Activation of autocrine and paracrine signalling by endothelin 1 (ET1) binding to its receptors elicits pleiotropic effects on tumour cells and on the host microenvironment. This activation modulates cell proliferation, apoptosis, migration, epithelial-to-mesenchymal transition, chemoresistance and neovascularization, thus providing a strong rationale for targeting ET1 receptors in cancer. In this Review, we discuss the advances in our understanding of the diverse biological roles of ET1 in cancer and describe the latest preclinical and clinical progress that has been made using small-molecule antagonists of ET1 receptors that inhibit ET1-driven signalling.

The connexins are the protein subunits of the gap junction intercellular channels. In the present study a new rat connexin was cloned by degenerate reverse transcription-polymerase chain reaction and its gene isolated from a mouse genomic library. The nucleotide sequence encodes a protein of 321 amino acids (called Cx36) with highly significant homology to the members of the connexin family. In situ hybridization analysis of rat brain and retina showed the strongest expression in neurons of the inferior olive, the olfactory bulb, the CA3/CA4 hippocampal subfields and several brain-stem nuclei. An intense expression was also found in the pineal gland and in the retinal ganglion cell and inner nuclear layers. Experiments with neurotoxins, locally injected in the hippocampus or specifically acting on inferior olivary neurons, confirmed the neuronal localization of Cx36. It is the first connexin to be expressed predominantly in mammalian neurons and its identification paves the way for a molecular approach in the study of the role played by gap junctions in the physiology and the pathology of the mammalian brain.

No specific funding was obtained for this study. None of the authors have any competing interests to declare.

Despite considerable efforts to improve early detection and advances in chemotherapy, metastatic relapses remain a major challenge in the management of ovarian cancer. The endothelin A receptor (ET A R)/endothelin-1 (ET-1) axis has been shown to have a significant role in ovarian carcinoma by promoting tumorigenesis. Here we show that the ET-1/ET A R autocrine pathway drives epithelial-to-mesenchymal transition (EMT) in ovarian tumor cells by inducing a fibroblastoid and invasive phenotype, down-regulation of E-cadherin, increased levels of B-catenin, Snail, and other mesenchymal markers, and suppression of E-cadherin promoter activity. Activation of ET A R by ET-1 triggers an integrin-linked kinase (ILK)-mediated signaling pathway leading to glycogen synthase kinase-3B (GSK-3B) inhibition, Snail and B-catenin stabilization, and regulation of transcriptional programs that control EMT. Transfection of dominant negative ILK or exposure to an ILK inhibitor suppresses the ET-1-induced phosphorylation of GSK-3B as well as Snail and B-catenin protein stability, activity, and invasiveness, indicating that ET-1/ ET A R-induced EMT-promoting effects depend on ILK. ET A R blockade by specific antagonists or reduction by ET A R RNA interference reverses EMT and cell invasion by inhibiting autocrine signaling pathways. In ovarian carcinoma xenografts, ABT-627, a specific ET A R antagonist, suppresses EMT determinants and tumor growth. In human ovarian cancers, ET A R expression is associated with E-cadherin downregulation, N-cadherin expression, and tumor grade. Collectively, these findings provide evidence of a critical role for the ET-1/ET A R axis during distinct steps of ovarian carcinoma progression and identify novel targets of therapeutic intervention. (Cancer Res 2005; 65(24): 11649-57)

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.

Angiogenesis is an essential prerequisite for tumor growth, invasion, and metastasis. In ovarian carcinoma cells, endothelin-1 (ET-1) stimulates the secretion of vascular endothelial growth factor (VEGF), a major mediator of tumor angiogenesis. In OVCA 433 and HEY ovarian carcinoma cell lines, ET-1 treatment increases VEGF mRNA expression and induces VEGF protein levels in a time-and dose-dependent fashion, and do so to a greater extent under hypoxic conditions. ET-1 also increases hypoxia-inducible factor-1␣ (HIF-1␣) accumulation and activates the HIF-1 transcription complex under both normoxic and hypoxic conditions, suggesting a role for HIF-1 in the induction of VEGF expression. These effects are inhibited by the selective ET A receptor (ET A R) antagonist, BQ123. The ET-1-induced increase in HIF-1␣ protein levels is due to the enhanced HIF-1␣ stabilization. These results implicate HIF-1␣ in the induction of VEGF expression in ET-1-stimulated ovarian carcinoma cells, and provide a mechanism whereby ET-1 acting selectively through ET A R can interact with the HIF-1␣-dependent machinery of angiogenesis. Our results suggest that new therapeutic strategies using specific ET A R antagonists could provide an additional approach to the treatment of ovarian carcinoma by inhibiting neovascularization as well as tumor cell growth.

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