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-associatedMCs, providing a favorable environment for SOC cell dissemination. However, factors and molecular mechanisms involved in this process are largely unexplored. Herewe investigated the tumor-related endothelin-1 (ET-1) as an inducer of changes inMCs supporting SOC progression. Here, we report a significant production of ET-1 from MCs associatedwith the expression of its cognate receptors, ETA and ETB, along with the protein β-arrestin1. ET-1 triggers MC proliferation via β-arrestin1-dependentMAPK 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, NFkB-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 ETB R or by β-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 prometastatic behaviour via MMTAssociazione Italiana Ricerca sul Cancro (AIRC) to LR grant number AIRC 21372 and partially by Agencia Estatal de Investigación Project to ML-P “PID 2019-110132RBI00/AEI/10.13039/50110001103

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

Abstract 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 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

Ovarian cancer-driven mesothelial-to-mesenchymal transition is triggered by the endothelin-1/β-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 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 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 β-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

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

: 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 (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