RAB4A acts as a negative feedback regulator of extracellular vesicle secretion during TGF ‐β signaling

Extracellular vesicles (EVs) secreted by cancer cells actively modulate the tumor microenvironment, thereby promoting cancer progression. Transforming growth factor-β (TGF-β) signaling has been implicated in the regulation of EV biogenesis, yet the molecular mechanisms underlying this process have only recently begun to emerge. In this study, we investigated TGF-β-responsive mediators that regulate EV release in lung, breast, and ovarian carcinoma cells by modulating the expression and activity of genes associated with EV biogenesis, including components of the ESCRT machinery, tetraspanins, and Rab GTPases. We found that TGF-β selectively enhances the mRNA expression of PDCD6IP (ALIX), CD81, ARF6, and RAB4A in a cell type-specific manner. R-SMAD silencing had clear negative effects on the regulation of ALIX or RAB4A, whereas AKT kinase inhibition suppressed the induction of ALIX and CD81. Additionally, TGF-β stimulation increased ALIX S-palmitoylation, consistent with enhanced ALIX-TSG101 complex formation on vesicular membranes. However, knockdown of ALIX or CD81 did not impair TGF-β-induced EV secretion. On the contrary, TGF-β-induced upregulation of RAB4A expression is functionally unique because RAB4A facilitates fast endosomal recycling, a process that limits EV release. Accordingly, silencing RAB4A significantly increased the fusion of multivesicular bodies with the plasma membrane followed by EV secretion, suggesting that TGF-β-induced RAB4A acts as a negative feedback regulator of EV release. Our findings reveal a novel mechanism by which RAB4A modulates TGF-β-driven EV production by cancer cells.