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Cell surface mucin (cs-mucin) glycoproteins are constitutively expressed at the surface of respiratory epithelia where pathogens such as influenza A virus (IAV) gain entry into cells. Different members of the cs-mucin family each express a large and heavily glycosylated extracellular domain that towers above other receptors on the epithelial cell surface, a transmembrane domain that enables shedding of the extracellular domain, and a cytoplasmic tail capable of triggering signaling cascades. We hypothesized that IAV can interact with the terminal sialic acids presented on the extracellular domain of cs-mucins, resulting in modulation of infection efficiency. Utilizing human lung epithelial cells, we found that IAV associates with the cs-mucin MUC1 but not MUC13 or MUC16. Overexpression of MUC1 by epithelial cells or the addition of sialylated synthetic MUC1 constructs, reduced IAV infection in vitro. In addition, Muc1 -/- mice infected with IAV exhibited enhanced morbidity and mortality, as well as greater inflammatory mediator responses compared to wild type mice. This study implicates the cs-mucin MUC1 as a critical and dynamic component of the innate host response that limits the severity of influenza and provides the foundation for exploration of MUC1 in resolving inflammatory disease.

The MUC1 cell-surface mucin is highly expressed on the gastric mucosal surface, while MUC13 is highly expressed on the intestinal mucosal surface. Polymorphisms in both MUC1 and MUC13 have been linked to inflammatory bowel diseases. MUC1 can act as a decoy molecule on the apical cell surface of epithelial cells and thereby limit bacterial adherence, infection, and inflammation. In this study, we examined whether and how MUC1 and MUC13 modulate infectious and inflammatory signaling. Using gastrointestinal tissue from Muc1- or Muc13-deficient mice in ex vivo culture, MUC1 small interfering RNA (siRNA) silencing in MKN7 gastric epithelial cells, and MUC13 siRNA silencing in LS513 intestinal epithelial cells, we showed that loss of MUC1 increased chemokine secretion, whereas loss of MUC13 decreased chemokine secretion in response to tumor necrosis factor-α. Anti-inflammatory activity of MUC1 and pro-inflammatory activity of MUC13 were also seen after exposure to pathogens, NOD1 (nucleotide-binding oligomerisation domain-containing protein-1), and Toll-like receptor ligands. MUC1 and MUC13 both regulate chemokine secretion in gastrointestinal epithelial cells through a nuclear factor-κB-dependent pathway, although MUC13 modulation could also involve other pathways. Our studies demonstrate that MUC1 and MUC13 are important components of gastrointestinal homeostasis and that disruption or inappropriate expression of these mucins could predispose to infectious and inflammatory disease and inflammation-induced cancer.

The MUC1 mucin (CD227) is a cell surface mucin originally thought to be restricted to epithelial tissues. We report that CD227 is expressed on human blood dendritic cells (DC) and monocyte-derived DC following in vitro activation. Freshly isolated murine splenic DC had very low levels of CD227; however, all DC expressed CD227 following in vitro culture. In the mouse spleen, CD227 was seen on clusters within the red pulp and surrounding the marginal zone in the white pulp. Additionally, we confirm CD227 expression by activated human T cells and show for the first time that the CD227 cytoplasmic domain is tyrosine-phosphorylated in activated T cells and DC and is associated with other phosphoproteins, indicating a role in signaling. The function of CD227 on DC and T cells requires further elucidation.

High molecular weight mucins represent a unique challenge as tumor markers by virtue of their complex array of epitopes. The list is dominated by the high molecular weight mucins MUC1, CEA and CA125. While the currently accepted role for these tumor markers is in the prediction and detection of relapse, it is possible that their sensitivity and specificity can be improved. Although immunoassays detecting the tumor marker MUC1 are both sensitive and specific for predicting relapse in breast cancer, so far they are not in widespread use in the follow-up of this disease. Are there new combinations of conventional reagents that could improve assay sensitivity, or should we be looking for more radical changes in assay design incorporating combinatorial technology?, (Copyright 2001 S. Karger AG, Basel)

