The activation of endothelin-A receptor (ETAR) by endothelin-1 (ET-1) has a critical role in ovarian tumorigenesis and progression. To define the molecular mechanism in ET-1-induced tumor invasion and metastasis, we focused on ␤-arrestins as scaffold and signaling proteins of G protein-coupled receptors. Here, we demonstrate that, in ovarian cancer cells, ␤-arrestin is recruited to ETAR to form two trimeric complexes: one through the interaction with Src leading to epithelial growth factor receptor (EGFR) transactivation and ␤-catenin Tyr phosphorylation, and the second through the physical association with axin, contributing to release and inactivation of glycogen synthase kinase (GSK)-3␤ and ␤-catenin stabilization. The engagement of ␤-arrestin in these two signaling complexes concurs to activate ␤-catenin signaling pathways. We then demonstrate that silencing of both ␤-arrestin-1 and ␤-arrestin-2 inhibits ETAR-driven signaling, causing suppression of Src, mitogen-activated protein kinase (MAPK), AKT activation, as well as EGFR transactivation and a complete inhibition of ET-1-induced ␤-catenin/TCF transcriptional activity and cell invasion. ETAR blockade with the specific ETAR antagonist ZD4054 abrogates the engagement of ␤-arrestin in the interplay between ETAR and the ␤-catenin pathway in the invasive program. Finally, ETAR is expressed in 85% of human ovarian cancers and is preferentially co-expressed with ␤-arrestin-1 in the advanced tumors. In a xenograft model of ovarian metastasis, HEY cancer cells expressing ␤-arrestin-1 mutant metastasize at a reduced rate, highlighting the importance of this molecule in promoting metastases. ZD4054 treatment significantly inhibits metastases, suggesting that specific ETAR antagonists, by disabling multiple signaling activated by ETAR/␤-arrestin, may represent new therapeutic opportunities for ovarian cancer.beta-arrestin ͉ beta-catenin ͉ endothelin A receptor ͉ metastasis ͉ ovarian cancer

Purpose: Emerging evidence suggests molecular and phenotypic association between chemoresistance and epithelial-mesenchymal transition (EMT) in cancer. Endothelin-1 (ET-1)/endothelin A receptor (ET A R) axis is implicated in the pathobiology of epithelial ovarian cancer (EOC) by driving tumorpromoting effects, including EMT. Here, we analyzed how ET A R regulates chemoresistance and EMT in EOC.Experimental Design: The effects of ET-1 axis on cell proliferation, drug-induced apoptosis, invasiveness, and EMT were analyzed in cultured EOC cells sensitive and resistant to cisplatinum and taxol. Tumor growth in response to ET A R antagonist was examined in EOC xenografts. ET A R expression was examined in 60 human EOC tumors by immunohistochemistry and correlated with chemoresistance and EMT.Results: In resistant EOC cells ET-1 and ET A R are upregulated, paralleled by enhanced mitogen activated protein kinase (MAPK) and Akt phosphorylation and cell proliferation. Moreover, in these cells the expression of E-cadherin transcriptional repressors, including Snail, Slug, and Twist, as well as of mesenchymal markers, such as vimentin and N-cadherin, were upregulated and linked with enhanced invasive behavior. Interestingly, ET A R blockade with zibotentan, a specific ET A R antagonist, or its silencing, downregulated Snail activity, restored drug sensitivity to cytotoxic-induced apoptosis, and inhibited the invasiveness of resistant cells. In vivo, zibotentan inhibited tumor growth of sensitive and resistant EOC xenografts, and sensitized to chemotherapy. Analysis of EOC human tissues revealed that ET A R is overexpressed in resistant tumors and is associated with EMT phenotype.Conclusions: Our data provide the first evidence that blockade of ET A R-driven EMT can overcome chemoresistance and inhibit tumor progression, improving the outcome of EOC patients' treatment. Clin Cancer Res; 17(8); 2350-60. Ó2011 AACR.

