Targeting tumor-stroma communication by blocking endothelin-1 receptors sensitizes high-grade serous ovarian cancer to PARP inhibition.

PARP inhibitors (PARPi) have changed the treatment paradigm of high-grade serous ovarian cancer (HG-SOC). However, the impact of this class of inhibitors in HG-SOC patients with a high rate of TP53 mutations is limited, highlighting the need to develop combinatorial therapeutic strategies to improve responses to PARPi. Here, we unveil how the endothelin-1/ET-1 receptor (ET-1/ET-1R) axis, which is overexpressed in human HG-SOC and associated with poor prognosis, instructs HG-SOC/tumor microenvironment (TME) communication via key pro-malignant factors and restricts the DNA damage response induced by the PARPi olaparib. Mechanistically, the ET-1 axis promotes the p53/YAP/hypoxia inducible factor-1α (HIF-1α) transcription hub connecting HG-SOC cells, endothelial cells and activated fibroblasts, hence fueling persistent DNA damage signal escape. The ET-1R antagonist macitentan, which dismantles the ET-1R-mediated p53/YAP/HIF-1α network, interferes with HG-SOC/stroma interactions that blunt PARPi efficacy. Pharmacological ET-1R inhibition by macitentan in orthotopic HG-SOC patient-derived xenografts synergizes with olaparib to suppress metastatic progression, enhancing PARPi survival benefit. These findings reveal ET-1R as a mechanistic determinant in the regulation of HG-SOC/TME crosstalk and DNA damage response, indicating the use of macitentan in combinatorial treatments with PARPi as a promising and emerging therapy.

Functional interaction between endothelin-1 and ZEB1/YAP signaling regulates cellular plasticity and metastasis in high-grade serous ovarian cancer.

BACKGROUND: Epithelial-to-mesenchymal transition (EMT) encompasses a highly dynamic and complex key process which leads to metastatic progression. In high-grade serous ovarian carcinoma (HG-SOC), endothelin-1 (ET-1)/endothelin A receptor (ETAR) signaling promotes EMT driving tumor progression. However, the complex nature of intertwined regulatory circuits activated by ET-1 to trigger the metastatic process is not fully elucidated. METHODS: The capacity of ET-1 pathway to guide a critical transcriptional network that is instrumental for metastatic growth was identified in patient-derived HG-SOC cells and cell lines through immunoblotting, q-RT-PCR, co-immunoprecipitation, in situ proximity ligation, luciferase reporter, chromatin immunoprecipitation assays and publicly available databases. Functional assays in HG-SOC cells and HG-SOC xenografts served to test the inhibitory effects of ET-1 receptors (ET-1R) antagonist in vitro and in vivo. RESULTS: We demonstrated that ET-1/ETAR axis promoted the direct physical ZEB1/YAP interaction by inducing their nuclear accumulation in HG-SOC cells. Moreover, ET-1 directed their engagement in a functional transcriptional complex with the potent oncogenic AP-1 factor JUN. This led to the aberrant activation of common target genes, including EDN1 (ET-1) gene, thereby creating a feed-forward loop that sustained a persistent ET-1/ZEB1 signaling activity. Notably, ET-1-induced Integrin-linked kinase (ILK) signaling mediated the activation of YAP/ZEB1 circuit driving cellular plasticity, invasion and EMT. Of therapeutic interest, treatment of HG-SOC cells with the FDA approved ET-1R antagonist macitentan, targeting YAP and ZEB1-driven signaling, suppressed metastasis in vivo in mice. High gene expression of ETAR/ILK/YAP/AP-1/ZEB1 was a strong predictor of poor clinical outcome in serous ovarian cancer patients, indicating the translational relevance of this signature expression. CONCLUSIONS: This study provides novel mechanistic insights of the ET-1R-driven mediators that support the ability of HG-SOC to acquire metastatic traits which include the cooperation of YAP and ZEB1 regulatory circuit paving the way for innovative treatment of metastatic ovarian cancer.

