MUC1 extracellular domain confers resistance of epithelial cancer cells to anoikis.

Anoikis, a special apoptotic process occurring in response to loss of cell adhesion to the extracellular matrix, is a fundamental surveillance process for maintaining tissue homeostasis. Resistance to anoikis characterises cancer cells and is a pre-requisite for metastasis. This study shows that overexpression of the transmembrane mucin protein MUC1 prevents initiation of anoikis in epithelial cancer cells in response to loss of adhesion. We show that this effect is largely attributed to the elongated and heavily glycosylated extracellular domain of MUC1 that protrudes high above the cell membrane and hence prevents activation of the cell surface anoikis-initiating molecules such as integrins and death receptors by providing them a mechanically 'homing' microenvironment. As overexpression of MUC1 is a common feature of epithelial cancers and as resistance to anoikis is a hallmark of both oncogenic epithelial-mesenchymal transition and metastasis, MUC1-mediated cell resistance to anoikis may represent one of the fundamental regulatory mechanisms in tumourigenesis and metastasis.

The expression pattern of MUC1 (EMA) is related to tumour characteristics and clinical outcome of invasive ductal breast carcinoma.

AIMS: To clarify MUC1 patterns in invasive ductal breast carcinoma and to relate them to clinicopathological parameters, coexpression of other biological markers and prognosis. METHODS AND RESULTS: Samples from 243 consecutive patients with primary ductal carcinoma were incorporated into tissue microarrays (TMAs). Slides were stained for MUC1, oestrogen receptor (ER), progesterone receptor (PR), Her2/neu, p53 and cyclin D1. Apical membrane MUC1 expression was associated with smaller tumours (P = 0.001), lower tumour grades (P < 0.001), PR positivity (P = 0.003) and increased overall survival (OS; P = 0.030). Diffuse cytoplasmic MUC1 expression was associated with cyclin D1 positivity (P = 0.009) and increased relapse-free survival (RFS; P = 0.034). Negativity for MUC1 was associated with ER negativity (P = 0.004), PR negativity (P = 0.001) and cyclin D1 negativity (P = 0.009). In stepwise multivariate analysis MUC1 negativity was an independent predictor of both RFS [hazard ratio (HR) 3.5, 95% confidence interval (CI) 1.5, 8.5; P = 0.005] and OS (HR 14.7, 95% CI 4.9, 44.1; P < 0.001). CONCLUSIONS: The expression pattern of MUC1 in invasive ductal breast carcinoma is related to tumour characteristics and clinical outcome. In addition, negative MUC1 expression is an independent risk factor for poor RFS and OS, besides 'classical' prognostic indicators.

Alkyl-lysophospholipid 1-O-octadecyl-2-O-methyl- glycerophosphocholine induces invasion through episialin-mediated neutralization of E-cadherin in human mammary MCF-7 cells in vitro.

1-O-octadecyl-2-O-methyl-glycerophosphocholine (ET-18-OMe) is an analogue of the naturally occurring 2-lysophosphatidylcholine belonging to the class of antitumor lipids. Previously, we demonstrated that ET-18-OMe modulates cell-cell adhesion of human breast cancer MCF-7 cells. In the present study, we tested the effect of ET-18-OMe on adhesion, invasion and localisation of episialin and E-cadherin in MCF-7/AZ cells expressing a functional E-cadherin/catenin complex. The MCF-7/6 human breast cancer cells were used as negative control since their E-cadherin/catenin complex is functional in cells grown on solid substrate but not in suspension. The function of E-cadherin, a calcium-dependent transmembrane cell-cell adhesion and signal-transducing molecule, is disturbed in invasive cancers by mutation, loss of mRNA stability, proteolytic degradation, tyrosine phosphorylation of associated proteins and large cell-associated proteoglycans or mucin-like molecules such as episialin. Episialin, also called MUC1, is an anti-adhesion molecule that by its large number of glycosylated tandem repeats can sterically hinder the adhesive properties of other glycoproteins. ET-18-OMe inhibited the E-cadherin functions of MCF-7/AZ cells as measured by inhibition of fast and slow aggregation and by the induction of collagen invasion. These effects were enhanced by MB2, an antibody against E-cadherin and blocked by monoclonal antibodies (MAbs) 214D4 or M8 against episialin. ET-18-OMe had no influence on tyrosine phosphorylation of beta-catenin and the E-cadherin/catenin complex remained intact. Transcription, translation, protein turnover and cell surface localisation of episialin were not altered. ET-18-OMe induced finger-like extensions with clustering of episialin together with E-cadherin and carcinoembryonic antigen but not with occludin. In cells in suspension, ET-18-OMe caused a shift in the flow-cytometric profile of episialin toward a lower intensity for MCF-7/AZ cells. In contrast with MCF-7/AZ cells, the adhesion-deficient and noninvasive MCF-7/6 cells showed neither morphotypic changes nor induction of aggregation nor invasion in collagen I upon treatment with ET-18-OMe. Co-localisation of episialin with E-cadherin was rarely observed. We conclude that in the human breast cancer cells MCF-7/AZ, E-cadherin and episialin are key molecular players in the regulation of promotion and suppression of cell-cell adhesion and invasion.

