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. 2017 Apr 18;114(16):4159-4164.
doi: 10.1073/pnas.1702913114. Epub 2017 Apr 3.

Bidirectional KCNQ1:β-catenin Interaction Drives Colorectal Cancer Cell Differentiation

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Free PMC article

Bidirectional KCNQ1:β-catenin Interaction Drives Colorectal Cancer Cell Differentiation

Raphael Rapetti-Mauss et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

The K+ channel KCNQ1 has been proposed as a tumor suppressor in colorectal cancer (CRC). We investigated the molecular mechanisms regulating KCNQ1:β-catenin bidirectional interactions and their effects on CRC differentiation, proliferation, and invasion. Molecular and pharmacologic approaches were used to determine the influence of KCNQ1 expression on the Wnt/β-catenin signaling and epithelial-to-mesenchymal transition (EMT) in human CRC cell lines of varying stages of differentiation. The expression of KCNQ1 was lost with increasing mesenchymal phenotype in poorly differentiated CRC cell lines as a consequence of repression of the KCNQ1 promoter by β-catenin:T-cell factor (TCF)-4. In well-differentiated epithelial CRC cell lines, KCNQ1 was localized to the plasma membrane in a complex with β-catenin and E-cadherin. The colocalization of KCNQ1 with adherens junction proteins was lost with increasing EMT phenotype. ShRNA knock-down of KCNQ1 caused a relocalization of β-catenin from the plasma membrane and a loss of epithelial phenotype in CRC spheroids. Overexpression of KCNQ1 trapped β-catenin at the plasma membrane, induced a patent lumen in CRC spheroids, and slowed CRC cell invasion. The KCNQ1 ion channel inhibitor chromanol 293B caused membrane depolarization, redistribution of β-catenin into the cytosol, and a reduced transepithelial electrical resistance, and stimulated CRC cell proliferation. Analysis of human primary CRC tumor patient databases showed a positive correlation between KCNQ1:KCNE3 channel complex expression and disease-free survival. We conclude that the KCNQ1 ion channel is a target gene and regulator of the Wnt/β-catenin pathway, and its repression leads to CRC cell proliferation, EMT, and tumorigenesis.

