上皮细胞粘附分子的细胞内结构域诱导Wnt受体转录以促进结直肠癌的进展

上皮细胞粘附分子（EPCAM）由于其在各种实体瘤中的表达而被广泛研究为肿瘤抗原。此外，糖蛋白通过其细胞外（EPEX）和细胞内（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已与Wnt信号通路有关，因为Epicd和β-catenin concortinal旋转到细胞核。一旦进入细胞核，EPICD转录调节Epcam靶基因；然而，尚不清楚Wnt信号是否通过EPICD活性调节。患者衍生的类器官（PDOS），患者衍生的异种移植物（PDXS），并使用各种CRC细胞系来研究EPCAM和EPICD在Wnt受体表达中的作用。荧光和共聚焦显微镜用于分析从PDX和其他异种移植模型以及CRC细胞系中分离出的肿瘤。 EPCAM信号传导与我们人源化的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的作用。进行了荧光素酶报告基因测定，以评估启动子，γ-分泌酶和Wnt活性。进行了功能测定，包括体内肿瘤形成，器官形成，球体形成实验，以研究与WNT相关的现象。 Hepab2-6抑制EPCAM的治疗潜力在人类CRC的异种移植物和原位模型中与Wnt受体的启动子（FZD6和LRP5/6）相互作用，因此上调了其转录活性诱导WNT信号传导。此外，在β-catenin破坏复合物（GSK3β和CK1）中与Wnt-pathway相关激酶的激活诱导了γ-分泌酶活性，以增强EPICD脱落，从而建立阳性反馈回路。我们的HEPAB2-6抗体阻止了EPICD介导的Wnt受体表达的上调，并在人类CRC的PDX和原位模型中赋予了治疗益处。这项研究发现了EPCAM的相关功能，其中Wnt受体通过Epicd的转录分子活性上调了Wnt受体。 WNT信号传导的结果增强诱导γ-分泌酶活性进一步刺激了EPICD裂解及其核易位。我们人源化的抗EPAM抗体HEPAB2-6阻止了这些机制，因此可能在CRC中提供治疗益处。

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.