Function Of Epithelial Cell Adhesion Molecule Research Articles

An Intrabody against B-Cell Receptor-Associated Protein 31 (BAP31) Suppresses the Glycosylation of the Epithelial Cell-Adhesion Molecule (EpCAM) via Affecting the Formation of the Sec61-Translocon-Associated Protein (TRAP) Complex.

The epithelial cell-adhesion molecule (EpCAM) is hyperglycosylated in carcinoma tissue and the oncogenic function of EpCAM primarily depends on the degree of glycosylation. Inhibiting EpCAM glycosylation is expected to have an inhibitory effect on cancer. We analyzed the relationship of BAP31 with 84 kinds of tumor-associated antigens and found that BAP31 is positively correlated with the protein level of EpCAM. Triple mutations of EpCAM N76/111/198A, which are no longer modified by glycosylation, were constructed to determine whether BAP31 has an effect on the glycosylation of EpCAM. Plasmids containing different C-termini of BAP31 were constructed to identify the regions of BAP31 that affects EpCAM glycosylation. Antibodies against BAP31 (165-205) were screened from a human phage single-domain antibody library and the effect of the antibody (VH-F12) on EpCAM glycosylation and anticancer was investigated. BAP31 increases protein levels of EpCAM by promoting its glycosylation. The amino acid region from 165 to 205 in BAP31 plays an important role in regulating the glycosylation of EpCAM. The antibody VH-F12 significantly inhibited glycosylation of EpCAM which, subsequently, reduced the adhesion of gastric cancer cells, inducing cytotoxic autophagy, inhibiting the AKT-PI3K-mTOR signaling pathway, and, finally, resulting in proliferation inhibition both in vitro and in vivo. Finally, we clarified that BAP31 plays a key role in promoting N-glycosylation of EpCAM by affecting the Sec61 translocation channels. Altogether, these data implied that BAP31 regulates the N-glycosylation of EpCAM and may represent a potential therapeutic target for cancer therapy.

EpCAM is essential for maintenance of the small intestinal epithelium architecture via regulation of the expression and localization of proteins that compose adherens junctions.

Epithelial cell adhesion molecule (EpCAM) is highly expressed in mammalian intestines, and is essential for maintaining the homeostasis of the intestinal epithelium. EpCAM protein is localized at tight junctions and the basolateral membrane of the intestinal epithelium, where it interacts with many cell adhesion molecules. To explore the molecular functions of EpCAM in regulating adherens junctions in the intestinal epithelium, EpCAM knockout embryos and newborn pups were analyzed. Hematoxylin and eosin staining was used to assess the histology of the duodenum, jejunum, ileum and colon from wild-type and EpCAM−/− mice at E18.5, P0 and P3. The expression and localization of adherens junction-associated genes and genes that encode the proteins that participate in the assembly of adherens junctions were measured at the mRNA and protein levels using qPCR, western blot analysis and immunofluorescence staining. The results showed that although there was no significant damage to the intestines of EpCAM−/− mice at E18.5 and P0, they were significantly damaged at P3 in mutant mice. The expression of adherens junction-associated genes in EpCAM mutant mice was normal at the mRNA level from E18.5 to P3, but their protein levels were gradually reduced and mislocalized from E18.5 to P3. The expression of nectin 1, which can regulate the assembly and adhesion activity of E-cadherin, was also gradually reduced at both the mRNA and protein levels in the intestinal epithelium of EpCAM mutant mice from E18.5 to P3. In summary, the loss of EpCAM may cause the reduction and mislocalization of proteins that compose adherens junctions partly via the downregulation of nectin 1 in the intestines.

Deglycosylation of epithelial cell adhesion molecule affects epithelial to mesenchymal transition in breast cancer cells.