Biosynthesis and shedding of epiglycanin: a mucin-type glycoprotein of the mouse TA3Ha mammary carcinoma cell.

Epiglycanin is a mucin-type glycoprotein present at the surface of TA3Ha mouse mammary tumour cells. It is a long rod-like glycoprotein with a molecular mass of 500 kDa. Its function has not yet been established but its overexpression can affect cell-cell and cell-matrix adhesion. To understand better the biological function of epiglycanin, we have studied the biochemical structure and biosynthesis of epiglycanin in TA3Ha cells. Pulse-chase labelling experiments with [(3)H]threonine revealed an early precursor with a molecular mass of approx. 300 kDa containing approx. 5-10 kDa of N-linked glycans. The precursor was gradually converted into a high-molecular-mass mature form, owing mainly, if not entirely, to O-glycosylation. The mature molecule consists of two major glycoforms that differ in sialylation. Unlike secreted mucins, epiglycanin did not form cysteine-bound multimers, providing further evidence that epiglycanin belongs to the class of membrane-associated mucins. The mature form, but not the precursor form, is shed from the cell surface. The half-life of epiglycanin on the cell surface was found to be approx. 60 h. These results provide the first detailed analysis of the biochemical structure and biosynthesis of epiglycanin.

A stat-responsive element in the promoter of the episialin/MUC1 gene is involved in its overexpression in carcinoma cells.

The mucin-like glycoprotein episialin (MUC1) is highly overproduced by a number of human carcinomas. We have shown previously in a variety of mammalian cell lines that overexpression of this very large transmembrane molecule diminishes cellular adhesion, suggesting that episialin/MUC1 overexpression may play an important role in tumor invasion and metastasis. By using in situ hybridization, we show here that episialin/MUC1 mRNA expression can be increased more than 10-fold in breast carcinoma cells relative to the expression in adjacent normal breast epithelium. In search of the molecular mechanism of this overexpression, we observed that the episialin/MUC1 promoter contains a candidate binding site for transcription factors of the STAT family approximately 500 base pairs upstream of the transcription start site. Cytokines and/or growth factors such as interleukin-6 or interferon-gamma can activate STATs. In the human breast carcinoma cell line T47D, both compounds are able to stimulate transcription of a luciferase reporter gene under the control of a 750-base pair MUC1 promoter fragment proximal to the transcription start site. The observed increase is entirely mediated by the single STAT-binding site, since mutation of this site abolishes stimulation of the reporter by interleukin-6 and interferon-gamma. In addition, mutation of the STAT site also decreased the promoter activity in nonstimulated T47D cells, suggesting that the STAT-binding site is among the elements that are involved in the overexpression of MUC1 in tumor cells.

MUC1/episialin: a critical barrier in the female reproductive tract.