The MUC1 epithelial mucin is expressed by glandular epithelial cells and is often highly expressed and associated with poor prognosis in adenocarcinomas. Tyrosine phosphorylation of the highly conserved cytoplasmic tail of MUC1 (MUC1-CT) has been demonstrated in MUC1 transfected cells and in one breast cancer cell line. In addition, associations between MUC1 and secondary signalling molecules have been demonstrated in breast cancer cell lines. MUC1 clearly plays a role in intracellular signalling, since we were able to demonstrate tyrosine phosphorylation of the MUC1-CT in breast and ovarian cancer cell lines and in primary cultures of serous ovarian cancers. We were unable to modulate MUC1-CT phosphorylation using conditioned media from cell lines showing the highest levels of signalling. However, in several breast and ovarian cancer cell lines, we clearly showed the highest levels of MUC1-CT tyrosine phosphorylation occurred during recolonisation of culture dishes or in low-density adherent cultures. We now hypothesise that phosphorylation changes may reflect either involvement of MUC1 in cell motility or a redistribution of MUC1 in the membrane during the course of cell-cell adhesion., (Copyright 2000 Wiley-Liss, Inc.)

Increased expression of the epithelial mucin MUC1 has been linked to tumor aggressiveness in human breast carcinoma. Recent studies have demonstrated that overexpression of MUC1 interferes with cell-substrate and cell-cell adhesion by masking cell surface integrins and E-cadherin. Additionally, the cytoplasmic tail of MUC1 is involved in signal transduction and interactions with catenins. In the present study, we have examined the in vitro expression of MUC1 mRNA and protein in a panel of 14 human breast cancer cell lines using northern blotting, western blotting, immunocytochemistry, and flow cytometry. Considerable variability of expression was noted not only between cell lines but also within several individual lines. Many cell lines such as BT 20, KPL-1, and T47D expressed abundant MUC1 whilst others such as MDA-MB-231 and MCF-7 showed intermediate expression, and MDA-MB-435 and MDA-MB-453 expressed very low levels. Low levels of MUC1 expression were associated with decreased expression of cytokeratin and increased expression of vimentin. Additionally, 12 of the cell lines were established as xenografts in immunocompromised (SCID) mice, and MUC1 expression in both the primary tumors as well as metastases was assessed immunohistochemically. In general, in vivo expression mirrored in vitro expression, although there was reduced in vivo expression in T47D and ZR-75-1 xenografts. Although we showed no correlation between tumorigenicity or metastasis and MUC1 expression, this study will assist development of experimental models to assess the influence of MUC1 of on breast cancer progression.

Antibodies reactive with peptide epitopes on the core protein of MUC1 epithelial mucin have been demonstrated in some patients with adenocarcinomas. Because these epitopes can be exposed on MUC1 in the serum of healthy women, we measured concentrations of MUC1-reactive antibodies in the serum of healthy pregnant and non-pregnant women, and in patients with benign and malignant ovarian tumours. Antibodies were measured in an enzyme-linked immunosorbent assay utilising a synthetic peptide corresponding to a 105-amino-acid segment of the MUC1 tandem repeat region (5.25 repeats). MUC1-reactive antibodies were always of an IgM isotype and concentrations were highest in young healthy women and declined progressively with age (P=0.0006) concomitantly with increasing serum MUC1 levels (P=0.003). Regardless of age, antibody levels were lower in cancer patients than in healthy women (P<0.0001), but MUC1 levels were much higher in cancer patients (P<0.0001). Although high antibody levels were associated with greater survival in ovarian cancer (P=0.015), multivariate regression analysis showed that this was not a significant independent prognostic indicator after consideration of the International Federation of Gynaecology and Obstetrics (FIGO) stage, histological type, serum MUC1 levels and age. Serial measurement of MUC1 and MUC1 antibodies during treatment in 18 patients with ovarian cancer and throughout pregnancy in 10 women showed a negative correlation between alterations in MUC1 and MUC1 antibodies. These results show that MUC1-peptide-reactive antibodies are present in the serum of healthy women and women with cancer and that they probably form immune complexes with MUC1, but provide no evidence for an augmentation of the humoral immune response to MUC1 in ovarian cancer

