Potassium channel activity controls breast cancer metastasis by affecting β-catenin signaling

Abstract

Potassium ion channels are critical in the regulation of cell motility. The acquisition of cell motility is an essential parameter of cancer metastasis. However, the role of K⁺ channels in cancer metastasis has been poorly studied. High expression of the hG1 gene, which encodes for Kv11.1 channel associates with good prognosis in estrogen receptor-negative breast cancer (BC). We evaluated the efficacy of the Kv11.1 activator NS1643 in arresting metastasis in a triple negative breast cancer (TNBC) mouse model. NS1643 significantly reduces the metastatic spread of breast tumors in vivo by inhibiting cell motility, reprogramming epithelial-mesenchymal transition via attenuation of Wnt/β-catenin signaling and suppressing cancer cell stemness. Our findings provide important information regarding the clinical relevance of potassium ion channel expression in breast tumors and the mechanisms by which potassium channel activity can modulate tumor biology. Findings suggest that Kv11.1 activators may represent a novel therapeutic approach for the treatment of metastatic estrogen receptor-negative BC. Ion channels are critical factor for cell motility but little is known about their role in metastasis. Stimulation of the Kv11.1 channel suppress the metastatic phenotype in TNBC. This work could represent a paradigm-shifting approach to reducing mortality by targeting a pathway that is central to the development of metastases.

Fig. 1. Kv11.1 stimulation inhibits primary tumor growth and metastasis in\ a xenograft model of breast cancer.

MDA-MB-231 cells were injected subcutaneously into the dorsal flank of NSG mice. When tumors were palpable, the mice were injected intraperitoneally with vehicle alone or Kv11.1 activator NS1643 at 6 mg/kg every 2 days. a Mean tumor volume in mice treated with either vehicle control (n = 12) or Kv11.1 activator (NS1643) (n = 12). b Barchart showing the absolute frequencies of liver macrometastases observed in mice treated with either vehicle control or NS1643. c Representative images of livers demonstrating macrometastases in mice treated with either vehicle control or NS1643. d Representative confocal images of human nuclear antigen (HNA; red) and nuclei (DAPI; blue) expression in liver tissue sections from mice treated with either vehicle control or NS1643. e Representative western blot images of HNA expression in liver and (f) lung homogenates from mice treated with either vehicle control or NS1643. Quantification of the levels of HNA protein was done using NIH ImageJ software. Data is expressed as mean ± SEM; *p < 0.05; **p < 0.001)

Fig. 2. Kv11.1 stimulation inhibits breast cancer cell motility.

a Representative images are showing the effect of Kv11.1 stimulation on breast cancer cell motility in wound healing scratch assays. Cells were treated with indicated concentrations of NS1643 (50 μm; n = 5; Dotted lines align with the center of the control border cell cluster). b Single-cell tracking of untreated control cells, vehicle control cells (DMSO) and NS1643-treated cells. Bar graphs show the effect of NS1643 on wound closure. Data are mean values ± S.E; *p < 0.01 or **p < 0.05 from WT and on cell speed. Data are mean values ± S.E; *p < 0.0001 or **p < 0.0001 from WT. c Representative images of the effect of Kv11.1 activator on breast cancer cell migration and invasion. Transwell migration and invasion assays were carried out. Student’s t test is used to compare two samples and analysis of variance with multiple testing corrections should be performed for comparing three or more groups of data. A P value < 0.05 is used to define statistical significance

Fig. 3. Kv11.1 stimulation inhibits reprograms EMT.

a Representative western blot images of EMT markers (N-cadherin, Vimentin, CD44, and E-cadherin) in MDA-MB-231, SKBR3, and MCF7 breast cancer cells treated with either vehicle control or NS1643. Cells were treated with 50 μm of NS1643 for 24 h, harvested and subjected to western blot analysis. Actin was used as a loading control. Quantification of the levels of Vimentin, CD44, N-Cadherin, and E-cadherin was done using NIH ImageJ software. Data are expressed as mean ± SEM; *p < 0.005; **p < 0.001; n = 3). b Representative western blot images of EMT markers, Vimentin and CD44, in MDA-MB-231-derived tumors from mice treated with either vehicle control or NS1643. c The mRNA levels of Snail, Slug, Twist, and Zeb were represented relative to β-actin transcripts. Cells were treated with 50 μm of NS1643 for 24 h, harvested, and subjected to qRT-PCR analysis. Data are expressed as mean ± SD of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.00005

