V‐ATPase‐mediated Activation of Cellular and Secreted Cathepsins: Exploring Their Role in Breast Cancer Metastasis

Abstract

Vacuolar H+‐ATPases are essential H+ pumps that acidify cellular compartments. V‐ATPases at the cell surface of MDA‐MB231 breast cancer cells may contribute to a highly metastatic phenotype. We hypothesize that cell surface V‐ATPases create an acidic microenvironment that activates secreted cathepsins, which leads to cleavage of extracellular matrix, facilitating tumor cell invasion. Less is known about the activation of secreted cathepsins thus we looked into the role of V‐ATPases in secretion and activation of cathepsins B and L (CatB and CatL). Cathepsin activity was measured using a substrate which fluoresces when cleaved. The assays were performed + or ‐ cathepsin‐specific inhibitors. Activity levels were corroborated by immunoblotting for each cathepsin in cell lysates and conditioned media. CatB activity is much higher than CatL inside the cell and CatL activity was much higher extracellularly. Inhibition of V‐ATPases with concanamycin A (a general V‐ATPase inhibitor) leads to a large reduction in the intracellular activity of both CatB and CatL. In conditioned media, inhibition of V‐ATPases leads to a large reduction in CatB activity and a modest reduction in CatL activity. From our results it appears that V‐ATPases are playing a role in activation of both intracellular and secreted cathepsins. These results provide evidence that cell surface V‐ATPases play a role in activation of secreted cathepsins. However, there may be specificity in which secreted cathepsins they act on. Concanamycin can penetrate the cell membrane so we cannot rule out that intracellular V‐ATPases are activating CatB and CatL in secretory vesicles. Experiments with subunit “a” isoform‐specific siRNAs will help define the role that intra‐ and extracellular V‐ATPases play in activation of secreted CatB and CatL.