Expression Of sialyl-Tn Antigen Research Articles

Role of KLF4 and SIAT7A interaction accelerates myocardial hypertrophy induced by Ang II.

Sialylation catalysed by sialyltransferase 7A (SIAT7A) plays a role in the development of cardiac hypertrophy. However, the regulatory mechanisms upstream of SIAT7A in this context remain poorly elucidated. Previous study demonstrated that KLF4 activates the SIAT7A gene in ischemic myocardium by binding to its promoter region. Nevertheless, the potential involvement of KLF4 in regulating SIAT7A expression in Ang II-induced hypertrophic cardiomyocytes remains uncertain. This study seeks to deepen the underlying mechanisms of the KLF4 and SIAT7A interaction in the progression of Ang II-induced cardiac hypertrophy. The results showed a concurrent increase in SIAT7A and KLF4 levels in hypertrophic myocardium of essential hypertension patients and in hypertrophic cardiomyocytes stimulated by Ang II. Invitro experiments revealed that reducing KLF4 levels led to a decrease in both SIAT7A synthesis and Sialyl-Tn antigen expression, consequently inhibiting Ang II-induced cardiomyocyte hypertrophy. Intriguingly, reducing SIAT7A levels also resulted in decreased KLF4 expression and suppression cardiomyocyte hypertrophy. Consistent with this, elevating SIAT7A levels increased KLF4 expression and exacerbated cardiomyocyte hypertrophy in both invivo and invitro experiments. Additionally, a time-course analysis indicated that KLF4 expression preceded that of SIAT7A. Luciferase reporter assays further confirmed that modulating SIAT7A levels directly influenced the transcriptional activity of KLF4 in cardiomyocytes. In summary, KLF4 expression is upregulated in cardiomyocytes treated with Ang II, which subsequently induces the expression of SIAT7A. The elevated levels of SIAT7A, in turn, enhance the transcription of KLF4. These findings suggest a positive feedback loop between KLF4 and SIAT7A-Sialyl-Tn, ultimately promoting Ang II-induced cardiac hypertrophy.

Siglec-15 recognition of sialoglycans on tumor cell lines can occur independently of sialyl Tn antigen expression.

Siglec-15 is a conserved sialic acid-binding Ig-like lectin expressed on osteoclast progenitors, which plays an important role in osteoclast development and function. It is also expressed by tumor-associated macrophages and by some tumors, where it is thought to contribute to the immunosuppressive microenvironment. It was shown previously that engagement of macrophage-expressed Siglec-15 with tumor cells expressing its ligand, sialyl Tn (sTn), triggered production of TGF-β. In the present study, we have further investigated the interaction between Siglec-15 and sTn on tumor cells and its functional consequences. Based on binding assays with lung and breast cancer cell lines and glycan-modified cells, we failed to see evidence for recognition of sTn by Siglec-15. However, using a microarray of diverse, structurally defined glycans, we show that Siglec-15 binds with higher avidity to sialylated glycans other than sTn or related antigen sequences. In addition, we were unable to demonstrate enhanced TGF-β secretion following co-culture of Siglec-15-expressing monocytic cell lines with tumor cells expressing sTn or following Siglec-15 cross-linking with monoclonal antibodies. However, we did observe activation of the SYK/MAPK signaling pathway following antibody cross-linking of Siglec-15 that may modulate the functional activity of macrophages.

Tn and Sialyl-Tn antigens in canine gastric tissues.

