Glycosylation Of MUC1 Research Articles

Differential Glycoform Analysis of MUC1 Derived from Biological Specimens Using an Antibody-Overlay Lectin Microarray.

The association between altered glycosylation of MUC1 and various disease events has sparked significant interest. However, analytical technologies to investigate the disease-related glycoforms of endogenous MUC1 in blood and tissue specimens are limited. Therefore, we devised a reliable technique for differential analysis of endogenous MUC1 glycoforms based on an antibody-assisted lectin microarray. Its highly sensitive detection aids in analyzing soluble MUC1 from relatively small amounts of serum via a simple enrichment process. Micro-/macro-dissection of the MUC1-positive region is combined with glycoform analysis of the membrane-tethered MUC1. Thus, we have optimized the protocol for sample qualification using immunohistochemistry, sample pretreatment for tissue sections, protein extraction, purification via immunoprecipitation, and the antibody-overlay lectin microarray, which are sequentially essential for differential glycoform analysis of endogenous MUC1.

Advances in MUC1-Mediated Breast Cancer Immunotherapy.

Breast cancer (BRCA) is the leading cause of death from malignant tumors among women. Fortunately, however, immunotherapy has recently become a prospective BRCA treatment with encouraging achievements and mild safety profiles. Since the overexpression and aberrant glycosylation of MUC1 (human mucin) are closely associated with BRCA, it has become an ideal target for BRCA immunotherapies. In this review, the structure and function of MUC1 are briefly introduced, and the main research achievements in different kinds of MUC1-mediated BRCA immunotherapy are highlighted, from the laboratory to the clinic. Afterward, the future directions of MUC1-mediated BRCA immunotherapy are predicted, addressing, for example, urgent issues in regard to how efficient immunotherapeutic strategies can be generated.

Aberrant glycosylation of MUC1 and tumour-associated macrophages (TAMs)

Aberrant glycosylation of MUC1 and tumour-associated macrophages (TAMs)

Core 2 β1,6-N-acetylglucosaminyltransferases accelerate the escape of choriocarcinoma from natural killer cell immunity

Hyperglycosylated human chorionic gonadotropin (H-hCG) is secreted from choriocarcinoma and contains a core2 O-glycan formed by core2 β1,6-N-acetylglucosaminyl transferase (C2GnT). Choriocarcinoma is considered immunogenic as it is gestational and contains paternal chromosomal components. Here we examined the function of C2GnT in the evasion of choriocarcinoma cells from natural killer (NK) cell-mediating killing. We determined that C2GnT is highly expressed in malignant gestational trophoblastic neoplasms. C2GnT KO downregulates core2 O-glycan expression in choriocarcinoma cells, which are more efficiently killed by NK cells than control cells. C2GnT KO cell containing tumor necrosis factor-related apoptosis inducing ligand have lower viability than control cells. Additionally, poly-N-acetyllactosamine in core2 branched oligosaccharides on MHC class I-related chain A (MICA) and mucin1 (MUC1) is significantly reduced in C2GnT KO cells. Meanwhile, the cumulative survival rate of nude mice inoculated with C2GnT KO tumors was higher than that of the control group. These findings suggest that choriocarcinoma cells may escape NK cell-mediated killing via glycosylation of MICA and MUC1.

Cross-talk between Colon Cells and Macrophages Increases ST6GALNAC1 and MUC1-sTn Expression in Ulcerative Colitis and Colitis-Associated Colon Cancer.

