AdipoRon Attenuates Wnt Signaling by Reducing Cholesterol-Dependent Plasma Membrane Rigidity

Abstract

The increasing prevalence of adult and adolescent obesity and its associated risk of colorectal cancer reinforces the urgent need to elucidate the underlying mechanisms contributing to the promotion of colon cancer in obese individuals. Adiponectin is an adipose tissue-derived adipokine, whose levels are reduced during obesity. Both epidemiological and preclinical data indicate that adiponectin suppresses colon tumorigenesis. We have previously demonstrated that both adiponectin and AdipoRon, a small-molecule adiponectin receptor agonist, suppress colon cancer risk in part by reducing the number of Lgr5⁺ stem cells in mouse colonic organoids. However, the mechanism by which the adiponectin signaling pathway attenuates colon cancer risk remains to be addressed. Here, we have hypothesized that adiponectin signaling supports colonic stem cell maintenance through modulation of the biophysical properties of the plasma membrane (PM). Specifically, we investigated the effects of adiponectin receptor activation by AdipoRon on the biophysical perturbations linked to the attenuation of Wnt-driven signaling and cell proliferation as determined by LEF luciferase reporter assay and colonic organoid proliferation, respectively. Using physicochemical sensitive dyes, Di-4-ANEPPDHQ and C-laurdan, we demonstrated that AdipoRon decreased the rigidity of the colonic cell PM. The decrease in membrane rigidity was associated with a reduction in PM free cholesterol levels and the intracellular accumulation of free cholesterol in lysosomes. These results suggest that adiponectin signaling plays a role in modulating cellular cholesterol homeostasis, PM biophysical properties, and Wnt-driven signaling. These findings are noteworthy because they may in part explain how obesity drives colon cancer progression.

Figure 1

AdipoRon suppresses Wnt-driven signaling. (A) Quantification of luciferase reporter activity from mouse fibroblasts (Leading Light 3T3) cells stably expressing an LEF luciferase reporter vestor. Cells were pretreated with 0.1% DMSO or indicated doses of AdipoRon for 12 h before stimulation, with 100 ng/mL of recombinant murine Wnt3a for 12 h in the presence of DMSO or AdipoRon (n = 3 wells per treatment). (B) Quantification of luciferase reporter activity. Mouse fibroblasts were pretreated with 0.1% DMSO or 10 μM AdipoRon for 12 h before stimulation with Wnt pathway activators (100 ng/mL Wnt3a, 500 ng/mL R-Spondin-1, 50 mM LiCl, or 2.5 μM CHIR99021) for 12 h in the presence of DMSO or AdipoRon (n = 3 wells per treatment). (C) Murine colonic organoid metabolic activity assessed by CellTiter-Blue Cell Viability Assay (n = 3 wells per treatment per time point). Unless otherwise indicated, data are mean ± SE; statistical significance between treatments (^∗p < 0.01) was determined using (A) one-way ANOVA with Dunnett’s multiple comparisons test or (B and C) an unpaired t-test. To see this figure in color, go online.

Figure 2

Plasma membrane (PM) biophysical properties are altered by AdipoRon. (A) Representative confocal images of young adult mouse colonic (YAMC) epithelial cell stained with Di-4-ANEPPDHQ: DIC, ordered, disordered, and generalized polarization (GP). (B) Quantification of cell membrane rigidity ΔGP (sample − control) values. Cells were treated with 0.1% DMSO or 10 μM AdipoRon for 24 h (n = 8–16 fields of view per treatment). (C) ΔGP values of cells treated for 48 h (n = 8–16 fields of view per treatment). (D) DKOB8 cells, (E) 24 h (n = 5–10 fields of view per treatment), and (F) 48 h (n = 7–10 fields of view per treatment). (G) Mouse colonic organoids, (H) 24 h (n = 30–62 organoids per treatment), and (I) 48 h (n = 73–91 organoids per treatment). Unless otherwise indicated, data are mean ± SE; statistical significance between treatments (^∗p < 0.01) was determined using an unpaired t-test. Scale bars, (A) 20 μm, (D) 50 μm, and (G) 100 μm. To see this figure in color, go online.

Figure 3

The effect of AdipoRon on PM rigidity is independent of cytoskeletal influences. YAMC cells were treated with (A) 0.1% DMSO or (B) 10 μM AdipoRon for 48 h, fixed in 4% PFA, permeabilized and stained for F-actin (phalloidin-Alexa Fluor 488). (C) A representative FlowSight image of GPMV generated from YAMC cells stained with Di-4-ANEPPDHQ: brightfield (BF), ordered, and disordered. (D) ΔGP values of YAMC GPMVs. Cells were treated for 24 h before GPMV generation (n = 5,797–24,448 GPMVs per treatment). (E) ΔGP values of DKOB8 GPMVs. Cells were treated for 24 h before GPMV generation (n = 5,797–24,448 GPMVs per treatment). (F) ΔGP values of 3T3 GPMVs. Cells were treated for 24 h before GPMV generation (n = 367–1,868 GPMVs per treatment). Unless otherwise indicated, data are mean ± SE; statistical significance between treatments (^∗p < 0.05, ^∗∗p < 0.01) was determined using an (D and E) unpaired t-test or (F) one-way ANOVA with Dunnett’s multiple comparisons test. Scale bars, (A and B) 100 μm, 10 μm (zoom), and (C) 20 μm. To see this figure in color, go online.

Figure 4

AdipoRon alters cholesterol trafficking. (A) A representative image of GPMV derived from YAMC cells and stained with Filipin III is shown. Filipin III intensity values of GPMVs derived from (B) YAMC and (C) DKOB8 cells treated with 0.1% DMSO or 10 μM AdipoRon for 24 h before GPMV generation (n = 1126–7910 GPMVs per treatment) are shown. (D) YAMC and (E) DKOB8 cells were treated with 0.1% DMSO or 10 μM AdipoRon for 24 h. Cells were then fixed in 4% PFA for 15 min, followed by labeling of free cholesterol with 50 μg/mL Filipin III for 45 min at room temperature in the dark (n = 9 fields of view per treatment). Unless otherwise indicated, data are mean ± SE; statistical significance between treatments (^∗p < 0.01) was determined using an unpaired t-test. Scale bars, (A) 20 μm, (D and E) 100, and 20 μm (zoom). To see this figure in color, go online.

Figure 5

AdipoRon reroutes PM and extracellular cholesterol into lysosomes. (A) YAMC cells were treated with 0.1% DMSO or 10 μM AdipoRon for 48 h before staining lysosomes with 50 nM LysoTracker Red DND-99 for 30 min before fixing with 4% PFA for 15 min. After fixation, free cholesterol was stained with 50 μg/mL Filipin III for 45 min at room temperature in the dark. Zoom scale bar, 2 μm. DKOB8 cells were treated with 0.1% DMSO or 10 μM AdipoRon for 24 h in either (B) 5% FBS or (C) 5% cholesterol-depleted FBS then stained and fixed as described above. To see this figure in color, go online.

Figure 6

Summary diagram highlighting the effect of AdipoRon on Wnt signaling. AdipoRon treatment attenuates canonical and R-Spondin-1 enhanced Wnt signaling by redirecting plasma membrane (PM) free cholesterol to intracellular pools, thereby decreasing PM rigidity. To see this figure in color, go online.