Endothelin-1 axis fosters YAP-induced chemotherapy escape in ovarian cancer.

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, ß-arrestin1 (ß-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 ETAR/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.

ß-arrestin1/YAP/mutant p53 complexes orchestrate the endothelin A receptor signaling in high-grade serous ovarian cancer.

The limited clinical response observed in high-grade serous ovarian cancer (HG-SOC) with high frequency of TP53 mutations (mutp53) might be related to mutp53-driven oncogenic pathway network. Here we show that ß-arrestin1 (ß-arr1), interacts with YAP, triggering its cytoplasmic-nuclear shuttling. This interaction allows ß-arr1 to recruit mutp53 to the YAP-TEAD transcriptional complex upon activation of endothelin-1 receptors (ET-1R) in patient-derived HG-SOC cells and in cell lines bearing mutp53. In parallel, ß-arr1 mediates the ET-1R-induced Trio/RhoA-dependent YAP nuclear accumulation. In the nucleus, ET-1 through ß-arr1 orchestrates the tethering of YAP and mutp53 to YAP/mutp53 target gene promoters, including EDN1 that ensures persistent signals. Treatment of patient-derived xenografts reveals synergistic antitumoral and antimetastatic effects of the dual ET-1R antagonist macitentan in combination with cisplatinum, shutting-down the ß-arr1-mediated YAP/mutp53 transcriptional programme. Furthermore, ETAR/ß-arr1/YAP gene signature correlates with a worst prognosis in HG-SOC. These findings support effective combinatorial treatment for repurposing the ET-1R antagonists in HG-SOC.

Targeting endothelin-1 receptor/ß-arrestin1 network for the treatment of ovarian cancer.

INTRODUCTION: Endothelin-1 receptor (ET-1R)/ß-arrestin1 (ß-arr1) signaling is dysregulated in ovarian cancer. This signaling circuit enables cancer cells to engage several signaling and transcriptional networks that are pervasively intertwined, and represent a potential therapeutic target for developing novel agents for ovarian cancer treatment. Areas covered: In this article, we discuss the role of the signaling network between ET-1R and key pathways mediated by the scaffold protein ß-arr1, as part of signaling complex, or as a transcription co-activator, promoting precise control of transcription of different genes, including ET-1. Therefore ET-1R/ß-arr1 is an actionable node involved in the activation of a persistent feedback loop that contributes to bypass signaling. Targeting ET-1R empowering this circuit can represent a necessary measure to reach clinical efficacy. Preclinical studies demonstrate that blocking ET-1R by FDA approved dual ETAR/ETBR antagonist prevents ß-arr1 network formation, offering a novel therapeutic strategy in ovarian cancer patients. Expert opinion: The information provided in this review about the ET-1R/ß-arr1 hub represents an invaluable tool for both identifying the interconnected pathways involved in ovarian cancer and targeting them more effectively. The new perspective arising from ET-1R therapeutics will likely prompt a valuable frame for the design of new promising combinatorial therapy, blocking compensatory networks.

Blocking endothelin-1-receptor/ß-catenin circuit sensitizes to chemotherapy in colorectal cancer.

