Wnt-driven LARGE2 mediates laminin-adhesive O-glycosylation in human colonic epithelial cells and colorectal cancer

Dietinger, Vanessa; de Durango, Cira R. Garcia; Wiechmann, Svenja; Boos, Sophie L.; Michl, Marlies; Neumann, Jens; Hermeking, Heiko; Kuster, Bernhard und Jung, Peter (2020): Wnt-driven LARGE2 mediates laminin-adhesive O-glycosylation in human colonic epithelial cells and colorectal cancer. In: Cell Communication and Signaling, Bd. 18, Nr. 1, 102

Abstract

Background: Wnt signaling drives epithelial self-renewal and disease progression in human colonic epithelium and colorectal cancer (CRC). Characterization of Wnt effector pathways is key for our understanding of these processes and for developing therapeutic strategies that aim to preserve tissue homeostasis. O-glycosylated cell surface proteins, such as alpha-dystroglycan (alpha-DG), mediate cellular adhesion to extracellular matrix components. We revealed a Wnt/LARGE2/alpha-DG signaling pathway which triggers this mode of colonic epithelial cell-to-matrix interaction in health and disease. Methods: Next generation sequencing upon shRNA-mediated silencing of adenomatous polyposis coli (APC), and quantitative chromatin immunoprecipitation (qChIP) combined with CRISPR/Cas9-mediated transcription factor binding site targeting characterized LARGE2 as a Wnt target gene. Quantitative mass spectrometry analysis on size fractionated, glycoprotein-enriched samples revealed functional O-glycosylation of alpha-DG by LARGE2 in CRC. The biology of Wnt/LARGE2/alpha-DG signaling was assessed by affinity-based glycoprotein enrichment, laminin overlay, CRC-to-endothelial cell adhesion, and transwell migration assays. Experiments on primary tissue, human colonic (tumor) organoids, and bioinformatic analysis of CRC cohort data confirmed the biological relevance of our findings. Results: Next generation sequencing identified the LARGE2 O-glycosyltransferase encoding gene as differentially expressed upon Wnt activation in CRC. Silencing of APC, conditional expression of oncogenic beta-catenin and endogenous beta-catenin-sequestration affected LARGE2 expression. The first intron of LARGE2 contained a CTTTGATC motif essential for Wnt-driven LARGE2 expression, showed occupation by the Wnt transcription factor TCF7L2, and Wnt activation triggered LARGE2-dependent alpha-DG O-glycosylation and laminin-adhesion in CRC cells. Colonic crypts and organoids expressed LARGE2 mainly in stem cell-enriched subpopulations. In human adenoma organoids, activity of the LARGE2/alpha-DG axis was Wnt-dose dependent. LARGE2 expression was elevated in CRC and correlated with the Wnt-driven molecular subtype and intestinal stem cell features. O-glycosylated alpha-DG represented a Wnt/LARGE2-dependent feature in CRC cell lines and patient-derived tumor organoids. Modulation of LARGE2/alpha-DG signaling affected CRC cell migration through laminin-coated membranes and adhesion to endothelial cells. Conclusions: We conclude that the LARGE2 O-glycosyltransferase-encoding gene represents a direct target of canonical Wnt signaling and mediates functional O-glycosylation of alpha-dystroglycan (alpha-DG) in human colonic stem/ progenitor cells and Wnt-driven CRC. Our work implies that aberrant Wnt activation augments CRC cell-matrix adhesion by increasing LARGE/alpha-DG-mediated laminin-adhesiveness.

• Dokumententyp: Zeitschriftenartikel
• Fakultät: Medizin
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Sprache: Englisch