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.

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.

Bcl-2-like protein-10 increases aggressive features of melanoma cells.

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.

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.