The limited clinical response to conventional chemotherapeutics observed in colorectal cancer (CRC) may be related to the connections between the hyperactivated ß-catenin signaling and other pathways in CRC stem-like cells (CRC-SC). Here, we show the mechanistic link between the endothelin-1 (ET-1)/ET-1 receptor (ET-1R) signaling and ß-catenin pathway through the specific interaction with the signal transducer ß-arrestin1 (ß-arr1), which initiates signaling cascades as part of the signaling complex. Using a panel of patient-derived CRC-SC, we show that these cells secrete ET-1 and express ETAR and ß-arr1, and that the activation of ETAR/ß-arr1 axis promotes the cross-talk with ß-catenin signaling to sustain stemness, epithelial-to-mesenchymal transition (EMT) phenotype and response to chemotherapy. Upon ETAR activation, ß-arr1 acts as a transcription co-activator that binds ß-catenin, thereby promoting nuclear complex with ß-catenin/TFC4 and p300 and histone acetylation, inducing chromatin reorganization on target genes, such as ET-1. The enhanced transcription of ET-1 increases the self-sustained ET-1/ß-catenin network. All these findings provide a strong rationale for targeting ET-1R to hamper downstream ß-catenin/ET-1 autocrine circuit. Interestingly, treatment with macitentan, a dual ETAR and ETBR antagonist, able to interfere with tumor and microenvironment, disrupts the ET-1R/ß-arr1-ß-catenin interaction impairing pathways involved in cell survival, EMT, invasion, and enhancing sensitivity to oxaliplatin (OX) and 5-fluorouracil (5-FU). In CRC-SC xenografts, the combination of macitentan and OX or 5-FU enhances the therapeutic effects of cytotoxic drugs. Together, these results provide mechanistic insight into how ET-1R coopts ß-catenin signaling and offer a novel therapeutic strategy to manage CRC based on the combination of macitentan and chemotherapy that might benefit patients whose tumors show high ETAR and ß-catenin expression.

Nuclear ß-arrestin1 is a critical cofactor of hypoxia-inducible factor-1α signaling in endothelin-1-induced ovarian tumor progression.

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 ß-arrestin1 (ß-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 ß-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, ß-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 ß-arr1 or HIF-1α silencing or by pharmacological treatment with the dual ET-1 receptor antagonist macitentan. Interestingly, ET(A)R/ß-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 ß-arr1, inhibits intravasation and metastasis formation. Collectively, these findings reveal the interplay of ß-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 ß-arr1/HIF-1α pathway by using macitentan may impair EOC progression.

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

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.

Endothelin-1/endothelin A receptor axis activates RhoA GTPase in epithelial ovarian cancer.

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 ß-arrestin-1 (ß-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 ß-arr1 silencing, suggesting that ET-1/ETAR regulate RhoA signaling through ß-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 ß-arr1 to sustain RhoA/ROCK signaling in response to ETAR activation in EOC.

miR-30a inhibits endothelin A receptor and chemoresistance in ovarian carcinoma.

Drug resistance remains the major clinical barrier to successful treatment in epithelial ovarian carcinoma (EOC) patients, and the evidence of microRNA involvement in drug resistance has been recently emerging. Endothelin-1 (ET-1)/ETA receptor (ETAR) axis is aberrantly activated in chemoresistant EOC cells and elicits pleiotropic effects promoting epithelial-to-mesenchymal transition (EMT) and the acquisition of chemoresistance. However, the relationship between ETAR and miRNA is still unknown. Hence, in this study we evaluated whether dysregulation of miRNA might enhance ETAR expression in sensitive and resistant EOC cells. Based on bioinformatic tools, we selected putative miRNA able to recognize the 3'UTR of ETAR. An inverse correlation was observed between the expression levels of miR-30a and ETAR in both EOC cell lines and tumor samples. miR-30a was found to specifically bind to the 3'UTR of ETAR mRNA, indicating that ETAR is a direct target of miR-30a. Overexpression of miR-30a decreased Akt and mitogen activated protein kinase signaling pathway activation, cell proliferation, invasion, plasticity, EMT marker levels, and vascular endothelial growth factor release. Interestingly, ectopic expression of miR-30a re-sensitized platinum-resistant EOC cells to cisplatinum-induced apoptosis. Consistently, resistant EOC xenografts overexpressing miR-30a resulted in significantly less tumor growth than controls. Together our study provides a new perspective on the regulatory mechanism of ETAR gene. Interestingly, our findings highlight that blockade of ETAR regulatory axis is the mechanism underlying the tumor suppressor function of miR-30a in chemoresistant EOC cells.

