Anna Bagnato

YAP signaling orchestrates the endothelin-1-guided invadopodia formation in high-grade serous ovarian cancer

Abstract 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, restores YAP phosphorylation. 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, halts 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.

The endothelin-1-driven tumor-stroma feed-forward loops in high-grade serous ovarian cancer

Abstract The high-grade serous ovarian cancer (HG-SOC) tumor microenvironment (TME) is constellated by cellular elements and a network of soluble constituents that contribute to tumor progression. In the multitude of the secreted molecules, the endothelin-1 (ET-1) has emerged to be implicated in the tumor/TME interplay; however, the molecular mechanisms induced by the ET-1-driven feed-forward loops (FFL) and associated with the HG-SOC metastatic potential need to be further investigated. The tracking of the patient-derived (PD) HG-SOC cell transcriptome by RNA-seq identified the vascular endothelial growth factor (VEGF) gene and its associated signature among those mostly up-regulated by ET-1 and down-modulated by the dual ET-1R antagonist macitentan. Within the ligand–receptor pairs concurrently expressed in PD-HG-SOC cells, endothelial cells and activated fibroblasts, we discovered two intertwined FFL, the ET-1/ET-1R and VEGF/VEGF receptors, concurrently activated by ET-1 and shutting-down by macitentan, or by the anti-VEGF antibody bevacizumab. In parallel, we observed that ET-1 fine-tuned the tumoral and stromal secretome toward a pro-invasive pattern. Into the fray of the HG-SOC/TME double and triple co-cultures, the secretion of ET-1 and VEGF, that share a common co-regulation, was inhibited upon the administration of macitentan. Functionally, macitentan, mimicking the effect of bevacizumab, interfered with the HG-SOC/TME FFL-driven communication that fuels the HG-SOC invasive behavior. The identification of ET-1 and VEGF FFL as tumor and TME actionable vulnerabilities, reveals how ET-1R blockade, targeting the HG-SOC cells and the TME simultaneously, may represent an effective therapeutic option for HG-SOC patients.

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

AbstractRecruitment 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 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/ETA/BR/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 ETA/BR or β-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/β-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.

Endothelin-1 receptor blockade impairs invasion patterns in engineered 3D high-grade serous ovarian cancer tumouroids

Abstract 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.

The interaction of β-arrestin1 with talin1 driven by endothelin A receptor as a feature of α5β1 integrin activation in high-grade serous ovarian cancer

AbstractDissemination 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 (ETAR) 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, ETAR/β-arr1-driven Intα5β1 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 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 ETAR/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 ETAR/β-arr1 regulates Intα5β1 functional pathway.

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, 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

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 (ET

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

Abstract 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.