The ß-arrestin1/endothelin axis bolsters ovarian fibroblast-dependent invadosome activity and cancer cell metastatic potential.

Recruitment of fibroblasts to tumors and their activation into cancer-associated fibroblasts (CAFs) is a strategy used by tumor cells to direct extracellular matrix (ECM) remodeling, invasion, and metastasis, highlighting the need to investigate the molecular mechanisms driving CAF function. Endothelin-1 (ET-1) regulates the communication between cancer and stroma and facilitates the progression of serous ovarian cancer (SOC). By binding to Endothelin A (ETA) and B (ETB) receptors, ET-1 enables the recruitment of ß-arrestin1 (ß-arr1) and the formation of signaling complexes that coordinate tumor progression. However, how ET-1 receptors might "educate" human ovarian fibroblasts (HOFs) to produce altered ECM and promote metastasis remains to be elucidated. This study identifies ET-1 as a pivotal factor in the activation of CAFs capable of proteolytic ECM remodeling and the generation of heterotypic spheroids containing cancer cells with a propensity to metastasize. An autocrine/paracrine ET-1/ETA/BR/ß-arr1 loop enhances HOF proliferation, upregulates CAF marker expression, secretes pro-inflammatory cytokines, and increases collagen contractility, and cell motility. Furthermore, ET-1 facilitates ECM remodeling by promoting the lytic activity of invadosome and activation of integrin ß1. In addition, ET-1 signaling supports the formation of heterotypic HOF/SOC spheroids with enhanced ability to migrate through the mesothelial monolayer, and invade, representing metastatic units. The blockade of ETA/BR or ß-arr1 silencing prevents CAF activation, invadosome function, mesothelial clearance, and the invasive ability of heterotypic spheroids. In vivo, therapeutic inhibition of ETA/BR using bosentan (BOS) significantly reduces the metastatic potential of combined HOFs/SOC cells, associated with enhanced apoptotic effects on tumor cells and stromal components. These findings support a model in which ET-1/ß-arr1 reinforces tumor/stroma interaction through CAF activation and fosters the survival and metastatic properties of SOC cells, which could be counteracted by ETA/BR antagonists.

A diagnostic circulating miRNA signature as orchestrator of cell invasion via TKS4/TKS5/EFHD2 modulation in human gliomas.

BACKGROUND: Altered microRNA profiles have been observed not only in tumour tissues but also in biofluids, where they circulate in a stable form thus representing interesting biomarker candidates. This study aimed to identify a microRNA signature as a non-invasive biomarker and to investigate its impact on glioma biology. METHODS: MicroRNAs were selected using a global expression profile in preoperative serum samples from 37 glioma patients. Comparison between serum samples from age and gender-matched controls was performed by using the droplet digital PCR. The ROC curve and Kaplan-Meier survival analyses were used to evaluate the diagnostic/prognostic values. The functional role of the identified signature was assessed by gain/loss of function strategies in glioma cells. RESULTS: A three-microRNA signature (miR-1-3p/-26a-1-3p/-487b-3p) was differentially expressed in the serum of patients according to the isocitrate dehydrogenase (IDH) genes mutation status and correlated with both patient Overall and Progression Free Survival. The identified signature was also downregulated in the serum of patients compared to controls. Consistent with these results, the signature expression and release in the conditioned medium of glioma cells was lower in IDH-wild type cells compared to the mutated counterpart. Furthermore, in silico analysis of glioma datasets showed a consistent deregulation of the signature according to the IDH mutation status in glioma tumour tissues. Ectopic expression of the signature negatively affects several glioma functions. Notably, it impacts the glioma invasive phenotype by directly targeting the invadopodia-related proteins TKS4, TKS5 and EFHD2. CONCLUSIONS: We identified a three microRNA signature as a promising complementary or even an independent non-invasive diagnostic/prognostic biomarker. The signature displays oncosuppressive functions in glioma cells and impacts on proteins crucial for migration and invasion, providing potential targets for therapeutic intervention.

The interaction of ß-arrestin1 with talin1 driven by endothelin A receptor as a feature of α5ß1 integrin activation in high-grade serous ovarian cancer.