The transmembrane glycoprotein epithelial cell adhesion molecule (EpCAM) is overexpressed in most epithelial cancers including breast cancer, where it plays an important role in cancer progression. Previous study has demonstrated that knockdown of EpCAM inhibits breast cancer cell growth and metastasis via inhibition of the Ras/Raf/ERK signaling pathway and matrix metallopeptidase-9 (MMP-9). Although glycosylation is believed to be associated with the function of EpCAM, the contribution of N-glycosylation to this function remains unclear. We constructed the N-glycosylation mutation plasmid of EpCAM and used it to treat breast cancer cells. Loss of N-glycosylation at all three sites EpCAM had no effect on its level of expression or membrane localization. However, mutation at glycosylation sites significantly reduced the ability of EpCAM to promote epithelial to mesenchymal transition in breast cancer. N-glycosylation mutation of EpCAM led to decrease phosphorylation of Raf, ERK, and Akt, and inhibited the Ras/Raf/ERK and PI3K/Akt signaling pathways. Furthermore, we demonstrated that N-glycosylation mutation of EpCAM-mediated invasion and metastasis of breast carcinoma cells required the downregulation of MMP-9 via inhibition of these two signaling pathways. Our results identified the characteristics and function of EpCAM glycosylation. These data could illuminate molecular regulation of EpCAM by glycosylation and promote our understanding of the application of glycosylated EpCAM as a target for breast cancer therapy.

Functions of EpCAM in physiological processes and diseases (Review)

EpCAM (epithelial cell adhesion molecule) is a type I transmembrane glycoprotein, which was originally identified as a tumor-associated antigen due to its high expression level in rapidly growing epithelial tumors. Germ line mutations of the human EpCAM gene have been indicated as the cause of congenital tufting enteropathy. Previous studies based on cell models have revealed that EpCAM contributes to various biological processes including cell adhesion, signaling, migration and proliferation. Due to the previous lack of genetic animal models, the in vivo functions of EpCAM remain largely unknown. However, EpCAM genetic animal models have recently been generated, and are useful for understanding the functions of EpCAM. The authors here briefly review the functions and mechanisms of EpCAM in physiological processes and different diseases.

Concise Review: Aggressive Colorectal Cancer: Role of Epithelial Cell Adhesion Molecule in Cancer Stem Cells and Epithelial-to-Mesenchymal Transition.

Colorectal cancer (CRC) is one of the most common malignancies worldwide. In spite of various attempts to ameliorate outcome by escalating treatment, significant improvement is lacking particularly in the adjuvant setting. It has been proposed that cancer stem cells (CSCs) and the epithelial‐to‐mesenchymal transition (EMT) are at least partially responsible for therapy resistance in CRC. The epithelial cell adhesion molecule (EpCAM) was one of the first CSC antigens to be described. Furthermore, an EpCAM‐specific antibody (edrecolomab) has the merit of having launched the era of monoclonal antibody treatment in oncology in the 1990s. However, despite great initial enthusiasm, monoclonal antibody treatment has not proven successful in the adjuvant treatment of CRC patients. In the meantime, new insights into the function of EpCAM in CRC have emerged and new drugs targeting various epitopes have been developed. In this review article, we provide an update on the role of EpCAM in CSCs and EMT, and emphasize the potential predictive selection criteria for novel treatment strategies and refined clinical trial design. stem cells translational medicine 2018;7:495–501

Differential expression of epithelial cell adhesion molecule in salivary gland neoplasms.

Epithelial cell adhesion molecule (EpCAM) is the epithelial-specific molecule expressed on various epithelial cell types. The function of EpCAM involves cellular adhesion, proliferation, and signaling in both normal tissues and cancers. The purposes of this study were to investigate the EpCAM expression in salivary gland neoplasms and examine its relationship with pathologic characteristics. Forty-two cases of salivary gland neoplasms, including 20 mucoepidermoid carcinomas (MECs), 11 adenoid cystic carcinomas (ACCs), 9 pleomorphic adenomas (PAs), and 2 polymorphous low-grade adenocarcinomas (PLGAs) were enrolled. Epithelial cell adhesion molecule expression was analyzed immunohistochemically using MOC-31 and BerEP4 antibodies. Results showed that the majority of MECs and all PLGAs showed EpCAM expression in more than 50% of neoplastic cells, whereas most PAs and ACCs did not express this protein. In MECs, most EpCAM-positive neoplastic cells were clear cells, glandular epithelial cells, and intermediate cells, whereas squamous cells and mucous cells were largely negative. The expression was limited to ductal epithelium in EpCAM-positive PAs and ACCs. The decreased EpCAM expression in MECs was significantly associated with microscopically diminished cystic components, the presence of small nest invasion at invasive front, cellular anaplasia, vascular invasion, and high pathologic grade. These data suggested that EpCAM showed different expression pattern among salivary gland neoplasms and in different grades of MECs.