The MUC1 epithelial mucin is a transmembrane glycoprotein that is frequently but variably over-expressed by adenocarcinomas. It is used as a diagnostic serum tumour marker and is a candidate target for tumour immunotherapy. Peritoneal fluid (PF) samples from ovarian cancer patients were investigated for their ability to modulate MUC1 expression in 6 ovarian cancer cell lines which showed a range from very low to high endogenous MUC1 expression. Cell lines were cultured in 20% PF for 4 days, fixed in situ and MUC1 assayed by ELISA. MUC1 expression was stimulated by some PF samples in 5 of 6 lines tested. MUC1 expression in the PE04 cell line (very low endogenous expression) was increased by 35 of 36 PFs tested (p < 0.05); stimulation varied between PFs but was greater than with 100 IU/mL hu-r-gamma-interferon. Western blotting confirmed the stimulation of MUC1 in PE04 cells and FACS showed an increase in the proportion of cells expressing MUC1. The active factor was partially purified by gel filtration and was shown to stimulate PE04 cells in a dose-dependent manner. Concentrations of IL1beta, IL4, IL6, IL8, IL10, TNF-alpha, TGF-beta and GM-CSF were often very high in PF and varied substantially between different PF samples but did not correlate with the degree of MUC1 stimulatory activity.

The effects of oestrogen and progesterone on expression of the MUC1 epithelial mucin were examined in MCF7 and ZR-75-1 breast cancer cells grown in steroid-free medium. Oestrogen caused an increase in cell growth and both cellular and secreted MUC1 in both cell lines. However, after correcting for increases in cellular protein, there was no clear changes. Progesterone, in combination with oestrogen, caused significant increases (over 2-fold, P<0.01) in cellular and secreted MUC1 when compared with oestrogen alone in both cell lines despite no or small increases in cellular protein. Growth of ZR-75-1 cells in a competitive inhibitor of O-glycosylation demonstrated that the increased detection by ELISA was not due to altered glycosylation. Progesterone may modulate expression of MUC1 in steroid-responsive breast cancer cells.

This is the first study to describe the association between expression of MUC1 and MUC2 mucins and prognosis in ovarian cancer. Paraffin sections of epithelial ovarian tumours (n = 182: 29 benign, 21 low malignant potential, and 132 invasive tumours) were analysed immunohistochemically for expression of MUC1 and MUC2 mucin core proteins. Most benign, low malignant potential, and invasive tumours showed high MUC1 expression in the cytoplasm. Low cytoplasmic expression of MUC1 was a predictor for good prognosis, particularly within stage III tumours. A minority of benign epithelial tumours, but most low malignant potential and invasive non-mucinous tumours, showed high MUC1 expression on the cell membrane. High apical MUC1 reactivity was associated with non-mucinous tumours. Low expression of MUC1 in the apical membrane was associated with early stage and good outcome for invasive tumours. Most benign and low malignant potential tumours, but only a minority of invasive tumours, showed MUC2 expression. MUC2 was found in non-mucinous as well as in mucinous tumours. The presence of MUC2 was inversely associated with high tumour grade but was not associated with altered survival. These results support experimental evidence that MUC1 influences the metastatic ability of ovarian cancer.

The MUC1 mucin produced by many adenocarcinomas has functions that may be of biological significance and is of importance clinically as a serum tumour marker and as a candidate target for immunotherapy. Previous studies of MUC1 production by ovarian cancers have been limited to immunohistochemical studies of tumour specimens and in vitro studies using cell lines. In this study the biosynthesis, secretion and glycosylation of MUC1 were studied in primary cultures of tumour cells obtained from 35 patients with stage 3 ovarian cancer. Although 34 of the 35 tumours produced MUC1 in vitro, the concentrations of intracellular and secreted MUC1, as measured by an ELISA using core protein-reactive antibodies, varied over a wide range. In addition, the amount of secreted MUC1 as a proportion of the intracellular concentration varied between tumours. Pulse/chase amino acid labelling studies of MUC1 biosynthesis also demonstrated variation in secretion rates. Multivariate regression analysis showed that of the variables tumour size, histological type, grade, ploidy status and intracellular and secreted MUC1 concentrations in vitro, only mucin secretion rate was significantly associated with serum mucin concentrations (p < 0.001). Culture of tumour cells for 4 days in the presence or absence of a competitive inhibitor of O-glycosylation, BAG, showed that the degree of glycosylation of MUC1 varied between tumours and that under-glycosylation was not correlated with production or secretion rates. Our study has shown heterogeneity in the production, secretion and glycosylation of MUC1 and a strong correlation between the secretion rate in vitro and the concentration in the serum of patients.