Dissemination of high-grade serous ovarian cancer (HG-SOC) in the omentum and intercalation into a mesothelial cell (MC) monolayer depends on functional α5ß1 integrin (Intα5ß1) activity. Although the binding of Intα5ß1 to fibronectin drives these processes, other molecular mechanisms linked to integrin inside-out signaling might support metastatic dissemination. Here, we report a novel interactive signaling that contributes to Intα5ß1 activation and accelerates tumor cells toward invasive disease, involving the protein ß-arrestin1 (ß-arr1) and the activation of the endothelin A receptor (ETAR) by endothelin-1 (ET-1). As demonstrated in primary HG-SOC cells and SOC cell lines, ET-1 increased Intß1 and downstream FAK/paxillin activation. Mechanistically, ß-arr1 directly interacts with talin1 and Intß1, promoting talin1 phosphorylation and its recruitment to Intß1, thus fueling integrin inside-out activation. In 3D spheroids and organotypic models mimicking the omentum, ETAR/ß-arr1-driven Intα5ß1 signaling promotes the survival of cell clusters, with mesothelium-intercalation capacity and invasive behavior. The treatment with the antagonist of ETAR, Ambrisentan (AMB), and of Intα5ß1, ATN161, inhibits ET-1-driven Intα5ß1 activity in vitro, and tumor cell adhesion and spreading to intraperitoneal organs and Intß1 activity in vivo. As a prognostic factor, high EDNRA/ITGB1 expression correlates with poor HG-SOC clinical outcomes. These findings highlight a new role of ETAR/ß-arr1 operating an inside-out integrin activation to modulate the metastatic process and suggest that in the new integrin-targeting programs might be considered that ETAR/ß-arr1 regulates Intα5ß1 functional pathway.

SEMA6A/RhoA/YAP axis mediates tumor-stroma interactions and prevents response to dual BRAF/MEK inhibition in BRAF-mutant melanoma.

BACKGROUND: Despite the promise of dual BRAF/MEK inhibition as a therapy for BRAF-mutant (BRAF-mut) melanoma, heterogeneous responses have been observed in patients, thus predictors of benefit from therapy are needed. We have previously identified semaphorin 6A (SEMA6A) as a BRAF-mut-associated protein involved in actin cytoskeleton remodeling. The purpose of the present study is to dissect the role of SEMA6A in the biology of BRAF-mut melanoma, and to explore its predictive potential towards dual BRAF/MEK inhibition. METHODS: SEMA6A expression was assessed by immunohistochemistry in melanoma cohort RECI1 (N = 112) and its prognostic potential was investigated in BRAF-mut melanoma patients from DFCI and TCGA datasets (N = 258). The molecular mechanisms regulated by SEMA6A to sustain tumor aggressiveness and targeted therapy resistance were investigated in vitro by using BRAF-mut and BRAF-wt melanoma cell lines, an inducible SEMA6A silencing cell model and a microenvironment-mimicking fibroblasts-coculturing model. Finally, SEMA6A prediction of benefit from dual BRAF/MEK inhibition was investigated in melanoma cohort RECI2 (N = 14). RESULTS: Our results indicate higher protein expression of SEMA6A in BRAF-mut compared with BRAF-wt melanoma patients and show that SEMA6A is a prognostic indicator in BRAF-mut melanoma from TCGA and DFCI patients cohorts. In BRAF-mut melanoma cells, SEMA6A coordinates actin cytoskeleton remodeling by the RhoA-dependent activation of YAP and dual BRAF/MEK inhibition by dabrafenib+trametinib induces SEMA6A/RhoA/YAP axis. In microenvironment-mimicking co-culture condition, fibroblasts confer to melanoma cells a proliferative stimulus and protect them from targeted therapies, whereas SEMA6A depletion rescues the efficacy of dual BRAF/MEK inhibition. Finally, in BRAF-mut melanoma patients treated with dabrafenib+trametinib, high SEMA6A predicts shorter recurrence-free interval. CONCLUSIONS: Overall, our results indicate that SEMA6A contributes to microenvironment-coordinated evasion of melanoma cells from dual BRAF/MEK inhibition and it might be a good candidate predictor of short-term benefit from dual BRAF/MEK inhibition.

