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. 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. 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 β-catenin and endogenous β-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. 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.

中文翻译：

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Wnt 驱动的 LARGE2 在人结肠上皮细胞和结直肠癌中介导层粘连蛋白粘附的 O-糖基化。

Wnt 信号驱动人类结肠上皮细胞和结直肠癌 (CRC) 的上皮自我更新和疾病进展。Wnt 效应通路的表征是我们理解这些过程和制定旨在保持组织稳态的治疗策略的关键。O-糖基化的细胞表面蛋白，如α-肌营养不良蛋白聚糖（α-DG），介导细胞与细胞外基质成分的粘附。我们揭示了 Wnt/LARGE2/α-DG 信号通路，它在健康和疾病中触发了这种结肠上皮细胞与基质相互作用的模式。shRNA 介导的腺瘤性息肉病 (APC) 沉默的下一代测序，以及定量染色质免疫沉淀 (qChIP) 结合 CRISPR/Cas9 介导的转录因子结合位点靶向，将 LARGE2 表征为 Wnt 靶基因。对大小分级、富含糖蛋白的样品进行定量质谱分析揭示了 CRC 中 LARGE2 对α-DG 的功能性 O-糖基化。Wnt/LARGE2/α-DG 信号传导的生物学通过基于亲和力的糖蛋白富集、层粘连蛋白覆盖、CRC 与内皮细胞粘附和 transwell 迁移测定进行评估。对原代组织、人类结肠（肿瘤）类器官的实验和 CRC 队列数据的生物信息学分析证实了我们研究结果的生物学相关性。下一代测序鉴定了 LARGE2 O-糖基转移酶编码基因在 CRC 中 Wnt 激活时差异表达。APC 的沉默、致癌 β-catenin 的条件表达和内源性 β-catenin 隔离影响 LARGE2 表达。LARGE2 的第一个内含子包含对 Wnt 驱动的 LARGE2 表达必不可少的 CTTTGATC 基序，显示被 Wnt 转录因子 TCF7L2 占据，Wnt 激活触发了 CRC 细胞中 LARGE2 依赖性 α-DG O-糖基化和层粘连蛋白粘附。结肠隐窝和类器官主要在富含干细胞的亚群中表达 LARGE2。在人腺瘤类器官中，LARGE2/α-DG 轴的活性是 Wnt 剂量依赖性的。LARGE2 表达在 CRC 中升高，并与 Wnt 驱动的分子亚型和肠干细胞特征相关。O-糖基化 α-DG 在 CRC 细胞系和患者来源的肿瘤类器官中代表 Wnt/LARGE2 依赖性特征。LARGE2/α-DG 信号的调节通过层粘连蛋白包被的膜影响 CRC 细胞迁移和与内皮细胞的粘附。我们得出结论，LARGE2 O-糖基转移酶编码基因代表经典 Wnt 信号传导的直接目标，并介导人结肠干/祖细胞和 Wnt 驱动的 CRC 中 α-dystroglycan (α-DG) 的功能性 O-糖基化。我们的工作意味着异常 Wnt 激活通过增加 LARGE/α-DG 介导的层粘连蛋白粘附来增强 CRC 细胞基质粘附。