Lewis(y) antigen (CD174) and apoptosis in gastric and colorectal carcinomas: correlations with clinical and prognostic parameters.

Lewis(y) (Le(y)), also designated CD174, represents a carbohydrate blood group antigen which is strongly expressed in neoplastic gastrointestinal tissues. Previous reports indicated an association between Le(y) expression and apoptosis. Therefore, we tried to elucidate its clinicopathological relevance in a series of 160 gastric and 215 colorectal carcinomas by immunohistochemical detection of Le(y) and visualization of apoptotic cells applying the in-situ-end labelling (ISEL) method, followed by semiquantitative scoring of the specimens. In both gastric as well as colorectal carcinomas, between 40 and 50% of the cases were Le(y) reactive. Signet-ring cell carcinomas of the stomach exhibited a significantly stronger Le(y) expression compared to other tumor types. In colorectal cancers, Le(y) was associated with increased tumor staging, showing the strongest positivity in stage IV. Further correlations with clinicopathological variables or prognosis were not observed. On the other hand, the amount of apoptotic cells was significantly reduced in mucinous adenocarcinomas of the colorectum compared to non-mucinous carcinomas. Scoring of apoptotic cells did not result in any other clinicopathologically relevant correlations. In addition, a significant association between Le(y) antigen expression and apoptosis score could not be established. Therefore, the hypothesis of a functional relationship between these two aspects of gastrointestinal tumor biology is not confirmed by our data.

ST6GalNAc I expression in MDA-MB-231 breast cancer cells greatly modifies their O-glycosylation pattern and enhances their tumourigenicity.

Sialyl-Tn is a carbohydrate antigen overexpressed in several epithelial cancers, including breast cancer, and usually associated with poor prognosis. Sialyl-Tn is synthesized by a CMP-Neu5Ac:GalNAcalpha2,6-sialyltransferase: CMP-Neu5Ac: R-GalNAcalpha1-O-Ser/Thr alpha2,6-sialyltransferase (EC 2.4.99.3) (ST6GalNAc I), which transfers a sialic acid residue in alpha2,6-linkage to the GalNAcalpha1-O-Ser/Thr structure. However, established breast cancer cell lines express neither ST6GalNAc I nor sialyl-Tn. We have previously shown that stable transfection of MDA-MB-231, a human breast cancer cell line, with ST6GalNAc I cDNA induces sialyl-Tn antigen (STn) expression. We report here the modifications of the O-glycosylation pattern of a MUC1-related recombinant protein secreted by MDA-MB-231 sialyl-Tn positive cells. We also show that sialyl-Tn expression and concomitant changes in the overall O-glycan profiles induce a decrease of adhesion and an increase of migration of MDA-MB-231. Moreover, STn positive clones exhibit an increased tumour growth in severe combined immunodeficiency (SCID) mice. These observations suggest that modification of the O-glycosylation pattern induced by ST6GalNAc I expression are sufficient to enhance the tumourigenicity of MDA-MB-231 breast cancer cells.

Design of a MUC1-based cancer vaccine.

The epithelial type 1 transmembrane mucin MUC1 is long-established as a marker for monitoring recurrence of breast cancer, and beyond its diagnostic marker qualities, it is a promising target for immunotherapeutic strategies to treat cancer by active specific immunization. The mucin is able to break tolerance and to induce humoral immune responses in healthy subjects and in cancer patients, but the response is generally weak. These natural responses to tumour-associated MUC1 glycoforms indicate that antibody reactivities are more directed to glycopeptide than to non-glycosylated peptide epitopes. To overcome the weak immunogenicity of heavily O-glycosylated MUC1, the question of whether O-linked glycans remain intact during processing in the MHC class II pathway was addressed. Attempts were made to define site-specific O-glycosylation and the structural requirements for efficient endosomal proteolysis by cathepsin L in dendritic cells. A fraction of glycopeptides survive the processing machinery, and have the capacity to bind to MHC class II and to activate sub-populations of glycopeptide-specific helper T-cell clones as a prerequisite for strong and long-lasting immune responses to MUC1-positive tumours. Moreover, studies on clusters of sequence-variant repeats, which are interspersed in the repeat domain of MUC1 at high frequency, have revealed that a limited set of concerted amino-acid replacements (Asp-Thr0-Arg1-Pro10 to Glu-Ser0-Arg1-Ala10) contributes considerably to increased peptide flexibility and to under-glycosylation of sequence-variant repeats which in concert modify immunological features of the mucin. Peptides and glycopeptides with the immunodominant DTR (Asp-Thr-Arg) or with the variant ESR (Glu-Ser-Arg) motif, and highly immunogenic peptides of the degenerate repeats that flank the repeat domain are currently evaluated as potential targets in multi-epitopic adjuvant-based vaccine strategies for their capacity to induce cytotoxic T-cell responses.