Malignant transformation is often associated with abnormal protein glycosylation expressed, amongst others, by the accumulation of simple mucin-type carbohydrates namely Tn and Sialyl-Tn (STn) antigens. These are usually limited in normal tissues and their increased expression has been associated with cancer progression and poor prognosis. This study aims to evaluate the role of Tn and STn antigens in the neoplastic transformation of the canine gastric mucosa and to correlate their putative immunoexpression alterations with some pathological features. Tn and STn antigens expression were immunohistochemically evaluated in canine normal gastric mucosa (n = 3), gastric polyps (n = 9) and gastric carcinomas (n = 25), neoplastic emboli (n = 12) and metastases (n = 8). In normal gastric mucosa, Tn antigen was detected in the gastric epithelial cells, while STn antigen was absent. Similarly, all gastric polyps expressed Tn antigen, but none displayed STn antigen immunostaining. In carcinomas, Tn antigen was expressed in 96% of the cases and STn antigen in 68% of the neoplasms. STn antigen was significantly higher in carcinomas compared with normal mucosa (P < .05). No correlation was found between each antigen and the different subtypes of tumours according to WHO classification, tumour differentiation, lymph vascular invasion or metastasis. All neoplastic emboli expressed both antigens, and the expression score was similar or higher than that displayed by the neoplastic cells of the primary tumour. The high prevalence of STn antigen in gastric carcinomas compared with normal mucosa highlights the cancer-associated nature of this antigen. Our results link STn antigen expression to neoplastic transformation and suggest that it may be a useful marker of gastric cancer progression in dogs.

Truncated O-glycans promote epithelial-to-mesenchymal transition and stemness properties of pancreatic cancer cells.

Aberrant expression of Sialyl‐Tn (STn) antigen correlates with poor prognosis and reduced patient survival. We demonstrated that expression of Tn and STn in pancreatic ductal adenocarcinoma (PDAC) is due to hypermethylation of Core 1 synthase specific molecular chaperone (COSMC) and enhanced the malignant properties of PDAC cells with an unknown mechanism. To explore the mechanism, we have genetically deleted COSMC in PDAC cells to express truncated O‐glycans (SimpleCells, SC) which enhanced cell migration and invasion. Since epithelial‐to‐mesenchymal transition (EMT) play a vital role in metastasis, we have analysed the induction of EMT in SC cells. Expressions of the mesenchymal markers were significantly high in SC cells as compared to WT cells. Equally, we found reduced expressions of the epithelial markers in SC cells. Re‐expression of COSMC in SC cells reversed the induction of EMT. In addition to this, we also observed an increased cancer stem cell population in SC cells. Furthermore, orthotopic implantation of T3M4 SC cells into athymic nude mice resulted in significantly larger tumours and reduced animal survival. Altogether, these results suggest that aberrant expression of truncated O‐glycans in PDAC cells enhances the tumour aggressiveness through the induction of EMT and stemness properties.

ST6GALNAC1 plays important roles in enhancing cancer stem phenotypes of colorectal cancer via the Akt pathway.

Colorectal cancer (CRC) is a mortal disease due to treatment resistance, recurrence and distant metastasis. Emerging evidence has revealed that a small sub-population of cancer cells termed cancer stem cells (CSCs)/ cancer-initiating cells (CICs) is endowed with high levels of tumor-initiating ability, self-renewal ability and differentiation ability and is responsible for treatment resistance, recurrence and distant metastasis. Eradication of CSCs/CICs is essential to improve current treatments. However, the molecular mechanisms by which CSCs/CICs are maintained are still elusive. In this study, we aimed to determine the molecular mechanisms by which colorectal (CR)-CSCs/CICs in are maintained human primary CRC cells. CR-CSCs/CICs were isolated by sphere-culture and the ALDEFLUOR assay, and transcriptome analysis revealed that the gene ST6 N-Acetylgalactosaminide Alpha-2,6-Sialyltransferase 1 (ST6GALNAC1) was expressed at high levels in CR-CSCs/CICs. Overexpression of ST6GALNAC1 enhanced the expression of sialyl-Tn (STn) antigen, which is carried by the CSC marker CD44, and increased the sphere-forming ability and resistance to a chemotherapeutic reagent. The opposite phenomena were observed by gene knockdown using siRNA. Furthermore, the Akt pathway was activated in ST6GANAC1-overexpressed cells, and activation of the pathway was cancelled by gene knockdown of galectin-3. The results indicate that ST6GALNAC1 has a role in the maintenance of CR-CSCs/CICs by activating the Akt pathway in cooperation with galectin-3 and that ST6GalNAC1 (or STn antigen) might be a reasonable molecule for CSC/CIC-targeting therapy.

