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 þ 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.

Roles of Adipokines in Digestive Diseases: Markers of Inflammation, Metabolic Alteration and Disease Progression

“…Consistent with these findings, adiponectin negatively regulates colorectal cell survival and migration [250]. Both adiponectin and AdipoRon, a small molecule adiponectin receptor agonist, were found to suppress colon cancer risk in part by reducing the number of leucine-rich repeat-containing G protein-coupled receptor 5+ (Lgr5+) stem cells in mouse colonic organoids [251]. In a study of 2412 cases, non-HMW but not HMW adiponectin was associated with CRC risk [252].…”

Membrane therapy using DHA suppresses epidermal growth factor receptor signaling by disrupting nanocluster formation

“…When incorporated into membrane phospholipids, including caveolae and lipid raft domains ( 45 , 46 ), the unique biophysical characteristics of DHA can influence membrane structure ( 33 , 47 , 48 ). Since the biophysical properties of the plasma membrane can modulate plasma membrane receptor function ( 36 ) and downstream cellular signaling ( 49 ), we initially explored the effects of LDL-DHA on plasma membrane rigidity. For this purpose, IMCE cells were labeled with a polarity-sensitive dye, Di-4-ANEPPDHQ (Di4) ( 34 , 35 ), and individually imaged ( Fig.…”

“…The acidic lipids PA and PIP 2 as well as cholesterol are key structural components of EGFR signaling proteolipid nanodomains ( 22 , 23 , 24 , 63 , 64 , 65 , 66 , 67 ). To investigate the role of PA, PIP 2 , and cholesterol in EGFR nanocluster formation in our models, we treated YAMC cells with 5-fluoro-2-indolyl des-chlorohalopemide (FIPI), phenylarsine oxide (PAO), or methyl-beta-cyclodextrin (MβCD) to reduce levels of PA, PIP 2 , and cholesterol, respectively ( 25 , 36 , 66 , 67 ). As expected, the suppression of PA, PIP 2 , and cholesterol reduced the size of EGFR nanoclusters ( Fig.…”

“…Although assessment of membrane rigidity provides information on the structure of the membrane itself, at present, it is difficult to predict its association with protein signaling. This is because the biophysical properties of the plasma membrane such as rigidity are the result of a myriad of variables, including lipid saturation, cholesterol content, lipid-protein interactions, and cytoskeletal dynamics ( 36 , 57 , 58 ). Similarly, we cannot rule out the contribution of a DHA bioactive metabolite, although to date there is no evidence that secreted lipid metabolites (in general) have membrane-altering properties.…”

“…Cells were stained with Di-4-ANEPPDHQ (Invitrogen, D36802) for membrane order determination as previously described ( 33 , 34 , 35 , 36 ). In brief, cells were stained with 1 μM Di4 and imaged via image-based flow cytometry, Amnis FlowSight ( 35 , 36 ). Laser light at 488 nm was used to excite Di4 and emission wavelengths and subsequently collected in two preset channels representing ordered (O: 480–560 nm) and disordered (D: 640–745 nm).…”