Raquel Almeida

Identification and characterization of large galactosyltransferase gene families: galactosyltransferases for all functions.

Enzymatic glycosylation of proteins and lipids is an abundant and important biological process. A great diversity of oligosaccharide structures and types of glycoconjugates is found in nature, and these are synthesized by a large number of glycosyltransferases. Glycosyltransferases have high donor and acceptor substrate specificities and are in general limited to catalysis of one unique glycosidic linkage. Emerging evidence indicates that formation of many glycosidic linkages is covered by large homologous glycosyltransferase gene families, and that the existence of multiple enzyme isoforms provides a degree of redundancy as well as a higher level of regulation of the glycoforms synthesized. Here, we discuss recent cloning strategies enabling the identification of these large glycosyltransferase gene families and exemplify the implication this has for our understanding of regulation of glycosylation by discussing two galactosyltransferase gene families.

Expression of intestine-specific transcription factors, CDX1 and CDX2, in intestinal metaplasia and gastric carcinomas.

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. Copyright

Role of the Human ST6GalNAc-I and ST6GalNAc-II in the Synthesis of the Cancer-Associated Sialyl-Tn Antigen

The Sialyl-Tn antigen (Neu5Acα2–6GalNAc-O-Ser/Thr) is highly expressed in several human carcinomas and is associated with carcinoma aggressiveness and poor prognosis. We characterized two human sialyltransferases, CMP-Neu5Ac:GalNAc-R α2,6-sialyltransferase (ST6GalNAc)-I and ST6GalNAc-II, that are candidate enzymes for Sialyl-Tn synthases. We expressed soluble recombinant hST6GalNAc-I and hST6GalNAc-II and characterized the substrate specificity of both enzymes toward a panel of glycopeptides, glycoproteins, and other synthetic glycoconjugates. The recombinant ST6GalNAc-I and ST6GalNAc-II showed similar substrate specificity toward glycoproteins and GalNAcα-O-Ser/Thr glycopeptides, such as glycopeptides derived from the MUC2 mucin and the HIVgp120. We also observed that the amino acid sequence of the acceptor glycopeptide contributes to the in vitro substrate specificity of both enzymes. We additionally established a gastric cell line, MKN45, stably transfected with the full length of either ST6GalNAc-I or ST6GalNAc-II and evaluated the carbohydrate antigens expression profile induced by each enzyme. MKN45 transfected with ST6GalNAc-I showed high expression of Sialyl-Tn, whereas MKN45 transfected with ST6GalNAc-II showed the biosynthesis of the Sialyl-6T structure [Galβ1–3 (Neu5Acα2–6)GalNAc-O-Ser/Thr]. In conclusion, although both enzymes show similar in vitro activities when Tn antigen alone is available, whenever both Tn and T antigens are present, ST6GalNAc-I acts preferentially on Tn antigen, whereas the ST6GalNAc-II acts preferentially on T antigen. Our results show that ST6GalNAc-I is the major Sialyl-Tn synthase and strongly support the hypothesis that the expression of the Sialyl-Tn antigen in cancer cells is due to ST6GalNAc-I activity.

Human MUC2 Mucin Gene Is Transcriptionally Regulated by Cdx Homeodomain Proteins in Gastrointestinal Carcinoma Cell Lines