Silencing of ST6GalNAc I suppresses the proliferation, migration and invasion of hepatocarcinoma cells through PI3K/AKT/NF-κB pathway.

ST6GalNAc I is the major Sialyl-Tn antigen (STn) synthase that is highly correlated with tumor invasion and metastasis. However, the roles and molecular mechanisms by which ST6GalNAc I mediates the malignant phenotypes of hepatocarcinoma cells still remain poorly unknown. In this study, we investigated the expression of STn and ST6GalNAc I in mouse hepatocarcinoma cell lines Hca-F, Hca-P, and Hepa1-6, which have different metastatic potential, as compared with normal mouse liver cell line IAR-20. The results showed that the expression of ST6GalNAc I and STn in Hca-F and Hca-P cells was much higher than that in Hepa1-6 and IAR20 cells. Knockdown of ST6GalNAc I by shRNA in Hca-F cells significantly decreased the expression of STn and inhibited the growth of tumor cells in vitro and in vivo. This reduction of ST6GalNAc I expression also led to the decreased migration and invasion of Hca-F cells. Furthermore, we found that ST6GalNAc I knockdown inhibited the expression levels of PI3k, p-Akt473, p-Akt308, NF-κB, and their downstream molecules. Together, our results suggest a role of ST6GalNAc I in promoting the growth and invasion of hepatocarcinoma cells through regulating PI3K/AKT signaling, and ST6GalNAc I might be a promising marker for the prognosis and therapy of hepatocarcinoma.

Abnormal Protein Glycosylation and Activated PI3K/Akt/mTOR Pathway: Role in Bladder Cancer Prognosis and Targeted Therapeutics

Muscle invasive bladder cancer (MIBC, stage ≥T2) is generally associated with poor prognosis, constituting the second most common cause of death among genitourinary tumours. Due to high molecular heterogeneity significant variations in the natural history and disease outcome have been observed. This has also delayed the introduction of personalized therapeutics, making advanced stage bladder cancer almost an orphan disease in terms of treatment. Altered protein glycosylation translated by the expression of the sialyl-Tn antigen (STn) and its precursor Tn as well as the activation of the PI3K/Akt/mTOR pathway are cancer-associated events that may hold potential for patient stratification and guided therapy. Therefore, a retrospective design, 96 bladder tumours of different stages (Ta, T1-T4) was screened for STn and phosphorylated forms of Akt (pAkt), mTOR (pmTOR), S6 (pS6) and PTEN, related with the activation of the PI3K/Akt/mTOR pathway. In our series the expression of Tn was residual and was not linked to stage or outcome, while STn was statically higher in MIBC when compared to non-muscle invasive tumours (p = 0.001) and associated decreased cancer-specific survival (log rank p = 0.024). Conversely, PI3K/Akt/mTOR pathway intermediates showed an equal distribution between non-muscle invasive bladder cancer (NMIBC) and MIBC and did not associate with cancer-specif survival (CSS) in any of these groups. However, the overexpression of pAKT, pmTOR and/or pS6 allowed discriminating STn-positive advanced stage bladder tumours facing worst CSS (p = 0.027). Furthermore, multivariate Cox regression analysis revealed that overexpression of PI3K/Akt/mTOR pathway proteins in STn+ MIBC was independently associated with approximately 6-fold risk of death by cancer (p = 0.039). Mice bearing advanced stage chemically-induced bladder tumours mimicking the histological and molecular nature of human tumours were then administrated with mTOR-pathway inhibitor sirolimus (rapamycin). This decreased the number of invasive lesions and, concomitantly, the expression of STn and also pS6, the downstream effector of the PI3K/Akt/mTOR pathway. In conclusion, STn was found to be marker of poor prognosis in bladder cancer and, in combination with PI3K/Akt/mTOR pathway evaluation, holds potential to improve the stratification of stage disease. Animal experiments suggest that mTOR pathway inhibition could be a potential therapeutic approach for this specific subtype of MIBC.