Patients with ulcerative colitis have an increased risk of developing colitis-associated colon cancer (CACC). Changes in glycosylation of the oncoprotein MUC1 commonly occur in chronic inflammation, including ulcerative colitis, and this abnormally glycosylated MUC1 promotes cancer development and progression. It is not known what causes changes in glycosylation of MUC1. Gene expression profiling of myeloid cells in inflamed and malignant colon tissues showed increased expression levels of inflammatory macrophage-associated cytokines compared with normal tissues. We analyzed the involvement of macrophage-associated cytokines in the induction of aberrant MUC1 glycoforms. A coculture system was used to examine the effects of M1 and M2 macrophages on glycosylation-related enzymes in colon cancer cells. M2-like macrophages induced the expression of the glycosyltransferase ST6GALNAC1, an enzyme that adds sialic acid to O-linked GalNAc residues, promoting the formation of tumor-associated sialyl-Tn (sTn) O-glycans. Immunostaining of ulcerative colitis and CACC tissue samples confirmed the elevated number of M2-like macrophages as well as high expression of ST6GALNAC1 and the altered MUC1-sTn glycoform on colon cells. Cytokine arrays and blocking antibody experiments indicated that the macrophage-dependent ST6GALNAC1 activation was mediated by IL13 and CCL17. We demonstrated that IL13 promoted phosphorylation of STAT6 to activate transcription of ST6GALNAC1. A computational model of signaling pathways was assembled and used to test IL13 inhibition as a possible therapy. Our findings revealed a novel cellular cross-talk between colon cells and macrophages within the inflamed and malignant colon that contributes to the pathogenesis of ulcerative colitis and CACC.See related Spotlight on p. 160.

Products of Chemoenzymatic Synthesis Representing MUC1 Tandem Repeat Unit with T-, ST- or STn-antigen Revealed Distinct Specificities of Anti-MUC1 Antibodies

Anti-mucin1 (MUC1) antibodies have long been used clinically in cancer diagnosis and therapy and specific bindings of some of them are known to be dependent on the differential glycosylation of MUC1. However, a systematic comparison of the binding specificities of anti-MUC1 antibodies was not previously conducted. Here, a total of 20 glycopeptides including the tandem repeat unit of MUC1, APPAHGVTSAPDTRPAPGSTAPPAHGV with GalNAc (Tn-antigen), Galβ1-3GalNAc (T-antigen), NeuAcα2-3Galβ1-3GalNAc (sialyl-T-antigen), or NeuAcα2-6GalNAc (sialyl-Tn-antigen) at each threonine or serine residue were prepared by a combination of chemical glycopeptide synthesis and enzymatic extension of carbohydrate chains. These glycopeptides were tested by the enzyme-linked immunosorbent assay (ELISA) for their capacity to bind 13 monoclonal antibodies (mAbs) known to be specific for MUC1. The results indicated that anti-MUC1 mAbs have diverse specificities but can be classified into a few characteristic groups based on their binding pattern toward glycopeptides in some cases having a specific glycan at unique glycosylation sites. Because the clinical significance of some of these antibodies was already established, the structural features identified by these antibodies as revealed in the present study should provide useful information relevant to their further clinical use and the biological understanding of MUC1.

Study of activated CIK armed with bispecific antibody for advanced liver cancer

Background & Aim Primary liver cancer is one of the most common malignant tumors in the world. The 5-year relative survival rate of patients with liver cancer is very low. At present, cytokine-induced killer cell (CIK) therapy is a mature clinical treatment method in cell therapy. CIK cells have both anti-tumor activity of T lymphocytes and non-MHC restrictive killing of NK cells. Abnormal glycosylation of MUC1 (a member of mucin family) was highly expressed in 60% of primary hepatocellular carcinoma and 90% of cholangiocarcinoma, but not in normal tissues. Methods, Results & Conclusion In this study, a nanotechnology was used to conjugate bispecific antibodies of MUC1 and CD3 with CIK cells for immunotherapy of primary hepatocellular carcinoma (NTC 03146637). MUC1-positive patients with advanced primary liver cancer received bispecific antibody armed with CIK cell immunotherapy every 4 weeks for 16 weeks. Biochemical indicators, tumor size, cytokines such as interleukin-6 and C-reactive protein, and tumor markers such as alpha fetoprotein, carbohydrate antigen 199 (CA199), CA125, CA724 were regularly detected. As a result, the subjects of biochemical indicators did not change significantly, no grade III adverse reactions, and the concentration of tumor markers were significantly decreased. According to RECIST 1.0, 67% of the patients had stable disease (SD) and 33% had disease progression (PD). Conclusion, bispecific antibody armed with CIK cell immunotherapy is safe and effective.

