Specific Endothelin A Receptor Antagonist Research Articles

Macitentan blocks endothelin-1 receptor activation required for chemoresistant ovarian cancer cell plasticity and metastasis

AimsIn 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 methodsThe 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 findingsWe 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. SignificanceOur 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.

Abstract 707: Acquisition of chemoresistance and epithelial to mesenchymal phenotype is linked with activation of the endothelin A receptor pathway in ovarian carcinoma cells

Abstract Understanding the molecular mechanisms underlying chemoresistance is of utmost importance for improving the treatment of epithelial ovarian cancer (EOC). Emerging evidence suggests molecular and phenotypic association between chemoresistance and epithelial-mesenchymal transition (EMT) in cancer cells. Endothelin-1 (ET-1) and the endothelin A receptor (ETAR) axis is implicated in the pathobiology of EOC by driving different tumor-promoting effects, including EMT. By employing two cisplatin- and taxol-resistant ovarian cancer cell lines, we demonstrated that in the resistant EOC cells ET-1 and ETAR are upregulated, at both mRNA and protein level, paralleled by enhanced MAPK and AKT phosphorylation and cell proliferation. To assess whether the chemoresistance in A2780 cells was associated with EMT molecular changes, we examined the expression of epithelial and mesenchymal markers. Enhanced Snail, Slug, Twist, N-cadherin and vimentin mRNA expression was observed in resistant cells, associated with a concomitant decrease in E-cadherin expression and promoter activity. Moreover, ET-1 induced a significant induction of Snail promoter activity in resistant cells, and the E-cadherin promoter sequences were detected bound to Snail in a time-dependent manner, as demonstrated by chromatin immunoprecipitation assays, suggesting that the ET-1-dependent and sustained binding of Snail in the E-cadherin promoter might account for EMT and chemoresistant phenotype of these cells. Moreover, the invasiveness and proteinase activity of chemoresistant cells was greater, compared with sensitive cells. Interestingly, ETAR blockade with the specific ETAR antagonist, zibotentan, or its silencing with siRNA, reverted EMT, restored drug sensitivity to cytotoxic-induced apoptosis, and inhibited the invasive potential of resistant cells. In vivo, zibotentan inhibited tumor growth of sensitive and resistant EOC xenografts, and sensitized to chemotherapy. Finally, analysis of EOC tissues, with different responses to chemotherapy, revealed that ETAR is overexpressed in resistant tumors and is associated with EMT marker expression. All these data indicate that blockade of ETAR-driven EMT can overcome chemoresistance in EOC, improving the outcome of EOC patients’ treatment. Supported by AIRC Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 707. doi:10.1158/1538-7445.AM2011-707

Abstract 698: β-arrestin-1 as nuclear signalling element essential for endothelin A receptor-induced epithelial to mesenchymal transition and chemoresistance

Abstract The onset of chemoresistance is the major challenge in epithelial ovarian cancer (EOC). Emerging evidence suggests molecular and phenotypic association between chemoresistance and epithelial-mesenchymal transition (EMT) in cancer. We recently demonstrated that β-arrestin-1, scaffold and signaling protein of G-protein-coupled receptors, is recruited to endothelin A receptor (ETAR) to form molecular signaling complexes which concur to activate β-catenin transcriptional activity, EMT and metastasis. Here, by using biochemical and imaging techniques, we provide evidence that in EOC cells, ETAR activation by endothelin-1 (ET-1) induces nuclear translocation of β-arrestin-1 and its association with β-catenin. ChIP assays demonstrated that following this translocation, β-arrestin-1 was selectively enriched at specific promoters of β-catenin target genes, such as ET-1, MMP-2, cyclin D1. Further experiments showed that ET-1 promotes the nuclear association between histone acetyltransferase p300 and β-arrestin and the recruitment of p300 on these promoters, resulting in H3 and H4 histone acetylation and enhanced gene transcription. In chemoresistant EOC cells, ET-1/ETAR axis is overexpressed associated with EMT marker expression. Most importantly, the transcriptional activity of β-catenin was upregulated in chemoresistant EOC cells, associated with enhanced ETAR and β-arrestin-1 expression, providing molecular evidence that the ETAR/β-arrestin-1-mediated EMT signaling in EOC can represent a “salvage pathway” occurring during chemoresistance development. Interestingly, ETAR blockade with zibotentan, a specific ETAR antagonist, or its silencing with siRNA, abrogates both the engagement of β-arrestin-1 in the nucleus and the interplay between ET-1 and the β-catenin in controlling gene transcription. In vivo, zibotentan inhibited tumor growth of sensitive and resistant EOC xenografts, associated with reduction of EMT markers, and sensitized to chemotherapy. Analysis of EOC human tissues revealed that ETAR is overexpressed in resistant tumors and is associated with EMT phenotype. Our data provide the first evidence that blockade of ETAR/β-arrestin-1-driven EMT can overcome chemoresistance and inhibit tumor progression, improving the outcome of EOC patients’ treatment. Supported by AIRC Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 698. doi:10.1158/1538-7445.AM2011-698