2160 GIV as a novel marker of recurrence risk in MMR proficient stage II colon cancer

s S387 Material and Methods: Formalin-fixed, paraffin-embedded tissue sections from 186 primary colorectal carcinomas and four normal colorectal tissues were immunostained using specific monoclonal antibodies against Tn and sialyl-Tn antigens. Baseline clinicopathological characteristics and overall 5-year survival were correlated with antigen expression. Results: Of the 186 colorectal carcinomas, Tn antigen is expressed in 70 (37.6%) and sialyl-Tn antigen is expressed in 141 (75.8%). There is no staining for these antigens in normal colorectal tissues. Immunostaining measurements for Tn antigen were significantly associated with the differentiation degree (p = 0.017) of the tumors, but independent of age, gender, tumor location, depth of penetration, status of lymph nodes, lymphovascular invasion, perineural invasion and TNM stage. The expression results of sialyl-Tn antigen revealed more significant associations with the degree of differentiation (p = 0.006) and perineural invasion (p = 0.041). The poor overall survival rate of patients was significantly associated with the positive expression of sialyl-Tn (p = 0.013) but not with the expression of Tn (p = 0.650). Survival at 5 years for patients with sialyl-Tn-negative versus sialyl-Tn-positive tumors was 72.1% versus 49.2%. Cox regression multivariate analysis confirmed that TNM stage (p = 0.001) and sialyl-Tn expression (p = 0.038) were the two most important variables of for predicting overall survival. Conclusions: These results indicate that Tn and sialyl-Tn antigens appear to be useful biomarkers of poorly differentiated adenocarcinomas and mucinous carcinomas. Moreover, the positive expression of sialyl-Tn antigen is an independent predictor of poor prognosis in human colorectal carcinoma. No conflict of interest.

CDX2 homeoprotein is involved in the regulation of ST6GalNAc-I gene in intestinal metaplasia

De novo expression of Sialyl-Tn (STn) antigen is one of the most common features of intestinal metaplasia (IM) and gastric carcinomas, and its biosynthesis has been mostly attributed to ST6GalNAc-I activity. However, the regulation of this glycosyltransferase expression is not elucidated. In IM lesions and in the intestine, CDX2 homeobox transcription factor is co-expressed with STn and ST6GalNAc-I. We therefore hypothesized that CDX2 might induce STn expression by positive regulation of ST6GalNAc-I. We showed that ST6GalNAc-I transcript levels and CDX2 have a coordinated expression upon Caco-2 in vitro differentiation, and overexpression of CDX2 in MKN45 gastric cells increases ST6GalNAc-I transcript levels. Nine putative CDX-binding sites in the ST6GalNAc-I-regulatory sequence were identified and analyzed by chromatin immunoprecipitation in Caco-2 cells and in IM. The results showed that CDX2 protein is recruited to all regions, being the most proximal sites preferentially occupied in vivo. Luciferase assays demonstrated that CDX2 is able to transactivate ST6GalNac-I-regulatory region. The induction was stronger for the regions mapped in the neighbourhood of ATG start codon and site-directed mutagenesis of these sites confirmed their importance. In conclusion, we show that CDX2 transcriptionally regulates ST6GalNAc-I gene expression, specifically in the preneoplastic IM lesion.

RNAi-mediated gene silencing of ST6GalNAc I suppresses the metastatic potential in gastric cancer cells.

ST6GalNAc I is a sialyltransferase controlling the expression of sialyl-Tn antigen (STn), which is overexpressed in several epithelial cancers, including gastric cancer, and is highly correlated with cancer metastasis. However, the functional contribution of ST6GalNAc I to development or progression of gastric cancer remains unclear. In this study, we investigated the effects of suppression of ST6GalNAc I on gastric cancer in vitro and in vivo. Gastric cancer cell lines were transfected with ST6GalNAc I siRNA and were examined by cell proliferation, migration, and invasion assays. We also evaluated the effect of ST6GalNAc I siRNA treatment in a peritoneal dissemination mouse model. The differences in mRNA levels of selected signaling molecules were analyzed by polymerase chain reaction (PCR) arrays associated with tumor metastasis in MKN45 cells. The signal transducer and activator of transcription 5b (STAT5b) signaling pathways that reportedly regulate the insulin-like growth factor-1 (IGF-1) were analyzed by Western blot. ST6GalNAc I siRNA inhibited gastric cancer cell growth, migration, and invasion in vitro. Furthermore, intraperitoneal administration of ST6GalNAc I siRNA- liposome significantly inhibited peritoneal dissemination and prolonged the survival of xenograft model mice with peritoneal dissemination of gastric cancer. PCR array confirmed that suppression of ST6GalNAc I caused a significant reduction in expression of IGF-1 mRNA. Decreased IGF-1 expression in MKN45 cells treated with ST6GalNAc I siRNA was accompanied by reduced phosphorylation of STAT5b. ST6GalNAc I may regulate the gene expression of IGF-1 through STAT5b activation in gastric cancer cells and may be a potential target for treatment of metastasizing gastric cancer.

