Inhibition of cholesterol transport impairs Cav-1 trafficking and small extracellular vesicles secretion, promoting amphisome formation in melanoma cells

Caveolin-1 (Cav-1) is a fundamental constituent of caveolae, whose functionality and structure are strictly dependent on cholesterol. In this work the U18666A inhibitor was used to study the role of cholesterol transport in the endosomal degradative-secretory system in a metastatic human melanoma cell line (WM266-4). We found that U18666A induces a shift of Cav-1 from the plasma membrane to the endolysosomal compartment, which is involved, through Multi Vesicular Bodies (MVBs), in the formation and release of small extracellular vesicles (sEVs). Moreover, this inhibitor induces an increase in the production of sEVs with chemical-physical characteristics similar to control sEVs but with a different protein composition (lower expression of Cav-1 and increase of LC3II) and reduced transfer capacity on target cells. Furthermore, we determined that U18666A affects mitochondrial function and also cancer cell aggressive features, such as migration and invasion. Taken together, these results indicate that the blockage of cholesterol transport, determining the internalization of Cav-1, may modify sEVs secretory pathways through an increased fusion between autophagosomes and MVBs to form amphisome, which in turn fuses with the plasma membrane releasing a heterogeneous population of sEVs to maintain homeostasis and ensure correct cellular functionality.

Automated summary

In this study, the role of cholesterol transport in the endosomal degradative-secretory system was explored using the U18666A inhibitor in a metastatic human melanoma cell line (WM266-4). The results showed that U18666A induced a shift of Cav-1 from the plasma membrane to the endolysosomal compartment, affecting the formation and release of small extracellular vesicles (sEVs). The inhibitor also altered the protein composition of sEVs and reduced their transfer capacity on target cells.