Endothelin A receptor/ß-arrestin signaling to the Wnt pathway renders ovarian cancer cells resistant to chemotherapy.

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 (ETAR, 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 ETAR/ß-arrestin-1 (ß-arr1, ARRB1) links Wnt signaling to acquire chemoresistant and EMT phenotype. We found that ETAR/ß-arr1 activity promoted nuclear complex with ß-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 ß-arr1 or pharmacologic treatment with the dual ETAR/ETBR 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 ß-arr1 and ß-catenin to ET-1 gene promoter. In these tissues, high expression of ETAR significantly associated with poor clinical outcome and chemoresistance. Collectively, our findings reveal the existence of a novel mechanism by which ETAR/ß-arr1 signaling is integrated with the Wnt/ß-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.

Endothelin-1 regulates hypoxia-inducible factor-1α and -2α stability through prolyl hydroxylase domain 2 inhibition in human lymphatic endothelial cells.

AIMS: Lymphangiogenesis, the formation of new lymphatic vessels, is thought to constitute a route for the tumor cells to metastasize. We previously demonstrated that endothelin-1 (ET-1) induces the expression of lymphangiogenic factors through hypoxia-inducible factor (HIF)-1α and HIF-2α. The stability of these transcriptional factors is essential for lymph/angiogenesis and tumor progression. Here we analyze the molecular mechanism through which ET-1 regulates HIF-1α and HIF-2α protein levels and how these transcriptional factors are implicated in controlling lymphatic endothelial cell (LEC) behavior. MAIN METHODS: Using Western blotting assay and a reporter gene containing the HIF-1α oxygen-dependent degradation domain we monitored the capacity of ET-1 to increase HIF-1α and HIF-2α stability and nuclear accumulation. In addition, using siRNA against HIF-1α or HIF-2α, we investigated the implication of these transcriptional factors in ET-1-mediated tube-like structure formation. As HIF-1α proteosomal degradation is controlled by site-specific hydroxylation carried out by HIF-prolyl hydroxylase domain (PHD) enzymes, we analyzed the expression of PHD-2 isoform. KEY FINDINGS: We show that ET-1 through its receptor, ETBR, controls HIF-α stability and nuclear accumulation by inhibiting prolyl hydroxylation and reduces PHD2 mRNA and protein levels. Transfection with HIF-1α or HIF-2α siRNA abrogated the capacity of ET-1 to induce tube-like structure formation. SIGNIFICANCE: These results reveal a PHD2-mediated mechanism through which ET-1 stabilizes HIF-1α and HIF-2α pathway thereby regulating LEC behavior and lymphangiogenesis.

ß-Arrestin 1 is required for endothelin-1-induced NF-κB activation in ovarian cancer cells.

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 ß-catenin, through specific cytosolic and nuclear scaffolding functions of ß-arrestin 1 (ß-arr1). Here, we have assessed the potential role of ET-1/ETAR in promoting NF-κB signalling in EOC cells through ß-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 ß-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 ß-arr1 was achieved by ß-arr1 silencing with shRNA and expression of ß-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 ß-arr-1 silencing, suggesting that ET-1 through ETAR promotes the recruitment of ß-arr1 to regulate NF-κB signalling. Moreover, the nuclear physical interaction between p65 and ß-arr1 indicates a nuclear function of ß-arr-1 in ETAR-driven NF-κB transcriptional activity. SIGNIFICANCE: Altogether these findings reveal a previously unrecognized pathway that depends on ß-arr1 to sustain NF-κB signalling in response to ETAR activation in ovarian cancer.

The interplay between hypoxia, endothelial and melanoma cells regulates vascularization and cell motility through endothelin-1 and vascular endothelial growth factor.

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.

Endothelin-1 induces the transactivation of vascular endothelial growth factor receptor-3 and modulates cell migration and vasculogenic mimicry in melanoma cells.

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 ß-arrestin-1 reduced ET-1-induced VEGFR-3 phosphorylation, demonstrating that, upon ET-1 stimulus, ß-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.