ST6GalNAc-I controls expression of sialyl-Tn antigen in gastrointestinal tissues

Sialyl-Tn is a simple mucin-type carbohydrate antigen aberrantly expressed in gastrointestinal adenocarcinomas and in the precursor lesion intestinal metaplasia. Sialyl-Tn tumour expression is an independent indicator of poor prognosis. We have previously shown in vitro that ST6GalNAc-I and ST6GalNAc-II sialyltransferases can synthesize sialyl-Tn. The aim of the present study was to establish whether ST6GalNAc-I is the major enzyme responsible for the expression of sialyl-Tn. We used a model of CHO-ldlD cells producing only MUC1-Tn glycoform and showed that ST6GalNAc-I is the key-enzyme leading to sialyl-Tn biosynthesis. We developed novel monoclonal antibodies specific for ST6GalNAc-I and evaluated its expression in gastrointestinal tissues. ST6GalNAc-I was detected in normal colon mucosa co-localized with O-acetylated sialyl-Tn. Expression was largely unaltered in colorectal adenocarcinomas. In contrast, we found that ST6GalNAc-I is weakly expressed in normal gastric mucosa, but over-expressed in intestinal metaplasia, co-localized with sialyl-Tn. In gastric carcinomas ST6GalNAc-I was also associated with sialyl-Tn, but with heterogeneous staining and partial co-localization. Our results showed ST6GalNAc-I as the major enzyme controlling the expression of cancer-associated sialyl-Tn antigen in gastrointestinal tissues.

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.

Expression of a Core 3 Disialyl-Lex Hexasaccharide in Human Colorectal Cancers: A Potential Marker of Malignant Transformation in Colon

Cancer-associated alterations in cell surface and secreted glycoproteins have been catalogued for many years but many of the studies of alterations in mucin carbohydrate have relied on histochemical or immunohistochemical methods, with little direct chemical analysis. In this study, we analyzed the O-glycosylation pattern of MUC2 glycoprotein isolated from colorectal carcinomas, transitional mucosa and resection margins from three patients with blood group A, B and O, respectively. After alkaline borohydride treatment, the released oligosaccharides were structurally characterized by nanoESI Q-TOF tandem mass spectrometry without prior fractionation or derivatization. As expected, we found an increased expression of sialyl-Tn antigen in the colonic cancer mucins. A more interesting feature was the increased expression of a core 3 sialyl-Le(x) hexasaccharide, NeuAcalpha2-3Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3(NeuAcalpha2-6)GalNAc in tumor, which appeared to compete with its sulfo-Le(x) counterpart in normal tissue, SO3-3Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3(NeuAcalpha2-6)GalNAc. This antigen, whose structure was confirmed by NMR experiments, is based on a core 3 glycan and may be a potential marker for the malignant transformation of colonic cells. Unexpectedly, most of the glycans recovered in normal and carcinomas extracts were based on a sialylated core 3, GlcNAcbeta1-3(NeuAcalpha2-6)GalNAcol. Moreover, the pattern of glycosylation was very similar between mucins isolated from each sample, the main differences related to the level of expression of the major oligosaccharides. The data obtained in this investigation may have value for future screening studies on colorectal cancer.