Acquisition of Chemoresistance and EMT Phenotype Is Linked with Activation of the Endothelin A Receptor Pathway in Ovarian Carcinoma Cells

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 tumor-promoting effects, including EMT. Here, we analyzed how ET(A)R regulates chemoresistance and EMT in EOC. 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. 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. 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.

Phase III study of the efficacy and safety of zibotentan (ZD4054) in patients with bone metastatic castration-resistant prostate cancer (CRPC).

117 Background: Endothelin-1 and the endothelin A (ETA) receptor have been implicated in prostate cancer progression in bone. Zibotentan, a specific ETA receptor antagonist, had a promising signal for prolonged overall survival (OS) in a phase II study of patients with CRPC and bone metastases who were pain free or mildly symptomatic for pain. The aim of this phase III study was to confirm the efficacy and safety of zibotentan in a similar but larger population. Methods: Patients with CRPC and bone metastases were randomized 1:1 to zibotentan 10 mg/day po or placebo, plus standard of care including chemotherapy. The primary endpoint was OS. Secondary endpoints included times to pain progression, chemotherapy use, new bone metastases, and safety. Efficacy endpoints were analyzed using a log-rank test. At least 263 deaths were required for formal analysis. If the true hazard ratio (HR) for zibotentan versus placebo was 0.67, the analysis would have 90% power to demonstrate a statistically significant effect in OS at the 5% level. Results: A total of 594 patients were randomized (299 to zibotentan; 295 to placebo). Baseline group demographics were similar. Mean age was ∼71 yrs, and 64% were Caucasian. Although median OS was longer in zibotentan-treated patients than those receiving placebo (median 24.5 vs 22.5 months), the difference did not reach significance (HR [95.2% confidence interval]; 0.87 [0.69–1.10]: P=0.240). No significant differences were observed for any secondary endpoints. The most commonly reported AEs in the zibotentan group, peripheral edema and headache, were consistent with the pharmacologic action of zibotentan as a vasodilator. Cardiac failure events, actively solicited following a phase II signal, were higher in the zibotentan group (any grade, 5.7%; Common Terminology Criteria for Adverse Events [CTCAE] grade ≥3, 3.0%) than placebo (any grade, 1.7%; CTCAE grade ≥3, 1.0%), but were manageable and reversible. Conclusions: In this placebo-controlled phase III trial treatment with zibotentan 10 mg/day did not lead to a significant improvement in OS in patients with CRPC and bone metastases. Zibotentan had an acceptable safety profile. [Table: see text]

Combination therapy of zibotentan with cisplatinum and paclitaxel is an effective regimen for epithelial ovarian cancerThis article is one of a selection of papers published in the two-part special issue entitled 20 Years of Endothelin Research.