An anti-MUC1-antibody-interleukin-2 fusion protein that activates resting NK cells to lysis of MUC1-positive tumour cells.

MUC1 mucin is aberrantly glycosylated and overexpressed in a number of epithelial malignancies and is therefore a promising tumour-associated antigen for target-directed immunotherapy of a panel of malignant diseases. In MUC1-positive tumours, MHC class I expression is frequently downregulated and MUC1-specific cytotoxic T cells (CTLs) are either not available or in a state of anergy allowing tumour growth without limitation by CTL control. To activate lymphocytes and natural killer (NK) cells, we here generated an anti-MUC1-scFv-IL2 fusion protein (C595scFv-Fc-IL2) that contains the C595 single-chain antibody for MUC1 binding, the human IgG1 CH2CH3 domain for protein dimerisation, and interleukin-2 (IL2) for activation of immunological effector cells. The fusion protein binds to MUC1-derived peptides and to MUC1-positive tumour cells with the same specificity as does the C595 monoclonal antibody. Bound to MUC1, the C595scFv-Fc-IL2 fusion protein stimulates proliferation of human activated lymphocytes in vitro. Upon binding to MUC1-positive MCF7 breast carcinoma cells, moreover, the fusion protein activates resting NK cells to tumour cell lysis. These properties make the C595scFv-Fc-IL2 fusion protein a suitable candidate for the immunotherapy of MUC1-positive tumours.

Comparative evaluation of the prognostic value of MUC1, MUC2, sialyl-Lewis(a) and sialyl-Lewis(x) antigens in colorectal adenocarcinoma.

AIMS: The significance of MUC1, MUC2 and sialylated Lewis blood group antigens as prognostic markers in colorectal adenocarcinoma was investigated in a large series of patients because previous investigations revealed inconsistent results due to unrelated tumour samples from different patient groups and methodological differences. METHODS AND RESULTS: Tissues from 243 patients with colorectal adenocarcinoma were stained immunohistochemically. MUC1 showed a strong immunoreactivity (in more than 35% of the tumour area) in 32.5%, MUC2 in 51.0%, sialyl-Lewis(x) in 67.9% and sialyl-Lewis(a) in 73.7% of the cases, respectively. MUC1 immunoreactivity displayed a significant correlation with tumour progression as reflected by advancing pTNM staging and poor differentiation. MUC2 expression was significantly stronger in mucinous adenocarcinomas. Sialyl-Lewis(x) immunostaining correlated with the extent of lymph node metastasis as well as low cytological differentiation. According to univariate and multivariate analysis (P < 0.0001) only MUC1 reactivity represented a marker of worse survival probability, opposed to the sialylated Lewis antigens that did not exert a predictive value. CONCLUSIONS: According to our data, MUC1 and sialyl-Lewis(x) immunoreactivity exhibit statistically significant correlations with established markers of tumour progression. However, only MUC1 presents as an independent prognostic factor of colorectal adenocarcinoma.

Immunoreactivity of Lewis blood group and mucin peptide core antigens: correlations with grade of dysplasia and malignant transformation in the colorectal adenoma-carcinoma sequence.

