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High-grade serous ovarian cancer (HG-SOC), accounting for 70-80% of ovarian cancer deaths, is characterized by a widespread and rapid metastatic nature, influenced by diverse cell types, cell-cell interactions, and acellular components of the tumour microenvironment (TME). Within this tumour type, autocrine and paracrine activation of the endothelin-1 receptors (ET-1R), expressed in tumour cells and stromal elements, drives metastatic progression. The lack of three-dimensional models that faithfully recapitulate the unique HG-SOC TME has been the bottleneck in performing drug screening for personalized medicine. Herein, we developed HG-SOC tumouroids by engineering a dense central artificial cancer mass (ACM) containing HG-SOC cells, nested within a compressed hydrogel recapitulating the stromal compartment comprising type I collagen, laminin, fibronectin, and stromal cells (fibroblasts and endothelial cells). ET-1-stimulated HG-SOC cells in the tumouroids showed an altered migration pattern and formed cellular aggregates, mimicking micrometastases that invaded the stroma. Compared with control cells, ET-1-stimulated tumouroids showed a higher number of invasive bodies, which were reduced by treatment with the dual ET-1 receptor (ET-1R) antagonist macitentan. In addition, ET-1 increased the size of the invading aggregates compared with control cells. This study establishes an experimental 3D multicellular model eligible for mechanical research, investigating the impact of matrix stiffness and TME interactions, which will aid drug screening to guide therapeutic decisions in HG-SOC patients., (© 2024 The Author(s).)

The high-grade serous ovarian cancer (HG-SOC) is a notoriously challenging disease, characterized by a rapid peritoneal dissemination. HG-SOC cells leverage actin-rich membrane protrusions, known as invadopodia, to degrade the surrounding extracellular matrix (ECM) and invade, initiating the metastatic cascade. In HG-SOC, the endothelin-1 (ET-1)/endothelin A receptor (ETAR)-driven signaling coordinates invadopodia activity, however how this axis integrates pro-oncogenic signaling routes, as YAP-driven one, impacting on the invadopodia-mediated ECM degradation and metastatic progression, deserves a deeper investigation. Herein, we observed that downstream of the ET-1/ET-1R axis, the RhoC and Rac1 GTPases, acting as signaling intermediaries, promote the de-phosphorylation and nuclear accumulation of YAP. Conversely, the treatment with the dual ETA/ETB receptor antagonist, macitentan, inhibits the ET-1-driven YAP activity. Similarly, RhoC silencing, or cell transfection with a dominant inactive form of Rac1, restore the YAP phosphorylated and inhibited state. Mechanistically, the ET-1R/YAP signal alliance coordinates invadopodia maturation into ECM-degrading structures, indicating how such ET-1R-guided protein network represents a route able to enhance the HG-SOC invasive potential. At functional level, we found that the interconnection between the ET-1R/RhoC and YAP signals is required for MMP-2 and MMP-9 proteolytic functions, cell invasion, and cytoskeleton architecture changes, supporting the HG-SOC metastatic strength. In HG-SOC patient-derived xenografts (PDX) macitentan, turning-off the invadopodia regulators RhoC/YAP, halt the metastatic colonization. ET-1R targeting, hindering the YAP activity, weakens the invadopodia machinery, embodying a promising therapeutic avenue to prevent peritoneal dissemination in HG-SOC., (Copyright 2024 The Author(s).)

