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Background: 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., Methods: 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., Results: 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., Conclusions: 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).)

Background: Addition of oxaliplatin to adjuvant 5-FU has significantly improved the disease-free survival and served as the first line adjuvant chemotherapy in advanced colorectal cancer (CRC) patients. However, a fraction of patients remains refractory to oxaliplatin-based treatment. It is urgent to establish a preclinical platform to predict the responsiveness toward oxaliplatin in CRC patients as well as to improve the efficacy in the resistant patients., Methods: A living biobank of organoid lines were established from advanced CRC patients. Oxaliplatin sensitivity was assessed in patient-derived tumor organoids (PDOs) in vitro and in PDO-xenografted tumors in mice. Based on in vitro oxaliplatin IC 50 values, PDOs were classified into either oxaliplatin-resistant (OR) or oxaliplatin-sensitive (OS) PDOs. The outcomes of patients undergone oxaliplatin-based treatment was followed. RNA-sequencing and bioinformatics tools were performed for molecular profiling of OR and OS PDOs. Oxaliplatin response signatures were submitted to Connectivity Map algorithm to identify perturbagens that may antagonize oxaliplatin resistance., Results: Oxaliplatin sensitivity in PDOs was shown to correlate to oxaliplatin-mediated inhibition on PDO xenograft tumors in mice, and parallelled clinical outcomes of CRC patients who received FOLFOX treatment. Molecular profiling of transcriptomes revealed oxaliplatin-resistant and -sensitive PDOs as two separate entities, each being characterized with distinct hallmarks and gene sets. Using Leave-One-Out Cross Validation algorithm and Logistic Regression model, 18 gene signatures were identified as predictive biomarkers for oxaliplatin response. Candidate drugs identified by oxaliplatin response signature-based strategies, including inhibitors targeting c-ABL and Notch pathway, DNA/RNA synthesis inhibitors, and HDAC inhibitors, were demonstrated to potently and effectively increase oxaliplatin sensitivity in the resistant PDOs., Conclusions: PDOs are useful in informing decision-making on oxaliplatin-based chemotherapy and in designing personalized chemotherapy in CRC patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Geevimaan, Guo, Shen, Jiang, Fann, Hwang, Shui, Lin, Wang, Shih, Li, Chang, Yang and Chen.)