上皮细胞粘附分子（EpCAM）胞内域诱导Wnt受体转录以促进结直肠癌（CRC）进展

上皮细胞粘附分子（EpCAM）作为一种在多种实体瘤中表达的肿瘤抗原，已被广泛研究。此外，该糖蛋白通过其胞外（EpEX）和胞内（EpICD）域在关键的癌症相关细胞功能中发挥作用。在结直肠癌（CRC）中，EpCAM被认为参与Wnt信号通路，因为EpICD和β-连环蛋白（β-Catenin）被协同转运到细胞核。一旦进入细胞核，EpICD可转录调控EpCAM的靶基因，但其是否调节Wnt信号仍不明确。我们使用患者源性类器官（PDOs）、患者源性异种移植（PDXs）及多种CRC细胞系，研究了EpCAM及EpICD在Wnt受体表达中的作用。通过荧光与共聚焦显微镜分析来自PDX和其他异种移植模型以及CRC细胞系的肿瘤。我们用人源化的EpCAM中和抗体hEpAb2-6干预了EpCAM信号。构建了Wnt受体启动子在荧光素酶报告基因中的表达系统，评估EpICD的影响。采用荧光素酶报告实验、γ-分泌酶和Wnt活性检测等功能性实验。还进行了体内肿瘤形成、类器官形成、球体和菌落形成等实验，以研究Wnt相关现象。评估了hEpAb2-6抑制EpCAM的潜在治疗效果。在研究中发现，EpICD与Wnt受体（FZD6和LRP5/6）启动子结合，增强它们的转录活性，从而激活Wnt信号。此外，β-连环蛋白降解复合体（GSK3β和CK1）中的Wnt相关激酶的激活促进γ-分泌酶活性，增强EpICD的裂解，形成正反馈环。hEpAb2-6抗体阻断了EpICD介导的Wnt受体上调，带来了治疗益处。该研究揭示了EpCAM在Wnt受体上调中的关键作用，Wnt信号增强促进γ-分泌酶活性，进一步促进EpICD的裂解和核转位。人源化抗EpCAM抗体hEpAb2-6可以阻断这些机制，可能为CRC治疗提供新策略。

Epithelial cell adhesion molecule (EpCAM) has been widely studied as a tumor antigen due to its expression in varieties of solid tumors. Moreover, the glycoprotein contributes to critical cancer-associated cellular functionalities via its extracellular (EpEX) and intracellular (EpICD) domains. In colorectal cancer (CRC), EpCAM has been implicated in the Wnt signaling pathway, as EpICD and β-Catenin are coordinately translocated to the nucleus. Once in the nucleus, EpICD transcriptionally regulates EpCAM target genes that; however, remains unclear whether Wnt signaling is modulated by EpICD activity.Patient-derived organoids (PDOs), patient-derived xenografts (PDXs), and various CRC cell lines were used to study the roles of EpCAM and EpICD in Wnt receptor expression. Fluorescence and confocal microscopy were used to analyze tumors isolated from PDX and other xenograft models as well as CRC cell lines. EpCAM signaling was intervened with our humanized form of EpCAM neutralizing antibody, hEpAb2-6. Wnt receptor promoters under luciferase reporters were constructed to examine the effects of EpICD. Luciferase reporter assays were performed to evaluate promoter, γ-secretase and Wnt activity. Functional assays including in vivo tumor formation, organoid formation, spheroid and colony formation experiments were performed to study Wnt related phenomena. The therapeutic potential of EpCAM suppression by hEpAb2-6 was evaluated in xenograft and orthotopic models of human CRC.EpICD interacted with the promoters of Wnt receptors (FZD6 and LRP5/6) thus upregulated their transcriptional activity inducing Wnt signaling. Furthermore, activation of Wnt-pathway-associated kinases in the β-Catenin destruction complex (GSK3β and CK1) induced γ-secretase activity to augment EpICD shedding, establishing a positive-feedback loop. Our hEpAb2-6 antibody blocked EpICD-mediated upregulation of Wnt receptor expressions and conferred therapeutic benefits in both PDX and orthotopic models of human CRC.This study uncovers relevant functions of EpCAM where Wnt receptors are upregulated via the transcriptional co-factor activity of EpICD. The resultant enhancement of Wnt signaling induces γ-secretase activity further stimulating EpICD cleavage and its nuclear translocation. Our humanized anti-EpCAM antibody hEpAb2-6 blocks these mechanisms and may thereby provide therapeutic benefit in CRC.