Cancer-associated mucins: role in immune modulation and metastasis.

Mucins (MUC) protect epithelial barriers from environmental insult to maintain homeostasis. However, their aberrant overexpression and glycosylation in various malignancies facilitate oncogenic events from inception to metastasis. Mucin-associated sialyl-Tn (sTn) antigens bind to various receptors present on the dendritic cells (DCs), macrophages, and natural killer (NK) cells, resulting in overall immunosuppression by either receptor masking or inhibition of cytolytic activity. MUC1-mediated interaction of tumor cells with innate immune cells hampers cross-presentation of processed antigens on MHC class I molecules. MUC1 and MUC16 bind siglecs and mask Toll-like receptors (TLRs), respectively, on DCs promoting an immature DC phenotype that in turn reduces T cell effector functions. Mucins, such as MUC1, MUC2, MUC4, and MUC16, interact with or form aggregates with neutrophils, macrophages, and platelets, conferring protection to cancer cells during hematological dissemination and facilitate their spread and colonization to the metastatic sites. On the contrary, poor glycosylation of MUC1 and MUC4 at the tandem repeat region (TR) generates cancer-specific immunodominant epitopes. The presence of MUC16 neo-antigen-specific T cell clones and anti-MUC1 antibodies in cancer patients suggests that mucins can serve as potential targets for developing cancer therapeutics. The present review summarizes the molecular events involved in mucin-mediated immunomodulation, and metastasis, as well as the utility of mucins as targets for cancer immunotherapy and radioimmunotherapy.

Assessment of tumor characteristics based on glycoform analysis of membrane-tethered MUC1

Clinical tissue specimens are useful for pathological diagnosis, which is, in some cases, supported by visualization of biomolecule localization. In general, diagnostic specificity in molecular pathology is increased by the acquisition of a probe to distinguish the modification of isomers. Although glycosylation is one of the candidate modifications in a protein, comparative glycan analysis of disease-associated proteins derived from a single tissue section is still challenging because of the lack of analytical sensitivity. Here we demonstrate a possible method for differential glycoform analysis of an endogenous tumor-associated glycoprotein MUC1 by an antibody-overlay lectin microarray. Tissue sections (5 μm thick) of patients with cholangiocarcinoma (CCA; n=21) and pancreatic ductal adenocarcinoma (PDAC; n=50) were stained with an anti-MUC1 antibody MY.1E12 that was established as a monoclonal antibody recognizing an MUC1 glycosylation isoform with a sialyl-core 1 structure (NeuAcα2-3galactosyl β1-3-N-acetylgalactosamine). MY.1E12-positive tissue areas (2.5 mm2) were selectively dissected with a laser capture microdissection procedure. The membrane MUC1 was enriched by immunoprecipitation with MY.1E12 and subjected to lectin microarray analysis. Even though the reactivities of MY.1E12 between CCA and PDAC were similar, the lectin-binding patterns varied. We found Maackia amurensis leukoagglutinin and pokeweed lectin distinguished MY.1E12-reactive MUC1 of CCA from that of PDAC. Moreover, MUC1 with M. amurensis hemagglutinin (MAH) reactivity potentially reflected the degree of malignancy. These results were confirmed with MAH-MY.1E12 double fluorescent immunostaining. These glycan changes on MUC1 were detected with high sensitivity owing to the cluster effect of immobilized lectins on a tandem repeat peptide antigen covered with highly dense glycosylation such as mucin. Our approach provides the information to investigate novel glycodynamics in biology, for example, glycoalteration, as well as diseases related to not only MUC1 but also other membrane proteins.