Fig. 4. Kv11.1 stimulation suppresses the stemness of breast cancer cells.

a Representative images are showing the effect of Kv11.1 stimulation on mammosphere forming efficiency (MFE) in SKBR3 and MCF7. Cells were seeded into Ultra-low attachment 6-well plate under DMSO or 50 μm of NS1643 for 7 days. b The mRNA levels of Oct4, Nanog, and Sox were represented relative to β-actin transcripts. Cells were treated with 50 μm of NS1643 for 24 h, harvested, and subjected to qRT-PCR analysis. Data are expressed as mean ± SD of three independent experiments. *p < 0.01; **p < 0.05; ***p < 0.0005

Fig. 5. Kv11.1 stimulation regulates the cellular localization of β-catenin.

a Representative western blot images are demonstrating subcellular localization of β-catenin. Cells were treated with 50 μm of NS1643 for 24 h, harvested, and subjected to cellular fractionation (nuclear and membrane fraction). Quantification of the levels of β-catenin protein was done using NIH ImageJ software. *p < 0.05; **p < 0.005; ***p < 0.001 b β-catenin TCF/LEF promoter activities in control group, NS1643-treated group, Wnt3a-treated group (positive control) and NS1643 + Wnt3a-treated group were measured using TopFlash and FopFlash vectors

Fig. 6. Kv11.1 controls β-catenin nuclear translocation signaling.

a Representative western blot images of phospho-AKT^T308, phospho-β-catenin^S552, and total AKT levels in cells or b tumors extracted from mice treated with control or NS1643. Cells were treated with 50 μm of NS1643 for 24 h, harvested, and subjected to western blot analysis. Actin was used as a loading control. Quantification of the levels of phospho-AKT and phospho-β-catenin were done using NIH ImageJ software. c Representative western blot images of phospho-GSK3⍰Ser9 and total GSK3⍰ levels in MDA-MB-231. d Representative western blot images of β-catenin levels in cells treated with cycloheximide alone or NS1643 for the indicated time and e quantification of the effects (n = 3). Quantification of the levels of β-catenin and phospho-β-catenin were done using NIH ImageJ software. f Confocal microscopy images are representing the accumulation of β-catenin at the surface membrane in MDA-MD-231 cells treated with control vehicle or NS1643 for 24 h. g Graph showing the effect of NS1643 on cell–cell contact measured by Dispase assay (n = 6; mean ± SD; *P < 0.05)

Fig. 7. KCNH2 expression and survival in patients with breast cancer.

Kaplan–Meier plots of relapse-free survival a, b and overall survival c, d, according to the level of KCNH2 gene expression (top vs. bottom tertiles). Results are presented for all breast cancer patients a, c and the estrogen receptor-negative subgroup b, d. Hazard ratios (HR) compare the hazard of relapse or death in the high expression vs. low expression groups

Fig. 8. Schematic representation of the antimetastatic signaling promoted by activation of Kv11.1.

1) In normal epithelial cells, β-catenin is in complex with adhesive proteins (e.g., E-cadherin) and promotes cell–cell contact. GSK3⍰-dependent phosphorylation controls β-catenin degradation while AKT-dependent phosphorylation controls β-catenin nuclear translocation. 2) In cancer cells, the Wnt-dependent signaling arrests β-catenin degradation by inhibiting GSK3β activity. Consequently, upon AKT phosphorylation β-catenin translocates to the nucleus and transcribes for genes controlling epithelial to mesenchymal transition (EMT) and migration. 3) Activation of the Kv11.1 promotes epithelial phenotype in mesenchymal cancer cells (MET) by inhibiting GSK3β-dependent β-catenin degradation and AKT-dependent nuclear translocation of β-catenin, producing β-catenin accumulation at the surface membrane and therefore, improving cell–cell contact