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

Expression of the voltage gated proton channel (H v 1) as identified by immunocytochemistry has been reported previously in breast cancer tissue. Increased expression of H V 1 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 H V 1 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 H V 1 expression in 3 out of 8 “triple negative” breast cancer cell lines (estrogen, progesterone, and HER2 receptor expression negative). We examine the function of H V 1 in breast cancer using MDA-MB-231 cells as a model by suppressing the expression of H V 1 using shRNA (knock-down; KD) and by eliminating H V 1 using CRISPR/Cas9 gene editing (knock-out; KO). Surprisingly, these two approaches produced incongruous effects. Knock-down of H V 1 using shRNA resulted in slower cell migration in a scratch assay and a significant reduction in H 2 O 2 release. In contrast, H V 1 Knock-out cells did not show reduced migration or H 2 O 2 release. H V 1 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 H V 1 KO cells. When we compared MDA-MB-231 xenograft growth rates in immunocompromised mice, tumors from H V 1 KO cells grew less than WT in mass, with lower staining for the Ki-67 marker for cell proliferation rate. Therefore, deletion of H V 1 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 H V 1 KO and KD affect certain cellular mechanisms differently, both implicate H V 1-mediated pathways for control of tumor growth in the MDA-MB-231 cell line.