上皮细胞粘附分子（EpCAM）由于其在多种实体瘤中的表达而作为肿瘤抗原被广泛研究。此外，糖蛋白通过其胞外 (EpEX) 和胞内 (EpICD) 结构域有助于关键的癌症相关细胞功能。在结直肠癌 (CRC) 中，EpCAM 与 Wnt 信号通路有关，因为 EpICD 和 β-Catenin 协同易位至细胞核。一旦进入细胞核，EpICD 就会转录调节 EpCAM 靶基因：然而，Wnt信号传导是否受EpICD活性调节仍不清楚。使用患者来源的类器官（PDO）、患者来源的异种移植物（PDX）和各种CRC细胞系来研究EpCAM和EpICD在Wnt受体表达中的作用。使用荧光和共聚焦显微镜分析从 PDX 和其他异种移植模型以及 CRC 细胞系中分离的肿瘤。 EpCAM 信号传导通过我们的人源化 EpCAM 中和抗体 hEpAb2-6 进行干预。构建了荧光素酶报告基因下的 Wnt 受体启动子来检查 EpICD 的效果。进行荧光素酶报告基因测定以评估启动子、γ-分泌酶和Wnt活性。进行了包括体内肿瘤形成、类器官形成、球体和集落形成实验在内的功能测定来研究Wnt相关现象。在人 CRC 的异种移植和原位模型中评估了 hEpAb2-6 抑制 EpCAM 的治疗潜力。EpICD 与 Wnt 受体启动子（FZD6 和 LRP5/6）相互作用，从而上调其诱导 Wnt 信号传导的转录活性。此外，β-连环蛋白破坏复合物（GSK3β和CK1）中Wnt通路相关激酶的激活诱导γ-分泌酶活性以增强EpICD脱落，建立正反馈回路。我们的 hEpAb2-6 抗体可阻断 EpICD 介导的 Wnt 受体表达上调，并在 PDX 和人类 CRC 原位模型中赋予治疗益处。这项研究揭示了 EpCAM 的相关功能，其中 Wnt 受体通过 EpICD 的转录辅因子活性上调。由此产生的 Wnt 信号传导增强会诱导 γ 分泌酶活性，进一步刺激 EpICD 裂解及其核转位。我们的人源化抗 EpCAM 抗体 hEpAb2-6 可阻断这些机制，从而可能为 CRC 提供治疗益处。© 2024。作者。

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.© 2024. The Author(s).