arrestin-1 (β-arr1) is an epigenetic regulator of endothelin (ET)-1-induced β-catenin signaling in epithelial ovarian cancer (EOC). In response to ET A receptor (ETAR) activation by ET-1, β-arr1 increases its nuclear translocation and direct binding to β-catenin. This in turn enhanced β-catenin nuclear accumulation and transcriptional activity, which was prevented by expressing a mutant β-arr1 incapable of nuclear distribution. β-arr1-β-catenin interaction controls β-catenin target gene expressions, such as ET-1, Axin 2, Matrix metalloproteinase 2, and Cyclin D1, by promoting histone deacetylase 1 (HDAC1) dissociation and the recruitment of p300 acetyltransferase on these promoter genes, resulting in enhanced H3 and H4 histone acetylation, and gene transcription, required for cell migration, invasion and epithelial-to-mesenchymal transition. These effects are abrogated by β-arr1 silencing or by mutant β-arr1, as well as by β-catenin or p300 silencing, confirming that nuclear β-arr1 forms a functional complex capable of regulating epigenetic changes in β-catenin-driven invasive behavior. In a murine orthotopic model of metastatic human EOC, silencing of β-arr1 or mutant β-arr1 expression, as well as ETAR blockade, inhibits metastasis. In human EOC tissues, β-arr1-β-catenin nuclear complexes are selectively enriched at β-catenin target gene promoters, correlating with tumor grade, confirming a direct in vivo β-arr1-β-catenin association at specific set of genes involved in EOC progression. Collectively, our study provides insights into how a β-arr1-mediated epigenetic mechanism controls β-catenin activity, unraveling new components required for its nuclear function in promoting metastasis.

Endothelin receptor B (ET(B)R) is a G-protein-coupled receptor overexpressed in melanoma, blood, and lymphatic endothelial cells. Given that aberrant signal transduction can be mediated through cross talk between receptors, here, we explore the functional relationship between ET(B)R and the vascular endothelial growth factor receptor (VEGFR)-3 system and how this cross talk might influence the aggressive behavior of melanoma cells. The expression of VEGFR-3 and its ligands, VEGF-C and VEGF-D, significantly increased after activating ET(B)R by ET-1 in primary and metastatic melanoma cell lines. These effects, similarly to those induced by hypoxia, were mediated by hypoxia-inducible factor (HIF)-1α and HIF-2α. ET-1 caused the phosphorylation of VEGFR-3, which was accompanied by the activation of the downstream signaling molecules, such as MAPK and AKT. Inhibition of c-Src activity or silencing of the scaffold protein β-arrestin-1 reduced ET-1-induced VEGFR-3 phosphorylation, demonstrating that, upon ET-1 stimulus, β-arrestin-1 is involved with c-Src in the ET(B)R-mediated VEGFR-3 transactivation. Moreover, ET-1 in combination with VEGF-C further increased VEGFR-3, MAPK, and AKT phosphorylation and markedly promoted cell migration and vasculogenic mimicry. Dual inhibition of ET(B)R and VEGFR-3 was required for the effective inhibition of these effects, as well as for VEGFR-3 phosphorylation, demonstrating that ET(B)R cross talk with VEGFR-3 enhances cell plasticity and motility. Finally, in melanoma xenografts, ET(B)R antagonist inhibited tumor growth and the activation of the VEGF-C/VEGFR-3 axis, indicating that targeting ET(B)R may improve melanoma treatment acting directly or indirectly by impairing ET(B)R cross talk with VEGFR-3.

The contribution of the endothelin-1 (ET-1)/ET A receptor (ETAR) axis in tumor growth and progression is investigated in many tumor types, including ovarian carcinoma. In ovarian cancer cells, ET-1 acts as an autocrine growth factor selectively through the ETAR triggering the concomitant activation of multiple pathways. In these cells, the involvement of beta-arrestin-1 as signal transducer in ET-1-dependent signalling pathways has been recently highlighted. Because several G protein-coupled receptors have been shown to activate signalling pathways in a beta-arrestin-dependent manner, in this study we explored whether beta-arrestin-1 is involved in a distinct signalling mechanism linking the ETAR to phosphoinositide 3-kinase (PI3K)/integrin-linked kinase (ILK)/Akt in HEY ovarian cancer cells. The inhibitory effects of ZD4054 (zibotentan), a specific ETAR antagonist, in ET-1-dependent phosphorylation of ILK, Akt, and glycogen synthase kinase (GSK-3beta) demonstrated the involvement of the ETAR in these effects. By using a kinase assay, we demonstrate that beta-arrestin-1 silencing inhibits the ET-1-induced ILK activity in a time-dependent manner and downstream Akt and GSK-3beta phosphorylation. These results reveal that beta-arrestin-1 is implicated as an ETAR-transducer in the activation of ILK and Akt and in the inactivation of GSK-3beta in response to ET-1 and further support the role of beta-arrestin-1 as a multifunctional adaptor facilitating interprotein interactions critically involved in ETAR-mediated signalling that regulate invasive and metastatic behaviour of ovarian cancer.