Hyperthermia is considered a promising strategy to boost the curative outcome of traditional chemotherapeutic treatments. However, this thermally mediated drug delivery is still affected by important limitations. First, the poor accumulation of the conventional anticancer formulations in the target site limits the bioavailability of the active ingredient and induces off-site effects. In addition, some tumoral scenarios, such as ovarian carcinoma, are characterized by cell thermotolerance, which induces tumoral cells to activate self-protecting mechanisms against high temperatures. To overcome these constraints, we developed thermoresponsive nanoparticles (NPs) with an upper critical solution temperature (UCST) to intracellularly deliver a therapeutic payload and release it on demand through hyperthermia stimulation. These NPs were synthesized via reversible addition-fragmentation chain transfer (RAFT) emulsion polymerization and combine polyzwitterionic stabilizing segments and an oligoester-based biodegradable core. By leveraging the pseudo-living nature of RAFT polymerization, important physicochemical properties of the NPs were controlled and optimized, including their cloud point ( T cp ) and size. We have tuned the T cp of NPs to match the therapeutic needs of hyperthermia treatments at 43 °C and tested the nanocarriers in the controlled delivery of paclitaxel, a common anticancer drug. The NPs released almost entirely the encapsulated drug only following 1 h incubation at 43 °C, whereas they retained more than 95% of the payload in the physiological environment (37 °C), thus demonstrating their efficacy as on-demand drug delivery systems. The administration of drug-loaded NPs to ovarian cancer cells led to therapeutic effects outperforming the conventional administration of non-encapsulated paclitaxel, which highlights the potential of the zwitterionic UCST-type NPs as an innovative hyperthermia-responsive drug delivery system.

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 (ET A ) and B (ET B ) receptors, ET-1 enables the recruitment of β-arrestin1 (β-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/ET A/B R/β-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 β1. 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 ET A/B R or β-arr1 silencing prevents CAF activation, invadosome function, mesothelial clearance, and the invasive ability of heterotypic spheroids. In vivo, therapeutic inhibition of ET A/B R 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/β-arr1 reinforces tumor/stroma interaction through CAF activation and fosters the survival and metastatic properties of SOC cells, which could be counteracted by ET A/B R antagonists., (© 2024. The Author(s).)

Staphylococcus aureus is a gram-positive bacterium that may cause intestinal inflammation by secreting enterotoxins, which commonly cause food-poisoning and gastrointestinal injuries. Staphylococcal enterotoxin B (SEB) acts as a superantigen (SAg) by binding in a bivalent manner the T-cell receptor (TCR) and the costimulatory receptor CD28, thus stimulating T cells to produce large amounts of inflammatory cytokines, which may affect intestinal epithelial barrier integrity and functions. However, the role of T cell-mediated SEB inflammatory activity remains unknown. Here we show that inflammatory cytokines produced by T cells following SEB stimulation induce dysfunctions in Caco-2 intestinal epithelial cells by promoting actin cytoskeleton remodelling and epithelial cell-cell junction down-regulation. We also found that SEB-activated inflammatory T cells promote the up-regulation of epithelial-mesenchymal transition transcription factors (EMT-TFs) in a nuclear factor-κB (NF-κB)- and STAT3-dependent manner. Finally, by using a structure-based design approach, we identified a SEB mimetic peptide (pSEB 116-132 ) that, by blocking the binding of SEB to CD28, dampens inflammatory-mediated dysregulation of intestinal epithelial barrier., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision, (Copyright © 2024 Amormino, Russo, Tedeschi, Fiorillo, Paiardini, Spallotta, Rosanò, Tuosto and Kunkl.)