Regulatory roles of grass carp EpCAM in cell morphology, proliferation and migration.

Epithelial cell adhesion molecule (EpCAM) is a Ca(2+)-independent and relatively weak adhesion molecule, which has been extensively investigated in mammalian models. However, the functional roles of its fish homolog are largely unknown. In the present study, we explored the biological properties of grass carp EpCAM (gcEpCAM) in a fish kidney cell line (CIK) via overexpression of gcEpCAM or gcEpCAM intracellular domain (gcEpICD) deletion mutant. Results showed that gcEpCAM overexpression significantly changed the cell morphology, and the proliferation of the cells transfected with gcEpCAM was significantly decreased when compared to the control cells, which is unexpectedly opposite to the increasing effects induced by its mammalian homolog. Moreover, overexpression of gcEpICD deletion mutant had no effect on cell proliferation, indicating gcEpICD's involvement in the cell growth control that is concerted with its role in mammalian model. Additionally, gcEpCAM overexpression increased cell migration which is at least partially consistent with the findings in mammalian cells in which EpCAM expression both positively and negatively affects cell migration. It is worth noting that gcEpICD was not essential to the stimulatory effect of gcEpCAM on cell migration, but overexpression of human EpICD in tumor cells increases cell migration, suggesting the functional discrepancy of EpICD in fish and mammals. In conclusion, we elucidated the cellular functionality of EpCAM in fish cells which will be of benefit to defining the functions of fish EpCAM and also provide rewarding information on the functional evolution of EpCAM in vertebrates.

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.

Crystal structure and its bearing towards an understanding of key biological functions of EpCAM.

EpCAM (epithelial cell adhesion molecule), a stem and carcinoma cell marker, is a cell surface protein involved in homotypic cell-cell adhesion via intercellular oligomerization and proliferative signalling via proteolytic cleavage. Despite its use as a diagnostic marker and being a drug target, structural details of this conserved vertebrate-exclusive protein remain unknown. Here we present the crystal structure of a heart-shaped dimer of the extracellular part of human EpCAM. The structure represents a cis-dimer that would form at cell surfaces and may provide the necessary structural foundation for the proposed EpCAM intercellular trans-tetramerization mediated by a membrane-distal region. By combining biochemical, biological and structural data on EpCAM, we show how proteolytic processing at various sites could influence structural integrity, oligomeric state and associated functionality of the molecule. We also describe the epitopes of this therapeutically important protein and explain the antigenicity of its regions.

EpCAM Is an Endoderm-Specific Wnt Derepressor that Licenses Hepatic Development

Mechanisms underlying cell-type-specific response to morphogens or signaling molecules during embryonic development are poorly understood. To learn how response to the liver-inductive Wnt2bb signal is achieved, we identify an endoderm-enriched, single transmembrane protein, epithelial-cell-adhesion-molecule (EpCAM), as an endoderm-specific Wnt derepressor in zebrafish. hi2151/epcam mutants exhibit defective liver development similar to prt/wnt2bb mutants. EpCAM directly binds to Kremen1 and disrupts the Kremen1-Dickkopf2 (Dkk2) interaction, which prevents Kremen1-Dkk2-mediated removal of Lipoprotein-receptor-related protein 6 (Lrp6) from the cell surface. These data lead to a model in which EpCAM derepresses Lrp6 and cooperates with Wnt ligand to activate Wnt signaling through stabilizing membrane Lrp6 and allowing Lrp6 clustering into active signalosomes. Thus, EpCAM cell autonomously licenses and cooperatively activates Wnt2bb signaling in endodermal cells. Our results identify EpCAM as the key molecule and its functional mechanism to confer endodermal cells the competence to respond to the liver-inductive Wnt2bb signal.

