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

Abstract

Extracellular 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.

Anti-Human CD9 Fab Fragment Antibody Blocks the Extracellular Vesicle-Mediated Increase in Malignancy of Colon Cancer Cells

“…As expected, the ER-associated calnexin was absent ( Figure 2 B). These data are in agreement with our previous studies, as well as studies by others [ 57 , 62 , 63 , 64 ]. The overexpression of CD9-GFP did not influence the presence or absence of these proteins in SW620 cell-derived EVs ( Figure 2 C).…”

“…Third, the distribution of tetraspanins among the EVs was determined at super-resolution using dSTORM ( Figure 2 D). Analysis of small EVs (<200 nm in diameter) revealed the heterogeneity of the EV population with a variable distribution of single, double, and triple positives ( Figure 2 E and Supplementary Figure S2A ), in agreement with previous studies [ 57 , 65 ]. Triple-positive EVs (CD9, CD63, and CD81) constituted the major population (>70%) of EVs.…”

“…In other experiments, cells were pre-treated with or without 10 µM PRR851, a drug synthesized in one of our laboratories (for detail, see Ref. [ 57 ]), in the presence or absence of CD9 Fab (25 µg/mL) for 30 min at 37 °C. CD9-GFP + EVs were then added for 5 h. DMSO alone was used as vehicle control.…”

“…Direct stochastic optical reconstruction microscopy (dSTORM) was applied on EVs derived from SW480, SW620, and CD9-GFP + SW620 cells. EVs were immunolabeled and imaged using the EasyVisi Single-Extracellular Vesicle Characterization kit from ONI (beta v1.0, Oxford Nanoimaging, UK) as described previously [ 57 ]. Briefly, EVs (3.5 × 10 7 particles) were incubated overnight at 4 °C with the fluorescently labeled Abs: CD9-Atto488, CD63-Cy3B, and CD81-AlexaFluor ® 647.…”

The Role of Lamins in the Nucleoplasmic Reticulum, a Pleiomorphic Organelle That Enhances Nucleo-Cytoplasmic Interplay

“…As a result of these findings, the VOR complex may become a novel drug target to impair the intercellular communication in the cancer microenvironment. Indeed, itraconazole treatment disrupts the VOR complex and inhibits EV-mediated pro-metastatic morphological changes and migratory properties of colon cancer cells ( Santos et al, 2021 ).…”