The female reproductive tract must resist microbial infections as well as support embryonic development, implantation and placentation. Reproductive tract mucins, in general, and Muc1/episialin, in particular, play key roles in implantation related events and in protection from microbial infection. High levels of mucin expression in the lower reproductive tract presumably affords protection against infection while down-regulation of uterine mucins has been suggested to provide access to the uterine surface. The present studies demonstrate that mucins, particularly Muc1, are effective barriers to embryo attachment. Furthermore, a strain of female Muc1 null mice in normal housing displays chronic infection and inflammation of the lower reproductive tract and markedly reduced fertility rates. This phenotype is not observed when Muc1 nulls are housed in a pathogen-free environment indicating that this phenotype results from chronic microbial exposure. Only normal endogenous flora were isolated from the reproductive tracts of affected Muc1 null mice, suggesting that these bacterial species become opportunistic with loss of the mucin barrier. Staphylococcal adherence to lower reproductive tract epithelia was found to be mediated by cell surface mucin carbohydrates. Collectively, these studies demonstrate a critical barrier role for Muc1 in various aspects of female reproductive tract physiology.

Depolarized expression of episialin (EMA, MUC1) in lung adenocarcinoma is associated with tumor progression.

Carcinoma cell detachment is an important step in tumor progression and metastasis. Episialin (EMA), if expressed throughout the entire cell surface, may inhibit cell-cell and cell-matrix adhesion. We investigated whether the cellular distribution of episialin in non-small cell lung cancer (NSCLC) is associated with tumor progression. We evaluated the expression of episialin by immunohistochemical staining, in surgical specimens from 122 adenocarcinomas and 99 squamous cell carcinomas. Episialin was present in most NSCLC, with a higher percentage of immunoreactive neoplastic cells in adenocarcinoma than in squamous cell carcinoma (p = 0.0001). In adenocarcinoma the depolarized pattern was significantly associated with nodal metastasis (p = 0.005) and with advanced stage (p = 0.007). In conclusion, nodal metastasis and advanced pathological stage in adenocarcinoma are associated with a depolarized cellular distribution of episialin, suggesting a possible involvement of the molecule in cancer metastasis.

Monoclonal antibodies against the nonmucin domain of MUC1/episialin.

We have tested the reactivity of the monoclonal antibodies submitted to the ISOBM TD-4 Workshop for reactivity with a hybrid molecule consisting of the bacterial beta-galactosidase and most of the extracellular nonrepeat domain of MUC1/episialin. Two monoclonal antibodies submitted to the Workshop, 232A1 and M29, were directed against the protein moiety of this domain as shown by immunoblotting and immunoprecipitation.

MUC1 (episialin) expression in non-small cell lung cancer is independent of EGFR and c-erbB-2 expression and correlates with poor survival in node positive patients.

AIM: To examine tumour samples immunohistochemically for MUC1 (episialin), epidermal growth factor receptor (EGFR), and c-erbB-2, since the disruption of the cell-cell adhesion system by MUC1 and the c-erbB oncoprotein family is known to be important in the development of metastasis in human cancers. METHODS: 93 tumour samples from patients with early stage non-small cell lung cancer treated with surgery alone were examined for episialin, EGFR, and c-erbB-2. RESULTS: Episialin depolarised expression did not correlate with any of the histopathological variables examined (T,N stage, grade, histology, Ki67 proliferation index). No correlation was observed between episialin and EGFR or c-erbB-2 expression. Survival analysis showed that episialin depolarised expression correlated with poor prognosis (p = 0.003), especially in squamous cell cases (p = 0.0003). Episialin expression defined a group of patients with poor prognosis in the node positive category (p = 0.003). In multivariate analysis episialin was the most significant independent prognostic factor (p = 0.007), followed by N stage (p = 0.04). CONCLUSIONS: Depolarised expression of episialin is associated with poor outcome in early stage non-small cell lung cancer. Despite the similar activity on the cadherin cell-cell adhesion system, the expression of episialin and c-erbB oncoproteins is likely to be activated within different pathogenic pathways.

Effect of paclitaxel (Taxol) on CA 125 expression and release by ovarian cancer cell lines.

Two ovarian cancer cell lines, OVC432 and the newly established CVU4I, were used to study the effect of Taxal on cell growth and simultaneous CA 125 antigen expression. Growth of both cell lines was effectively inhibited by drug concentrations of 0.1 microM and higher. Complete inhibition of cell growth may result from high concentrations of Cremophor EL present in the Taxol formulation. Immunohistochemical analysis demonstrated that both cell lines retained the CA 125 expression on the cell surface during exposure to paclitaxel. This was reflected in a constant statistically significant correlation between cell numbers and CA 125 concentrations found in cell lysates. CA 125 levels in the culture medium showed a significant relation to cell numbers and, consequently, to the response of the cell line to the administered anticancer drug. It may be concluded from this study that CA 125 seems to be a reliable tumor marker in monitoring tumor response during paclitaxel treatment.

