Effects of Vacuolar H+‐ATPase Inhibition and the a1 and a3 Isoforms on Secretion and Activation of Cathepsins in MDA‐MB231 Breast Cancer Cells

Abstract

V‐ATPases are multi‐subunit H+ pumps that acidify intracellular compartments and are essential for bone degradation and synaptic signaling. 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 in a localized environment which promotes tumor cell invasion by activating secreted cathepsins. Studies indicate that cathepsins secreted by cancer cells cleave extracellular matrix proteins and activate other proteases, facilitating tumor cell invasion. Our lab is investigating a connection between cell surface V‐ATPases, activation of secreted cathepsins and the metastatic phenotype of MDA‐MB231 cells. Previous data from our lab indicates that inhibition of V‐ATPases by a general inhibitor reduced the activity of secreted cathepsin B, but not cathepsin L. However, although there was a trend, the difference in activity for secreted cathepsin B after V‐ATPase inhibition was not statistically significant. Our current data supports the conclusion that there is a significant difference in the effects of V‐ATPase inhibition on secreted cathepsins. Inhibiting V‐ATPases does not appear to affect the activity of secreted cathepsin L, however it reduces the activity of secreted cathepsins B and D To investigate the role of V‐ATPases in the activation of cathepsins further we are using CRISPR/Cas9 to knockout the gene for two of the four V‐ATPase “a” subunit isoforms, a1 and a3. Published data indicates that the a3 and a4 isoforms are found in V‐ATPases at the cell surface of MDA‐MB231 cells. The a1 isoform is found in intracellular compartments. We hypothesize that the a3 isoform knockout cells will have reduced activation of secreted cathepsins B and D while not effecting activation of cathepsin L. We predict that the a1 isoform knockout will affected the activity of intracellular cathepsins, but not affect the activity of secreted cathepsin B, D or L. Separate abstracts submitted by our lab are focused on the a1 and a3 isoform knockouts.Support or Funding InformationDenison University Anderson AssistantshipDenison University Research Fund