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

Abstract

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.

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

“…We took advantage of the 3D models to compare the therapeutic anti-metastatic potential of different ET-1 receptor antagonists as well as β-arr1 silencing in vitro and in vivo. Although previous data have shown that ambrisentan prevents peritoneal metastasis in SOC xenografts [ 30 ], our study is the first to demonstrate that bosentan is effective in controlling tumor dissemination by targeting the metastatic potential of heterotypic spheroids, associated with enhanced apoptosis in cancer cells, fibroblasts and endothelial cells expressing ET B R, placing them as the cornerstone of peritoneal metastasis. These data support previously published findings demonstrating that the dual ET-1R receptor antagonist macitentan interrupts ET-1-driven pro-survival signals, affecting cancer cells and the feed-forward loops in the TME [ 32 ].…”

“…Considering the role of invadosome in fibroblast-driven tissue invasion and matrix remodeling [ 33 ], and that ET-1/β-arr1 promotes invadopodia in cancer cells [ 29 , 30 ], we evaluated the ET-1 axis as a regulator of invadosome. As shown by confocal laser scanning microscopy (CLSM) analysis, while untreated HOFs did not form degradative cortactin clusters, under the addition of ET-1, F-actin/cortactin puncta became visible within the cell body, displaying invadosome properties, as degradative activity, but not when cells were treated with AMB or BQ788, or BOS (Fig.…”

“…Since F-actin cores are surrounded by “adhesive rings” enriched in integrins, and that β-arr1 regulates both adhesion ring formation around invadopodia, and intβ1 signaling [ 29 , 30 , 34 – 36 ], we evaluated intβ1-linked molecular effectors to ECM remodeling. HOFs express intβ1 and intα5 (Supplementary Fig.…”

“…Mice were injected intraperitoneally (i.p.) with 200 μL PBS containing 3 × 10 6 viable of SKOV3-Luc+HOFs-Luc (3:1, SKOV3:HOFs) [ 30 ], following the guidelines for animal experimentation. One week after, mice, were randomized into three different groups undergoing the following treatments for 5 weeks: (i) 200 μL Metocell (vehicle oral gavage, CTR), (ii) 200 μL Bosentan (10 mg/kg, oral gavage daily), and Ambrisentan (5 mg/kg, oral gavage daily).…”

“…The ET A R/β-arr1 signaling promotes invadopodia-dependent invasion [ 29 ]. Moreover, ET A R/β-arr1 drives integrin α5β1 (intα5β1) inside-out activation and promotes the survival of 3D spheroids, with mesothelium-intercalation capacity and invasive behavior [ 30 ]. However, the role of ET-1/β-arr1 in ovarian fibroblast activation and ECM remodeling is completely unknown.…”