A mechanism for inhibition of E-cadherin-mediated cell-cell adhesion by the membrane-associated mucin episialin/MUC1.

Episialin (MUC1, PEM, EMA, CA15-3 antigen) is a sialylated, membrane-associated glycoprotein with an extended mucin-like ectodomain. This domain mainly consists of 30-90 homologous 20-amino acid repeats that are rich in O-glycosylation sites (serines and threonines). It is likely that this part forms a polyproline beta-turn helix. As a result, the ectodomain can protrude more than 200 nm above the cell surface, whereas most cell surface molecules do not exceed a length of 35 nm. Normally, episialin is present at the apical side of glandular epithelial cells. On carcinoma cells, however, it can be strongly overexpressed and it is often present over the entire cell surface. We have previously shown that episialin, if it is interspersed between adhesion molecules, nonspecifically reduces cell-cell and cell-extracellular matrix interactions in vitro and in vivo, presumably by steric hindrance caused by the extreme length and high density of the episialin molecules at the cell surface. To analyze the molecular mechanism for this anti-adhesion effect in more detail, we have now deleted an increasing number of repeats in the episialin cDNA and transfected the resulting mutants into murine L929 cells expressing the homophilic adhesion molecule E-cadherin. Here we show that the length of episialin is the dominant factor that determines the inhibition of E-cadherin-mediated cell-cell interactions. For the anti-adhesive effect mediated by the full length episialin, charge repulsion by negatively charged sialylated O-linked glycans is far less important.

Immunoscintigraphy of small-cell lung cancer xenografts with anti neural cell adhesion molecule monoclonal antibody, 123C3: improvement of tumour uptake by internalisation.

The efficacy of three murine monoclonal antibodies (MAbs) for immunoscintigraphy of small-cell lung cancer (SCLS) xenografts was studied in a Balb/c nu/nu mouse model. These Mabs, 123C3, 123A8 and MOC191, belong to cluster 1 of anti-SCLC MAbs and bind to the neural cell adhesion molecule (NCAM) with similar affinity. After intraperitoneal injection of these MAbs, labelled with 125I, the highest uptake in tumour tissue was obtained with MAb 123C3. Seven days after the administration of this MAb 13.9% of the injected dose per gram of tumour tissue was retained in the tumour. The corresponding tumour tissue ratios ranged from 3.97 for blood to 31.03 for colon. The imaging results and the tumour uptake were less favourable for the two other MAbs, 123A8 and MOC191 (fractions of injected dose respectively 6.7% and 9.2%), although affinity, biological activity after labelling and uptake in non-tumour tissues were very similar for all three MAbs. These results may be explained by the differences in the interaction between the MAbs and the tumour cells. Mab 123C3 is internalised into tumour cells, whereas both other anti-NCAM Mabs are not. Internalisation into NCI H69 cells was demonstrated in vitro by radioimmunoassay, confocal laser scanning microscopy and electron microscopy. The internalised fraction of MAb 123C3 was 22.3% after 24h, whereas this fraction was only 7.5% for MAb 123A8. Although the internalised radiolabeled Mabs are usually degraded and dehalogenated intracellularly, the retained radioactivity is high. Apparently, intracellular degradation of radiolabelled MAb 123C3 and subsequent secretion of radioactive iodine did not prevent the accumulation of intracellular radioactivity. In conclusion, accumulation and retention of radioactivity in the tumour tissue, due to internalisation of radiolabelled MAbs, may improve the results immunoscintigraphy.

Radioimmunotherapy of small-cell lung cancer xenografts using 131I-labelled anti-NCAM monoclonal antibody 123C3.