ST6GalNAc-I controls expression of sialyl-Tn antigen in gastrointestinal tissues

ST6GalNAc-I controls expression of sialyl-Tn antigen in gastrointestinal tissues

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.

Stable expression of sialyl-Tn antigen in T47-D cells induces a decrease of cell adhesion and an increase of cell migration

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: GalNAc alpha2,6-sialyltransferase: ST6GalNAc I, which catalyzes the transfer of a sialic acid residue in alpha2,6-linkage to the GalNAcalpha1-O-Ser/Thr structure. The resulting disaccharide (Neu5Acalpha2-6GalNAcalpha1-O-Ser/Thr) cannot be further elongated and sialyl-Tn expression results therefore in a shortening of the O-glycan chains. However, usual breast cancer cell lines express neither ST6GalNAc I nor sialyl-Tn antigen. We have previously shown that stable transfection of MDA-MB-231 cells with the hST6GalNAc I cDNA induces the sialyl-Tn antigen expression at the cell surface and leads to a decreased cell growth and an increased cell migration. We describe herein the generation of new T47-D clones expressing sialyl-Tn antigen after hST6GalNAc I cDNA stable transfection. sialyl-Tn antigen is carried by several high molecular weight membrane bound O-glycoproteins, including MUC1. We show that sialyl-Tn expression induces a decrease of cell growth and adhesion, and an increase of cell migration in sialyl-Tn positive clones compared to mock transfected cells. These observations show that the alteration of the O-glycans pattern is sufficient to modify the biological features of cancer cells. These T47-D sialyl-Tn expressing clones might allow further in vivo investigation to determine precisely the impact of such O-glycosylation modifications on breast cancer development.

Colon carcinogenesis in inflammatory bowel disease: applying molecular genetics to clinical practice.

Although both sporadic colorectal cancer (CRC) and colitis-associated CRC arise from dysplastic precursor lesions and share several molecular alterations, the nature of the dysplasia and the frequency and timing of several of the key molecular changes differ enough to consider colitis-associated CRC a rather unique entity. To date, cancer surveillance in both ulcerative colitis and Crohn's colitis rests upon the detection of dysplasia. However, because there are considerable limitations to the detection and interpretation of dysplasia, there is a need for other molecular markers to complement the histologic analysis of dysplasia. Because patients with inflammatory bowel disease (IBD) undergo repeated, periodic surveillance colonoscopies, it affords an opportunity to study marker expression over time. Of the few markers that have been studied chronologically, aneuploidy, p53, and mucin-associated sialyl-Tn antigen expression each hold promise as markers of CRC risk in IBD. It will be important to study whether these markers, or other panels of gene or protein expression, can identify patients at highest risk for developing CRC in future clinical studies.

SIALYL-Tn ANTIGEN DISTRIBUTION IN HELICOBACTER PYLORI CHRONIC GASTRITIS IN CHILDREN: AN IMMUNOHISTOCHEMICAL STUDY

Sialyl-Tn antigen (STn) is a mucin-type carbohydrate normally present in goblet cells of small and large bowel. STn expression has been demonstrated to occur in complete and incomplete intestinal metaplasia as well as in many carcinomas but in no normal gastric cell. The aim of our present study was to evaluate the distribution of STn in Helicobacter pylori chronic gastritis (HpCG) of pediatric patients. Eighteen gastric biopsies from 15 children (mean age: 11.5 years) with HpCG, 9 gastric biopsies from 9 children without H. pylori infection, and 1 heterotopic gastric mucosa in Meckel's diverticulum were immunostained using the anti-STn antibody STn1 (18/18), NCL-MUC-1 (7/18), and NCL-MUC-2 (18/18) antibodies. Also, sulfated mucosubstances were investigated with the Alcian Blue-Periodic Acid Schiff (AB-PAS), pH 1.0 stain. Although with different intensity (weak in 5/18, moderate 9/18, and intense 4/18) all cases with HpCG exhibited STn immunoreactivity. The expression of STn was found to be located mainly to the supranuclear region of the epithelial cells at the foveolae and glandular necks, with occasional cells showing diffuse cytoplasmic staining. When reactivity was intense, it was for the most part found in the cells at the neck of the glands. The mucus out of the luminal border above the positive cells was usually also stained. MUC-1 was negative (2/7) or weakly positive (5/7) in a few surface mucous cells. MUC-2 was negative (16/18) or occasionally detected in some foveolar and surface cells (2/18). AB-PAS pH 1.0 revealed the presence of sulfomucins in the cytoplasm of isolated cells of gastric pits and glands of most cases (11/15). None of these findings was observed in the control group. We conclude that STn can be identified in gastric cells of pediatric patients with HpCG and that this does not correlate with other mucosubtances markers. The findings could indicate that minimal intestinal metaplasia takes place in children with HpCG.

