Caveolin-1-overexpression induces extracellular vesicle secretion in a model of rhabdomyosarcoma

Caveolin-1 (CAV-1) is an integral membrane protein required to generate caveolae and cholesterol-enriched lipid rafts of the plasma membrane. Moreover, CAV-1 binds to many proteins, controls cholesterol homeostasis, and regulates various cell functions. CAV-1 has a controversial role in cancer, it is widely accepted that loss of CAV-1 correlates with early-stage tumor progression, while its over-expression and phosphorylation are associated with recurrence and metastatic disease. For example, it has been shown that CAV-1 cooperates to tumor growth and metastatic potential in rhabdomyosarcoma (RD). A considerable body of evidence suggests the possibility that extracellular CAV-1 may be relevant in cancer cell metastasis. The present work aims to investigate if the increased aggressiveness of RD cells overexpressing CAV-1 correlates with an altered extracellular vesicle (EV) release. EVs were isolated by differential ultracentrifugation and density gradient separation methods from conditioned media of control (RD-Ctrl) and metastatic RD cell lines overexpressing CAV1 (RD-CAV1). Collected small (sEVs) and large (lEVs) extracellular vesicles were characterized by NanoparticleTracking Analysis (NTA), transmission electron microscopy (TEM) and western blot analysis (WB). The applied ultracentrifugation protocol allowed the isolation of two EV subpopulations, called sEVs and lEVs, of about 93 nm and 170 nm in diameter, respectively. The obtained data showed that RD-CAV1 cells release 3-4 fold more EVs compared to control cells; however, NTA revealed that CAV-1 overexpression seems not to affect the sEV versus lEV production ratio with sEVs about 10 times more abundant than lEVs in both RD cell models. The efficient separation between sEVs and lEVs was supported by western blot analysis: small vesicles exhibit the exosomal markers Alix, Flot-1, Syntenin-1, and TSG101, whereas large EVs were positive for Calnexin, an endoplasmic reticulum marker. Interestingly, RD-CAV1 EVs do not express the tetraspanins CD63, CD81 and CD9, which instead were detected in RD-Ctrl EVs, suggesting that CAV-1 over-expression induces an alteration of the EV biogenesis. Proteomic analysis confirmed the absence of tetraspanins in the sEVs released from RD-CAV-1 and showed that the lack of tetraspanin is associated with a significant decrease in EV protein loading; in fact, we identified about 1000 different proteins in RD-Ctrl sEVs, whereas only 547 proteins were detectable in RD-CAV-1 sEVs. The reported data suggest that, in addition to its well-established structural role, CAV-1 is a key regulatory factor potentially involved in the remodelling of the tumor microenvironment by stimulating the release of EVs deeply altered in protein composition.