Previous studies on the immunoreactivity of various mucin peptide and carbohydrate antigens in neoplastic colorectal tissues led to at least in part contradictory results. Therefore, we investigated a series of 42 adenomas and 44 carcinomas applying monoclonal antibodies (mabs) directed against Lewis blood group antigens (sialyl-Le(a), Le(x), sialyl-Le(x), Le(y)) as well as mucin peptide cores (MUC1, MUC2 and MUC5AC) by immunohistochemistry. A statistically significant positive correlation between the development of high-grade dysplasia in colorectal adenomas and the immunoreactivity of Le(y) and MUC1 epitopes was observed, whereas MUC2 exhibited a significant negative correlation. The reactivity of the other epitopes did not show an association with the progression of malignant transformation. Colorectal carcinomas were subdivided according to their histopathological subtype. The immunohistochemical staining resulted in a significantly stronger MUC2 reactivity of mucinous vs. tubular adenocarcinomas. Immunoreactivity of the MUC1-specific mab, which does not react with the fully glycosylated peptide core, showed a statistically non-significant inverse tendency, whereas all carbohydrate antigens displayed a strong expression in both tumor subtypes. Furthermore, correlations between mucin peptide and carbohydrate epitope labelling were evaluated. Progression of the adenoma-carcinoma sequence was accompanied by an increase of Le(y) as well as MUC1 antigen and an increase of all Lewis antigens compared to MUC2 immunoreactivity. On the other hand, mucinous carcinomas exhibited an inverse pattern. In conclusion, these results demonstrate that Le(y) and MUC1 immunoreactivity correlate with malignant transformation in the colorectum, whereas MUC2 represents a marker for low-grade dysplasia and the subtype of mucinous carcinomas.

Evidence for glycosylation-dependent activities of polypeptide N-acetylgalactosaminyltransferases rGalNAc-T2 and -T4 on mucin glycopeptides.

We present evidence that site-specific O-glycosylation by recombinant polypeptide N-acetylgalactosaminyltransferases rGalNAc-T2 and -T4 is controlled by the primary sequence context, as well as by the position and structure of previously introduced O-glycans. Synthetic mucin-type (glyco)peptides corresponding to sections of the tandem repeat regions of MUC1, MUC2, and MUC4 were used as substrates for recombinant polypeptide N-acetylgalactosaminyltransferases, rGalNAc-T2 and -T4. By concerted and sequential action the two transferases are able to fully glycosylate MUC1 but only partially MUC2 and MUC4 tandem repeat peptides. GalNAc residues on MUC1 acceptor peptides trigger activity of rGalNAc-T4 directed to Ser in VTSA and Thr in PDTR and of rGalNAc-T2 to Ser/Thr within the GSTA motif of variant MUC1 peptides. However, elongation of GalNAc by beta3-galactosylation inhibits rGalNAc-T4 activity completely and rGalNAc-T2 activity with respect to the acceptor site GSTA. These findings are in accord with the inhibition of rGalNAc-T2 and -T4 by fully GalNAc-substituted MUC1 repeat peptide and support a glycosylation-dependent activity induction or enhancement of both enzymes.

Immunoreactivity of monoclonal antibody BW835 represents a marker of progression and prognosis in early gastric cancer.

The Thomsen-Friedenreich (TF) antigen is a well-known human pan-carcinoma antigen. It represents a carbohydrate core disaccharide (Gal beta 1-3GalNAc) which is predominantly bound to mucin peptide cores. Its immunoreactivity depends on changes in glycosylation which lead to a reduction in the carbohydrate chain length and the exposure of core carbohydrates. In the present study, we investigated 208 gastric adenocarcinomas with respect to their immunohistochemical reactivity applying two monoclonal antibodies (MAbs). MAb specifically detecting TF antigen (A78-G/A7) and MAb BW835 were included. The latter reacts with a certain glycoform of the MUC1 peptide core, characterized by core-type glycans like TF. A78-G/A7 epitopes were detected in 68.8% and BW835 epitopes in 57.7% of the carcinomas. BW835 immunoreactivity correlated with the presence of lymph node metastases. Both A78-G/A7 and BW835 staining were significantly stronger in tubular/papillary cancer (WHO classification) and intestinal-type cancer according to Laurén. In univariate survival analyses of all patients studied, BW835 immunoreactivity was a marker of an unfavorable prognosis (p < 0.05). The presence of A78-G/A7 and BW835 epitopes exerted a negative effect on the subgroup of pTNM stage I carcinomas. These results indicate that TF and MUC1-TF immunoreactivity defines a 'high-risk' subgroup of stage I patients in gastric cancer.

Structural characterization of human recombinant and bone-derived bone sialoprotein. Functional implications for cell attachment and hydroxyapatite binding.