Abstract 4126: Validation of hypoglycosylated MUC1-CIN85 protein-protein interaction as a new therapeutic target for prevention of cancer invasion and metastasis

Abstract Overexpression and abnormal glycosylation of the epithelial glycoprotein MUC1 in cancer cells promotes metastasis. We have identified CIN85 (Cbl-interacting protein 85 KDa), as a binding partner of MUC1 in tumors. MUC1/CIN85 complex is found in early as well as advanced clinical stages of breast, ovarian, colon and prostate cancers among others. Co-localization of MUC1 and CIN85 on invadopodia structures enhances invasion and migration of cancer cells. Our hypothesis is that preventing the formation or dissociating existing CIN85/MUC1 complexes may result in a less aggressive tumor by decreasing local invasion and preventing distant metastases. To test this hypothesis, we have already identified and tested two novel compounds that significantly reduce the association between hypoglycosylated form of MUC1 and CIN85, and at 10μM concentration drastically reduce the migratory activity of mouse and human epithelial cancer cells. Our preliminary data also suggest that dissociation of the MUC1 and CIN85 complex decreases expression of hypoglycosylated MUC1, restoring the expression of its normal, fully glycosylated form. We are currently investigating the MUC1/CIN85-dependent signaling pathway with a particular focus on the ability of CIN85 to modulate MUC1 glycosylation by controlling its plasma membrane-Golgi trafficking. Ongoing experiments in an in vivo mouse model will confirm the ability of these drug compounds to decrease tumor growth and metastasis and validate CIN85/MUC1 complex as a viable therapeutic target and support development and testing of more potent antagonists. Citation Format: Sandra Cascio, Jacque Faylo, Raahul Sriram, Anda Vlad, Carlos Camacho, Olivera J. Finn. Validation of hypoglycosylated MUC1-CIN85 protein-protein interaction as a new therapeutic target for prevention of cancer invasion and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4126. doi:10.1158/1538-7445.AM2017-4126

Abstract 4554: An evaluation of the role of Mucin in drug resistant ovarian cancer

Abstract MUC1 is a transmembrane glycoprotein that is overexpressed in epithelial ovarian cancer. Due to extensive O-glycosylation, mucins form a physical barrier that limits the intracellular accumulation and cytotoxic effect of chemotherapeutic drugs. Our investigation represents a key step in determining if drug resistance in ovarian cancer develops due to altered MUC1 expression and glycosylation. Immunoblotting and ELISA techniques were utilized to characterize the extent of membrane-bound and shed MUC1 expression in drug-resistant and drug-sensitive ovarian cancer cells. An (anti-MUC1) antibody-based in vitro model was employed to determine if the inhibition of O-glycosylation enhanced functional access of small molecule drugs using FACS and fluorescence microscopic analyses. Expression of MUC1 was detected in both the drug resistant ovarian cancer cell line SK-OV-3 (MDR) and the parent cell line SK-OV-3 (WT), with the highest levels observed in the drug resistant sub-clone. The greatest improvement in functional anti-MUC1 antibody access following inhibition of O-glycosylation was observed in the drug-resistant ovarian cancer cells. Our results provide evidence to support drug resistance in ovarian cancer due in part to a mucin barrier mesh. The inhibition of mucin O-glycosylation may be a viable strategy to improve drug delivery and efficacy of small molecule delivery to ovarian carcinomas. Citation Format: Steven M. Richards, Robert B. Campbell. An evaluation of the role of Mucin in drug resistant ovarian cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4554. doi:10.1158/1538-7445.AM2015-4554

Abstract 2886: Epigenetic regulation of MUC1 in NF-kB target gene expression of pro-inflammatory cytokines in colon cancer

