Effects of Vacuolar H+‐ATPase Inhibition and the a2 Isoform on Secretion and Activation of Cathepsin B in MDA‐MB231 Breast Cancer Cells

Abstract

V‐ATPases are multi‐subunit H+ pumps that acidify intracellular compartments and are important for immune functionality, synaptic signaling, and other processes. 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. Studies indicate that cathepsins secreted by cancer cells are involved in metastasis. Cathepsins cleave extracellular matrix proteins and activate other proteases, facilitating tumor invasion. We are interested in 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 did not cathepsin L. However, although there was a trend, difference in activity for secreted cathepsin B after V‐ATPase inhibition was not statistically significant. Our current data builds on that trend to show a significant difference in the activity of secreted cathepsin B. To investigate the role of V‐ATPases in the activation of cathepsins further we are using CRISPR/Cas9 to knockout the gene for the V‐ATPase a2 isoform of subunit “a”. Published data indicates that the a3 and a4 isoforms are found in V‐ATPases at the cell surface of MDA‐MB231 cells. Additionally, it was found that the a1 and a2 isoforms are not present on the cell surface, thus we hypothesize that the a2 isoform knockout will not affect the activity of secreted cathepsin B, but will affect the activity of cathepsin B in the cell interior. Separate abstracts submitted by our lab are focused on the a1, a3, and a4 isoform knockouts.Support or Funding InformationDenison University Anderson Summer Research Assistantship