V‐ATPase‐mediated activation of cathepsins in breast cancer cells: investigating the role of vacuolar H+‐ATPases in breast cancer metastasis (934.8)

Abstract

V‐ATPases are multi‐subunit H+ pumps that acidify intracellular compartments and are essential for bone degradation synaptic signaling, etc. They are at the cell surface of the highly metastatic breast cancer cell line MDA‐MB231, and may contribute to its metastatic phenotype. We hypothesize that V‐ATPases create a low pH environment that promotes tumor cell invasion by activating secreted cathepsins. Cathepsins cleave extracellular matrix proteins and activate other proteases, facilitating local tumor invasion. We are studying the role of subunit the V‐ATPase in secretion and activation of cathepsins B and L. Inhibition of V‐ATPases with concanamycin A (a general V‐ATPase inhibitor) leads to a large reduction in the activity of intracellular cathepsin B. In addition, inhibition of V‐ATPases leads to a large reduction in the activity of extracellular cathepsin L. Thus it seems that V‐ATPases are playing a role in activation of both intracellular and extracellular cathepsins. It is known that V‐ATPases are required for activation of proteases such as cathepsins within lysosomes, however, not as much is known about the activation of secreted cathepsins. Our findings support the hypothesis that cell surface V‐ATPases are important for activation of cathepsins in the extracellular space. Since concanamycin can penetrate the cell membrane we cannot rule out the alternative hypothesis that V‐ATPases within the cell are activating pools of cathepsin L within secretory vesicles. Further experiments with immunoblotting and cathepsin activity assays are being used to differentiate between the role V‐ATPases are playing in activation of cathepsin L within secretory vesicles versus activation of cathepsin L in the extracellular space. This study increases our understanding of the role V‐ATPases are playing in the activation of secreted cathepsins in the tumor microenvironment of MB231 cells.