In human ovarian carcinoma, the endothelin-1 (ET-1) / endothelin A receptor (ETAR) axis is overexpressed, correlating with tumor grade. Moreover, ETAR activation by ET-1 affects cell proliferation, survival, angiogenesis, and invasion. ETAR blockade with zibotentan (ZD4054), a specific ETAR antagonist, significantly inhibits ovarian cancer growth in vitro and in vivo, underscoring the relevance of this pathway as a target for cancer therapy. Since clinical trial results have defined the combination of platinum and taxane as the standard of care in the management of ovarian cancer, here we explored the therapeutic efficacy of the integration of zibotentan with cytotoxic drugs having different modes of action. We found that the combination of zibotentan with cisplatinum as well as zibotentan with paclitaxel was more effective at inhibiting ovarian cancer HEY cell proliferation induced by endogenous ET-1 than were the single agents alone. However, a significantly enhanced efficacy was observed when we combined zibotentan, cisplatinum, and paclitaxel. Accordingly, in HEY xenografts the coadministration of zibotentan with cisplatinum enhanced the efficacy of the cytotoxic drug alone in controlling tumor growth, associated with reduction in proliferation index and microvessel density. Remarkably, the combination of zibotentan with both cisplatinum and paclitaxel was very effective in inhibiting tumor growth, neovascularization, and cell proliferation, representing a preclinical endpoint to guide combination therapy in clinical trials.

Abstract 2551: Acquisition of epithelial-mesenchymal transition phenotype of chemoresistant ovarian cancer cells is linked with activation of the endothelin signaling pathway

Abstract Chemoresistance is a major challenge in ovarian cancer (OC). High-throughput analysis has shown that the endothelin A receptor (ETAR) is overexpressed in post-chemotherapy OCs, suggesting a role in the chemoresistance onset. To identify ETAR-driven molecular determinants involved in chemoresistance to cisplatin and paclitaxel, we used the A2780 WT, cisplatin-resistant A2780 CIS, and taxol-resistant A2780 TAX human OC cells. ET-1 and ETAR mRNA and protein expression as well as ET-1 secretion were higher in the resistant OC cells, associated with increased ET-1-induced MAPK and AKT activation and cell proliferation. These effects were reverted by treatment with zibotentan (ZD4054), a specific ETAR antagonist. In sensitive OC cells, zibotentan enhanced the number of early apoptotic cells, similar to cisplatinum. Furthermore, zibotentan sensitized resistant A2780 CIS cells to cisplatinum-induced apoptosis, confirming the role of ET-1 as survival factor. Given the documented role of the epithelial to mesenchymal transition (EMT) in the chemoresistance development and the ability of the ET-1/ETAR axis to regulate EMT effectors, such as Snail and β-catenin, we analyzed whether ETAR-driven EMT molecular changes were linked with the chemoresistance in EOC cells. Decreased expression of the epithelial adhesion molecule E-cadherin and an increased expression of mesenchymal markers, such as Snail and Slug, the principal transcriptional repressor of E-cadherin, as well as Snail promoter activity were observed in sensitive OC cells after ET-1 treatment. Moreover, these effects were upregulated in resistant OC cells and were blocked in the presence of zibotentan, providing molecular evidence that the ETAR-mediated EMT signaling in EOC can represent a “salvage pathway” occurring during chemoresistance development. In sensitive A2780, as well as in resistant A2780 CIS and TAX xenografts, zibotentan induced a significant tumor growth inhibition, associated with reduction of Snail and upregulation of E-cadherin, suggesting a specific therapeutic window in which combination therapy of zibotentan with cytotoxic drugs is effective also in resistant OC. Finally immunohistochemical analysis of human OC, with different responses to chemotherapy, showed that ET-1 and ETAR are overexpressed in the resistant tumors, associated with upregulation of E-cadherin and with downregulation of N-cadherin and β-catenin. In conclusion, our study suggest that ETAR blockade with the specific antagonist zibotentan can revert EMT and increase the sensitivity to chemotherapeutic agents, representing a potential strategy to overcome chemoresistance in OC. Supported by AIRC, Ministero della Salute, AstraZeneca. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2551.

