Dysfunction in IGF2R Pathway and Associated Perturbations in Autophagy and WNT Processes in Beckwith-Wiedemann Syndrome Cell Lines

Abstract

Beckwith-Wiedemann Syndrome (BWS) is an imprinting disorder characterized by overgrowth, stemming from various genetic and epigenetic changes. This study delves into the role of IGF2 upregulation in BWS, focusing on insulin-like growth factor pathways, which are poorly known in this syndrome. We examined the IGF2R, the primary receptor of IGF2, WNT, and autophagy/lysosomal pathways in BWS patient-derived lymphoblastoid cell lines, showing different genetic and epigenetic defects. The findings reveal a decreased expression and mislocalization of IGF2R protein, suggesting receptor dysfunction. Additionally, our results point to a dysregulation in the AKT/GSK-3/mTOR pathway, along with imbalances in autophagy and the WNT pathway. In conclusion, BWS cells, regardless of the genetic/epigenetic profiles, are characterized by alteration of the IGF2R pathway that is associated with the perturbation of the autophagy and lysosome processes. These alterations seem to be a key point of the molecular pathogenesis of BWS and potentially contribute to BWS's characteristic overgrowth and cancer susceptibility. Our study also uncovers alterations in the WNT pathway across all BWS cell lines, consistent with its role in growth regulation and cancer development.

Keywords: Beckwith–Wiedemann Syndrome (BWS); IGF2; IGF2R; WNT pathway; autophagy; imprinting.

Figure 1

Localization and expression of IGF2R in control and BWS cell lines. (A,B) Immunofluorescence analysis in controls (A) and BWS (B) cell lines with a 100× objective using the antibodies against IGF2R (green signal) and lysosomal-associated membrane protein 1 (LAMP1) with a lysosomal marker protein (red signal). Nuclei were stained with DAPI (blue signal). Csu-W1 Nikon spinning disk confocal microscopy (C) Immunofluorescence analysis with a 40× objective using the antibodies against IGF2R (green signal), nuclei were stained with DAPI (blue signal) in CTRL2 and BWS UPD. Arrows indicate the two cellular subpopulations for UPD cells, UPD-A and UPD-B. LEICA SP8 confocal microscope. (D) IGF2R expression was evaluated by Western blot in CTRL, SRS, and BWS LCLs. Protein loading was normalized to β-actin and the shown images are representative of three independent experiments.

Figure 2

IGF2R distribution analysis. (A) Representative images of line scan of fluorescence intensity (yellow bars in figure’s miniatures) for CTRL 1 and CTRL 4, and BWS IC1 and BWS IC2 cell lines by using ImageJ software (version 1.54). The image of the analyzed cell is shown in miniature. (B) Graph shows the centroid and center of mass distance of almost 30 cells from 3 independent experiments for each cell line. Boxes include 50% of data points, lines represent the median distance, and whiskers report the minimum and maximum values. Differences (two-way ANOVA test and t-test) are indicated by asterisks (*** < 0.0001 and * < 0.05, respectively).

Figure 3

Analysis of possible targets of the IGF2R pathway in control and BWS cell lines. (A) Expression of the phosphorylation levels of AKT, ERK1/2, and S6 were analyzed by Western blot in controls (CTRL1 and CTRL2), SRS, and BWS cell lines. (B) Representative images of Western blot for phosphorylation and expression of GSK-3α/β and CREB are shown. In (A,B), protein loading was normalized to β-actin and the shown images are representative of three independent experiments.

Figure 4

Evaluation of autophagy in control and BWS LCLs. Phosphorylation and expression of ULK and Beclin1 were analyzed by Western blot in control, SRS, and BWS cell lines. β-tubulin was used as loading control. The images are representative of three independent experiments.

Figure 5

WNT pathway analysis in BWS and control cell lines. (A) Volcano plot of DEGs BWS compared to control cell lines. Upregulated genes are highlighted by red dots, while downregulated genes by blue dots. FDR Benjamini–Hochberg adjusted p values and unadjusted p value < 0.05 are indicated by horizontal lines. The VolcaNoser tool was used for creating volcano plots. (B) Principal Component Analysis distributed samples according to the first principal components in three BWS LCLs (gray dots) and four controls LCLs (orange dots). (C) Analysis of WNT panel’s sub-pathways. Left: trend plot of pathway scores vs. sample types (CTRLs and BWS). This image shows the differences of the expression of the genes belonging to the different sub-pathways of the Vantage 3DTM RNA WNT Pathways Panel between controls and BWS. Right: the analysis of the two most dysregulated sub-pathways (canonical WNT and the transcription factor) is depicted also as box plots. (D) Schematic representation of DEGs in the BWS cell lines belonging to the three main WNT pathways. Pathway nodes shown in white have no genes in the Vantage 3DTM RNA WNT Pathways Panel. Pathway nodes in gray have corresponding genes in the panel. However, no significant differential expression is observed. Nodes in blue and orange denote downregulation or upregulation in BWS compared to CTRLs. The nodes of the pathways that were found to be dysregulated by Pathview (nSolver Advanced Analysis Software 4.0) were p53 (DEG: TP53), Frizzle d (DEG: FZD2), WNT (DEG: WNT10A), GBP (DEG: FRAT1), JNK (DEGs: MAPK9 and MAPK10), BAMBI (DEG: BAMBI), DKK (DEG: DKK4), and cycD (DEG: CCND1). PCA, pathway enrichment analysis, box plots, and schematic representation of DEGs were performed by nSolver software (Figures rendered by Pathview, nSolver Advanced Analysis Software 4.0).