Tejeda-Muñoz N et al. (2024), Na,K-ATPase activity promotes macropinocytosis ...

XB-ART-60701

Biol Open 2024 May 15;135:. doi: 10.1242/bio.060269.

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Na,K-ATPase activity promotes macropinocytosis in colon cancer via Wnt signaling.

Tejeda-Muñoz N , Azbazdar Y , Sosa EA , Monka J , Wei PS , Binder G , Mei KC , Kurmangaliyev YZ , De Robertis EM .

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Recent research has shown that membrane trafficking plays an important role in canonical Wnt signaling through sequestration of the β-catenin destruction complex inside multivesicular bodies (MVBs) and lysosomes. In this study, we introduce Ouabain, an inhibitor of the Na,K-ATPase pump that establishes electric potentials across membranes, as a potent inhibitor of Wnt signaling. We find that Na,K-ATPase levels are elevated in advanced colon carcinoma, that this enzyme is elevated in cancer cells with constitutively activated Wnt pathway and is activated by GSK3 inhibitors that increase macropinocytosis. Ouabain blocks macropinocytosis, which is an essential step in Wnt signaling, probably explaining the strong effects of Ouabain on this pathway. In Xenopus embryos, brief Ouabain treatment at the 32-cell stage, critical for the earliest Wnt signal in development-inhibited brains, could be reversed by treatment with Lithium chloride, a Wnt mimic. Inhibiting membrane trafficking may provide a way of targeting Wnt-driven cancers.

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Species referenced: Xenopus laevis
Genes referenced: atp1a1 calr cd63 mrc1 nodal3 otx2 rax sia1 sox2 wnt3a
GO keywords: embryo development [+]

