Intestinal metaplasia (IM) is part of a stepwise sequence of alterations of the gastric mucosa, leading ultimately to gastric cancer, and is strongly associated with chronic Helicobacter pylori infection. The molecular mechanisms underlying the onset of IM remain elusive. The aim of this study was to assess the putative involvement of two intestine-specific transcription factors, CDX1 and CDX2, in the pathogenesis of gastric IM and gastric carcinoma. Eighteen foci of IM and 46 cases of gastric carcinoma were evaluated by immunohistochemistry for CDX1 and CDX2 expression. CDX1 was expressed in all foci of IM and in 41% of gastric carcinomas; CDX2 was expressed in 17/18 foci of IM and in 54% of gastric carcinomas. In gastric carcinomas, a strong association was observed between the expression of CDX1 and CDX2, as well as between the intestinal mucin MUC2 and CDX1 and CDX2. No association was observed between the expression of CDX1 and CDX2 and the histological type of gastric carcinoma. In conclusion, these results show that aberrant expression of CDX1 and CDX2 is consistently observed in IM and in a subset of gastric carcinomas. The association of CDX1 and CDX2 with expression of the intestinal mucin MUC2, both in IM and in gastric carcinoma, indirectly implies that CDX1 and CDX2 may be involved in intestinal differentiation along the gastric carcinogenesis pathway.

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SUMMARYTo investigate the expression of MUC6 mucin in gastric carcinomas, we generated a novel monoclonal antibody (MAb CLH5) using an MUC6 synthetic peptide. MAb CLH5 reacted exclusively with the MUC6 peptide and with native and deglycosylated mucin extracts from gastric tissues. MAb CLH5 immunoreactivity was observed in normal gastric mucosa restricted to pyloric glands of the antrum and mucopeptic cells of the neck zone of the body region. In a series of 104 gastric carcinomas, 31 (29.8%) were immunoreactive for MUC6. The expression of MUC6 was not associated with histomorphological type or with clinicopathological features of the carcinomas. Analysis of the co-expression of MUC6 with other secreted mucins (MUC5AC and MUC2) in 20 gastric carcinomas revealed that different mucin core proteins are co-expressed in 55% of the cases. MUC6 was co-expressed and co-localized with MUC5AC in 45% and with MUC2 in 5% of the cases. Expression of MUC2 alone was observed in 25% of the cases. All carcinomas expressing MUC2 mucin in more than 50% of the cells were of the mucinous type according to the WHO classification. The co-expression of mucins was independent of the histomorphological type and stage of the tumors. In conclusion, we observed, using a novel well-characterized MAb, that MUC6 is a good marker of mucopeptic cell differentiation and is expressed in 30% of gastric carcinomas, independent of the clinicopathological features of the cases. Furthermore, we found that co-expression and co-localization of mucins in gastric carcinomas is independent of histomorphology and staging. Finally, we observed that intestinal mucin MUC2 is expressed as the most prominent mucin of the mucins tested in mucinous-type gastric carcinomas.

Exposure for 24 h of mucus-secreting HT-29 cells to the sugar analogue GalNAc-α-O-benzyl results in inhibition of Galβ1-3GalNAc:α2,3-sialyltransferase, reduced mucin sialylation, and inhibition of their secretion (Huet, G., I. Kim, C. de Bolos, J.M. Loguidice, O. Moreau, B. Hémon, C. Richet, P. Delannoy, F.X. Real., and P. Degand. 1995. J. Cell Sci. 108:1275–1285). To determine the effects of prolonged inhibition of sialylation, differentiated HT-29 populations were grown under permanent exposure to GalNAc-α-O-benzyl. This results in not only inhibition of mucus secretion, but also in a dramatic swelling of the cells and the accumulation in intracytoplasmic vesicles of brush border–associated glycoproteins like dipeptidylpeptidase-IV, the mucin-like glycoprotein MUC1, and carcinoembryonic antigen which are no longer expressed at the apical membrane. The block occurs beyond the cis-Golgi as substantiated by endoglycosidase treatment and biosynthesis analysis. In contrast, the polarized expression of the basolateral glycoprotein GP 120 is not modified. Underlying these effects we found that (a) like in mucins, NeuAcα2-3Gal-R is expressed in the terminal position of the oligosaccharide species associated with the apical, but not the basolateral glycoproteins of the cells, and (b) treatment with GalNAc-α-O-benzyl results in an impairment of their sialylation. These effects are reversible upon removal of the drug. It is suggested that α2-3 sialylation is involved in apical targeting of brush border membrane glycoproteins and mucus secretion in HT-29 cells.

