Expression and function of voltage gated proton channels (H(v)1) 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.