Isoform-specific gene disruptions reveal a role for the V-ATPase subunit a4 isoform in the invasiveness of 4T1-12B breast cancer cells

J Biol Chem. 2019 Jul 19;294(29):11248-11258. doi: 10.1074/jbc.RA119.007713. Epub 2019 Jun 5.

Abstract

The vacuolar H+-ATPase (V-ATPase) is an ATP-driven proton pump present in various intracellular membranes and at the plasma membrane of specialized cell types. Previous work has reported that plasma membrane V-ATPases are key players in breast cancer cell invasiveness. The two subunit a-isoforms known to target the V-ATPase to the plasma membrane are a3 and a4, and expression of a3 has been shown to correlate with plasma membrane localization of the V-ATPase in various invasive human breast cancer cell lines. Here we analyzed the role of subunit a-isoforms in the invasive mouse breast cancer cell line, 4T1-12B. Quantitation of mRNA levels for each isoform by quantitative RT-PCR revealed that a4 is the dominant isoform expressed in these cells. Using a CRISPR/Cas9-based approach to disrupt the genes encoding each of the four V-ATPase subunit a-isoforms, we found that ablation of only the a4-encoding gene significantly inhibits invasion and migration of 4T1-12B cells. Additionally, cells with disrupted a4 exhibited reduced V-ATPase expression at the leading edge, suggesting that the a4 isoform is primarily responsible for targeting the V-ATPase to the plasma membrane in 4T1-12B cells. These findings suggest that different subunit a-isoforms may direct V-ATPases to the plasma membrane of different invasive breast cancer cell lines. They further suggest that expression of V-ATPases at the cell surface is the primary factor that promotes an invasive cancer cell phenotype.

Keywords: a-subunit isoforms; breast cancer; cell invasion; cell migration; metastasis; plasma membrane; protein trafficking; proton pump; vacuolar ATPase.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Breast Neoplasms / enzymology*
  • Breast Neoplasms / genetics
  • Breast Neoplasms / pathology
  • CRISPR-Cas Systems
  • Cell Line, Tumor
  • Cell Membrane / enzymology
  • Enzyme Inhibitors / pharmacology
  • Female
  • Humans
  • Isoenzymes / antagonists & inhibitors
  • Isoenzymes / genetics
  • Isoenzymes / metabolism*
  • Neoplasm Invasiveness*
  • Neoplasm Metastasis
  • RNA, Messenger / genetics
  • Vacuolar Proton-Translocating ATPases / antagonists & inhibitors
  • Vacuolar Proton-Translocating ATPases / genetics
  • Vacuolar Proton-Translocating ATPases / metabolism*

Substances

  • Enzyme Inhibitors
  • Isoenzymes
  • RNA, Messenger
  • Vacuolar Proton-Translocating ATPases