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	Fig. 1.Na,K-ATPase subunit α is overexpressed in colon cancer. (A) ATP1A1 RNA overexpression is significantly associated with colon adenocarcinoma. Bean plot analyses of ATP1A1 expression in 596 adenocarcinomas and 373 healthy colon samples reveal an approximately threefold difference in expression. The width of the horizontal lines represents the number of samples at each value, while the pink and yellow lines indicate the average ATP1A1 expression in normal colon and adenocarcinoma, respectively. The triple black asterisk indicates a P-value of 2.33×10−23 obtained via a two-tailed Student's t-test. (B) Quantification of the colocalization between Na,K-ATPase, and β-catenin in normal colon and in advanced stages of cancer from human array sections. (C-E″) Immunohistochemistry from B shows strong colocalization between Na,K-ATPase and β-catenin in advanced colon cancers (inset). Error bars denote SEM (n≥3) (**P<0.01). Scale bars: 10 μm. Human array sections were quantified in triplicate fields.
	Fig. 2.The Na,K-ATPase is required for Wnt signaling and its regulation is via GSK3. (A-A‴) 3T3 cells transfected with the stabilized constitutively active forms of β-catenin-GFP show increased levels of Na,K-ATPase compared to untransfected neighbor cells used as a control. Quantification is shown on the right side. (B-B‴) Overexpression of WT-GSK3-GFP (demarcated by a stippled line) in CRC SW480 cells blocks the Na,K-ATPase stabilization by Wnt. (C-C‴) Transfecting DN-GSK3-GFP (DN-GSK3-GFP) in 3T3 cells increases Na,K-ATPase levels. Note that the accumulation is observed in vesicles (indicated by the stippled box). (D) HEK293BR (BAR/Renilla) cells stained with Na,K-ATPase antibody. (E) HEK293BR cells treated with the Na,K-ATPase inhibitor Ouabain (1 µM) overnight showed no changes in Na,K-ATPase levels. (F) Overnight treatment with Wnt3a protein (100 ng/ml from Peprotech) treatment stabilized Na,K-ATPase. (G) Ouabain blocked Na,K-ATPase stabilization due to Wnt3a. (H) Quantification of the fluorescence intensity of the Na,K-ATPase from D-G. (I) Western blot showing that endogenous levels of Na,K-ATPase increase after activating the canonical Wnt pathway. (J) Diagram showing that Na,K-ATPase is positively regulated by Wnt via macropinocytosis in lysosomes. EIPA blocks macropinocytosis; Ouabain blocks the Na,K-ATPase, and lysosomes enhance Wnt. All experiments with cultured cells were biological triplicates. Scale bars: 10 μm. Error bars denote s.e.m. (n≥3) (P<0.01, *P<0.001).
	Fig. 3.Ouabain inhibits β-catenin signaling, macropinocytosis, and cell proliferation. (A) β-catenin transcriptional activity stimulated by Wnt3a was strongly inhibited by Ouabain in HEK293BR cells. (B) Untreated HEK293BR (BAR/Renilla) cells stained with total β-catenin antibody. (C) HEK293BR (BAR/Renilla) cells treated with Ouabain showed no changes in β-catenin levels. (D) LiCl (40 mM) activates the β-catenin transcriptional response as shown in the BAR-luciferase assay. This effect was blocked with Ouabain (1 µM) treatment. (E) The increase of β-catenin levels due to LiCl, which increases macropinocytosis (Albrecht et al., 2020), was blocked by Ouabain. (F) Quantification of the β-catenin levels from B and E. (G) Quantification of the macropinocytosis uptake of TMR-dextran 70 kDa into SW480 cells and its reduction by Ouabain treatment. (H) TMR-dextran 1 (1 mg/ml) uptake into SW480 cells after 1 h of incubation. (I) TMR-dextran uptake after 1 h of incubation is reduced with Ouabain (200 nM) treatment. (J-K) SW480 cells transfected with membrane-GFP display sustained macropinocytic vesicles (arrowhead and inset) that were rapidly blocked within 30 min of Ouabain treatment (still images from Movie 1). (L) Cell proliferation in SW480 cells is decreased by Ouabain treatment (200 nM), only viable cells were scored. All experiments with cultured cells were biological triplicates. Error bars denote SEM (n≥3) (P<0.01, **P<0.0001). Scale bar: 10 μm.
	Movie 1.The Sustained Plasma Membrane Macropinosome Ruffles Characteristic of Cancer Cells with Activating Wnt Pathway Mutation Stop within Minutes of Addition of Ouabain; Related to Fig. 3. SW480 cells have sustained macropinocytosis [5] that involves the uptake of significant extracellular fluid and solutes by forming large vesicles (>0.2 μm) called macropinosomes (arrow) that were reduced rapidly after treatment with Ouabain (200 nM). Since macropinocytosis is required for Wnt signaling [50]–[54], blocking macropinocytosis explains the potent inhibitory effects of Ouabain on canonical Wnt signaling.
	Fig. 4.Ouabain inhibits anterior CNS development in Xenopus embryos. (A-B) Incubation of Xenopus embryos at the 32-cell stage in 10 μM Ouabain for 7 min results in embryos with a ventralized (small head) phenotype. A, anterior; P, posterior. (A′-B′) Ouabain incubation reduced the pan-neural marker sox2 in Xenopus embryos. (A″-B″) Ouabain incubation reduces the forebrain and midbrain marker Otx2 in Xenopus embryos (arrowhead). (A‴-B‴) Ouabain incubation reduces the eye marker Rx2a in Xenopus embryos. The numbers of embryos analyzed were as follows: A: 130, 100%; A′: 138, 97%; five independent experiments; A′: 32, 100%; B′: 36; 97%; A″: 32, 100%; B″: 28, 92%; A‴: 30, 100%; B‴: 27, 93%. Scale bar: 500 μm.
