Intracellular domain of epithelial cell adhesion molecule induces Wnt receptor transcription to promote colorectal cancer progression.

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 ß-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 ß-Catenin destruction complex (GSK3ß 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.

EpEX, the soluble extracellular domain of EpCAM, resists cetuximab treatment of EGFR-high head and neck squamous cell carcinoma.

OBJECTIVES: Cetuximab (Cmab) is a molecularly targeted monoclonal antibody drug for head and neck squamous cell carcinoma (HNSC), although cetuximab resistance is a serious challenge. Epithelial cell adhesion molecule (EpCAM) is an established marker for many epithelial tumors, while the soluble EpCAM extracellular domain (EpEX) functions as a ligand for epidermal growth factor receptor (EGFR). We investigated the expression of EpCAM in HNSC, its involvement in Cmab action, and the mechanism by which soluble EpEX activated EGFR and played key roles in Cmab resistance. MATERIALS AND METHODS: We first examined EPCAM expression in HNSCs and its clinical significance by searching gene expression array databases. We then examined the effects of soluble EpEX and Cmab on intracellular signaling and Cmab efficacy in HNSC cell lines (HSC-3 and SAS). RESULTS: EPCAM expression was found to be enhanced in HNSC tumor tissues compared to normal tissues, and the enhancement was correlated with stage progression and prognosis. Soluble EpEX activated the EGFR-ERK signaling pathway and nuclear translocation of EpCAM intracellular domains (EpICDs) in HNSC cells. EpEX resisted the antitumor effect of Cmab in an EGFR expression-dependent manner. CONCLUSION: Soluble EpEX activates EGFR to increase Cmab resistance in HNSC cells. The EpEX-activated Cmab resistance in HNSC is potentially mediated by the EGFR-ERK signaling pathway and the EpCAM cleavage-induced nuclear translocation of EpICD. High expression and cleavage of EpCAM are potential biomarkers for predicting the clinical efficacy and resistance to Cmab.

Subcellular differential expression of Ep-ICD in oral dysplasia and cancer is associated with disease progression and prognosis.

BACKGROUND: Identification of patients with oral dysplasia at high risk of cancer development and oral squamous cell carcinoma (OSCC) at increased risk of disease recurrence will enable rigorous personalized treatment. Regulated intramembranous proteolysis of Epithelial cell adhesion molecule (EpCAM) resulting in release of its intracellular domain Ep-ICD into cytoplasm and nucleus triggers oncogenic signaling. We analyzed the expression of Ep-ICD in oral dysplasia and cancer and determined its clinical significance in disease progression and prognosis. METHODS: In a retrospective study, immunohistochemical analysis of nuclear and cytoplasmic Ep-ICD and EpEx (extracellular domain of EpCAM), was carried out in 115 OSCC, 97 oral dysplasia and 105 normal oral tissues, correlated with clinicopathological parameters and disease outcome over 60 months for oral dysplasia and OSCC patients. Disease-free survival (DFS) was determined by Kaplan-Meier method and multivariate Cox regression analysis. RESULTS: In comparison with normal oral tissues, significant increase in nuclear Ep-ICD and membrane EpEx was observed in dysplasia, and OSCC (p = 0.013 and < 0.001 respectively). Oral dysplasia patients with increased overall Ep-ICD developed cancer in short time period (mean = 47 months; p = 0.044). OSCC patients with increased nuclear Ep-ICD and membrane EpEx had significantly reduced mean DFS of 33.7 months (p = 0.018). CONCLUSIONS: Our study provided clinical evidence for Ep-ICD as a predictor of cancer development in patients with oral dysplasia and recurrence in OSCC patients, suggesting its potential utility in enhanced management of those patients detected to have increased risk of progression to cancer and recurrence in OSCC patients.

Predominant expression of truncated EpCAM is associated with a more aggressive phenotype and predicts poor overall survival in colorectal cancer.

Regulated intramembrane proteolysis (RIP) has been shown to be an important mechanism for oncogenic activation of EpCAM through nuclear translocation of the intracellular domain EpICD. Recently, we identified two different membranous EpCAM variants namely EpCAM(MF) (full-length) and EpCAM(MT) (truncated) to be expressed in the majority of human epithelial tumors. The aim of our study was to evaluate the potential role of these two protein variants as additional prognostic biomarkers in colorectal cancer. In most studies only one antibody targeting the extracellular domain of EpCAM (EpEX) has been used, whereas in the present study additionally an antibody which detects the intracellular domain (EpICD) was applied to discriminate between different EpCAM variants. Using immunohistochemistry, we analyzed the expression of EpCAM(MF) and EpCAM(MT) variants in 640 patients with colorectal cancer and determined their correlations with other prognostic factors and clinical outcome. A statistically significant association was observed for EpCAM(MT) with advanced tumor stage (p < 0.001), histological grade (p = 0.01), vascular (p < 0.001) and marginal (p = 0.002) invasion. Survival analysis demonstrated reduced overall survival (p < 0.004) in patients with tumors expressing the EpCAM(MT) phenotype when compared to patients with tumors expressing the EpCAM(MF) variant. In conclusion, this study for the first time indicates that expression of EpCAM(MT) is associated with a more aggressive phenotype and predicts poor survival in patients with colorectal cancer.

Circulating tumor cells isolation: the "post-EpCAM era".

Circulating tumor cells (CTCs) represent a submicroscopic fraction detached from a primary tumor and in transit to a secondary site. The prognostic significance of CTCs in metastatic cancer patients was demonstrated for the first time more than ten years ago. To date, it seems clear enough that CTCs are highly heterogeneous and dynamically change their shape. Thus, the inadequacy of epithelial cell adhesion molecule (EpCAM) as universal marker for CTCs detection seems unquestionable and alternative methods able to recognize a broader spectrum of phenotypes are definitely needed. In this review the pleiotropic functions of EpCAM are discussed in detail and the role of the molecule in the biology of CTCs is critically dissected.

Loss of membranous expression of the intracellular domain of EpCAM is a frequent event and predicts poor survival in patients with pancreatic cancer.

AIMS: Epithelial cell adhesion molecule (EpCAM) is a widely used immunohistochemical marker for epithelial human malignancies. Antibodies to target EpCAM are usually directed against its ectodomain (EpEX), but do not detect the intracellular domain (EpICD). The aim of this study was to compare membranous EpEX versus EpICD expression by immunohistochemistry. METHODS AND RESULTS: Concurrent EpEX and EpICD expression was investigated retrospectively in cancerous and matched non-neoplastic tissue samples from patients with pancreatic adenocarcinoma. In total, 317 paired samples of pancreatic tissue from 88 patients were analysed and correlated with clinicopathological parameters. In non-cancerous tissue, a high concordance of membranous EpEX and EpICD expression was observed and defined as the expression of the full-length EpCAM (EpEX(+)/EpICD(+) phenotype, EpCAM(MF)), which was highly predominant. In contrast, while most tumour samples were EpEX positive, loss of membranous EpICD expression (EpEX(+)/EpICD(-) phenotype, EpCAM(MT)) was observed in one-third of cases, and these patients had a significantly shortened disease-free and overall survival. CONCLUSIONS: This study demonstrates for the first time that loss of membranous EpICD expression is a frequent event and predicts poor prognosis in patients with pancreatic cancer. Additional studies evaluating the predictive and prognostic value of the expression of different membranous EpCAM variants are warranted in epithelial cancers.