Abstract 2363: Positive inter-regulation between β-catenin and endothelin signaling in ovarian cancer cells: Epigenetic regulation of gene transcription

Abstract Although endothelin-1 (ET-1) and the endothelin A receptor (ETAR) regulate different steps of ovarian tumor progression, the molecular mechanism controlling the expression of ET-1 in this tumor is unknown. We previously demonstrated that ET-1 activates β-catenin/TCF4 transcriptional activity to promote cell invasion and metastasis. Here, we show that ET-1 gene is directly regulated by β-catenin in ovarian cancer cells. Thus, inhibition of β-catenin signalling results in lowered ET-1 promoter activity and expression, while enhanced β-catenin signalling leads to further activation of this gene. Chromatin immunoprecipitation (ChIP) demonstrated that, after ET-1 stimulus, β-catenin and its cognate DNA binding partner, TCF4, are recruited on the specific DNA element within the ET-1 promoter. Unravelling the role of the scaffold protein β-arrestin-1 as nucleus chaperone controlling β-catenin transcription activity, we demonstrated that β-arrestin-1 is required for the activation of ET-1 promoter, and for the recruitment of β-catenin/TCF4 on this promoter. In agreement with these findings, immunoprecipitation assay demonstrated that ET-1 promotes the interaction between endogenous β-catenin and β-arrestin-1 both in the cytoplasm and in the nucleus. Moreover, β-arrestin-1 siRNA leads to the loss of ET-1 mRNA expression and ET-1 secretion, as well as a significant inhibition of ET-1 promoter activity, thus suggesting the critical role of β-arrestin-1 in the β-catenin-dependent ET-1 gene expression. Further experiments showed that ET-1 promotes the nuclear association between histone acetyltransferase p300 and β-arrestin and the recruitment of p300 on the ET-1 promoter, resulting in H3 and H4 histone acetylation and enhanced ET-1 transcription. Besides ET-1, β-arrestin-1 represents a platform for achieving signal specificity that converges on β-catenin-mediated transcription of defined genes, such as cyclin D1 and matrix metalloprotease (MMP)-2, but not MMP-9. Moreover, ETAR blockade with the specific ETAR antagonist, zibontentan (ZD4054), abrogates both the engagement of β-arrestin and the interplay between ET-1 and the β-catenin in controlling gene transcription. In an intraperitoneal model of ovarian cancer metastasis, HEY cells expressing the mutant β-arrestin-1, or treatment with zibontentan, significantly reduced peritoneal tumor-cell dissemination and β-catenin expression, highlighting the importance of β-arrestin-mediated signaling in metastasis formation. Altogether these results reveal a positive inter-regulation between β-catenin and ET-1 that amplify the ET-1/ETAR autocrine loop in ovarian cancer cells, in which β-arrestin-1 acts as a nuclear messenger mediating epigenetic mechanism in β-catenin-mediated ET-1 transcription. Supported by AIRC, AstraZeneca Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2363.

ETAR antagonist ZD4054 exhibits additive effects with aromatase inhibitors and fulvestrant in breast cancer therapy, and improves in vivo efficacy of anastrozole

Endothelin-1 (ET-1) and endothelin A receptor (ETAR) contribute to the development and progression of breast carcinomas by modulating cell proliferation, angiogenesis, and anti-apoptosis. We investigated antitumoral effects of the specific ETAR antagonist ZD4054 in breast cancer cells and xenografts, and assessed antitumoral efficacy of the combinations of ZD4054 with aromatase inhibitors and fulvestrant. Gene expression changes were assessed by quantitative real-time PCR. Cell proliferation was measured using alamarBlue; migration and invasion assays were performed using modified Boyden chambers. Evaluating the antitumoral efficacy of ZD4054 in vivo, different breast cancer models were employed using nude mice xenografts. ZD4054 reduced ET-1 and ETAR expression in MCF-7, MDA-MB-231, and MDA-MB-468 breast cancer cells in a concentration-dependent manner. ZD4054 inhibited invasion by up to 37.1% (P = 0.022). Combinations of ZD4054 with either anastrozole or letrozole produced significant reductions in migration of aromatase-overexpressing MCF-7aro cells (P < 0.05). Combination of ZD4054 with fulvestrant reduced MCF-7 cell migration and invasion by 36.0% (P = 0.027) and 56.7% (P < 0.001), respectively, with effects significantly exceeding those seen with either compound alone. Regarding tumor volume reduction in vivo, ZD4054 (10 mg/kg) was equipotent to fulvestrant (200 mg/kg) and exhibited additive effects with anastrozole (0.5 mg/kg). These data are the first indicating that selective ETAR antagonism by ZD4054 displays antitumoral activity on breast cancer cells in vitro and in vivo. Our data strongly support a rationale for the clinical use of ZD4054 in combination with endocrine therapies.