Keywords: KCNQ1; adherens junctions; colon cancer; epithelial–mesenchymal transition; β-catenin.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
KCNQ1:β-catenin interactions and EMT phenotype. (A) Western blotting analysis of KCNQ1, E- cadherin, and N-cadherin protein expression in a panel of CRC cell lines of varying EMT differentiation state. β-actin was used as a loading control; images are representative of three experiments. (B) Quantitative expression of KCNQ1 mRNA determined by qPCR showing KCNQ1 expression in CRC cells. Data were normalized to GAPDH (n = 3). (C) Subcellular distribution of KCNQ1 (green) and β-catenin (red) imaged by confocal immunofluorescence microscopy. (Scale bars, 10 μm.) n = 3 in CRC cells. (D) In isolated rat colonic crypts, n = 20 crypts form 5 rats (F-actin in magenta). Merge images indicate colocalization between KCNQ1 and β-catenin staining (yellow).
Fig. 2.
Fig. 2.
β-catenin:TCF4 transcriptional repression of KCNQ1. (A) Western blot and densitometry analysis of KCNQ1 expression in HT29cl.19A cells after treatment with two GSK3-β inhibitors AR-A014418 (AR-A 20 µM) or GSK3-inhibitor X (GSK3-iX 5 µM) (n = 5; ***P < 0.001). (B) ChIP assay revealed the physical interaction of β-catenin and (C) TCF4 with the KCNQ1 promoter after pharmacologic activation of β-catenin in SW480 (n = 3; *P < 0.05). (D) Luciferase assay showing transcriptional activity at the KCNQ1 promoter after pharmacologic activation of β-catenin by GSK3-iX (40 nM) in transfected CHO cells (n = 5; **P < 0.01). (E) Western blot and densitometry analysis of KCNQ1 expression in DLD-1 and (F) SW480 cell lines, transfected for 48 h with a plasmid carrying a dominant negative TCF4 (h∆N-TCF4). C-myc-tag antibody was used to monitor hΔN-TCF4 expression. Bar graphs show expression of KCNQ1 in empty vector (EV: pcDNA3.1) and h∆N-TCF4 transfected cells (n = 4; **P < 0.01).
Fig. 3.
Fig. 3.
KCNQ1:β-Catenin and E-cadherin interactions at the plasma membrane. (A) PLA showing interaction (red signal) between KCNQ1 and β-catenin in isolated rat colonic crypts (12 crypts from 3 rats). The negative control lacking primary antibodies is shown. Nuclear DAPI staining is in blue. (Scale bars, 10 µm.) (B) PLA showing interaction (red signal) between KCNQ1 and β-catenin and also between KCNQ1 and E-Cadherin in HT29cl.19A cells. Nuclear DAPI staining is in blue (n = 3). (Scale bars, 15 μm.) (C) PLA showing interaction between β-catenin and E-cadherin in HT29cl.19A stably expressing a nontargeting ShRNA (ShRD) or a ShRNA targeting KCNQ1 mRNA (ShQ1-1). (Scale bars, 15 µm.) PLA dots quantification is shown on the bar graph (n = 3; *P < 0.05). (D) TEER in ShRNA KCNQ1 (ShQ1-1; ShQ1-2) HT29cl.19A monolayers (n = 6, ***P < 0.001). Insert shows the protein expression of KCNQ1 by Western blot analysis. (E) Western blot and densitometry analysis of different phosphorylated β-catenin residues in HT29cl.19A KCNQ1 knockdown cells (n = 3–5. *P < 0.05; **P < 0.01; ***P < 0.001).
Fig. 4.
Fig. 4.
KCNQ1 expression and epithelial cell phenotype (A) Western blot and densitometry analysis showing the effect of the molecular silencing of KCNQ1 on the expression of epithelial and mesenchymal markers in HT29cl.19A control cells (ShRD) or in HT29cl.19A ShKCNQ1 (ShQ1-1 and ShQ1-2). Bar graph shows protein expression normalized to tubulin (n = 3–10). *P < 0.05; **P < 0.01; ***P < 0.001. (B) Confocal immunofluorescence images of HT29cl.19A (ShRD) and KCNQ1 knock-down cells spheroids grown in Matrigel showing β-catenin (green), F-actin (red), nuclear staining (DAPI-blue). (Scale bar, 20 μm.) The bar graph shows the frequency of lumen formation in control ShRD and in ShKCNQ1 HT29cl.19A spheroids (n = 13–25; P = 0.000005; Fisher's exact test).
Fig. 5.
Fig. 5.
Overexpression of KCNQ1 in HCT116 promotes epithelial phenotype. (A) Western blot and densitometry analysis of epithelial and mesenchymal protein markers in HCT116 cells transfected with a control empty vector (HCT116) or a KCNQ1 overexpression construct (HCT116-Q1). Bar graph shows protein expression normalized to GAPDH (n = 4; *P < 0.05; **P < 0.01). (B) Confocal immunofluorescence images of HCT116 and HCT116-Q1 spheroids grown in Matrigel showing β-catenin (red), F-actin (magenta), and KCNQ1 (green). (Scale bar, 20 μm.) The bar graph shows the frequency of lumen formation in control HCT116 spheroids and in HCT116 spheroids overexpressing KCNQ1 (n = 16; P = 0.01; Fisher's exact test).
Fig. 6.
Fig. 6.
KCNQ1 ion channel function and β-catenin membrane localization. (A) Confocal immunofluorescence images showing the subcellular distribution of β-catenin (red) in HCT116 control cells or in HCT116 cells stably expressing a KCNQ1 construct (HCT116-Q1). KCNQ1 is shown in green, (n = 3). (B) Membrane resting potential of HCT116 control cells and in HCT116-Q1 cells. KCNQ1 inhibitor chromanol 293B (C293B) was used as a control (n = 6; *P < 0.05). (C) Confocal immunofluorescence images of HT29cl.19A cells showing the subcellular distribution of β-catenin (red) and KCNQ1 (green) after 5 min treatment with high K+ (140 mM) Krebs solution. Images are representative of 3 independent experiments. (D) Confocal immunofluorescence images of HT29cl.19A cells showing the subcellular distribution of β-catenin (red) and KCNQ1 (green) after treatment by the specific KCNQ1 channel blocker C293B (10 μM). Images are representative of 3 independent experiments.
Fig. 7.
Fig. 7.
Correlation of KCNQ1:KCNE3 and TCF4 expression with CRC patient survival. Kaplan-Meier analysis of relapse-free survival from 286 colon cancer patients. KCNQ1 (A) and KCNE3 (B) expression significantly correlated with relapse-free survival (P = 0.022; P = 0.034). Patients with high KCNQ1 and KCNE3 expression had significantly better survival rates than those with low KCNQ1 and KCNE3. (C) Low TCF4 expression significantly correlated with longer relapse-free survival (P = 0.044).

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