Human bone sialoprotein (BSP) comprises 15% of the total noncollagenous proteins in bone and is thought to be involved in bone mineralization and remodeling. Recent data suggest a role for BSP in breast cancer and the development of bone metastases. We have produced full-length recombinant BSP in a human cell line and purified the protein from human bone retaining the native structure with proper folding and post-translational modifications. Mass spectrometry of bone-derived BSP revealed an average mass of 49 kDa and for recombinant BSP 57 kDa. The post-translational modifications contribute 30-40%. Carbohydrate analysis revealed 10 different complex-type N-glycans on both proteins and eight different O-glycans on recombinant BSP, four of those were found on bone-derived BSP. We could identify eight threonines modified by O-glycans, leaving the C terminus of the protein free of glycans. The recombinant protein showed similar secondary structures as bone-derived BSP. BSP was visualized in electron microscopy as a globule linked to a thread-like structure. The affinity for hydroxyapatite was higher for bone-derived BSP than for recombinant BSP. Cell adhesion assays showed that the binding of BSP to cells can be reversibly diminished by denaturation.

Recombinant human tumor antigen MUC1 expressed in insect cells: structure and immunogenicity.

MUC1, a member of the mucin family of molecules, is a transmembrane glycoprotein abundantly expressed on human ductal epithelial cells and tumors originating from those cells. MUC1 expressed by malignant cells is aberrantly O-glycosylated. Differences in O-glycosylation of the tandem repeat region of MUC1 make tumor and normal forms of this antigen immunologically distinct. The tumor-specific glycoform is, therefore, expected to be a good target for immunotherapy and a good immunogen for generation of antitumor immune responses. We have generated a renewable source of this glycoform by expressing MUC1 cDNA in Sf-9 insect cells using a baculovirus vector. This form of MUC1 (BV-MUC1) is O-glycosylated at a very low level, approximately 0.3% (w/w), and this is not due to the lack of appropriate glycosylotransferases in insect cells. Peptidyl GalNAc-transferases isolated from Sf-9 cells were able to glycosylate in vitro a synthetic MUC1 peptide as efficiently as the transferases isolated from human milk. Neither preparation of peptidyl GalNAc-transferases, however, was able to glycosylate BV-MUC1. This underglycosylated recombinant MUC1 mimics underglycosylated MUC1 on human tumor cells and could serve as an immunogen to stimulate responses that would recognize MUC1 on tumor cells. To test this we immunized mice with Sf-9 cells expressing BV-MUC1. Sera from immunized mice recognized MUC1 on human tumor cells. We also generated MUC1-specific T cells that proliferated in response to synthetic MUC1 peptide.

Identification and topology of variant sequences within individual repeat domains of the human epithelial tumor mucin MUC1.

This study shows for the first time that the tandemly repeated icosapeptide of human MUC1 underlies a genetic sequence polymorphism at three positions (underlined): PDTRPAPGSTAPPAHGVTSA. The concerted replacement DT-->ES (sequence variation 1) and the single replacements P-->Q (sequence variation 2), P-->A (sequence variation 3), and P-->T (sequence variation 4) were identified by sequencing of polymerase chain reaction products and studied by minisatellite variant repeat analysis for their incidence and topology in the 5' and 3' peripheral regions of the variable number of tandem repeats domain. Minisatellite variant repeat analyses were performed with 27 individual samples of genomic DNA from human cells and tissues covering 30-60% of the domain. Within the peripheral regions, sequence variations 1-4 occur at high incidence and show a nearly constant repeat topology in all individual normal and tumor samples. Also, individuals who were non-Caucasian or of different ethnic background were found to have the same set of replacements with identical topology. The repeat variant 1 replacing the established tumor target motif DTR with ESR was found in all individuals and appears predominantly in repeat clusters (diads and triads). The largely constant topology of variant repeats is interpreted by the assumption that the variable number of tandem repeats domain has evolved as a recent expansion of sequence variable super-repeats.

O-glycosylation of the mucin type.