A phase I study of zibotentan (ZD4054) in patients with metastatic, castrate-resistant prostate cancer

To assess the maximum well-tolerated dose (MWTD), dose limiting toxicity (DLT), pharmacokinetics (PK) and pharmacodynamics of zibotentan, a novel specific endothelin-A receptor antagonist, in patients with metastatic prostate cancer. Patients with metastatic, castrate-resistant prostate cancer (CRPC) were treated with escalating doses of oral zibotentan (ZD4054) 10-200 mg once daily. The initial cohort received 28 daily doses (Period 1). Patients who had evidence of clinical benefit and who had not met any of the criteria for withdrawal were allowed to receive zibotentan at their current dose level until they no longer derived clinical benefit (Period 2). PK of zibotentan and changes in prostate-specific antigen and bone markers were also assessed. Sixteen patients were evaluable for the safety and single-dose PK analyses. Eleven patients completed Period 1, and nine patients proceeded to Period 2. DLTs were encountered at 22.5 mg; one patient had grade 3 dyspnea and peripheral edema and a second patient had grade 3 headache and intraventricular hemorrhage. Enrollment was expanded at the 15 mg dose level to further determine the safety and tolerability of zibotentan. No DLTs were seen at 15 mg, and the most frequent adverse events were headache, peripheral edema, fatigue, nasal congestion and nausea. The MWTD for zibotentan was 15 mg orally daily. The predominant adverse events observed were consistent with those reported for this class of drugs, and prolonged stable disease was noted in some patients. Phase III studies with zibotentan in men with metastatic CRPC are ongoing.

New endothelin-A receptor antagonist prolongs survival

Original article James, N. D. et al. Safety and efficacy of the specific endothelin-A receptor antagonist ZD4054 in patients with hormone-resistant prostate cancer and bone metastases who were pain free or mildly symptomatic: a double-blind, placebo-controlled, randomised, phase 2 trial. Eur. Urol. 55, 1112–1123 (2009). PubMed

In vitro metabolism of the specific endothelin-A receptor antagonist ZD4054 and clinical drug interactions between ZD4054 and rifampicin or itraconazole in healthy male volunteers

ZD4054 is an oral specific endothelin-A receptor antagonist in development for the treatment of hormone-resistant prostate cancer. Both renal and metabolic processes contribute to its overall clearance.Two preclinical in vitro studies investigated the metabolism of ZD4054 using human liver microsomes, individual cytochrome P450 (CYP) isozymes, and flavin-containing monooxygenase isoforms. Two Phase I open-label crossover volunteer studies subsequently investigated in vivo drug interactions between ZD4054 and the CYP450 inducer rifampicin or CYP3A4 inhibitor itraconazole.The most abundant metabolite produced in in vitro incubations accounted for 12.8% of radioactivity after ZD4054 was incubated with CYP3A4. No significant flavin-containing monooxygenase metabolism of ZD4054 was observed. In the in vivo studies, rifampicin co-administration reduced the area under the concentration–time curve and maximum plasma concentration of ZD4054 by 68% and 29%, respectively, whilst co-administration with itraconazole was associated with an increase in ZD4054 area under the curve of approximately 28%.While co-administration of CYP450 inducers might be associated with reduced efficacy of ZD4054, dose reduction is unlikely to be required with concomitant administration of CYP3A4 inhibitors.

β-Arrestin links endothelin A receptor to β-catenin signaling to induce ovarian cancer cell invasion and metastasis