Ovarian Cancer-Driven Mesothelial-to-Mesenchymal Transition is Triggered by the Endothelin-1/ß-arr1 Axis.

Transcoelomic spread of serous ovarian cancer (SOC) results from the cooperative interactions between cancer and host components. Tumor-derived factors might allow the conversion of mesothelial cells (MCs) into tumor-associated MCs, providing a favorable environment for SOC cell dissemination. However, factors and molecular mechanisms involved in this process are largely unexplored. Here we investigated the tumor-related endothelin-1 (ET-1) as an inducer of changes in MCs supporting SOC progression. Here, we report a significant production of ET-1 from MCs associated with the expression of its cognate receptors, ETA and ETB, along with the protein ß-arrestin1. ET-1 triggers MC proliferation via ß-arrestin1-dependent MAPK and NF-kB pathways and increases the release of cancer-related factors. The ETA/ETB receptor activation supports the genetic reprogramming of mesothelial-to-mesenchymal transition (MMT), with upregulation of mesenchymal markers, as fibronectin, α-SMA, N-cadherin and vimentin, NF-kB-dependent Snail transcriptional activity and downregulation of E-cadherin and ZO-1, allowing to enhanced MC migration and invasion, and SOC transmesothelial migration. These effects are impaired by either blockade of ETAR and ETBR or by ß-arrestin1 silencing. Notably, in peritoneal metastases both ETAR and ETBR are co-expressed with MMT markers compared to normal control peritoneum. Collectively, our report shows that the ET-1 axis may contribute to the early stage of SOC progression by modulating MC pro-metastatic behaviour via MMT.

Endothelin-1 drives invadopodia and interaction with mesothelial cells through ILK.

Cancer cells use actin-based membrane protrusions, invadopodia, to degrade stroma and invade. In serous ovarian cancer (SOC), the endothelin A receptor (ETAR) drives invadopodia by a not fully explored coordinated function of ß-arrestin1 (ß-arr1). Here, we report that ß-arr1 links the integrin-linked kinase (ILK)/ßPIX complex to activate Rac3 GTPase, acting as a central node in the adhesion-based extracellular matrix (ECM) sensing and degradation. Downstream, Rac3 phosphorylates PAK1 and cofilin and promotes invadopodium-dependent ECM proteolysis and invasion. Furthermore, ETAR/ILK/Rac3 signaling supports the communication between cancer and mesothelial cells, favoring SOC cell adhesion and transmigration. In vivo, ambrisentan, an ETAR antagonist, inhibits the adhesion and spreading of tumor cells to intraperitoneal organs, and invadopodium marker expression. As prognostic factors, high EDNRA/ILK expression correlates with poor SOC clinical outcome. These findings provide a framework for the ET-1R/ß-arr1 pathway as an integrator of ILK/Rac3-dependent adhesive and proteolytic signaling to invadopodia, favoring cancer/stroma interactions and metastatic behavior.

Tumor Cellular and Microenvironmental Cues Controlling Invadopodia Formation.

During the metastatic progression, invading cells might achieve degradation and subsequent invasion into the extracellular matrix (ECM) and the underlying vasculature using invadopodia, F-actin-based and force-supporting protrusive membrane structures, operating focalized proteolysis. Their formation is a dynamic process requiring the combined and synergistic activity of ECM-modifying proteins with cellular receptors, and the interplay with factors from the tumor microenvironment (TME). Significant advances have been made in understanding how invadopodia are assembled and how they progress in degradative protrusions, as well as their disassembly, and the cooperation between cellular signals and ECM conditions governing invadopodia formation and activity, holding promise to translation into the identification of molecular targets for therapeutic interventions. These findings have revealed the existence of biochemical and mechanical interactions not only between the actin cores of invadopodia and specific intracellular structures, including the cell nucleus, the microtubular network, and vesicular trafficking players, but also with elements of the TME, such as stromal cells, ECM components, mechanical forces, and metabolic conditions. These interactions reflect the complexity and intricate regulation of invadopodia and suggest that many aspects of their formation and function remain to be determined. In this review, we will provide a brief description of invadopodia and tackle the most recent findings on their regulation by cellular signaling as well as by inputs from the TME. The identification and interplay between these inputs will offer a deeper mechanistic understanding of cell invasion during the metastatic process and will help the development of more effective therapeutic strategies.

