Laura Rosanò

Zwitterionic nanoparticles for thermally activated drug delivery in hyperthermia cancer treatment

Through RAFT emulsion polymerization, zwitterionic nanoparticles (NPs) were synthesized as thermally activated drug release systems. Following hyperthermia, drug-loaded NPs outperforms the therapeutic effect of the free drug in ovarian cancer cells.

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.

The extracellular matrix protein type I collagen and fibronectin are regulated by β-arrestin-1/endothelin axis in human ovarian fibroblasts

Abstract Background The invasive and metastatic spread of serous ovarian cancer (SOC) results from the cooperative interactions between cancer and stroma, which include extracellular matrix (ECM) and cellular components, including cancer-associated fibroblasts (CAFs). Soluble factors secreted by cancer and stromal cells contribute to stroma remodeling through the secretion of ECM proteins, providing a favorable environment for cancer cell dissemination. The peptide endothelin-1 (ET-1), through two G protein-coupled receptors (GPCR), endothelin receptor type A (ETAR) and B (ETBR), acts on both cancer and stromal cells, engaging the protein β-arrestin1 (β-arr1), to bolster SOC progression. However, its role in the regulation of the ECM proteins by ovarian fibroblasts is not understood. This study delves into the role of ET-1 as a regulator of type I collagen (Col1) and fibronectin (FN). Methods We used human primary ovarian fibroblasts (HOFs) and CAFs. The expression of Col1 (COL1A1) and FN (FN1) were detected by western blotting (WB), quantitative real time-polymerase chain reaction (qRT-PCR), immunofluorescence (IF), and confocal laser scanning microscopy (CLSM) in cells and tumor tissue sections from mice xenografts, while the transcription of COL1A1 was detected by luciferase reporter gene assay. The nuclear function of β-arr1 was evaluated by silencing and rescue expression with wild-type (WT) and nuclear mutant plasmid constructs, RNA seq and differential gene expression and gene sets enrichment analyses. The prognostic role of COL1A1, FN1, EDN1 (ET-1) and ARRB1 (β-arr1) gene expression was evaluated using the Kaplan–Meier plotter database and clinical ovarian cancer tissue samples. Results We demonstrated that ET-1 boosts Col1 and FN expression in HOFs, akin to ovarian CAF levels. Both receptors are implicated, evident from inhibitory effects after ETAR or ETBR antagonist treatments and notably with bosentan, a dual antagonist, in vitro and in vivo. At the molecular level, ET-1 triggers the activation of COL1A1 promoter activity and its enhanced expression via β-arr1 nuclear function. Transcriptome analysis of β-arr1-silenced HOFs confirms the nuclear role of β-arr1 in collagen and ECM remodeling-related protein transcriptional regulation. Accordingly, a high level of EDN1/ARRB1 expression in combination with either COL1A1 or FN1 is associated with the poor prognosis of SOC patients. Conclusions These findings hint at ET-1 involvement in ECM remodeling and early SOC stages by modulating the expression of Col1 and FN. Targeting ET-1 signaling with ETAR/ETBR antagonists might interfere with the ability of CAFs to produce key ECM proteins in this tumor.

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.