The activation of endothelin-A receptor (ET(A)R) 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 beta-arrestins as scaffold and signaling proteins of G protein-coupled receptors. Here, we demonstrate that, in ovarian cancer cells, beta-arrestin is recruited to ET(A)R to form two trimeric complexes: one through the interaction with Src leading to epithelial growth factor receptor (EGFR) transactivation and beta-catenin Tyr phosphorylation, and the second through the physical association with axin, contributing to release and inactivation of glycogen synthase kinase (GSK)-3beta and beta-catenin stabilization. The engagement of beta-arrestin in these two signaling complexes concurs to activate beta-catenin signaling pathways. We then demonstrate that silencing of both beta-arrestin-1 and beta-arrestin-2 inhibits ET(A)R-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 beta-catenin/TCF transcriptional activity and cell invasion. ET(A)R blockade with the specific ET(A)R antagonist ZD4054 abrogates the engagement of beta-arrestin in the interplay between ET(A)R and the beta-catenin pathway in the invasive program. Finally, ET(A)R is expressed in 85% of human ovarian cancers and is preferentially co-expressed with beta-arrestin-1 in the advanced tumors. In a xenograft model of ovarian metastasis, HEY cancer cells expressing beta-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 ET(A)R antagonists, by disabling multiple signaling activated by ET(A)R/beta-arrestin, may represent new therapeutic opportunities for ovarian cancer.

Preclinical anticancer activity of the specific endothelin A receptor antagonist ZD4054

Endothelins are a family of small peptides (ET-1, ET-2, and ET-3) that mediate various physiological processes of mitogenesis, repair, and tissue differentiation by binding to endothelin A (ETA) and endothelin B (ETB) cell surface receptors. Activation of the ETA receptor by ET-1 has emerged as an important factor promoting tumor cell proliferation, survival, angiogenesis, migration, invasion, and metastasis in several tumor types including prostate, ovary, colon, cervix, breast, and lung. As activation of the ETB receptor has an opposing effect, inducing cell death by apoptosis, a rationale exists for specific antagonism of the ETA receptor as a treatment strategy for cancer. ZD4054 is a specific ETA receptor antagonist currently being evaluated in hormone-resistant prostate cancer in phase III clinical trials. In vitro, ZD4054 reversed ET-1-mediated inhibition of apoptosis in serum-deprived rat A10 and human VLTR-16 cells in a concentration-dependent manner. ZD4054 inhibited ET-1-mediated survival signaling pathways and decreased proliferation in ovarian OVCA 433 and HEY cells and in prostate PPC-1 and LAPC-4 cells. In A673 rhabdomyosarcoma cells, ET-1-induced phosphorylation of FAK, FAK, and paxillin was reversed with ZD4054, inhibiting the invasive phenotype mediated by these adhesion factors. In vivo, ZD4054 led to a significant reduction in tumor growth in animals bearing ovarian tumor xenografts, and significantly inhibited tumor angiogenesis. Pretreatment with ZD4054 also significantly delayed the onset of metastatic events after intracardiac injection of bladder TSU-Pr1-B1 cells in mice. These preclinical data show the potential anticancer effects of the specific blockade of the ETA receptor with ZD4054, supporting a program of clinical investigation.

Safety and Efficacy of the Specific Endothelin-A Receptor Antagonist ZD4054 in Patients with Hormone-Resistant Prostate Cancer and Bone Metastases Who Were Pain Free or Mildly Symptomatic: A Double-Blind, Placebo-Controlled, Randomised, Phase 2 Trial

BackgroundThe endothelin-A receptor (ETAR) has been implicated in the progression of prostate cancer. ObjectivesTo investigate the safety and efficacy of the specific ETAR antagonist ZD4054 in patients with metastatic hormone-resistant prostate cancer (HRPC). Design, setting, and participantsDouble-blind, placebo-controlled, randomised, parallel-group, multicentre, phase 2 trial in patients attending cancer centres with HRPC and bone metastases who were pain free or mildly symptomatic for pain. InterventionPatients were randomised to receive once-daily oral tablets of ZD4054 10mg, or ZD4054 15mg, or placebo. MeasurementsThe primary end point was time to progression, defined as clinical progression, requirement for opiate analgesia, objective progression of soft-tissue metastases, or death in the absence of progression. Secondary end points included overall survival, time to prostate-specific antigen (PSA) progression, and safety. Statistical significance was preset at 20%. Results and limitationsA total of 312 patients were randomised (ZD4054 10mg, n=107; ZD4054 15mg, n=98; placebo, n=107). At the primary analysis, median time to progression was 3.6 mo, 4.0 mo, and 3.8 mo in the placebo, ZD4054 10mg, and ZD4054 15mg groups, respectively, with no statistically significant difference between ZD4054 groups and placebo (hazard ratio [HR] vs placebo for the ZD4054 10mg group: 0.88 [80% CI: 0.71–1.09]; HR vs placebo for the ZD4054 15mg group: 0.83 [80% CI: 0.66–1.03]). However, a signal for prolonged overall survival was observed in the ZD4054 treatment groups versus placebo, based on 40 deaths. At a subsequent analysis after 118 deaths, this survival benefit was confirmed (HR vs placebo for the ZD4054 10mg group, 0.55 [80% CI: 0.41–0.73], p=0.008; HR vs placebo for the ZD4054 15mg group, 0.65 [80% CI: 0.49–0.86], p=0.052) but the differences in time to progression remained nonsignificant. Median overall survival was 17.3 mo, 24.5 mo, and 23.5 mo in the placebo group, the ZD4054 10mg group, and the ZD4054 15mg group, respectively. Discordance between results for time to progression and overall survival may be due to the sensitivity of the definition of progression. Adverse events were in line with the expected pharmacologic effects of an ETAR antagonist. ConclusionsThe primary end point of time to progression was not achieved in this study, but an improvement was seen in overall survival in both active treatment arms. ZD4054 was well tolerated. Trial registrationClinicaltrials.gov NCT00090363.