Pathways of mucin O-glycosylation in normal and malignant rat colonic epithelial cells reveal a mechanism for cancer-associated Sialyl-Tn antigen expression.

The Sialyl-Tn antigen (Sialyl alpha-Ser/Thr) is expressed as a cancer-associated antigen on the surface of cancer cells. Its presence is associated with a poor prognosis in patients with colorectal and other cancers. We previously reported that Sialyl-Tn expression in LSC human colon cancer cells could be explained by a specific lack of the activity of core 1 beta3-Gal-transferase (Brockhausen et al., Glycoconjugate J. 15, 595-603, 1998) and an inability to synthesize the common O-glycan core structures. To support this mechanism, or find other mechanisms to explain Sialyl-Tn antigen expression, we investigated the O-glycosylation pathways in clonal rat colon cancer cell lines that were selected for positive or negative expression of Sialyl-Tn antigen, and compared these pathways to those in normal rat colonic mucosa. Normal rat colonic mucosa had very active glycosyltransferases synthesizing O-glycan core structures 1 to 4. Several sialyl-, sulfo- and fucosyltransferases were also active. An M type core 2 beta6-GlcNAc-transferase was found to be present in rat colon mucosa and all of the rat colon cancer cells. O-glycosylation pathways in rat colon cancer cells were significantly different from normal rat colonic mucosa; for example, rat colon cancer cells lost the ability to synthesize O-glycan core 3. All rat colon cancer cell lines, regardless of the Sialyl-Tn phenotype, expressed glycosyltransferases assembling complex O-glycans of core 1 and core 2 structures (unlike human LSC colon cancer cells which lack core 1 beta3-Gal-transferase activity). It was the activity of CMP-sialic acid:GalNAc-mucin alpha6-sialyltransferase that coincided with Sialyl-Tn expression. Sialyl-Tn negative cells had a several fold higher activity of core 2 beta6-GlcNAc-transferase which synthesizes complex O-glycans that may mask adjacent Sialyl-Tn epitopes. The results suggest a new mechanism controlling Sialyl-Tn expression in cancer cells.

Expression of sialyl-Tn antigen in breast cancer cells transfected with the human CMP-Neu5Ac: GalNAc alpha2,6-sialyltransferase (ST6GalNac I) cDNA.

Sialyl-Tn antigen (STn) is a cancer associated carbohydrate antigen over-expressed in several cancers including breast cancer, and currently associated with more aggressive diseases and poor prognosis. However, the commonly used breast cancer cell lines (MDA-MB-231, T47-D and MCF7) do not express STn antigen. The key step in the biosynthesis of STn is the transfer of a sialic acid residue in alpha2,6-linkage to GalNAc alpha-O-Ser/Thr. This reaction is mainly catalyzed by a CMP-Neu5Ac GalNAc alpha2,6-sialyltransferase: ST6GalNAc I. In order to generate STn-positive breast cancer cells, we have cloned a cDNA encoding the full-length human ST6GalNAc I from HT-29-MTX cells. The stable transfection of MDA-MB-231 with an expression vector encoding ST6GalNAc I induces the expression of STn antigen at the cell surface. The expression of STn short cuts the initial O-glycosylation pattern of these cell lines, by competing with the Core-1 beta1,3-galactosyltransferase, the first enzyme involved in the elongation of O-glycan chains. Moreover, we show that STn expression is associated with morphological changes, decreased growth and increased migration of MDA-MB-231 cells.