While only about ten percent of the databank entries are defined as glycoproteins, it has been estimated recently that more than half of all proteins are glycoproteins. Mucin-type O-glycosylation is a widespread post-translational modification of proteins found in the entire animal kingdom, but also in higher plants. The structural complexity of the chains initiated by O-linked GalNAc exceeds that of N-linked chains by far. The process during which serine and threonine residues of proteins become modified is confined to the cis to trans Golgi compartments. The initiation of this process by peptidyl GalNAc-transferases is ruled by the sequence context of putative O-glycosylation sites, but also by epigenetic regulatory mechanisms, which can be mediated by enzyme competition. The cellular repertoir of glycosyltransferases with their distinct donor sugar and acceptor sugar specificities, their sequential action at highly-ordered surfaces, and their localizations in subcompartments of the Golgi finally determine the cell-specific O-glycosylation profile. Dramatic alterations of the glycosylation machinery are observed in cancer cells, resulting in aberrantly O-glycosylated proteins that expose previously masked peptide motifs and new antigenic targets. The functional aspects of O-linked glycans, which comprise among many others their potential role in sorting and secretion of glycoproteins, their influence on protein conformation, and their multifarious involvement in cell adhesion and immunological processes, appear as complex as their structures.

Biochemical characterization of the soluble form of tumor antigen MUC1 isolated from sera and ascites fluid of breast and pancreatic cancer patients.

Transmembrane glycoprotein tumor antigen MUC1 that is overexpressed on pancreatic and breast tumor cells can be found in large amounts in soluble form in serum and ascites fluid. MUC1 has been identified as a target of human antitumor antibody and CTL responses that are generated in the absence of helper T cells. The soluble form of MUC1 should support generation of helper T cells, but we have found recently that this form, although effectively endocytosed by dendritic cells, remains trapped in early endosomes and is not trafficked to antigen-processing compartments. The exact biochemical structure of this form of MUC1 has not been elucidated to date, and it is thus not clear what structural characteristics may be responsible for its retention in early endosomes. We have purified soluble MUC1 from ascites fluid of breast/pancreatic cancer patients (ASC-MUC1) and quantitated O-linked carbohydrates. We have altered ASC-MUC1 by enzymatic treatment: trypsin or clostripain digestion, desialylation, and further in vitro glycosylation. We have found that desialylated ASC-MUC1 was further glycosylated by peptidyl N-acetylgalactosamine transferases and was not when sialic acid was present. These alterations created new forms of ASC-MUC1 that might be handled more efficiently by antigen-presenting cells to generate better tumor-specific immunity and used to identify structures that are directly involved in retention of this antigen in early endosomes.

Epitope-dependent differential immunoreactivities of anti-MUC1 monoclonal antibodies in human carcinomas.

According to studies on a variety of malignant tumors from different organs MUC1 mucin antigen presents as a valuable marker of cancer progression and prognosis. During recent years, a great number of monoclonal antibodies (mabs) directed to MUC1 was generated. Their epitopes can be classified according to their position within the tandem repeat domain of the mucin and with respect to effects exerted by site-specific glycosylation. In this study, eight mabs from different clusters were selected to correlate their epitope specificity with their binding pattern in human cancer specimens. By applying an immunohistochemical ABC-peroxidase method, ten carcinomas derived from breast, pancreas, stomach and colon were characterized. A positive reaction of all mabs could be observed in the majority of the carcinomas, however, the extent of the stained tumor area varied significantly. In general, mabs M38, VA1 and BC3 exhibited the strongest staining reaction. Mab BW835 showed a similar binding intensity, especially in pancreatic and gastric carcinomas. It is tempting to speculate that the different binding patterns may reflect differences in epitope specificity. In conclusion, future immunohistochemical, immunoserological and therapeutic studies involving MUC1 antigen should prefer well-characterized and highly reactive mabs detecting defined peptide epitopes.

Glycoprotein identification and localization of O-glycosylation sites by mass spectrometric analysis of deglycosylated/alkylaminylated peptide fragments.

In-gel digestion of densely O-glycosylated proteins, an essential step in proteome analysis, is often hampered by steric hindrance of the proteases. To overcome this technical problem a simple and convenient method has been developed, which combines several advantages: (1) Approximately 70% of the oligosaccharides are cleaved without significant protein hydrolysis at the optimal reaction conditions of 70% ethylamine, and quantitative cleavage is achieved with 40% methylamine, at 50 degrees C. (2) To the unsaturated derivatives of Ser and Thr the alkylamine is added as a label of previous O-glycosylation sites. (3) The alkylaminylated protein is effectively cleaved by proteolysis. (4) The modified peptides are identified by MALDI mass spectrometry under consideration of incremental mass increases. (5) The alkylamine label is stable under MALDI post-source-decay analysis as well as in collision-induced dissociation experiments allowing sequencing and peptide localization of O-glycosylation sites. Applicability of the method is evaluated with a series of synthetic glycopeptides, the densely O-glycosylated human glycophorin A, and with the mucin MUC1 from human milk fat globule membranes.

