Caveolin-1-overexpression alters extracellular vesicle secretion and cargo in a model of rhabdomyosarcoma

Background. Caveolin-1 (CAV1) is an integral membrane protein required to generate caveolae and cholesterol-enriched lipid rafts of the plasma membrane. It is widely accepted that loss of CAV1 correlates with early-stage tumor progression, while its overexpression and phosphorylation are associated with metastatic disease. Extracellular vesicles (EVs) are lipid-bound vesicles secreted by cells into the extracellular space; it is well-known that EVs have a pivotal role in cancer disease. Aim. The present work aims to investigate if the increased aggressiveness of RD cells overexpressing CAV1 (RD-CAV1) correlates with an altered extracellular vesicle release and cargo and if RD-EVs contribute to the cancer dissemination. Results. The obtained data show that RD-CAV1 cells release more EVs compared to RD-Ctrl cells. Western Blot analysis highlighted that small extracellular vesicles (sEVs) exhibit the exosomal markers Alix, Flot-1, Syntenin-1, and TSG101, whereas large extracellular vesicles (lEVs) are positive for Calnexin. Interestingly, the tetraspanins CD63, CD81, and CD9 were not detectable in RD-CAV1 EVs, which instead are detected in RD-Ctrl EVs, suggesting that CAV1 overexpression induces an alteration of the EVs biogenesis. These data are all supported and extended by the Proteomic Analysis. Moreover, the treatment of HUVEC with RD-CAV1 EVs shows an increase in cell proliferation and migration, both in a dose-dependent manner. Conclusions. Taken together, these data confirm that CAV1 overexpression critically affects RD-EV release and cargo, allowing them to alter the behavior of target cells of the tumor niche.