Integrin-linked kinase functions as a downstream mediator of endothelin-1 to promote invasive behavior in ovarian carcinoma

The endothelin-1 (ET-1) axis represents a novel target in several malignancies, including ovarian carcinoma. Upon being activated, the endothelin A receptor (ET(A)R) mediates multiple tumor-promoting activities, including mitogenesis, escape from apoptosis, angiogenesis, metastasis-related protease activation, epithelial-mesenchymal transition, and invasion. Integrin-linked kinase (ILK) is a multidomain focal adhesion protein that conveys intracellular signaling elicited by beta1-integrin and growth factor receptors. In this study, we investigate whether the signaling triggered by ET(A)R leading to an aggressive phenotype is mediated by an ILK-dependent mechanism. In HEY and OVCA 433 ovarian carcinoma cell lines, activation of ET(A)R by ET-1 enhances the expression of alpha2beta1 and alpha3beta1 integrins. ILK activity increases as ovarian cancer cells adhere to type I collagen through beta1 integrin signaling, and do so to a greater extent on ET-1 stimulation. ET-1 increases ILK mRNA and protein expression and activity in a time- and concentration-dependent manner. An ILK small-molecule inhibitor (KP-392) or transfection with a dominant-negative ILK mutant effectively blocks the phosphorylation of downstream signals, Akt and glycogen synthase kinase-3beta. The blockade of ET-1/ET(A)R-induced ILK activity results in an inhibition of matrix metalloproteinase activation as well as of cell motility and invasiveness in a phosphoinositide 3 kinase-dependent manner. In ovarian carcinoma xenografts, ABT-627, a specific ET(A)R antagonist, suppresses ILK expression, Akt and glycogen synthase kinase-3beta phosphorylation, and tumor growth. These data show that ILK functions as a downstream mediator of the ET-1/ET(A)R axis to potentiate aggressive cellular behavior. Thus, the ILK-related signaling cascade can be efficiently targeted by pharmacologic blockade of ET(A)R.

Endothelin-1 Promotes Epithelial-to-Mesenchymal Transition in Human Ovarian Cancer Cells

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 beta-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-3beta (GSK-3beta) inhibition, Snail and beta-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-3beta as well as Snail and beta-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 down-regulation, 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.

Changes of Vasoregulatory Gene Expression Following Hepatic Ischemia/Reperfusion and Treatment with Endothelin-A Receptor Blockade