Background: Peritoneal metastasis, which accounts for 85% of all epithelial ovarian carcinoma (EOC) metastases, is a multistep process that requires the establishment of adhesive interactions between cancer cells and the peritoneal membrane. Interrelations between EOC and the mesothelial stroma are critical to facilitate the metastatic process. No data is available so far on the impact of histone acetylation/deacetylation, a potentially relevant mechanism governing EOC metastasis, on mesothelial cells (MCs)-mediated adhesion., Methods: Static adhesion and peritoneal clearance experiments were performed pretreating mesenchymal-like MCs and platinum-sensitive/resistant EOC cell lines with MS-275-a Histone deacetylase (HDAC)1-3 pharmacological inhibitor currently used in combination trials. Results were acquired by confocal microscopy and were analyzed with an automated Opera software. The role of HDAC1/2 was validated by genetic silencing. The role of α4-, α5-α1 Integrins and Fibronectin-1 was validated using specific monoclonal antibodies. Quantitative proteomic analysis was performed on primary MCs pretreated with MS-275. Decellularized matrices were generated from either MS-275-exposed or untreated cells to study Fibronectin-1 extracellular secretion. The effect of MS-275 on β1 integrin activity was assessed using specific monoclonal antibodies. The role of Talin-1 in MCs/EOC adhesion was analyzed by genetic silencing. Talin-1 ectopic expression was validated as a rescue tool from MS-275-induced phenotype. The in vivo effect of MS-275-induced MC remodeling was validated in a mouse model of peritoneal EOC dissemination., Results: Treatment of MCs with non-cytotoxic concentrations of MS-275 caused a consistent reduction of EOC adhesion. Proteomic analysis revealed several pathways altered upon MC treatment with MS-275, including ECM deposition/remodeling, adhesion receptors and actin cytoskeleton regulators. HDAC1/2 inhibition hampered actin cytoskeleton polymerization by downregulating actin regulators including Talin-1, impairing β1 integrin activation, and leading to abnormal extracellular secretion and distribution of Fibronectin-1. Talin-1 ectopic expression rescued EOC adhesion to MS-275-treated MCs. In an experimental mouse model of metastatic EOC, MS-275 limited tumor invasion, Fibronectin-1 secretion and the sub-mesothelial accumulation of MC-derived carcinoma-associated fibroblasts., Conclusion: Our study unveils a direct impact of HDAC-1/2 in the regulation of MC/EOC adhesion and highlights the regulation of MC plasticity by epigenetic inhibition as a potential target for therapeutic intervention in EOC peritoneal metastasis., (© 2024. The Author(s).)

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., (© 2023. The Author(s).)

Dissemination of high-grade serous ovarian cancer (HG-SOC) in the omentum and intercalation into a mesothelial cell (MC) monolayer depends on functional α5β1 integrin (Intα5β1) activity. Although the binding of Intα5β1 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 Intα5β1 activation and accelerates tumor cells toward invasive disease, involving the protein β-arrestin1 (β-arr1) and the activation of the endothelin A receptor (ET A R) by endothelin-1 (ET-1). As demonstrated in primary HG-SOC cells and SOC cell lines, ET-1 increased Intβ1 and downstream FAK/paxillin activation. Mechanistically, β-arr1 directly interacts with talin1 and Intβ1, promoting talin1 phosphorylation and its recruitment to Intβ1, thus fueling integrin inside-out activation. In 3D spheroids and organotypic models mimicking the omentum, ET A R/β-arr1-driven Intα5β1 signaling promotes the survival of cell clusters, with mesothelium-intercalation capacity and invasive behavior. The treatment with the antagonist of ET A R, Ambrisentan (AMB), and of Intα5β1, ATN161, inhibits ET-1-driven Intα5β1 activity in vitro, and tumor cell adhesion and spreading to intraperitoneal organs and Intβ1 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 ET A R/β-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 ET A R/β-arr1 regulates Intα5β1 functional pathway., (© 2023. The Author(s).)

Conventional batch syntheses of polymer-based nanoparticles show considerable shortcomings in terms of scarce control over nanomaterials morphology and limited lot-to-lot reproducibility. Droplet-based microfluidics represents a valuable strategy to overcome these constraints, exploiting the formation of nanoparticles within discrete microdroplets. In this work, we synthesized nanogels (NGs) composed of hyaluronic acid and polyethyleneimine using a microfluidic flow-focusing device endowed with a pressure-driven micro-actuator. The actuator achieves real-time modulation of the junction orifice width, thereby regulating the microdroplet diameter and, as a result, the NG size. Acting on process parameters, NG hydrodynamic diameter could be tuned in the range 92-190 nm while preserving an extremely low polydispersity (0.015); those values are hardly achievable in batch syntheses and underline the strength of our toolbox for the continuous in-flow synthesis of nanocarriers. Furthermore, NGs were validated in vitro as a drug delivery system in a representative case study still lacking an effective therapeutic treatment: ovarian cancer. Using doxorubicin as a chemotherapeutic agent, we show that NG-mediated release of the drug results in an enhanced antiblastic effect vs . the non-encapsulated administration route even at sublethal dosages, highlighting the wide applicability of our microfluidics-enabled nanomaterials in healthcare scenarios.

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., (© 2022. The Author(s).)