Bidirectional modulation of endogenous EpCAM expression to unravel its function in ovarian cancer

Background:The epithelial cell adhesion molecule (EpCAM) is overexpressed on most carcinomas. Dependent on the tumour type, its overexpression is either associated with improved or worse patient survival. For ovarian cancer, however, the role of EpCAM remains unclear.Methods:Cell survival of ovarian cancer cell lines was studied after induction or repression of endogenous EpCAM expression using siRNA/cDNA or artificial transcription factors (ATF) consisting of engineered zinc-fingers fused to either a transcriptional activator or repressor domain.Results:Two ATFs were selected as the most potent down- and upregulator, showing at least a two-fold alteration of EpCAM protein expression compared with control. Downregulation of EpCAM expression resulted in growth inhibition in breast cancer, but showed no effect on cell growth in ovarian cancer. Induction or further upregulation of EpCAM expression decreased ovarian cancer cell survival.Conclusion:The bidirectional ATF-based approach is uniquely suited to study cell-type-specific biological effects of EpCAM expression. Using this approach, the oncogenic function of EpCAM in breast cancer was confirmed. Despite its value as a diagnostic marker and for immunotherapy, EpCAM does not seem to represent a therapeutic target for gene expression silencing in ovarian cancer.

A novel function of EpCAM in oral squamous cell carcinoma cells under anchorage-independent conditions

Epithelial cell adhesion molecule (EpCAM), involved in Ca2+-independent homotypic cell-cell adhesion in epithelial tissues, is overexpressed in several cancer types. Although studies investigating the function of EpCAM in cancers have shown that it plays a role in cell proliferation, invasion and metastasis, the overall function of EpCAM in cancer cells has remained elusive. Here, we report a novel function of EpCAM in multicellular aggregates (MCAs). EpCAM inhibition using RNA interference (RNAi) did not affect cell morphology, proliferation or expression of certain genes, including cyclin D1 in monolayer cultures of the human oral squamous cell carcinoma cell lines HSC-3 or HSC-4. However, in HSC-4 cells cultured as MCAs, suppression of EpCAM significantly reduced the expression levels of cyclin D1. Nuclear localization of the cyclin D1 protein was observed in MCAs of HSC-4 cells but not in MCAs of EpCAM knockdown HSC4 cells, suggesting that EpCAM regulates cyclin D1 expression and localization in HSC-4 cells under anchorage-independent conditions. We propose that targeting EpCAM might result in more efficient therapies under certain conditions of oral squamous cell carcinoma.

The tumor-associated EpCAM regulates morphogenetic movements through intracellular signaling

Epithelial cell adhesion molecule (EpCAM) is best known as a tumor-associated protein highly expressed in carcinomas. The function of this cell surface protein during embryonic development and its potential role in cancer are still poorly understood. We identified EpCAM in a gain-of-function screen for inducers of abnormal tissue mixing during gastrulation. Elevated EpCAM levels in either the ectoderm or the mesoderm confer "invasive" properties to cells in both populations. We found that this phenotype represents an "overstimulation" of an essential activity of EpCAM in controlling cell movements during embryonic development. Surprisingly, this property is independent of the putative adhesive function of EpCAM, and rather relies on a novel signaling function that operates through down-regulation of PKC activity. We show that inhibition of novel PKCs accounts entirely for the invasive phenotype induced by abnormally high levels of EpCAM as well as for its normal function in regulating cell rearrangement during early development.

A newly identified epithelial cell adhesion molecule (EpCAM) from grass carp ( Ctenopharyngodon idellus): Cloning, tissue distribution and lipopolysaccharide-induced expression in head kidney leucocytes

Epithelial cell adhesion molecule (EpCAM) was initially identified as a tumor-associated antigen in human. Recent studies show that EpCAM is a transmembrane glycoprotein with diverse functions in cell adhesion and other processes such as cellular signaling, cell migration, proliferation and differentiation. In fish, the knowledge on the structure and function of EpCAM is limited as only zebrafish EpCAM has been characterized and shown to play a role in embryonic development. In the present study, we isolated a cDNA encoding a homologue of zebrafish EpCAM from grass carp. The deduced grass carp EpCAM (gcEpCAM) protein of 306 amino acids shared 59-85% of similarity with the EpCAMs in other vertebrates. Protein sequence analysis revealed a transmembrane domain and two epidermal growth factor (EGF)-like repeats in the extracellular region of gcEpCAM which were both conserved in EpCAM from fish to mammal. RT-PCR analysis showed that gcEpCAM transcript was expressed with high abundance in skin, pituitary, kidney, head kidney, gill, thymus and head kidney leucocyte (HKL). Furthermore, real time PCR assay was developed to assess gcEpCAM mRNA expression in HKLs stimulated by lipopolysaccharide (LPS). Results showed that LPS significantly increased gcEpCAM mRNA expression in a time- and dose-dependent manner and the peak response was observed at 12 h after treatment with 30 μg/ml of LPS. These results indicated that gcEpCAM may be a LPS-induced gene involved in the immune response in fish head kidney.

