Protease activity at invadopodial focal digestive areas is dependent on NHE1-driven acidic pHe

Degradation of the extracellular matrix (ECM) is a critical step of tumor cell invasion and requires protease-dependent proteolysis focalized at the invadopodia where the proteolysis of the ECM takes place. Most of the extracellular proteases belong to serine- or metallo-proteases and the invadopodia is where protease activity is regulated. While recent data looking at global protease activity in the growth medium has reported that their activity and role in invasion is dependent on Na+/H+ exchanger (NHE1)-driven extracellular acidification, there is no data on this aspect at the invadopodia, an open question remains whether this acid pHe activation of proteases in tumor cells occurs preferentially at invadopodia. We have previously reported that the NHE1 is expressed in breast cancer invadopodia and that the NHE1-dependent acidification of the peri-invadopodial space is critical for ECM proteolysis. Here, using, for the first time an in situ zymography analysis, we demonstrate that a concordance between NHE1 activity, extracellular acidification and protease activity at invadopodia to finely regulate ECM digestion. We demonstrate that (i) ECM proteolysis taking place at invadopodia is driven by acidification of the peri-invadopodia microenvironment; (ii) that the proteases have a functional pHe optimum that is acidic; (iii) more than one protease is functioning to digest the ECM at these invadopodial sites of ECM proteolysis and (iv) lowering pHe or inhibiting the NHE1 increases protease secretion while blocking protease activity changes NHE1 expression at the invadopodia.