In intestinal metaplasia and 30% of gastric carcinomas, MUC2 intestinal mucin and the intestine-specific transcription factors Cdx-1 and Cdx-2 are aberrantly expressed. The involvement of Cdx-1 and Cdx-2 in the intestinal development and their role in transcription of several intestinal genes support the hypothesis that Cdx-1 and/or Cdx-2 play important roles in the aberrant intestinal differentiation program of intestinal metaplasia and gastric carcinoma. To clarify the mechanisms of transcriptional regulation of the MUC2 mucin gene in gastric cells, pGL3 deletion constructs covering 2.6 kb of the human MUC2 promoter were used in transient transfection assays, enabling us to identify a relevant region for MUC2 transcription in all gastric cell lines. To evaluate the role of Cdx-1 and Cdx-2 in MUC2 transcription we performed co-transfection experiments with expression vectors encoding Cdx-1 and Cdx-2. In two of the four gastric carcinoma cell lines and in all colon carcinoma cell lines we observed transactivation of the MUC2 promoter by Cdx-2. Using gel shift assays we identified two Cdx-2 binding sites at –177/–171 and –191/–187. Only simultaneous mutation of the two sites resulted in inhibition of Cdx-2-mediated transactivation of MUC2 promoter, implying that both Cdx-2 sites are active. Finally, stable expression of Cdx-2 in a gastric cell line initially not expressing Cdx-2, led to induction of MUC2 expression. In conclusion, this work demonstrates that Cdx-2 activates the expression of MUC2 mucin gene in gastric cells, inducing an intestinal transdifferentiation phenotype that parallels what is observed both in intestinal metaplasia and some gastric carcinomas.

MUC2 mucin is a major carrier of the cancer-associated sialyl-Tn antigen in intestinal metaplasia and gastric carcinomas

Changes in mucin protein expression and in glycosylation are common features in pre-neoplastic lesions and cancer and are therefore used as cancer-associated markers. De novo expression of intestinal mucin MUC2 and cancer-associated sialyl-Tn antigen are frequently observed in intestinal metaplasia (IM) and gastric cancer. However, despite that these antigens often co-localize, MUC2 has not been demonstrated to be a carrier of sialyl-Tn. By using the in situ proximity ligation assay (in situ PLA), we herein could show that MUC2 is a major carrier of the sialyl-Tn antigen in all IM cases and in most gastric carcinoma cases. The requirement by in situ PLA for the presence of both antigens in close proximity increases the selectivity compared to measurement of co-localization, as determined by immunohistochemistry. Identification of the mucin which is the carrier of a carbohydrate structure offers unique advantages for future development of more accurate diagnostic and prognostic markers.

Differential expression of α-2,3-sialyltransferases and α-1,3/4-fucosyltransferases regulates the levels of sialyl Lewis a and sialyl Lewis x in gastrointestinal carcinoma cells

Sialyl Lewis x and sialyl Lewis a expression depends on sialyltransferases and fucosyltransferases. In this study, we screened for major variations of sialyltransferases and fucosyltransferases involved in the synthesis and regulation of sialyl Lewis x and sialyl Lewis a epitopes in gastrointestinal carcinoma cells. Our results show that expression of ST3Gal IV in several gastrointestinal cell lines is correlated with the expression of sialyl Lewis x at the cell surface. ST3Gal IV overexpressed in the gastric MKN45 cell line, showed exclusive enzymatic activity towards glycoproteins containing terminal Galbeta1-4GlcNAc structure. On the other hand, when ST3Gal III was overexpressed in MKN45, an increase in the expression levels of both sialyl Lewis epitopes was observed. ST3Gal III and ST3Gal IV lead to de novo synthesis of sialyl Lewis x determinant on different molecular weight glycoproteins of MKN45 cells suggesting that each enzyme used different substrates within the available glycoproteome. The final glycosylation step in sialyl Lewis x and sialyl Lewis a biosynthesis in MKN45 cell line was shown to be associated to FUT5, which efficiently fucosylated sialyl Lewis precursors on glycoproteins. Moreover we demonstrate that the expression of sialyl Lewis epitopes in the MKN45 was induced by cell confluence, which can be regarded as a model to study altered glycosylation during tumour progression. This increase was observed together with an increase in mRNA levels of ST3GAL3, FUT5 and FUT6, and a decrease in FUT4 transcript levels in MKN45 confluent cells, suggesting a possible control at the transcriptional level.

c-erb B-2 Expression Is Associated with Tumor Location and Venous Invasion and Influences Survival of Patients with Gastric Carcinoma:

The HER-2/neu gene or c-erb B-2, localized on chromosome 17q, belongs to a family of tyrosine kinase receptors and shares extensive homology with the epidermal growth factor receptor. c-erb B-2 gene amplification and protein overexpression have been reported in several human cancers. The prognostic value of this genetic alteration in gastric carcinoma is far from being established. In the present study, formalin-fixed, paraffin-embedded gastric carcinoma tissues from 157 patients were evaluated for c-erb B-2 overexpression, by immunohistochemistry using a polyclonal antibody. c-erb B-2 expression was evaluated according to clinical and pathological parameters, and to the survival of the patients. Our results show that: (1) c-erb B-2 was overexpressed in 15.3% of gastric carcinoma cases; (2) c-erb B-2 overexpression was significantly more frequent in cardia (23.8%) and fundus/body (25.0%) carcinomas than in antrum (7.2%) carcinomas; (3) c-erb B-2 overexpression was significantly associated with venous invasion; (4) c-erb B-2 is a prognostic factor for gastric carcinoma.

MUC1 gene polymorphism in the gastric carcinogenesis pathway

MUC1 like most mucin genes shows extensive length polymorphism in the central core region. In a previous study it was shown that individuals with small MUC1 alleles/genotypes have an increased risk for development of gastric carcinoma. Our aim was to see if MUC1 gene polymorphism was involved in susceptibility for the development of conditions that precede gastric carcinoma: chronic atrophic gastritis (CAG) and intestinal metaplasia (IM). We evaluated MUC1 polymorphism in a population of 174 individuals with chronic gastritis (CG) displaying (CAG) and/or intestinal metaplasia (IM). The population of patients with CG shows MUC1 allele frequencies significantly different from the gastric carcinoma patients and blood donors population. A significantly lower frequency of CAG and IM was observed in MUC1 VNTR heterozygotic patients. Within the group of patients with IM, MUC1 large VNTR homozygotes show a significantly higher frequency of complete IM while small VNTR homozygotes show a significantly higher frequency of incomplete IM. These findings show that MUC1 polymorphism may define different susceptibility backgrounds for the development of conditions that precede gastric carcinoma: chronic atrophic gastritis (CAG) and intestinal metaplasia (IM).

Identification of new cancer biomarkers based on aberrant mucin glycoforms by in situ Proximity Ligation

Mucin glycoproteins are major secreted or membrane‐bound molecules that, in cancer, show modifications in both the mucin proteins expression and in the O‐glycosylation profile, generating some of the most relevant tumour markers in clinical use for decades. Thus far, the identification of these biomarkers has been based on the detection of either the protein or the O‐glycan modifications. We therefore aimed to identify the combined mucin and O‐glycan features, that is, specific glycoforms, in an attempt to increase specificity of these cancer biomarkers. Using in situ proximity ligation assays (PLA) based on existing monoclonal antibodies directed to MUC1, MUC2, MUC5AC and MUC6 mucins and to cancer‐associated carbohydrate antigens Tn, Sialyl‐Tn (STn), T, Sialyl‐Lea (SLea) and Sialyl‐Lex (SLex) we screened a series of 28 mucinous adenocarcinomas from different locations (stomach, ampulla of Vater, colon, lung, breast and ovary) to detect specific mucin glycoforms. We detected Tn/STn/SLea/SLex‐MUC1 and STn/SLea/SLex‐MUC2 glycoforms in

Key elements of the BMP/SMAD pathway co-localize with CDX2 in intestinal metaplasia and regulate CDX2 expression in human gastric cell lines

50% of the cases, with a variable distribution among organs. Some new glycoforms‐T/SLea‐MUC2, STn/T/SLea/SLex‐MUC5AC and STn/T/SLea/SLex‐MUC6‐were identified for the first time in the present study in a variable percentage of cases from different organs. In conclusion, application of the PLA technique allowed sensitive detection of specific aberrant mucin glycoforms in cancer, increasing specificity to the use of antibodies either to the mucin protein backbone or to the O‐glycan haptens alone.