We have studied the therapeutic efficacy of 131I-labelled monoclonal antibody 123C3 in human small-cell lung carcinoma xenografts established from the NCI-H69 cell line in nude mice. Several radiation doses were administered intraperitoneally and different treatment schedules were tested. The maximal tolerated dose, 2 x 500 microCi, resulted in complete remission of tumours smaller than 200 mm3 and long-lasting remission (more than 135 days) of the larger tumours. In control experiments, treatment with unlabelled monoclonal antibody 123C3 did not affect the tumour growth rate, while the effect of radiolabelled non-relevant, isotype-matched, monoclonal antibody M6/1 was minor and transient. Regrowth of the tumours occurred in all cases and could not be attributed to loss of neural cell adhesion molecule (NCAM) expression. Tumour recurrence is probably caused by insufficient radiation dosage. Radiation-induced toxicity was monitored by assessment of weight and bone marrow examination. Weight loss was observed in all treatment groups, but the mice regained their initial weight within 14 days, except for the group receiving the highest radiation dose (3 x 600 microCi). In this group all mice died as a result of radiotoxicity. Of the mice injected with 600 microCi radiolabelled control antibody, 50% died within 2 weeks after administration. Apparently the higher uptake of the radiolabelled monoclonal antibody in the tumour reduced systemic radiation toxicity.

Episialin (MUC1) overexpression inhibits integrin-mediated cell adhesion to extracellular matrix components.

Episialin (MUC1) is a transmembrane molecule with a large mucin-like extracellular domain protruding high above the cell surface. The molecule is located at the apical side of most glandular epithelial cells, whereas in carcinoma cells it is often present at the entire surface and it is frequently expressed in abnormally large quantities. We have previously shown that overexpression of episialin reduces cell-cell interactions. Here we show that the integrin-mediated adhesion to extracellular matrix of transfectants of a melanoma cell line (A375), a transformed epithelial cell line (MDCK-ras-e) and a human breast epithelial cell line (HBL-100) is reduced by high levels of episialin. This reduction can be reversed by inducing high avidity of the beta 1 integrins by mAb TS2/16 (at least for beta 1-mediated adhesion). The adhesion can also be restored by redistribution of episialin on the cell surface by monoclonal antibodies into patches or caps. Similarly, capping of episialin on ZR-75-1 breast carcinoma cells, growing in suspension, caused adherence and spreading of these cells. We propose that there is a delicate balance between adhesion and anti-adhesion forces in episialin expressing cells, which can be shifted towards adhesion by strengthening the integrin-mediated adhesion, or towards anti-adhesion by increasing the level of expression of episialin.

Is episialin/MUC1 involved in breast cancer progression?

Episialin, also designated MUC1, CA 15-3 antigen and PEM, is an established serum marker for breast cancer. Its function and possible involvement in tumor progression has not yet been completely established. The molecule is an extended rod-like molecule protruding high above the cell surface. It is often highly overexpressed in breast cancer relative to normal breast epithelium cells. Overexpression of episialin on cells in vitro reduces cell-cell and cell-extracellular matrix adhesion, because the rod-like molecule masks the adhesion receptors. Episialin also exerts its anti-adhesion effect in vivo. In certain human tumors, where episialin was present at the basal side of the cell, abnormal contacts between the plasma membrane and the stroma were observed. As a consequence of its anti-adhesion properties, episialin overexpression reduces the sensitivity of the cells for cytotoxic lymphocytes. This might be one of the reasons why episialin transfected cells are more potent to form experimental metastases after i.v. injection into nude mice.

Characterization and molecular cloning of a novel MUC1 protein, devoid of tandem repeats, expressed in human breast cancer tissue.

The human breast cancer marker protein, MUC1, is a polymorphic transmembrane molecule containing a large extracellular domain that is primarily composed of a variable number of highly conserved 20-amino-acid tandem repeats. We report here the detection of a novel invariantly sized 1.2-kb MUC1 mRNA, in addition to the large polymorphic mRNAs, by probing Northern blots with MUC1-cDNA-unique-sequence probes. The nucleotide sequence of this novel MUC1 mRNA demonstrates that it is identical to the MUC1 cDNA sequences downstream and upstream to the tandem-repeat array of the transmembrane form of MUC1. However, it contains neither the central tandem repeat array itself nor its directly flanking sequences that are deleted by a differential splicing event utilizing splice acceptor and donor sequences 5' and 3' to the tandem-repeat array. The splice event retains, downstream to the splice acceptor site, an open reading frame identical to that of the repeat-array-containing MUC1 thereby generating the novel MUC1/Y protein. Cells transiently transfected with the novel MUC1/Y cDNA express the MUC1/Y protein that is modified by glycosylation. The MUC1/Y protein is also readily detected in human breast cancer cells grown in vitro. Furthermore, primary breast cancer tissue samples demonstrate significant levels of the MUC1/Y protein whereas expression in tissue adjacent to the tumor is undetectable. Molecular characterization presented here, of the novel MUC1/Y molecule lacking the repeat array, suggests that it is likely to play a role distinct to that of the polymorphic repeat-array-positive MUC1 protein and that it may act as a new marker protein for human breast cancer.