	Fig. 5.Order-of-addition experiment showing that Wnt/β-catenin activation at the 32-cell stage is inhibited by Ouabain and can be rescued by subsequent GSK3 inhibition in Xenopus embryos. (A) Untreated embryo. (B) Ouabain incubation (10 μM in 20% L-15 medium for 7 min at the 32-cell stage) resulted in ventralized embryos. (C) LiCl (300 mM for 7 min) dorsalized embryos resulting in expanded heads and reduced trunks. (D) The dorsalized phenotype due to LiCl treatment was blocked after subsequent incubation with Ouabain. (A′-D′) In situ hybridization of Sox2 expression at the gastrula stage showing reduction of CNS development by Ouabain and its expansion by LiCL in dorsal views. The radial expanded CNS phenotype by treating first with LiCl incubation was inhibited by secondary Ouabain incubation. (A″-D″) Anterior views of the same embryos shown in A′-D′. (E-H) Embryos at the 32-cell stage were treated with Ouabain for 7 min (10 µM in 20% L-15 culture medium without serum), washed twice in 0.1 MMR saline, then immersed in 300 mM LiCl in MMR for an additional 7 min, all within the 32-cell stage. Note that treatment with LiCL secondly restored head structures. (E′-H′) In situ hybridizations of the neural marker Sox2 at the gastrula stage show that the ventralized phenotype due to Ouabain was rescued by subsequent LiCl treatment. (E″-H″) Anterior views from the same embryos are shown in panels E′ to H′. (I-I′) Quantitative RT-PCR (qPCR) for the Wnt target genes Siamois and Xnr3 at blastula stage 9 (about 7 h of development), shows that the phenotypic effects are due to the early activation of the Wnt pathway. The numbers of embryos analyzed are as follows: A: 82, 100%; B: 95, 96%; C: 110, 99%; D: 87, 94%; five independent experiments; A′: 33, 100%; B′: 29, 92%; C: 34, 100%; D: 31, 92%; E: 78, 100%; F: 83, 95%; G: 92, 99%; H: 85, 94%; E′: 28, 100%; F′: 33, 96%; G′: 30, 100%; H′: 35, 95%. Scale bars: 500 μm. Error bars denote SEM (n≥3) (**P<0.01).
	Fig. 6.Model of Ouabain blocking the Na,K-ATPase pump and macropinocytosis, resulting in inhibition of Wnt signaling. The Na,K-ATPase transmembrane protein is a member of the P-type ATPase family that pumps three Na+ ions out of cells while pumping two K+ ions into cells, establishing the electrical potential across membranes. The cardiotonic steroid Ouabain is a selective Na,K-ATPase inhibitor targeting the α1-subunit that blocks macropinocytosis, lysosomes, and Wnt signaling.
	Fig. S1 The Na,K-ATPase is Accumulated in Lysosomes by Wnt Pathway Activation and Depends on Macropinocytosis; Related to Figure 2 (A-A’’) The human SW480 colon adenocarcinoma cell line, which constitutive Wnt, was stained with Na,K-ATPase and CD63 antibodies, which colocalized. (B-B’’) SW480 cells treated with the EIPA (40 µM) macropinocytosis inhibitor have reduced levels of Na,K-ATPase, and CD63. (C-C’’) The Na,K-ATPase inhibitor Ouabain (200 nM) reduces CD63 levels, presumably through the inhibition of macropinocytosis. (D-D’’) SW480 cells stained with Na,K-ATPase and -catenin antibodies. (E-E’’) The macropinocytosis inhibitor EIPA reduces Na,K-ATPase levels and blocks Wnt signaling. (F-F’’) Ouabain decreases Na,K-ATPase and β-catenin accumulation. (G) Diagram illustrating that activation of Wnt causes the endocytosis of the Na,K-ATPase in lysosomes. (H) Control HEK293 cells stained with CD63 and Na,K-ATPase antibodies. (I) Ouabain (1 M) overnight treatment reduced basal levels of Na,K-ATPase. (J) The activation of the Wnt pathway via GSK3 inhibition with LiCl (40 mM) strongly increased Na,K-ATPase and CD63 levels. (K) Ouabain treatment strongly reduced the levels of CD63 and Na,K-ATPase that were enhanced by LiCl. (L) A second GSK3 inhibitor CHIR99021 (8 nM) was used to validate the increased levels of CD63 and Na,K-ATPase caused by GSK3 inhibition (which is a potent inducer of macropinocytosis). (M) Ouabain blocked the stabilization of Na,K-ATPase (and CD63) induced by CHIR99021. Data from four independent experiments; Scale bars 10 μm.
	Fig. S2. Ouabain Blocks Macropinocytosis in CRC SW480 Spheroids; Related to Figure 3 (A) Rostafuroxin/PST2238, which binds specifically to the extracellular domain of ATP1A1, used as a second Na,K-ATPase inhibitor, decreased β-catenin signaling stimulated by LiCl. (B) The depletion of ATP1A1 (A1-subunit of the NA,K-ATPase pump) using a siRNA in SW480 cells. This results in decreased β-catenin transcriptional activity as measured by the BAR-luciferase assay. (C) Flow cytometry analysis shows that overexpression of ATP1A1 increases β-catenin levels in HeLa cells. Black: untransfected control HeLa cells; blue: HeLa cells overexpressing ATP1A1; red: untransfected HeLa cells treated with LiCl; green: HeLa cells overexpressing ATP1A1 and treated with LiCl. Each experiment was conducted three or more times. The overexpression of ATP1A1 (blue) shows no significant changes in -catenin levels compared to controls. The treatment by LiCl (red) increases the -catenin level 1.7-fold compared to controls (unpaired ttest, t = 8.437, df = 4, p = 0.0011). The overexpression ATP1A1 combined with LiCl treatment (green) increases the -catenin level 2.2-fold compared to controls (Unpaired t-test, t = 8.563, df = 4, p = 0.0010) and 1.3-fold compared to LiCl treatment only (Unpaired t-test, t = 3.429, df = 4, p = 0.0266). Thus, we conclude that the overexpression of ATP1A1 enhances the activation of the Wnt pathway. (D) SW480 spheroids after 96 hours in inverted drop culture. (E) Ouabain (40 M) treatment affects the ability to form spheroids in 3D cultures. (F) Quantification of the spheroid area after Ouabain treatment. (G) Spheroids incubated with the macropinocytosis marker TMR-dextran 70kDa (1 mg/mL) for 1 hour. (H) Ouabain treatment reduces macropinocytosis of TMR-dextran 70 kDa. (I) Quantification of macropinocytic uptake inhibition caused Ouabain treatment. All experiments with cultured cells were biological triplicates. Eight spheroids were plated per condition, in triplicate. Error bars denote SEM (n ≥ 3) ( p < 0.001, p < 0.01, and * p < 0.05). Scale bars, 500 μm.
	Movie 1 The Sustained Plasma Membrane Macropinosome Ruffles Characteristic of Cancer Cells with Activating Wnt Pathway Mutation Stop within Minutes of Addition of Ouabain; Related to Fig. 3 SW480 cells have sustained macropinocytosis [5] that involves the uptake of significant extracellular fluid and solutes by forming large vesicles (> 0.2 µm) called macropinosomes (arrow) that were reduced rapidly after treatment with Ouabain (200 nM). Since macropinocytosis is required for Wnt signaling [50]-[54], blocking macropinocytosis explains the potent inhibitory effects of Ouabain on canonical Wnt signaling.

References [+] :

Albrecht, GSK3 Inhibits Macropinocytosis and Lysosomal Activity through the Wnt Destruction Complex Machinery. 2020, Pubmed, Xenbase