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

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 α-dystroglycan (α-DG), mediate cellular adhesion to extracellular matrix components. We revealed a Wnt/LARGE2/α-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 α-DG by LARGE2 in CRC. The biology of Wnt/LARGE2/α-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 ß-catenin and endogenous ß-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 α-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/α-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 α-DG represented a Wnt/LARGE2-dependent feature in CRC cell lines and patient-derived tumor organoids. Modulation of LARGE2/α-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 α-dystroglycan (α-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/α-DG-mediated laminin-adhesiveness. Video abstract.

Disease Modeling on Tumor Organoids Implicates AURKA as a Therapeutic Target in Liver Metastatic Colorectal Cancer.

BACKGROUND & AIMS: Patient-derived tumor organoids recapitulate the characteristics of colorectal cancer (CRC) and provide an ideal platform for preclinical evaluation of personalized treatment options. We aimed to model the acquisition of chemotolerance during first-line combination chemotherapy in metastatic CRC organoids. METHODS: We performed next-generation sequencing to study the evolution of KRAS wild-type CRC organoids during adaptation to irinotecan-based chemotherapy combined with epidermal growth factor receptor (EGFR) inhibition. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 protein (Cas9)-editing showed the specific effect of KRASG12D acquisition in drug-tolerant organoids. Compound treatment strategies involving Aurora kinase A (AURKA) inhibition were assessed for their capability to induce apoptosis in a drug-persister background. Immunohistochemical detection of AURKA was performed on a patient-matched cohort of primary tumors and derived liver metastases. RESULTS: Adaptation to combination chemotherapy was accompanied by transcriptomic rather than gene mutational alterations in CRC organoids. Drug-tolerant cells evaded apoptosis and up-regulated MYC (c-myelocytomatosis oncogene product)/E2F1 (E2 family transcription factor 1) and/or interferon-α-related gene expression. Introduction of KRASG12D further increased the resilience of drug-persister CRC organoids against combination therapy. AURKA inhibition restored an apoptotic response in drug-tolerant KRAS-wild-type organoids. In dual epidermal growth factor receptor (EGFR)- pathway blockade-primed CRC organoids expressing KRASG12D, AURKA inhibition augmented apoptosis in cases that had acquired increased c-MYC protein levels during chemotolerance development. In patient-matched CRC cohorts, AURKA expression was increased in primary tumors and derived liver metastases. CONCLUSIONS: Our study emphasizes the potential of patient-derived CRC organoids in modeling chemotherapy tolerance ex vivo. The applied therapeutic strategy of dual EGFR pathway blockade in combination with AURKA inhibition may prove effective for second-line treatment of chemotolerant CRC liver metastases with acquired KRAS mutation and increased AURKA/c-MYC expression.