Simultaneously targeting extracellular vesicle trafficking and TGF-β receptor kinase activity blocks signaling hyperactivation and metastasis

Metastasis is the leading cause of cancer-related deaths.Transforming growth factor beta(TGF-β)signaling drives metastasis and is strongly enhanced during cancer progression.Yet,the use of on-target TGF-βsignaling inhibitors in the treatment of cancer patients remains unsuccessful,highlighting a gap in the understanding of TGF-βbiology that limits the establishment of efficient anti-metastatic therapies.Here,we show that TGF-βsignaling hyperactivation in breast cancer cells is required for metastasis and relies on increased small extracellular vesicle(sEV)secretion.Demonstrating sEV’s unique role,TGF-βsignaling levels induced by sEVs exceed the activity of matching concentrations of soluble ligand TGF-β.Further,genetic disruption of sEV secretion in highlymetastatic breast cancer cells impairs cancer cell aggressiveness by reducing TGF-βsignaling to nearly-normal levels.Otherwise,TGF-βsignaling activity in non-invasive breast cancer cells is inherently low,but can be amplified by sEVs,enabling invasion and metastasis of poorly-metastatic breast cancer cells.Underscoring the translational potential of inhibiting sEV trafficking in advanced breast cancers,treatment with dimethyl amiloride(DMA)decreases sEV secretion,TGF-βsignaling activity,and breast cancer progression in vivo.Targeting both the sEV trafficking and TGF-βsignaling by combining DMA and SB431542 at suboptimal doses potentiated this effect,normalizing the TGF-βsignaling in primary tumors to potently reduce circulating tumor cells,metastasis,and tumor self-seeding.Collectively,this study establishes sEVs as critical elements in TGF-βbiology,demonstrating the feasibility of inhibiting sEV trafficking as a new therapeutic approach to impair metastasis by normalizing TGF-βsignaling levels in breast cancer cells.