V‐ATPase‐mediated secretion and activation of cathepsins in breast cancer cells: investigating the role of vacuolar H + ‐ATPases subunit A isoforms in breast cancer metastasis (934.4)

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 a isoforms in secretion and activation of cathepsin B and L. The 4 subunit a isoforms are responsible for cellular localization of V‐ATPases. Treatment with concanamycin A (a general V‐ATPase inhibitor) affects the activity of intracellular and secreted cathepsins B and L. Concanamycin A treamtment also affects the secretion of cathepsins B and L. siRNAs were used to reduce mRNA expression of each subunit a isoform. We have previously shown that knockdown of the a4 isoform decreased the level of processed cathepsin L in conditioned media, but not the unprocessed form. Our previous findings also indicate a4 targets V‐ATPases to the cell surface, thus we hypothesize that the a4 isoform will have the largest effect on secretion and activation of cathepsin B and/or L. Following isoform‐specific knockdown, immunoblotting and cathepsin activity assays are being used to analyze cathepsin B and L expression and activity in conditioned media and cell lysates of MB231 cells. 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. Grant Funding Source : Funded by Denison University Anderson Fellowship