Abstract MUC1 is a transmembrane glycoprotein normally expressed at low levels in a fully glycosylated form on the apical surface of epithelial cells but it is found overexpressed and hypoglycosylated in human adenocarcinomas as well as sites of chronic epithelial inflammations. Aberrant glycosylation of MUC1 in cancer cells alters its normal function and affects the behavior of cancer cells. The MUC1 extracellular domain also participates in activation of the NF-kB family members such as phospho-IkBa and phospho-p65. Our data showed that the tumor form of MUC1, in association with p65, regulates the transcriptional activity of pro-inflammatory cytokines, including IL-6 and TNF-alpha, by binding to their promoter regions in both mouse and human cancer cells. Because proteomic analysis showed that MUC1 is able to interact directly with histone proteins, we hypothesized that MUC1 might influence the transcriptional activity of cytokines by inducing epigenetic modification. Here, we explored MUC1/p65-regulated transcription of IL-6 and TNF-alpha in colon cancer cells and discovered that MUC1/p65 complex modulated the expression and interaction of histone methyltransferase Enhancer of Zeste protein-2 (EZH2) with pro-inflammatory cytokine promoters. Interestingly, the contribution of EzH2 in controlling the MUC1/p65-modulated cytokines expression was independent of its histone H3K27 methyltransferase activity but it shows novel properties in regulating chromatin remodeling that are currently under detailed investigation To investigate the in vivo significance of MUC1/p65-induced cytokines expression in the tumor microenvironment, we used human MUC1 transgenic mice that express MUC1 on the colonic epithelial cells, treated with azoxymethane to initiate and dextran sulfate sodium (AOM/DSS) to promote colorectal carcinogenesis. MUC1.Tg mice showed higher tumor incidence and decreased survival when compared with wild-type mice. Consistent with the in vitro data, NF-kB family members and EzH2 expression were higher in MUC1.Tg mice when compared to wild-type mice. In addition, the number of infiltrating macrophages strongly increased in AOM/DSS-treated colon tissues of MUC1.Tg mice. These are important findings that may explain the mechanism by which MUC1 and NF-kB regulate cancer progression. Citation Format: Sandra Cascio, Joshua Sciurba, Olivera Finn. Epigenetic regulation of MUC1 in NF-kB target gene expression of pro-inflammatory cytokines in colon cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2886. doi:10.1158/1538-7445.AM2015-2886

G.P.53: KL-6/MUC1 is a novel diagnostic marker for GNE-myopathy

GNE-myopathy is a genetic myopathy characterized clinically by distal muscle weakness, pathologically by the formation of inclusions and rimmed vacuoles (RVs). Mutations in the causative gene GNE result in the decrease of sialic acid that is necessary for glycosylation of MUC1. The extracellular domain of MUC1 (MUC1-N) has the highly glycosylated tandem repeat domain containing precursor of sialylated carbohydrate antigen Krebs von den lungen 6 (KL-6). In this study, we examined KL-6/MUC1 expression in muscle biopsy specimens, and serum KL-6 levels. The muscle biopsy specimens and laboratory data of GNE-myopathy (n = 6), sporadic inclusion body myositis (sIBM; n = 2), polymyositis (PM: n = 8), and normal control (NC: n = 5) were examined. These specimens were subjected to immunohistochemistry, immunofluorescent technique, and western blot analysis, using antibodies against phosphorylated TDP-43 (pTDP-43), MUC1-C, and KL-6. In GNE-myopathy, inclusions and RVs were immunopositive for pTDP-43, MUC1-C and KL-6, but the sarcolemma was not. On the other hand, the sarcolemma was also immunopositive for MUC1-C and KL-6 in sIBM. In PM and NC, the pale immunoreactivity for MUC1-C and KL-6 was observed in the sarcolemma and cytoplasm. Western blot demonstrated the decrease of MUC1-C and KL-6 in the skeletal muscle of GNE-myopathy. Serum KL-6 levels of GNE myopathy patients were lower than those of other neuromuscular disease patients (p

Increased expression of GCNT1 is associated with altered O-glycosylation of PSA, PAP, and MUC1 in human prostate cancers.

