Effects of Vacuolar H+‐ATPase Inhibition and the a4 Isoforms on Secretion and Activation of Cathepsin D in MDA‐MB231 Breast Cancer Cells

Abstract

V‐ATPases are multi‐subunit H+ pumps that acidify intracellular compartments and are important for acid‐base balance in the blood and sperm maturation. They are at the cell surface of the highly metastatic breast cancer cell line MDA‐MB231 and may contribute to its metastatic phenotype. V‐ATPases are hypothesized to create a localized low pH environment that promotes tumor cell invasion by activating secreted cathepsins. Studies indicate that cathepsins secreted by cancer cells are involved in metastasis by cleaving extracellular matrix proteins and activating other proteases, which facilitates 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 showed a trend of reducing the activity of secreted cathepsin B, but not cathepsin L. However, that trend, was not statistically significant. Cathepsin D is another protease that is secreted by cancer cells including metastatic breast cancer. For this study we looked at the activity of cathepsin D following inhibition of V‐ATPases. Our preliminary data indicates that inhibition of V‐ATPases reduces the activity of secreted cathepsin D. To investigate the role of V‐ATPases in the activation of cathepsins further we are using CRISPR/Cas9 to knockout the gene for the a4 isoform of the V‐ATPase “a” subunit. Published data indicates that the a3 and a4 isoforms are found in V‐ATPases at the cell surface of MDA‐MB231 cells. We hypothesize that the a4 isoform knockout cells will have reduced activation of secreted cathepsin D while not effecting activation of cathepsin L. Separate abstracts submitted by our lab are focused on the a1, a2 and a3 isoform knockouts.Support or Funding InformationAnderson Summer Research Assistantship