Aim: B-cell lymphoma-2 (Bcl-2)-like protein-10 (Bcl2L10) is the less studied member of Bcl-2 family proteins, with the controversial role in different cancer histotypes. Very recently, Bcl2L10 expression in melanoma tumor specimens and its role in melanoma response to therapy have been demonstrated. Here, the involvement of Bcl2L10 on the in vitro and in vivo properties associated with melanoma aggressive features has been investigated., Methods: Endogenous Bcl2L10 protein expression was detected by western blotting analysis in a panel of patient-derived and commercially available human melanoma cells. In vitro assays to evaluate clonogenicity, cell proliferation, cell migration, cell invasion, and in vitro capillary-like structure formation [vasculogenic mimicry (VM)] have been performed by using human melanoma cells stably overexpressing Bcl2L10 or transiently transfected for loss/gain function of Bcl2L10, grown under two- or three-dimensional (3D) conditions Xenograft melanoma model was employed to evaluate in vivo tumor growth and angiogenesis., Results: Results demonstrated that Bcl2L10 acts as an inducer of in vitro cell migration, invasion, and VM, while in vitro cell proliferation, in vivo tumor growth, as well as colony formation properties were not affected. Dissecting different signaling pathways, it was found that Bcl2L10 positively affects the phosphorylation of extracellular-signal-regulated kinase (ERK) and the expression of markers of cell invasion, such as urokinase plasminogen activator receptor (uPAR) and matrix metalloproteinases (MMPs). Of note, Bcl2L10-dependent in vitro migration, invasion, and VM are linked to uPAR. Bcl2L10 also negatively regulates the intracellular calcium level. Finally, reduced invasion capability in 3D spheroid invasion assay of melanoma cells transiently overexpressing Bcl2L10 was observed after treatment with inhibitors of MMPs and uPAR., Conclusions: Overall, data reported in this paper provide evidence supporting a positive role of Bcl2L10 in melanoma aggressive features., Competing Interests: The authors declare that there are no conflicts of interest., (© The Author(s) 2022.)

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, ET A and ET B , along with the protein β-arrestin1. ET-1 triggers MC proliferation via β-arrestin1-dependent MAPK and NF-kB pathways and increases the release of cancer-related factors. The ET A /ET B 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 ET A R and ET B R or by β-arrestin1 silencing. Notably, in peritoneal metastases both ET A R and ET B R 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Del Rio, Masi, Caprara, Spadaro, Ottavi, Strippoli, Sandoval, López-Cabrera, Sainz de la Cuesta, Bagnato and Rosanò.)

Cancer cells use actin-based membrane protrusions, invadopodia, to degrade stroma and invade. In serous ovarian cancer (SOC), the endothelin A receptor (ET A R) drives invadopodia by a not fully explored coordinated function of β-arrestin1 (β-arr1). Here, we report that β-arr1 links the integrin-linked kinase (ILK)/βPIX 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, ET A R/ILK/Rac3 signaling supports the communication between cancer and mesothelial cells, favoring SOC cell adhesion and transmigration. In vivo, ambrisentan, an ET A R 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/β-arr1 pathway as an integrator of ILK/Rac3-dependent adhesive and proteolytic signaling to invadopodia, favoring cancer/stroma interactions and metastatic behavior., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Identification of regulatory mechanisms underlying the poor prognosis of ovarian cancer is necessary for diagnostic and therapeutic implications. Here we show that endothelin A receptor (ET A R) and ZEB1 expression is upregulated in mesenchymal ovarian cancer and correlates with poor prognosis. Notably, the expression of ET A R and ZEB1 negatively correlates with miR-200b/c. These miRNAs, besides targeting ZEB1, impair ET A R expression through the 3'UTR binding. ZEB1, in turn, restores ET A R levels by transcriptionally repressing miR-200b/c. Activation of ET A R drives the expression of ZEB1 integrating the miR-200/ZEB1 double negative feedback loop. The ET A R-miR-200b/c-ZEB1 circuit promotes epithelial-mesenchymal transition, cell plasticity, invasiveness and metastasis. Of therapeutic interest, ET A R blockade with macitentan, a dual ET A R and ET B R antagonist, increases miR-200b/c and reduces ZEB1 expression with the concomitant inhibition of metastatic dissemination. Collectively, these findings highlight the reciprocal network that integrates ET A R and ZEB1 axes with the miR-200b/c regulatory circuit to favour metastatic progression 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, β-arrestin1 (β-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 ET A R/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., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2020 Masi, Caprara, Bagnato and Rosanò.)