Protein glycosylation is a common posttranslational modification and glycan structural changes have been observed in several malignancies including prostate cancer. We hypothesized that altered glycosylation could be related to differences in gene expression levels of glycoprotein synthetic enzymes between normal and malignant prostate tissues. We interrogated prostate cancer gene expression data for reproducible changes in expression of glycoprotein synthetic enzymes. Over-expression of GCNT1 was validated in prostate samples using RT-PCR. ELISA was used to measure core 2 O-linked glycan sialyl Lewis X (sLe(x) ) of prostate specific antigen (PSA), Mucin1 (MUC1), and prostatic acidic phosphatase (PAP) proteins. A key glycosyltransferase, GCNT1, was consistently over-expressed in several prostate cancer gene expression datasets. RT-PCR confirmed increased transcript levels in cancer samples compared to normal prostate tissue in fresh-frozen prostate tissue samples. ELISA using PSA, PAP, and MUC1 capture antibodies and a specific core 2 O-linked sLe(x) detection antibody demonstrated elevation of this glycan structure in cancer compared to normal tissues for MUC1 (P = 0.01), PSA (P = 0.03) and near significant differences in PAP sLe(x) levels (P = 0.06). MUC1, PSA and PAP protein levels alone were not significantly different between paired normal and malignant prostate samples. GCNT1 is over-expressed in prostate cancer and is associated with higher levels of core 2 O-sLe(x) in PSA, PAP and MUC1 proteins. Alterations of O-linked glycosylation could be important in prostate cancer biology and could provide a new avenue for development of prostate cancer specific glycoprotein biomarkers.

Helicobacter pylori dwelling on the apical surface of gastrointestinal epithelium damages the mucosal barrier through direct contact.

Epithelial junctions and mucins compose a major portion of the mucosal barrier. Helicobacter pylori (H. pylori) infections induce alterations of the tight junctions and adherens junctions in epithelial cells, although the precise mechanisms underlying this process are not fully understood. The expression of adhesion molecules and MUC1 was systematically investigated in gastrointestinal epithelial cells infected with H. pylori in vitro and in vivo. Furthermore, we developed several new in vitro methods to study the relationships between the bacterium and the dysfunction of tight junctions using Boyden Chambers. The expression of a series of junctional molecules and MUC1 decreased in the cultured cells that were infected with H. pylori. According to the degree of damage at the tight junctions, direct contact of H. pylori with the apical membrane of the cells resulted in the greatest increase in permeability compared to basal membrane binding or non-binding of H. pylori to the cells. Similarly, we noted that H. pylori infection could reduce the expression and glycosylation of MUC1. Helicobacter pylori dwelling on the apical surface of the gastrointestinal epithelium could directly induce serious injury of the mucosal barrier, and the new methods outlined here, based on the Boyden Chamber system, could be very useful for studying the relationships between bacteria and their target cells.

845 Glycosylation of MUC1 expressing on prostate cancer cell has crucial impact on susceptibility to natural killer cell immunity in TRAIL dependent manner

845 Glycosylation of MUC1 expressing on prostate cancer cell has crucial impact on susceptibility to natural killer cell immunity in TRAIL dependent manner

Inhibition of KL-6/MUC1 glycosylation limits aggressive progression of pancreatic cancer.

To evaluate the significance of KL-6/MUC1 (a type of MUC1) glycosylation in pancreatic cancer progression. KL-6/MUC1 expression was detected by immunohistochemistry in 48 patients with pancreatic duct cell carcinoma. The N-/O-glycosylation inhibitors (tunicamycin and benzyl-N-acetyl-α-galactosaminide) were then used to interfere with KL-6/MUC1 glycosylation in two pancreatic carcinoma cell lines, and the effects on KL-6/MUC1 expression, and cell adhesion and invasion were determined. In addition, protein expression of epithelial-mesenchymal transition markers, E-cadherin and vimentin, were evaluated in cells after treatment with glycosylation inhibitors. Overexpression of KL-6/MUC1 was found in all pancreatic cancer tissues, but not in the surrounding normal pancreatic tissues. The expression profile of KL-6/MUC1 was significantly decreased after treatment with the inhibitors. The adhesion and invasive ability of cancer cells were significantly decreased after drug treatment, and increased E-cadherin and decreased vimentin expression were found. KL-6/MUC1 glycosylation is involved in pancreatic cancer metastasis and invasion. Therapeutic strategies which target this may help control the aggressive behavior of pancreatic cancer cells.

