Dottorato in MORFOGENESI E INGEGNERIA TISSUTALE

Titolo della tesi: The interaction between integrin β1 and endothelin A receptor drives intraperitoneal dissemination and stromal invasion of ovarian cancer

RATIONALE: Intraperitoneal dissemination is the primary means of high-grade serous ovarian cancer (HG-SOC) metastasis, requiring molecular mechanisms different from hematogenous metastasis. When establishing secondary tumors, single cells or spheroids must attach to the mesothelium covering peritoneal organs, and the underlying basement membrane, while in the final metastatic step, tumor cells must penetrate mesothelial surfaces through a process called mesothelial cell (MC) clearance and remodel the sub-mesothelial interstitial extracellular matrix (ECM), containing fibroblasts and other stromal cells. Emerging data indicate that ECM remodeling and degradation might be operated by actin-based membrane invasive structures, the invadopodia, used by both cancer and stromal cells. Cues from the ECM, heterotypic interactions between tumor and stromal cells, and growth factor signaling are drivers of these processes. As bidirectional signal transducers expressed on tumor/stromal cells, integrins, the main ECM receptors of the cell adhesion with the primacy of the β1 subunit, regulate many of these processes. The prevailing view of integrin activation is that called inside-out activation, whereby intracellular signals can be induced by the binding of talin that rapidly regulates integrin affinity for ligands, highlighting that the binding of talin to β1 integrin is a common step of outside or inside integrin activation. In this framework, G protein-coupled receptors (GPCRs) might employ G-proteins in initiating “inside-out” signaling and cell adhesion. In this tumor histotype, a member of the GPCR family, the receptor A (ETAR) for endothelin-1 (ET-1) provides an active signaling network that controls pro-invasive signaling and metastatic features, including cytoskeletal activity, changes in shape, and invasive protrusions, through scaffold protein -arrestin1 (β-arr1). However, the direct interaction of ETAR with integrins remains unknown. GENERAL OBJECTIVES: Considering the interplay between GPCR and integrin signaling, and the ability of β-arr1 to interact with components of the multiprotein signaling platform forming the adhesome, we aimed to study: (i) the existence of ETAR-dependent Intβ1-linked signaling and the putative role of β-arr1 in 2D and 3D models of HG-SOC; (ii) how this interaction between different receptors regulates the invasive capacity of cancer cells and the interaction with stromal cells, specifically mesothelial cells and cancer-associated fibroblasts in 3D organotypic models of HG-SOC; (iii) the efficacy of ETAR and Intβ1 blockade as an anti-metastatic approach in preclinical models. RESULTS: The role of β-arr1 as a scaffold protein in ETAR-dependent integrin signaling has emerged. Indeed, we report that β-arr1 links 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. These findings provide a framework for ET-1R/β-arr1 pathway as an integrator of ILK/Rac3-dependent adhesive and proteolytic signaling to invadopodia, favoring metastatic behavior. We also demonstrated that in primary HG-SOC cells and cell lines ET-1 increased Intβ1 and talin1 phosphorylation. Mechanistically, β-arr1 directly interacts with talin1 and Int1 and promotes the recruitment of talin1 to Intβ1, thus fueling integrin inside-out activation. In 3D spheroids and 3D organotypic models mimicking the omentum, ETAR/β-arr1-driven Intα5β1 signaling promotes the survival of cell clusters, with mesothelium-intercalation capacity and invasive behavior. On the other hand, paracrine/autocrine ET-1 activates a CAF phenotype in HOFs and enhances their ability to degrade a gelatin matrix in an invadopodia-dependent manner. As therapeutic proof of concept, the combination of Ambrisentan (AMB) and ATN161, antagonists of ETAR and Intα5β1, respectively, inhibits ET-1-driven Intα5β1 activity in vitro, and adhesion, spreading, and stromal invasion of tumor cells to intraperitoneal organs in vivo. As a prognostic factor, high EDNRA/ITGB1 expression correlates with poor HG-SOC clinical outcomes. CONCLUSIONS: Collectively, these data establish a novel interaction between ETAR/β-arr1 and Intβ1 signaling to direct HG-SOC invasive behavior supporting ECM remodeling and the metastatic process and suggest that the co-targeting of Intα5β1 and ETAR/β-arr1 might be considered in the future as new anti-metastatic approach.