Anti-infectious properties of the human milk fat globule membrane.

Secretory immunoglobulin A (sIgA), the predominant antibody fraction of human milk, represents a major protective factor against neonatal infection. Until now, sIgA had been identified only in the humoral fraction of human milk. For bovine milk an association between sIgA and the milk fat globule (MFG) membranes has been demonstrated. The aim of our study was to assess whether sIgA is associated with the MFG membranes in human milk. Using anti-sIgA-agglutinated human MFG and immune fluorescence microscopy, we demonstrated that sIgA is, in fact, associated with human MFG. Subsequently, by electrophoretic separation of human MFG membranes and Western blotting, we demonstrated specific sIgA bands, suggesting that sIgA is truly an integral part of the human MFG membrane. This may be of physiological relevance, as undigested and functional human MFG are found in the stools of the newborn.

Increased galectin-3 expression in gastric cancer: correlations with histopathological subtypes, galactosylated antigens and tumor cell proliferation.

Galectin-3 represents an endogenous galactoside-binding lectin which may be involved in tumor cell adhesion and proliferation. In order to evaluate its biological significance in human gastric cancer, we investigated its expression in the stomach of a large series of patients (n = 193) by immunohistochemical staining with the monoclonal antibody Mac-2. Compared to normal tissues, primary gastric adenocarcinomas showed a slight increase in galectin-3 expression. However, there was no correlation of membrane-bound and cytoplasmic galectin-3 with histopathological differentiation parameters (according to the WHO and Laurén classifications) or tumor progression (as documented by pTNM staging). Nuclear galectin-3 reactivity was significantly stronger in diffuse-type cancer compared to the intestinal-type tumors. Galectin-3 binds to terminal GalNAcalpha(1-3) bound to polylactosamine chains and related glycotopes. Therefore, the strong coexpression of membrane/cytoplasmic galectin-3 with Griffonia simplicifolia agglutinin I (GSA I) binding sites (Galalpha1-3Gal-, GalNAcalpha-) on carcinoma cells seems to be interesting. On the other hand, nuclear galectin-3 immunoreactivity did not correlate with the incidence of Ki-67-positive tumor cells. A prognostic value of galectin-3 regarding patient survival could not be established.

Thomsen-Friedenreich antigen presents as a prognostic factor in colorectal carcinoma: A clinicopathologic study of 264 patients.

BACKGROUND: Up to now, the expression of the tumor-associated Thomsen-Friedenreich (TF) antigen in colorectal carcinoma has not been thoroughly investigated with particular emphasis on its correlation with established clinicopathologic characteristics and classifications as well as its prognostic relevance. METHODS: Formalin fixed, paraffin embedded specimens from 264 patients with colorectal carcinoma were stained using an avidin-biotin complex-peroxidase assay. As primary monoclonal antibodies (MAbs), A78-G/A7, which binds to TFalpha and TFbeta antigen irrespective of its carrier, and BW835, which detects TFalpha on MUC1 repeat peptide, were applied. RESULTS: MAbs A78-G/A7 and BW835 labeled 64.8% and 58. 0%, respectively, of carcinomas. None of the binding patterns correlated with gender, tumor localization, or growth type. Only BW835 reactivity exhibited a significant correlation with increasing pTNM staging and histologic grading. Staining of the MAb A78-G/A7 was significantly stronger in carcinomas that contained a mucinous component. In univariate survival analysis, in addition to pTNM staging and histologic grading, reactivity with A78-G/A7 as well as BW835 were significantly correlated with lower survival probability. Multivariate analysis according to the Cox proportional hazards model revealed only pTNM staging, histologic grading, and A78-G/A7 staining to be independent prognostic factors. CONCLUSIONS: According to these results, TF disaccharide represents a cancer-associated antigen in colorectal carcinoma that exhibits qualities of a prognostic marker. As demonstrated by BW835 staining, it is obviously coexpressed with MUC1 peptide core in a great number of cases. These results suggest that TF, in addition to MUC1, might also serve as a useful target antigen in the treatment of patients with colorectal carcinoma.