Regulation of extracellular matrix degradation and metastatic spread by IQGAP1 through endothelin-1 receptor signalling in ovarian cancer.

The invasive phenotype of serous ovarian cancer (SOC) cells is linked to the formation of actin-based protrusions, invadopodia, operating extracellular matrix (ECM) degradation and metastatic spread. Growth factor receptors might cause engagement of integrin-related proteins, like the polarity protein IQ-domain GTPase-activating protein 1 (IQGAP1), to F-actin core needed for invadopodia functions. Here, we investigated whether IQGAP1 forms a signalosome with endothelin-1 (ET-1)/ß-arrestin1 (ß-arr1) network, as signal-integrating module for adhesion components, cytoskeletal remodelling and ECM degradation. In SOC cells, ET-1 receptor (ET-1R) activation, besides altering IQGAP1 expression and localization, coordinates the binding of IQGAP1 with ß-arr1, representing a "hotspot" for ET-1R-induced invasive signalling. We demonstrated that the molecular interaction of IQGAP1 with ß-arr1 affects relocalization of focal adhesion components, as vinculin, and cytoskeleton dynamics, through the regulation of invadopodia-related pathways. In particular, ET-1R deactivates Rac1 thereby promoting RhoA/C activation for the correct functions of invasive structures. Silencing of either IQGAP1 or ß-arr1, or blocking ET-1R activation with a dual antagonist macitentan, prevents matrix metalloproteinase (MMP) activity, invadopodial function, transendothelial migration and cell invasion. In vivo, targeting ET-1R/ß-arr1 signalling controls the process of SOC metastasis, associated with reduced levels of IQGAP1, as well as other invadopodia effectors, such as vinculin, phospho-cortactin and membrane type 1-MMP. High expression of ETAR/ß-arr1/IQGAP1 positively correlates with poor prognosis, validating the clinical implication of this signature in early prognosis of SOC. These data establish the ET-1R-driven ß-arr1/IQGAP1 interaction as a prerequisite for the dynamic integration of pathways in fostering invadopodia and metastatic process in human SOC.

hMENA is a key regulator in endothelin-1/ß-arrestin1-induced invadopodial function and metastatic process.

Aberrant activation of endothelin-1 receptors (ET-1R) elicits pleiotropic effects relevant for tumor progression. The network activated by this receptor might be finely, spatially, and temporarily orchestrated by ß-arrestin1 (ß-arr1)-driven interactome. Here, we identify hMENA, a member of the actin-regulatory protein ENA/VASP family, as an interacting partner of ß-arr1, necessary for invadopodial function downstream of ET-1R in serous ovarian cancer (SOC) progression. ET-1R activation by ET-1 up-regulates expression of hMENA/hMENAΔv6 isoforms through ß-arr1, restricted to mesenchymal-like invasive SOC cells. The interaction of ß-arr1 with hMENA/hMENAΔv6 triggered by ET-1 leads to activation of RhoC and cortactin, recruitment of membrane type 1-matrix metalloprotease, and invadopodia maturation, thereby enhancing cell plasticity, transendothelial migration, and the resulting spread of invasive cells. The treatment with the ET-1R antagonist macitentan impairs the interaction of ß-arr1 with hMENA and inhibits invadopodial maturation and tumor dissemination in SOC orthotopic xenografts. Finally, high ETAR/hMENA/ß-arr1 gene expression signature is associated with a poor prognosis in SOC patients. These data define a pivotal function of hMENA/hMENAΔv6 for ET-1/ß-arr1-induced invadopodial activity and ovarian cancer progression.

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.

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.

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.