Itraconazole inhibits nuclear delivery of extracellular vesicle cargo by disrupting the entry of late endosomes into the nucleoplasmic reticulum.

Mark F Santos,Tony Huynh,Maria Antonietta Di Bella,Denis Corbeil,Patrizia Diana,Marshall R Pope,Aurelio Lorico,Riccardo Alessandro,Kevin Huang,Stella Maria Cascioferro,Jana Karbanová,Marta Moschetti,Gyunghwi Woo,Barbara Parrino,Germana Rappa,Goffredo O Arena,Girolamo Cirrincione,Giulio Vistoli,Simona Fontana

doi:10.1002/jev2.12132

Abstract

ABSTRACTExtracellular vesicles (EVs) are mediators of intercellular communication under both healthy and pathological conditions, including the induction of pro‐metastatic traits, but it is not yet known how and where functional cargoes of EVs are delivered to their targets in host cell compartments. We have described that after endocytosis, EVs reach Rab7+ late endosomes and a fraction of these enter the nucleoplasmic reticulum and transport EV biomaterials to the host cell nucleoplasm. Their entry therein and docking to outer nuclear membrane occur through a tripartite complex formed by the proteins VAP‐A, ORP3 and Rab7 (VOR complex). Here, we report that the antifungal compound itraconazole (ICZ), but not its main metabolite hydroxy‐ICZ or ketoconazole, disrupts the binding of Rab7 to ORP3–VAP‐A complexes, leading to inhibition of EV‐mediated pro‐metastatic morphological changes including cell migration behaviour of colon cancer cells. With novel, smaller chemical drugs, inhibition of the VOR complex was maintained, although the ICZ moieties responsible for antifungal activity and interference with intracellular cholesterol distribution were removed. Knowing that cancer cells hijack their microenvironment and that EVs derived from them determine the pre‐metastatic niche, small‐sized inhibitors of nuclear transfer of EV cargo into host cells could find cancer therapeutic applications, particularly in combination with direct targeting of cancer cells.

Highlights

Extracellular vesicles (EVs), including exosomes, microvesicles and other types of membrane particles act as mediators of intercellular communication in tissues and organs under healthy and pathological conditions (Maas et al, 2017; Mathieu et al, 2019; Raposo & Stoorvogel, 2013)
Compared to SW480 cells, highly metastatic SW620 cells have a more rounded shape, associated with amoeboid motility and high plasma membrane blebbing activity. These pro-metastatic properties can be transferred to SW480 cells by EVs derived from SW620 cells (Schillaci et al, 2017). To assess whether these cellular alterations are related to the nuclear transfer of the EV cargo, we exposed SW480 cells as well as those silenced for VAP-A, VAP-A homolog, VAP-B, and ORP3 for 5 h to EVs prepared from SW620 cells
By studying EV-cell communication using biochemical, loss-of-function and high-resolution microscopy techniques, we have demonstrated that FDA-approved ICZ and a novel small compound PRR851 interfere with EV-mediated pro-metastatic morphological transformation and migration behaviour of cancer cells by acting as inhibitors of nuclear transfer of late endosome-carried EV cargo

Summary

Introduction

Extracellular vesicles (EVs), including exosomes, microvesicles and other types of membrane particles act as mediators of intercellular communication in tissues and organs under healthy and pathological conditions (Maas et al, 2017; Mathieu et al, 2019; Raposo & Stoorvogel, 2013). Their role in modulation of immune responses, cell differentiation, and epithelial-mesenchymal transition has been demonstrated. We described a novel intracellular pathway whereby EV-containing late endosomes enter in the nucleoplasmic reticulum through type II nuclear envelope invaginations (NEI) and transfer EV cargoes into the nucleoplasm of recipient cells (Rappa et al, 2017). Silencing VAP-A or ORP3 abrogated the localization of Rab7+ late endosomes in NEI, and the subsequent nuclear transport of endocytosed EV-derived components (Santos et al, 2018)

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