The high mortality of epithelial ovarian cancer (EOC) is mainly caused by resistance to the available therapies. In EOC, the endothelin-1 (ET-1, EDN1)-endothelin A receptor (ET A R, EDNRA) signaling axis regulates the epithelial-mesenchymal transition (EMT) and a chemoresistant phenotype. However, there is a paucity of knowledge about how ET-1 mediates drug resistance. Here, we define a novel bypass mechanism through which ET A R/b-arrestin-1 (b-arr1, ARRB1) links Wnt signaling to acquire chemoresistant and EMT phenotype. We found that ET A R/b-arr1 activity promoted nuclear complex with b-catenin and p300, resulting in histone acetylation, chromatin reorganization, and enhanced transcription of genes, such as ET-1, enhancing the network that sustains chemoresistance. Silencing of b-arr1 or pharmacologic treatment with the dual ET A R/ET B R antagonist macitentan prevented core complex formation and restored drug sensitivity, impairing the signaling pathways involved in cell survival, EMT, and invasion. In vivo macitentan treatment reduced tumor growth, vascularization, intravasation, and metastatic progression. The combination of macitentan and cisplatinum resulted in the potentiation of the cytotoxic effect, indicating that macitentan can enhance sensitivity to chemotherapy. Investigations in clinical specimens of chemoresistant EOC tissues confirmed increased recruitment of b-arr1 and b-catenin to ET-1 gene promoter. In these tissues, high expression of ET A R significantly associated with poor clinical outcome and chemoresistance. Collectively, our findings reveal the existence of a novel mechanism by which ET A R/b-arr1 signaling is integrated with the Wnt/b-catenin pathway to sustain chemoresistance in EOC, and they offer a solid rationale for clinical evaluation of macitentan in combination with chemotherapy to overcome chemoresistance in this setting. Cancer Res; 74(24); 7453-64. Ó2014 AACR.

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 β-arrestin-1 (β-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 β-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 β-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 β-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 β-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 β-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.

Despite the fundamental pathophysiological importance of b-catenin in tumor progression, the mechanism underlying its final transcriptional output has been partially elucidated. Here, we report that b-arrestin-1 (b-arr1) is an epigenetic regulator of endothelin (ET)-1-induced b-catenin signaling in epithelial ovarian cancer (EOC). In response to ET A receptor (ET A R) activation by ET-1, b-arr1 increases its nuclear translocation and direct binding to b-catenin. This in turn enhanced b-catenin nuclear accumulation and transcriptional activity, which was prevented by expressing a mutant b-arr1 incapable of nuclear distribution. b-arr1-b-catenin interaction controls b-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 b-arr1 silencing or by mutant b-arr1, as well as by b-catenin or p300 silencing, confirming that nuclear b-arr1 forms a functional complex capable of regulating epigenetic changes in b-catenindriven invasive behavior. In a murine orthotopic model of metastatic human EOC, silencing of b-arr1 or mutant b-arr1 expression, as well as ET A R blockade, inhibits metastasis. In human EOC tissues, b-arr1-b-catenin nuclear complexes are selectively enriched at b-catenin target gene promoters, correlating with tumor grade, confirming a direct in vivo b-arr1-b-catenin association at specific set of genes involved in EOC progression. Collectively, our study provides insights into how a b-arr1-mediated epigenetic mechanism controls b-catenin activity, unraveling new components required for its nuclear function in promoting metastasis.Oncogene ( 1,2 b-arrs, consisting of b-arr1 and b-arr2, are involved in G-protein-coupled receptor signaling, and guide the receptor signals also in malignant cells. [3][4][5][6][7][8][9][10][11][12] A global proteomics analysis of b-arr1-interacting proteins demonstrated that b-arr1 interactions take place not only in the cytoplasm, but also in the nucleus, 13 suggesting that b-arr1 has a role in transcriptional regulation in different human cells.14-16 Previous studies indicate that under d-or k-opioid receptor stimulations, nuclear b-arr1 specifically accumulates at p27 and c-fos promoters through scaffolding cyclic adenosine monophosphate responseelement (CRE) binding protein and histone acetyltransferase p300, thus enhancing local histone acetylation and gene transcription.