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Mucins are glycoproteins normally synthesized by a variety of secretory epithelial cells. The aim of this study was to investigate the expression of mucins (MUC1, MUC2, MUC4, MUC5AC, MUCB, MUC6, MUC7) in mucoepidermoid carcinomas, the most frequent malignant tumor of salivary glands. Forty mucoepidermoid carcinomas and twenty-two normal salivary glands were studied for these mucins by immunohistochemistry from formalin-fixed and paraffin-embedded material. Normal salivary glands frequently expressed MUC1 and MUC4, mainly in ductal cells; MUC5B and MUC7 stained mucous and serous acini respectively of submandibular and minor salivary glands; and MUC5AC and MUC2 were poorly detected in excretory ducts. All mucoepidermoid carcinomas expressed MUC1, and 38/40 tumors expressed MUC4. Both membrane-bound mucins stained membranes and cytoplasm of all cell types (epidermoid, intermediate, mucous, clear and columnar). MUC5AC and MUC5B stained glandular differentiated cells in most tumors (29/40 and 33/40 cases, respectively). MUC6 was positive in 13/40 tumors, and both MUC2 and MUC7 in only 2/40 tumors. The high expression of MUC1 was related to high histologic grades, high recurrence and metastasis rates and a shorter disease-free interval (P < 0.05). Conversely, MUC4 high expression was mainly related to low-grade tumors, lower recurrence rates and a longer disease-free interval (P < 0.05). In conclusion, mucoepidermoid carcinomas of salivary glands usually express MUC1, MUC4, MUC5AC and MUC5B; less frequently MUC6; and rarely MUC2 and MUC7. This mucin expression pattern can be useful for diagnostic purposes. Therefore, MUC1 expression is related to tumor progression and worse prognosis, whereas MUC4 expression is related to a better prognosis.

Gastric MUC5AC and MUC6 mucins were studied using polyclonal antibodies. Immunohistochemistry showed MUC5AC to originate from the surface epithelium, whereas MUC6 was produced by the glands. Mucins from the surface epithelium or glands of corpus and antrum were purified using CsCl/4M guanidinium chloride density-gradient centrifugation. MUC5AC appeared as two distinct populations at 1.4 and 1.3g/ml, whereas MUC6, which was enriched in the gland tissue, appeared at 1.45g/ml. Reactivity with antibodies against the Leb structure (where Le represents the Lewis antigen) followed the MUC5AC distribution, whereas antibodies against the Ley structure and reactivity with the GlcNAc-selective Solanum tuberosum lectin coincided with MUC6, suggesting that the two mucins are glycosylated differently. Rate-zonal centrifugation of whole mucins and reduced subunits showed that both gastric MUC5AC and MUC6 are oligomeric glycoproteins composed of disulphide-bond linked subunits and that oligomeric MUC5AC was apparently smaller than MUC6. A heterogeneous population of ‘low-density’ MUC5AC mucins, which were smaller than the ‘high-density’ ones both before and after reduction, reacted with an antibody against a variable number tandem repeat sequence within MUC5AC, suggesting that they represent precursor forms of this mucin. Following ion-exchange HPLC, both MUC5AC and MUC6 appeared as several distinct populations, probably corresponding to ‘glycoforms’ of the mucins, the most highly charged of which were found in the gland tissue.

BackgroundCell surface sialylation is emerging as an important feature of cancer cell metastasis. Sialyltransferase expression has been reported to be altered in tumours and may account for the formation of sialylated tumour antigens. We have focused on the influence of alpha-2,3-sialyltransferase ST3Gal III in key steps of the pancreatic tumorigenic process.Methodology/Principal FindingsST3Gal III overexpressing pancreatic adenocarcinoma cell lines Capan-1 and MDAPanc-28 were generated. They showed an increase of the tumour associated antigen sialyl-Lewisx. The transfectants' E-selectin binding capacity was proportional to cell surface sialyl-Lewisx levels. Cellular migration positively correlated with ST3Gal III and sialyl-Lewisx levels. Moreover, intrasplenic injection of the ST3Gal III transfected cells into athymic nude mice showed a decrease in survival and higher metastasis formation when compared to the mock cells.ConclusionIn summary, the overexpression of ST3Gal III in these pancreatic adenocarcinoma cell lines underlines the role of this enzyme and its product in key steps of tumour progression such as adhesion, migration and metastasis formation.