The role of PAM4 in the management of pancreatic cancer: diagnosis, radioimmunodetection, and radioimmunotherapy.

PAM4, a new monoclonal antibody (MAb) known as clivatuzumab, is highly reactive with pancreatic cancer and precursor lesions. It is absent from the normal tissues and has limited reactivity with nonpancreatic cancer. The detailed characteristic of the PAM4 epitope is unknown but recent studies have shown that it is dependent on MUC1 glycosylation status. The limited PAM4 expression pattern makes it an attractive candidate for management of pancreatic adenocarcinoma. In addition, PAM4 is a serum biomarker for diagnosis of pancreatic cancer. Several different radiolabeled immunodiagnostic and immunotherapeutic agents of PAM4 have been developed and some are being evaluated in preclinical and/or clinical studies. The review will focus on PAM4 and its potential utility for the diagnosis, radioimmunodetection, and radioimmunotherapy of pancreatic cancer.

Altered glycosylation of MUC1 influences its association with CIN85: the role of this novel complex in cancer cell invasion and migration

MUC1 is a transmembrane glycoprotein abnormally expressed in human adenocarcinomas. The extracellular domain of MUC1 contains a variable number of tandem repeats (VNTR) region that is extensively O-glycosylated in normal epithelia and underglycosylated in tumor cells. This change in posttranslational modification of MUC1 leads to changes in its normal functions including, we hypothesized, its interaction with other molecules. We identified CIN85, an adaptor protein involved in multiple cellular processes including signal transduction, cytoskeletal remodeling and cancer cell invasion, as one of several proteins that associate with MUC1 in tumor cells. CIN85 associates with both the cytosolic tail and the extracellular VNTR of MUC1. Co-immunoprecipitation and confocal immunofluorescence confirmed that MUC1 and CIN85 co-localize primarily at the plasma membrane but the complex can be found also in the cytosol and on the cytoskeleton. MUC1 and CIN85 are both over-expressed in early as well as advanced clinical stages of breast cancer and co-localize on invadopodia-like structures implicated in cell invasion. siRNA-mediated silencing of CIN85 and/or MUC1 revealed that MUC1 enhances CIN85-dependent breast cancer cell migration and invasion in vitro. However, ectopic expression of MUC1 enhances the motility induced by CIN85. When tested in vivo in a tumor metastasis model of B16 melanoma, mice injected with CIN85-depleted melanoma cells exhibited few or no lung metastasis and, similarly to the in vitro results, overexpression of MUC1 recovered the shCIN85-reduced metastatic process. Our findings implicate this newly identified CIN85/MUC1 complex associated with invadopodia-related molecules in promoting the invasive and metastatic potential of breast cancer.

Apical MUC1 expression revealed on the foveolar epithelium in H. pylori gastritis

Background:The membrane mucin MUC1 is altered in its pattern of expression in cancer, and also in other pathological situations, including Helicobacter pylori gastritis. Here we investigate the basis for the loss of apical staining of the gastric foveolar epithelium in H. pylori gastritis.Methods:MUC1 was examined in the gastric antrum from cases of H. pylori gastritis and normal controls. We used tissue sections that were either treated or not treated with periodate to effect deglycosylation, and the monoclonal antibodies LICRLonM8, MUSE-11, CT2 and BC2.Results:We show that the epitopes on the TR domain of MUC1 are partially cryptic due to glycosylation and that MUC1 is present on the apical surface of the gastric foveolar epithelium of gastritis patients.Conclusion:This observation suggests that there is no substantial loss of the mucin domain of MUC1 from the apical surface in gastritis, as suggested by others, but rather the H. pylori influences the glycosylation of MUC1. This paper highlights the issue of epitope specificity of monoclonal antibodies directed against disease-associated markers, specifically when they are glycoproteins, as is the case for many cancer markers.