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. 2012 Apr 20;287(17):13877-88.
doi: 10.1074/jbc.M112.345280. Epub 2012 Feb 24.

Clinicopathological and biological significance of human voltage-gated proton channel Hv1 protein overexpression in breast cancer

Yifan Wang  1 , Shu Jie Li, Xingye Wu, Yongzhe Che, Qiang Li
Affiliations

Clinicopathological and biological significance of human voltage-gated proton channel Hv1 protein overexpression in breast cancer

Yifan Wang et al. J Biol Chem. .

Abstract

In our previous work, we showed for the first time that the voltage-gated proton channel Hv1 is specifically expressed in highly metastatic human breast tumor tissues and cell lines. However, the contribution of Hv1 to breast carcinogenesis is not well known. In this study, we showed that Hv1 expression was significantly correlated with the tumor size (p = 0.001), tumor classification (p = 0.000), lymph node status (p = 0.000), clinical stage (p = 0.000), and Her-2 status (p = 0.045). High Hv1 expression was associated significantly with shorter overall (p = 0.000) and recurrence-free survival (p = 0.000). In vitro, knockdown of Hv1 expression in the highly metastatic MDA-MB-231 cells decreased the cell proliferation and invasiveness, inhibited the cell proton secretion and intracellular pH recovery, and blocked the cell capacity of acidifying extracellular milieu. Furthermore, the gelatinase activity in MDA-MB-231 cells that suppressed Hv1 was reduced. In vivo, the breast tumor size of the implantation of the MDA-MB-231 xenografts in nude mice that were knocked down by Hv1 was dramatically smaller than that in the control groups. The results demonstrated that the inhibition of Hv1 function via knockdown of Hv1 expression can effectively retard the cancer growth and suppress the cancer metastasis by the decrease of proton extrusion and the down-regulation of gelatinase activity. Based on these results, we came to the conclusion that Hv1 is a potential biomarker for prognosis of breast cancer and a potential target for anticancer drugs in breast cancer therapy.

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Figures

FIGURE 1.
FIGURE 1.
Up-regulation of voltage-gated proton channel Hv1 protein is illustrated in tissues from patients with breast cancer. Representative immunohistochemical staining of Hv1 is shown (A–I). Tissue samples from patients with breast cancer were stained with anti-Hv1 antibody. Hv1 staining was negative or weak in normal breast (A) and hyperplasia of breast tissues (B and C), but there was moderate or strong Hv1 staining in breast cancer (D–I). The Hv1 is stained in brown, and the background nuclei are in blue. A, B, D, F, and H, 200; C, E, G, and I, 500. A negative control was performed to treat with nonimmune mouse serum as the primary antibody instead of anti-Hv1 antibody.
FIGURE 2.
FIGURE 2.
Kaplan-Meier overall and disease-free survival curves were calculated according to voltage-gated proton channel Hv1 expression levels. A, differences in cumulative overall survival are observed between patients with high and low levels of Hv1 expression. B, differences in cumulative recurrence-free survival are observed between patients with high and low levels of Hv1 expression. p values were obtained using the log-rank test.
FIGURE 3.
FIGURE 3.
Decreased cell proliferation and invasiveness are illustrated in voltage-gated proton channel Hv1-suppressed cells. MDA-MB-231 and MCF-7 cells were infected with Hv1-siRNA lentivirus and nonspecific Hv1-scr control lentivirus, respectively. A and B, quantitative real time PCR analysis was conducted in Hv1-suppressed breast cancer cells. Values are means ± S.D. (n = 3). p < 0.05, compared with normal MDA-MB-231 cells (nor). C, proliferation of Hv1-suppressed cells was decreased. ●, MDA-MB-231 cells infected with Hv1-siRNA lentivirus; ■, MDA-MB-231 cells infected with nonspecific Hv1-scr control lentivirus. Values are means ± S.D. (n = 5). p < 0.05, compared with MDA-MB-231-scr cells. D, invasiveness was significantly inhibited by Hv1-siRNA in the highly (MDA-MB-231) metastatic cells. Values are means ± S.D. (n = 3). p < 0.05, compared with normal MDA-MB-231 cells (nor).
FIGURE 4.
FIGURE 4.
Voltage-gated proton channel Hv1 regulates breast cancer cytosolic pH. A, Hv1 is mainly expressed in plasma membrane in MDA-MB-231 cells. Panel a, observation for FITC fluorescence (green); panel b, DAPI stain to visualize the nuclei (blue); panel c, images merged the fluorescence of FITC and DAPI. B, H+ channel activity of Hv1. The proton secretion activity of Hv1 in breast cancer cells was inhibited by down-regulation Hv1 expression. Panel a, membrane depolarization induces proton extrusion in MDA-MB-231 cells; panel b, inhibition of Hv1 expression by Hv1-siRNA notably suppresses the proton extrusion. The arrows in panels a and b indicate the time when the high K+ solution was added into the culture dish, without interruptions in the recordings. C, Hv1 regulates intracellular pH of breast cancer cells. Suppression of Hv1 by Hv1-siRNA notably induces a decrease in intracellular pH in the highly metastatic MDA-MB-231 cells. Values are means ± S.D. (n = 3). p < 0.05, compared with MDA-MB-231 cells (nor) for MDA-MB-231 and MCF-7 cells (nor) for MCF-7. D, highly metastatic MDA-MB-231 cells have a high capacity to acidify the extracellular milieu, whereas down-regulation of Hv1 expression in the cells obviously inhibited acidifying the extracellular milieu. MCF-7 and MDA-MB-231 cells infected with Hv1-siRNA lentivirus and nonspecific Hv1-scr control lentivirus, respectively, were grown at a confluence, and then changed into unbuffered DMEM for 24 h at 37 °C in a water-saturated atmosphere devoid of CO2. Values are means ± S.D. (n = 3). ▴, MCF7-scr; ●, MCF7-si; ■, MDA-MB-231-scr; ▾, MDA-MB-231-si. E, activity of gelatinase was apparently reduced in the supernatant of MDA-MB-231 cells suppressed Hv1 by Hv1-siRNA compared with MDA-MB-231 cells nonsuppressed Hv1.
FIGURE 5.
FIGURE 5.
Down-regulation of Hv1 expression reduces the rate of xenograft tumor growth. A and B, average size of xenografts in MDA-MB-231-si group was dramatically smaller than that of the MDA-MB-231-scr group. ●, MDA-MB-231 cells infected with Hv1-siRNA lentivirus; ■, MDA-MB-231 cells infected with nonspecific Hv1-scr control lentivirus. C, immunohistochemical analysis of tumor sections from mice injected with MDA-MB-231-si and MDA-MB-231-scr by H&E, Ki-67, and Hv1 staining.
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