Recent studies imply a key role of endothelin-1 receptor (ET-1R), belonging to the largest family of G protein-coupled receptors (GPCR), in the regulation of a plethora of processes involved in tumorigenesis and metastatic progression. β-arrestin-1 (β-arr1) system has been recognized as a critical hub controlling GPCR signaling network, directing the GPCR's biological outcomes. In ovarian cancer, ET-1R/β-arr1 axis enables cancer cells to engage several integrated signaling, and represents an actionable target for developing novel therapeutic approaches. Preclinical research studies demonstrate that ET-1R blockade by the approved dual ET A R/ET B R antagonist macitentan counteracts β-arr1-mediated signaling network, and hampers the dialogue among cancer cells and the tumor microenvironment, interfering with metastatic progression and drug response. In light of major developments in the ET-1R signaling paradigm, this review article discusses the emerging evidence of the dual ET-1R antagonist treatment in cancer, and outlines our challenge in preclinical studies warranting the repurposing of ET-1R antagonists for the design of more effective clinical trials based on combinatorial therapies to overcome, or prevent, the onset of drug resistance.

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)/β-arrestin1 (β-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 β-arr1, representing a "hotspot" for ET-1R-induced invasive signalling. We demonstrated that the molecular interaction of IQGAP1 with β-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 β-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/β-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 ET A R/β-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 β-arr1/IQGAP1 interaction as a prerequisite for the dynamic integration of pathways in fostering invadopodia and metastatic process in human SOC., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

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 β-arrestin1 (β-arr1), interacts with YAP, triggering its cytoplasmic-nuclear shuttling. This interaction allows β-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, β-arr1 mediates the ET-1R-induced Trio/RhoA-dependent YAP nuclear accumulation. In the nucleus, ET-1 through β-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 β-arr1-mediated YAP/mutp53 transcriptional programme. Furthermore, ET A R/β-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.

Tumor cells acquire invasive and metastatic behavior by sensing changes in the localization and activation of signaling pathways, which in turn determine changes in actin cytoskeleton. The core-scaffold machinery associated to β-arrestin (β-arr) is a key mechanism of G-protein coupled receptors (GPCR) to achieve spatiotemporal specificity of different signaling complexes driving cancer progression. Within different cellular contexts, the scaffold proteins β-arr1 or β-arr2 may now be considered organizers of protein interaction networks involved in tumor development and metastatic dissemination. Studies have uncovered the importance of the β-arr engagement with a growing number of receptors, signaling molecules, cytoskeleton regulators, epigenetic modifiers, and transcription factors in GPCR-driven tumor promoting pathways. In many of these molecular complexes, β-arrs might provide a physical link to active dynamic cytoskeleton, permitting cancer cells to adapt and modify the tumor microenvironment to promote the metastatic spread. Given the complexity and the multidirectional β-arr-driven signaling in cancer cells, therapeutic targeting of specific GPCR/β-arr molecular mechanisms is an important avenue to explore when considering future new therapeutic options. The focus of this review is to integrate the most recent developments and exciting findings of how highly connected components of β-arr-guided molecular connections to other pathways allow precise control over multiple signaling pathways in tumor progression, revealing ways of therapeutically targeting the convergent signals in patients.

