Expression and function of voltage gated proton channels (Hv1) in MDA-MB-231 cells

PLoS One. 2020 May 6;15(5):e0227522. doi: 10.1371/journal.pone.0227522. eCollection 2020.

Abstract

Expression of the voltage gated proton channel (Hv1) as identified by immunocytochemistry has been reported previously in breast cancer tissue. Increased expression of HV1 was correlated with poor prognosis and decreased overall and disease-free survival but the mechanism of its involvement in the disease is unknown. Here we present electrophysiological recordings of HV1 channel activity, confirming its presence and function in the plasma membrane of a breast cancer cell line, MDA-MB-231. With western blotting we identify significant levels of HV1 expression in 3 out of 8 "triple negative" breast cancer cell lines (estrogen, progesterone, and HER2 receptor expression negative). We examine the function of HV1 in breast cancer using MDA-MB-231 cells as a model by suppressing the expression of HV1 using shRNA (knock-down; KD) and by eliminating HV1 using CRISPR/Cas9 gene editing (knock-out; KO). Surprisingly, these two approaches produced incongruous effects. Knock-down of HV1 using shRNA resulted in slower cell migration in a scratch assay and a significant reduction in H2O2 release. In contrast, HV1 Knock-out cells did not show reduced migration or H2O2 release. HV1 KO but not KD cells showed an increased glycolytic rate accompanied by an increase in p-AKT (phospho-AKT, Ser473) activity. The expression of CD171/LCAM-1, an adhesion molecule and prognostic indicator for breast cancer, was reduced in HV1 KO cells. When we compared MDA-MB-231 xenograft growth rates in immunocompromised mice, tumors from HV1 KO cells grew less than WT in mass, with lower staining for the Ki-67 marker for cell proliferation rate. Therefore, deletion of HV1 expression in MDA-MB-231 cells limits tumor growth rate. The limited growth thus appears to be independent of oxidant production by NADPH oxidase molecules and to be mediated by cell adhesion molecules. Although HV1 KO and KD affect certain cellular mechanisms differently, both implicate HV1-mediated pathways for control of tumor growth in the MDA-MB-231 cell line.

Publication types

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

MeSH terms

  • Animals
  • CRISPR-Cas Systems / genetics
  • Cell Movement / genetics
  • Cell Proliferation / genetics*
  • Gene Expression Regulation, Neoplastic / drug effects
  • Gene Knockdown Techniques
  • Gene Knockout Techniques
  • Heterografts
  • Humans
  • Hydrogen Peroxide / pharmacology
  • Immunohistochemistry
  • Ion Channels / genetics*
  • Membrane Proteins / genetics*
  • Mice
  • NADPH Oxidases / genetics
  • RNA, Small Interfering / genetics
  • Triple Negative Breast Neoplasms / genetics*
  • Triple Negative Breast Neoplasms / pathology

Substances

  • HVCN1 protein, human
  • Ion Channels
  • Membrane Proteins
  • RNA, Small Interfering
  • Hydrogen Peroxide
  • NADPH Oxidases