The objective of this study was to investigate the effect of a specific endothelin-A receptor antagonist on mRNA expression of genes encoding vasoactive mediators and proinflammatory cytokines following complete vascular exclusion of the porcine liver. Fourteen adult German Landrace pigs were subjected to 120 minutes of warm hepatic ischemia by total vascular exclusion. The animals were divided into two groups: the control group received saline solution and the therapy group was given the selective endothelin-A receptor antagonist BSF 208075. Liver tissue samples were collected 1 hour after reperfusion and mRNA expression for preproendothelin-1, prointerleukin-1beta, prointerleukin- 6, pro-tumor necrosis factor-alpha and endothelial nitric oxide synthase was analyzed quantitatively using the TaqMan system. Additionally, immunohistochemical analysis using a semiquantitative score for endothelin-1 and endothelin-A receptor was performed. One hour after reperfusion, quantitative reverse transcriptase-polymerase chain reaction revealed significantly lower expression of preproendothelin-1, pro-tumor necrosis factor-alpha, and prointerleukin-6 in the therapy group compared to controls. Immunohistochemical analysis demonstrated significantly reduced endothelin-1 immunostaining after therapy. Treatment with the selective endothelin-A receptor antagonist exerts a protective effect on the microcirculation after liver ischemia and reperfusion. We were able to show that the endothelin-A receptor antagonist not only has effects on the expression of vasoactive genes, it also decreases gene expression of proinflammatory cytokines such as tumor necrosis factor-alpha and interleukin-6.

Targeting Endothelin Receptor Type A in Human Cervical Carcinoma Cells

Human papilloma viruses are associated with cervical cancer and enhance signal transduction of growth factors. In human papilloma virus-positive cervical cancer cells, the endothelin-A receptor mediates the endothelin-1-induced mitogenic effect, and sustains the basal growth rate of unstimulated cervical tumor cells. In this study, the action of a specific endothelin-A receptor antagonist (atrasentan) and a truly 'balanced' endothelin-A/endothelin-B antagonist (A-182086), was analysed in the human cervical carcinoma cells, CaSki and C33A. CaSki cells are human papilloma virus-16-positive, produce endothelin-1 and possess endothelin-A and endothelin-B receptors, whereas the C33A line is human papilloma virus-negative, does not secrete endothelin-1 and has only endothelin-B receptors. In human papilloma virus-positive cancer cells both antagonists caused a similar drastic reduction in BrdU incorporation and in the growth rate. These data clearly demonstrate that A-182086 and atrasentan show similar potency and also indicate that blocking of the endothelin-B receptor by A- 182086 does not increase the anti-proliferative effect. This finding suggests that the endothelin-B receptor does not participate in the growth control of CaSki cells. Results from C33A cells reinforce this previous conclusion as both compounds are ineffective in altering the BrdU uptake of these cells in basal and endothelin-1 stimulated conditions. In conclusion, targeting the endothelin-A receptor, but not the endothelin-B receptor, may be a valid tool in the therapy of cervical carcinoma.

Endothelin-1 mediates the alcohol-induced reduction of pancreatic capillary blood flow

Increased plasma endothelin-1 (ET-1) levels in rats after alcohol administration increased endothelin receptor expression in the pancreas in chronic alcoholic pancreatitis have led to the hypothesis that ET-1 may play a critical role in the pathogenesis of ethanol-induced pancreatic injury through impairment of perfusion. To further test the hypothesis that ET-1 mediates an alcohol-induced reduction of pancreatic perfusion, the present study compares the effect of intravenous alcohol and ET-1 on pancreatic capillary blood flow (PCBF) and investigates whether endothelin receptor blockade prevents the alcohol-induced reduction in PCBF. Anesthetized rats were randomly assigned to receive one of the following: a 1-hour infusion of 2 g/kg alcohol or the volume equivalent of saline solution plus ET-1 (1.25 μg/kg), a specific endothelin-A receptor antagonist (50 mg/kg), or saline solution (volume equivalent). The pancreas was exposed for intravital microscopy; PCBF was determined at the same location before the test solutions were given, after the infusion, and 1 hour thereafter. Alcohol and ET-1 significantly decreased PCBF from 2.0 nl/min/cap to 1.7 nl/min/cap. The reduction in PCBF was even more pronounced when alcohol and ET-1 were combined (1.5 nl/min/cap), whereas the ET receptor antagonist increased PCBF in saline-treated rats to 2.2 nl/min/cap and maintained stable PCBF in alcohol-treated animals. The observation that PCBF is reduced by both alcohol and ET-1 and that the alcohol-induced reduction of PCBF can be aggravated by ET-1 and prevented by a specific endothelin-1 antagonist supports the hypothesis that ET-1 is the mediator of the alcohol-associated reduction of pancreatic perfusion.