Endothelin-1 (ET-1), which acts through the endothelin A receptor (ET A R) or ET B R, belonging to the large family of G-protein coupled receptors (GPCR), is involved in physiopathological processes, such as cancer. In epithelial ovarian cancer, a pervasively activated ET-1/ET A R axis drives different steps of tumor progression and confers drug resistance. In this malignancy, one major aspect associated with the ET A R signaling machinery resides in the fact that this receptor may use β-arrestin-1 (β-arr1) function to spatially and temporally activate key oncogenic pathways. This results in specificity of ET-1/ET A R signal transduction mechanisms and downstream signaling pathways. As such, β-arr1 has been recognized as an important signal transducer involved in multiple cross talks with other signaling pathways, including those activated by tyrosine kinase receptors. The interaction with diverse sets of partners positions β-arr1 as a critical regulator of GPCR signal transduction and permits the integration of ET A R-mediated signals with other cytoplasmic or nuclear inputs. In particular, the scaffolding function of β-arr1 provides an essential link in translating ET A R function by altering β-catenin localization and function, promoting β-catenin-related transcriptional activity and gene transcription relevant to tumor progression. This chapter outlines the methodologies for the measurement of β-arr1/β-catenin protein interactions and functional activity in tumor cells.

Metastatic progression is strongly influenced by the connection between hyperactivated signaling pathways. G-protein coupled receptors (GPCRs) through β-arrestins (β-arrs), which serve as intracellular signaling molecules, integrate different pathways to control multiple aspects of metastatic process. As primary component of a core-scaffold, β-arr-dependent signaling represents a mean to direct spatiotemporal specificity of multi-protein complexes in invasion and extracellular matrix (ECM) degradation. Under this paradigm, β-arrs engage a growing number of signaling molecules and organizing protein networks controlling multiple pathways, and cytoskeleton modifications, permitting adaptation to the tumor microenvironment to sustain metastatic dissemination. These findings implicate GPCR/β-arr function as a regulatory tethering hub to orchestrate diverse cellular mechanisms of cancer cell migration and invasion that are critical for metastatic progression. In this chapter, we outline the most recent findings on GPCR/β-arr-guided molecular interactions in specific intracellular compartments to drive metastasis, while discussing new perspectives for the selection of most effective therapeutic options for a personalized medicine., (© 2019 Elsevier Inc. All rights reserved.)

Metastatization is a complex multistep process requiring fine-tuned regulated cytoskeleton re-modeling, mediated by the cross-talk of actin with interacting partners, such as the Rho GTPases. Our expanding knowledge of invadopodia, small invasive membrane protrusions composed of a core of F-actin, actin regulators and actin-binding proteins, and hotspots for secretion of extracellular matrix (ECM) proteinases, contributes to clarify critical steps of the metastatic program. Growth factor receptors and their intermediate signaling molecules, along with matrix adhesion and rigidity, pH and hypoxia, act as drivers of cytoskeleton changes and invadopodia formation. We recently pro-posed a novel route map by which cancer cells regulates invadopodia dynamics supporting metastasis as response to the endothelin A receptor (ET A R), among the highly druggable G-protein coupled receptors in cancer. The metastatic behavior exhibited by ovarian cancer cells overe-xpressing ET A R is now explained by the interplay with β-arrestin1 (β-arr1), a scaffold protein acting as signal-integrating module of RhoC and cofilin signaling for specific invadopodia formation, accomplished by its interaction with a Rho guanine nucleotide exchange factor (GEF), PDZ-RhoGEF, in a G-protein independent manner. Here, we summarize this novel activation of the RhoC pathway from ET A R/β-arr1 signaling that may be exploited therapeutically and discuss new perspectives for future directions of investigations.

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 β-arrestin1 (β-arr1)-driven interactome. Here, we identify hMENA, a member of the actin-regulatory protein ENA/VASP family, as an interacting partner of β-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 β-arr1, restricted to mesenchymal-like invasive SOC cells. The interaction of β-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 β-arr1 with hMENA and inhibits invadopodial maturation and tumor dissemination in SOC orthotopic xenografts. Finally, high ET A R/hMENA/β-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/β-arr1-induced invadopodial activity and ovarian cancer progression., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Introduction: Endothelin-1 receptor (ET-1R)/β-arrestin1 (β-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 β-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/β-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 ET A R/ET B R antagonist prevents β-arr1 network formation, offering a novel therapeutic strategy in ovarian cancer patients. Expert opinion: The information provided in this review about the ET-1R/β-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.