NCAM and lung cancer.

The presence of the neural cell adhesion molecule, NCAM, is indicative for a poor prognosis in lung-cancer patients. Using MAb 735, we have investigated the expression of polysialic acid, PSA, on NCAM in a spectrum of neuro-endocrine lung tumors, ranging from the slowly growing typical carcinoids via the atypical carcinoids with clinically unpredictable behavior to the highly aggressive small-cell lung carcinomas. Our immunohistochemical findings indicate a significant association between the presence of PSA on the tumor cells and an aggressive and immature sub-type of the tumor. This might be related to impairment of cell-cell and cell-matrix interactions by the presence of PSA, as we demonstrated in vitro, since detachment is one of the first steps in the metastatic process. The NCAM-MAb 123C3 used in these studies appeared extremely useful in immunoscintigraphy and immunotherapy of SCLC xenografts in nude mice, and for immunoscintigraphy of a SCLC patient. This may be explained by internalization of the 123C3 antibody, which we demonstrated in vitro. 123C3 is the only Cluster-I SCLC MAb studied thus far that becomes internalized.

The epithelial sialomucin, episialin, is sialylated during recycling.

Episialin, the sialomucin encoded by the MUC1 gene is abundantly present at the surface of various epithelial cells. It is a transmembrane molecule that matures through several intermediate forms generated by proteolysis and sequential addition and processing of numerous O-linked glycans. Here we demonstrate that one of the biosynthetic intermediates, the premature form, which is incompletely glycosylated appears at the cell surface. By neuraminidase protection assays we show that cell surface-associated forms of episialin are constitutively internalized (0.9% of surface fraction/min) and recycled, with the intracellular residence time and total cycle time being approximately 66 and 143 min, respectively. During recycling, sialic acid residues are added to the premature form of episialin resulting in complete maturation of the molecule. Complete sialylation of episialin requires several rounds of recycling which continues after sialylation is apparently completed. The recycling of episialin may serve to achieve and maintain a high degree of sialylation.

Immunohistochemical study of mucin carbohydrates and core proteins in hepatolithiasis and cholangiocarcinoma.

The expression of mucin carbohydrates [Tn, sialosyl-Tn(STn), and T antigens] and core proteins [MUCI-apomucin-related antigen (ARA) and MUC2-ARA] was examined immunohistochemically in tissues from 40 patients with hepatolithiasis and 26 patients with intrahepatic bile-duct carcinoma. Tn and STn antigens were expressed in most of the carcinomas, and were also often expressed in the atypical bile-duct epithelium of the patients with hepatolithiasis or carcinoma, whereas they were rarely or never expressed in the normal bile duct, suggesting that they are effective tumor markers. T antigen was less useful as a marker for intrahepatic bile-duct carcinoma or the atypical epithelium, because it was expressed in normal bile-duct of some cases. Regarding the expression of ARAs in the carcinomas, non-invasive bile-duct cyst adenocarcinomas with favorable prognosis either expressed no MUCI-ARA with [DF3(-), MUSEII(-) and 139H2(-)] staining pattern or expressed MUCI-ARA with [DF3(-), MUSEII(+) and 139H2(+)] staining pattern. However these tumors often expressed MUC2-ARA with [anti-MRP(+) and CCP58(+)] staining pattern. In contrast, most invasive non-papillary cholangiocarcinomas with poor prognosis expressed MUCI-ARA with [DF3(+), MUSEII(+) and 139H2(+)] staining pattern, but expressed no MUC2-ARA with [anti-MRP(-) and CCP58(-)] staining pattern. These results suggests that different apomucins are produced by bile-duct cystadenocarcinomas and cholangiocarcinomas with differing prognosis. Furthermore, expression of Tn and STn antigens is a useful indicator of malignancy in the intrahepatic duct.