Expression pattern of epithelial cell adhesion molecule on normal and malignant colon tissues

To investigate the expression pattern of epithelial cell adhesion molecule (Ep-CAM) on normal and malignant colon tissues to evaluate its diagnostic and therapeutic significance. cDNA encoding Ep-CAM extracellular domain was cloned by reverse transcription-polymerase chain reaction (RT-PCR) from excised malignant colon tissues and inserted into a glutathione S-transferase (GST)-tagged vector. Ep-CAM-GST fusion protein was induced by isopropyl-beta-D-thiogalactopyranoside (IPTG) and purified with glutathione-sepharose. The Ep-CAM-GST fusion protein was mixed with Freund's adjuvant and Balb/c mice were immunized with it. Sp2/0 myeloma cells were fused with the spleen cells of the immunized mice. After having selected by indirect ELISA, the anti-Ep-CAM monoclonal antibodies (MAbs) were generated and the corresponding ascites were obtained. Finally, the human colon carcinoma tissue array prepared from seventy individual patients was stained with the anti-Ep-CAM MAbs. The isolated Ep-CAM cDNA sequence was identical to the data in GenBank. The expressed fusion protein was almost soluble and had a molecular weight (MW) of 53 ku. Four MAbs against Ep-CAM were obtained and designated as FMU-Ep1, FMU-Ep2, FMU-Ep3 and FMU-Ep4 respectively. Among them, FMU-Ep4 could recognize the natural Ep-CAM on Colo205 and SW480 cells, and all of them could be used for immunohistochemical staining of tissue sections. It was found that Ep-CAM was distributed differently in normal and various malignant colon tissues, including squamous cell carcinoma, signet-ring cell carcinoma and adenocarcinoma. In normal colon gland epithelia, Ep-CAM antigen was mainly distributed on the basolateral membrane and in the region between the basolateral membrane and the cytoplastic part near the nuclei, whereas the expression pattern of colon malignancies was mainly on the whole surface of epithelia and the expression was much higher than the normal colon tissues. The staining pattern of tissue array showed in adenocarcinoma and papillary adenocarcinoma, and the expression of Ep-CAM was increased from grade I to grade III. MAbs against Ep-CAM might be useful for research on the structure and function of Ep-CAM and may have diagnostic and therapeutic value to various colon carcinomas.

KSA antigen Ep-CAM mediates cell-cell adhesion of pancreatic epithelial cells: morphoregulatory roles in pancreatic islet development.

Cell adhesion molecules (CAMs) are important mediators of cell-cell interactions and regulate cell fate determination by influencing growth, differentiation, and organization within tissues. The human pancarcinoma antigen KSA is a glycoprotein of 40 kD originally identified as a marker of rapidly proliferating tumors of epithelial origin. Interestingly, most normal epithelia also express this antigen, although at lower levels, suggesting that a dynamic regulation of KSA may occur during cell growth and differentiation. Recently, evidence has been provided that this glycoprotein may function as an epithelial cell adhesion molecule (Ep-CAM). Here, we report that Ep-CAM exhibits the features of a morphoregulatory molecule involved in the development of human pancreatic islets. We demonstrate that Ep-CAM expression is targeted to the lateral domain of epithelial cells of the human fetal pancreas, and that it mediates calcium-independent cell-cell adhesion. Quantitative confocal immunofluorescence in fetal pancreata identified the highest levels of Ep-CAM expression in developing islet-like cell clusters budding from the ductal epithelium, a cell compartment thought to comprise endocrine progenitors. A surprisingly reversed pattern was observed in the human adult pancreas, displaying low levels of Ep-CAM in islet cells and high levels in ducts. We further demonstrate that culture conditions promoting epithelial cell growth induce upregulation of Ep-CAM, whereas endocrine differentiation of fetal pancreatic epithelial cells, transplanted in nude mice, is associated with a downregulation of Ep-CAM expression. In addition, a blockade of Ep-CAM function by KS1/4 mAb induced insulin and glucagon gene transcription and translation in fetal pancreatic cell clusters. These results indicate that developmentally regulated expression and function of Ep-CAM play a morphoregulatory role in pancreatic islet ontogeny.