The Prostate-Specific Antigen (PSA) and the Cancer-Associated Serum Antigen (CASA) assay for the MUC1 mucin were compared in the serum of 303 patients with malignant or benign prostatic disease. Using cutpoints of 4, 10, and 20 micrograms/l, PSA was elevated in 93%, 81%, and 64% of patients with prostate cancer (n = 113), with corresponding specificities of 55%, 84%, and 96% in benign prostate disease (prostatic hyperplasia or prostatitis, n = 190). Using the recommended cutpoint of 4 Units/ml, CASA was elevated in 38% of patients with prostate cancer, with a specificity of 91% in benign disease. PSA and CASA showed a poor correlation in prostate cancer (r = 0.367) and benign disease (r = 0.158), and CASA was elevated in some PSA negative samples. Used together, PSA > or = 20 micrograms/l and CASA > or = 4 kU/l gave perfect specificity in benign disease, with a corresponding sensitivity of 29% (positive and negative predictive values of 100% and 70%, respectively). However, this combination gave no improvement over the use of PSA alone, with sensitivity 47% when the cutpoint was raised to give perfect specificity. These data suggest that CASA is of little use as an adjunct to PSA in the differentiation of benign and malignant prostate disease.

This is the first comparison of the three mucin based tests CA15-3, CASA, and MSA, and the cytokeratin-related TPS assay in breast cancer. The mucin markers were superior to TPS in receiver-operator analysis, though no marker was of use in the diagnosis of malignancy due to low sensitivity. Using cutpoints that gave 95% specificity in benign disease (n = 83), corresponding sensitivities in pre-treatment breast cancer (n = 123: 13 in situ, 54 stage I, 45 stage II, 4 stage III, 7 stage IV) were 17% (CA15-3), 16% (CASA), 13% (MSA), and 8% (TPS), with a strong relationship between marker levels and disease stage. These assays did not always detect the same patients, and the use of CA15-3 combined with CASA gave the highest sensitivity (23%), though this was not significantly better than the use of CA15-3 alone. Despite detecting similar antigens, these assays can show markedly different responses in some patients, indicating that one mucin-based test cannot be substituted for another.

The serum MUC1 markers CASA and CA 15-3 were compared with CA 125 in the serum of patients with ovarian cancer and in pregnant women. Used individually, CASA and CA 15-3 gave sensitivities of 54 and 56% in pre-operative ovarian carcinoma (n = 50), though these were lower than with CA 125 (84%). CASA levels were elevated in 3 women with a negative CA 125, while CA 15-3 was elevated in 2 of these women. The combined use of CA 125 with CASA or CA 15-3 led to the preclinical detection of recurrence in 4/5 patients, with mean lead times of 3.6 and 4.3 months, respectively. Of particular interest was the marked difference in reactivity observed with CASA and CA 15-3 in some patients, despite both assays utilising monoclonal antibodies (MAbs) that react with the MUC1 mucin. CA 15-3 and CASA showed a lower than expected correlation in patients with ovarian cancer (r = 0.70), with some patients having high concentrations of one mucin marker and low concentrations of another. Furthermore, different marker profiles were observed when monitoring the progress of patients with these markers. Marked differences between CA 15-3 and CASA were also observed in the serum of pregnant women (n = 10), where CASA showed marked elevation (mean 33.6 times cutpoint) and CA 15-3 did not (mean 0.88 times cutpoint). These data suggest that the specificities of the MAbs used in these assays affect the glycoform of MUC1 detected, and that it should not be assumed that all MUC1 assays will behave in the same manner.