Stable overexpression of the epithelial sodium channel alpha subunit reduces migration and proliferation in breast cancer cells

Abstract

Purpose: Breast cancer is the most common cancer diagnosed in women worldwide. Ion channels have emerged as novel regulators of cancer cell functions, including proliferation and migration. The epithelial sodium channel (ENaC) has a key role in blood pressure regulation, and ENaC levels affect the characteristics of several types of cancer. In breast cancer, a role for αENaC has not been investigated in migration previously nor the effect of stable overexpression of αENaC on proliferation.

Methods: Correlations of the mRNA levels for the four ENaC subunits and breast cancer survival outcomes were assessed in publicly available data and the association between αENaC and migration-related genes. Three isogenic monoclonal derivatives of MDA-MB-231 breast cancer cell lines were created with stable αENaC overexpression. Migration assays (scratch wound assay and Boyden chamber assays) and a proliferation assay (EdU) were used to determine the effect of αENaC overexpression compared to control MDA-MB-231 cells.

Results: Higher α- or δENaC expression was correlated with improved patient survival. Higher αENaC expression correlated with lower expression of migration-associated genes. Stable overexpression of αENaC in MDA-MB-231 cells resulted in reduced in vitro migration and proliferation of all three clones compared to parental control cells.

Conclusion: Higher αENaC expression correlates with improved patient outcomes, and overexpression in breast cancer cells reduces both cell migration and proliferation. These results highlight the possibility of ENaC as a target for future breast cancer treatments.

Keywords: Breast cancer; Epithelial sodium channel (ENaC); Migration; Proliferation.

Fig. 1

Correlations between ENaC subunits and survival or migration. A-D Survival analysis with the genes of the four subunits of ENaC ((A) SCNN1A: alpha, (B) SCNN1B: beta, (C) SCNN1G: gamma, and (D) SCNN1D: delta). Red denotes high expression of the gene of interest, and black is the low expression. Kaplan–Meier plots created using KM plotter for breast cancer [21, 22]. E Scatter plot showing the correlation between αENaC mRNA level and expression of migration metagene in SCAN-B dataset. Spearman rank method was used to calculate correlation, with the orange line showing the line of best fit

Fig. 2

αENaC protein expression in breast cancer cell lines and stable αENaC-overexpressing clones. A Representative western blot showing protein expression of αENaC and beta-actin as a loading control for T-47D and MDA-MB-231 cell lines. B Semi-quantitative densitometry using ImageJ was undertaken to compare the protein expression of αENaC relative to the loading control of beta-actin. Student’s t test performed ****p < 0.001, N = 5. C Representative Western blot showed an increase in αENaC protein expression in three MDA-MB-231 clones, visualized with anti-αENaC antibody and with beta-actin used as a loading control. Lane 1: control, 2: clone A, 3: clone G, and 4: clone I. D αENaC protein bands were semi-quantified using densitometry using ImageJ, and αENaC bands were normalized to beta-actin. One-way ANOVA was performed with Dunnett’s multiple comparisons test, **p < 0.01, *p < 0.05. Data shown as mean ± SEM. N = 7. E αENaC mRNA expression levels measured by quantitative RT-PCR in control and αENaC-overexpressing clones. One-way ANOVA performed with Dunnett’s multiple comparisons test, ***p < 0.001 **p < 0.01, *p < 0.05, N = 4

Fig. 3

Stable overexpression of αENaC reduces the migration ability of MDA-MB-231 cells. A Representative images of control, A, G, and I clone MDA-MB-231 breast cancer cells at time points 0 h and 24 h. Scale bar 200 μm. The yellow outlines indicate the uncovered area used for quantification using ImageJ. B All clones showed a statistically significant difference at time points 12 and 24 h in the percentage of area of the scratch that remained uncovered, compared to the control. Data are shown as mean ± SEM. Two-way ANOVA with Tukey’s post hoc test, resulted in a statistically significant difference, * for clone A, # clone G, + clone I, * # + p < 0.05, ** ## +  + p < 0.01, N = 4

Fig. 4

Stable αENaC overexpression reduced cell migration in a Boyden Chamber assay. Cells were seeded above a porous membrane in a Boyden chamber and left to migrate for 5 h. A–D Representative images of control, A, G, and I clone MDA-MB-231 breast cancer cells post-staining and fixing after 5-h incubation. Scale bar 200 μm. E Reduction in the number of cells migrated in the 5-h incubation in all three clones compared to the control cells, significant reduction for clones A and I. Eight images were taken per well and each cell type was run with two technical repeats. Images were turned to black (B) and white (W) pixels and the B:W ratio was determined using ImageJ, with the average of the eight images reported. One-way ANOVA with Dunnett’s post hoc test, resulted in a statistically significant difference, *p < 0.05, N = 3, N = 6. Data are shown as mean ± SEM

Fig. 5

Stable αENaC overexpression reduces cell proliferation. MDA-MB-231 clones and control cells were incubated with EdU and prepared for imaging as per the manufacturer’s protocol for the EdU Click Kit. A Example images of control and three αENaC MDA-MB-231 overexpressing clones (A, G, I). Green is EdU (proliferating cells) and blue is the counterstain of DAPI (cell nuclei). Scale bar 50 μm. B Overexpression of αENaC in clones led to a significant reduction of breast cancer cells proliferating. One-way ANOVA with multiple comparisons to control, **p < 0.01, *p < 0.05. Data are shown as mean ± SEM, N = 3