Endothelin-1 cooperates with hypoxia to induce vascular-like structures through vascular endothelial growth factor-C, -D and -A in lymphatic endothelial cells.

AIMS: Lymphangiogenesis refers to the formation of new lymphatic vessels and is thought to constitute conduits for the tumor cells to metastasize. We previously demonstrated that endothelin (ET)-1 through its binding with ETB receptor (ET(B)R) expressed on lymphatic endothelial cells (LEC), induced cell growth and invasiveness. Since vascular endothelial growth factor (VEGF)-A/-C/-D, and hypoxia play key role in lymphatic differentiation, in this study we investigated the involvement of these growth factors and hypoxia in ET-1-induced lymphangiogenesis. MAIN METHODS: Real time PCR and ELISA were used to quantify VEGF-A/-C/-D. LEC morphological differentiation was analyzed by tube formation assay on Matrigel. KEY FINDINGS: Hypoxia, as well as ET-1, induced an increase in VEGF-A/-C and -D expression that was reduced in the presence of a selective ET(B)R antagonist, BQ788, and enhanced when ET-1 was administered under hypoxic conditions. We analyzed the role of hypoxia on LEC morphological differentiation, and found that hypoxia increased the formation of vascular-like structures on Matrigel and that in combination with ET-1 this effect was markedly enhanced. The use of specific antibodies neutralizing VEGF-A, or recombinant VEGFR-3/(Flt-4)/Fc that block VEGF-C/-D, inhibited the effect of ET-1 as well that of hypoxia. SIGNIFICANCE: These results demonstrated that ET-1 and hypoxia act, at list in part, through VEGF to induce lymphangiogenic events and that these two stimuli may cooperate to induce VEGF-A/-C/-D expression and lymphatic differentiation. These data further support the role of ET-1 as potent lymphangiogenic factor that relies on the interplay with hypoxic microenvironment and with VEGF family members.

The endothelin A receptor and epidermal growth factor receptor signaling converge on ß-catenin to promote ovarian cancer metastasis.

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 ß-catenin signalling. Here, we evaluated ß-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 ß-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 ß-catenin at the residue Y654, its dissociation from E-cadherin complexes and the accumulation as an active form. This pool of Tyr-ß-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 ß-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.

Acquisition of chemoresistance and EMT phenotype is linked with activation of the endothelin A receptor pathway in ovarian carcinoma cells.

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 tumor-promoting 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.

Endothelin axis induces metalloproteinase activation and invasiveness in human lymphatic endothelial cells.

The molecular mechanisms involved in lymphangiogenesis were unknown until recently. We previously demonstrated that the endothelin-1 (ET-1) axis stimulates lymphatic endothelial cells (LEC) and lymphatic vessels to grow and invade. Here we further investigated the effect of ET-1 on lymphatic vessels and evaluated whether ET-1 actions result in the functional activation of lymphangiogenesis. Using highly purified human LEC, characterized for the expression of ET-1 axis members by quantitative real-time PCR, we found that the endothelin B receptor (ETB), upon activation by ET-1, induced matrix-metalloproteinase activation, demonstrating that ET-1 influenced the activity of the proteolytic enzymes required for LEC invasion. Functional assays performed by using intradermal lymphangiography demonstrated that ET-1 promoted the formation of lymphatic vessels and that these vessels were capable of lymphatic flow. ETB blockade with the specific antagonist BQ788 inhibited matrix-metalloproteinase activation and dye transport within the lymphatic vessels, demonstrating that ETB is involved in the regulation of the growth of and in the formation of functional vessels upon activation by ET-1. Our results suggest that ET-1 is a lymphangiogenic mediator and that targeting pharmacologically ETB may be therapeutically exploited in a variety of diseases, including cancer.

Beta-arrestin-1 mediates the endothelin-1-induced activation of Akt and integrin-linked kinase.

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.