The limited clinical response to conventional chemotherapeutics observed in colorectal cancer (CRC) may be related to the connections between the hyperactivated β-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 β-catenin pathway through the specific interaction with the signal transducer β-arrestin1 (β-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 ET A R and β-arr1, and that the activation of ET A R/β-arr1 axis promotes the cross-talk with β-catenin signaling to sustain stemness, epithelial-to-mesenchymal transition (EMT) phenotype and response to chemotherapy. Upon ET A R activation, β-arr1 acts as a transcription co-activator that binds β-catenin, thereby promoting nuclear complex with β-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/β-catenin network. All these findings provide a strong rationale for targeting ET-1R to hamper downstream β-catenin/ET-1 autocrine circuit. Interestingly, treatment with macitentan, a dual ET A R and ET B R antagonist, able to interfere with tumor and microenvironment, disrupts the ET-1R/β-arr1-β-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 β-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 ET A R and β-catenin expression.

New effective treatments are needed to improve outcomes for multiple myeloma (MM) patients. Receptors with restricted expression on plasma cells (PCs) represent attractive new therapeutic targets. The endothelin-1 (EDN1) axis, consisting of EDN1 acting through EDN-receptor A (EDNRA) and B (EDNRB), was previously shown to be overexpressed in several tumours, including MM. However, there is incomplete understanding of how EDN1 axis regulates MM growth and response to therapy. Besides EDNRA, the majority of MM cell lines and primary malignant PCs express high levels of EDNRB and release EDN1. Similarly, bone-marrow microenvironment cells also secrete EDN1. Investigating the extent of epigenetic dysregulation of EDNRB gene in MM, we found that hypermethylation of EDNRB promoter and subsequent down-regulation of EDNRB gene was observed in PCs or B lymphocytes from healthy donors compared to EDNRB-expressing malignant PCs. Pharmacological blockade with the dual EDN1 receptor antagonist bosentan decreased cell viability and MAPK activation of U266 and RPMI-8226 cells. Interestingly, the combination of bosentan and the proteasome inhibitor bortezomib, currently approved for MM treatment, resulted in synergistic cytotoxic effects. Overall, our data has uncovered EDN1-mediated autocrine and paracrine mechanisms that regulate malignant PCs growth and drug response, and support EDN1 receptors as new therapeutic targets in MM., (© 2017 John Wiley & Sons Ltd.)

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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 β-arrestin-1 (β-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 β-arr1 silencing, suggesting that ET-1/ETAR regulate RhoA signaling through β-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 β-arr1 to sustain RhoA/ROCK signaling in response to ETAR activation in EOC., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The advent of targeted therapeutics in human cancer has begun to find novel druggable targets and, in this context, the endothelin-1 receptor (ET-1R), namely ETA receptor (ETAR) and ETB receptor, among the GPCR family represents a class of highly druggable molecules in cancer. ET-1R are aberrantly expressed in human malignancies, potentially representing prognostic factors. Their activation by ligand stimulation initiate signaling cascades activating different downstream effectors, allowing precise control over multiple signaling pathways. ET-1R regulates cell proliferation, survival, motility, cytoskeletal changes, angiogenesis, metastasis as well as drug resistance. The molecular events underlying these responses are the activation of transcriptional factors and coactivators, and downstream genes, acting as key players in tumor growth and progression. ET-1R represent crucial cancer targets that have been exploited for ET-1R therapeutics. Importantly, efforts to explore new information of ETAR in cancer have uncovered that their functions are crucially regulated by multifunctional scaffold protein β-arrestins (β-arrs) which orchestrate the multidimensionality of ETAR signaling into highly regulated and distinct signaling complexes, a property that is highly advantageous for tumor signaling. Moreover, the role of β-arr1 in ET-1 signaling in cancer highlights why the pleiotropic effects of ET-1 and its dynamic signaling are more complex than previously recognized. In order to improve therapeutic strategies that interfere with the widespread effects of ET-1R, it is important to consider antagonists able to turn the receptors "off" selectively controlling β-arr1-dependent signaling, highlighting the possibility that targeting ETAR/β-arr1 may display a large therapeutic window in cancer.