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Abstract
Aberrant mucin O-glycosylation is often observed in cancer and is characterized by the expression of immature simple mucin-type carbohydrate antigens. UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase-6 (ppGalNAc-T6) is one of the enzymes responsible for the initial step in O-glycosylation. This study evaluated the expression of ppGalNAc-T6 in human gastric mucosa, intestinal metaplasia, and gastric carcinomas. Our results showed that ppGalNAc-T6 is expressed in normal gastric mucosa and in intestinal metaplasia. A heterogeneous expression and staining pattern for this enzyme was observed in gastric carcinomas. ppGalNAc-T6 was expressed in 79% of the cases, and its expression level was associated with the presence of venous invasion. Our results provide evidence that ppGalNAc-T6 is an IHC marker associated with venous invasion in gastric carcinoma and may contribute to the understanding of the molecular mechanisms that underlie aberrant glycosylation in gastric carcinogenesis and in gastric carcinoma.
Mucins areO-glycosylated glycoproteins produced by most glandular epithelial tissues, representing the main component of the mucus layer on the surface of epithelial cells (Lesuffleur et al. 1994; Hollingsworth and Swanson 2004). Recently, mucins and mucin O-glycosylation have attracted attention for their role in the adhesion of bacteria, cell-cell adhesion, and cancer cell metastization (Hollingsworth and Swanson 2004). The expression of mucins is often altered in cancer, with frequent aberrant glycosylation, resulting in the formation of immature structures and exposure of the peptide backbone (Reis et al. 1998a; Ferreira et al. 2006). These structures are useful markers of premalignant and malignant cells. Changes in mucin O-glycosylation in the Golgi apparatus are determined by the differential expression of the enzymes that initiate O-glycosylation: UDP-GalNAc:polypeptide N-acetylgalactosaminyl-transferases (ppGalNAc-Ts) (for a review, see Hassan et al. 2000a; Ten Hagen et al. 2003). This initial key step controlling mucin O-glycosylation is performed by a family of ppGalNAc-Ts that catalyze the transfer of GalNAc from the sugar donor UDP-GalNAc to serine and threonine residues on the protein synthesizing the Tn antigen (Clausen and Bennett 1996). Until now, 15 distinct members of the mammalian ppGalNAc-T family have been identified and characterized (Homa et al. 1993; White et al. 1995; Bennett et al. 1996, 1998, 1999a, b; Hagen et al. 1997; Ten Hagen et al. 1998, 1999, 2001; Guo et al. 2002; Schwientek et al. 2002; Wang et al. 2003; Zhang et al. 2003; Cheng et al. 2004), and in silico analysis indicates that as many as 20 ppGalNAc-Ts may exist (Ten Hagen et al. 2003). The members of ppGalNAc-Ts family, although catalyzing the same enzymatic step, are tissue specific and have different kinetic properties and acceptor substrate specificities that may determine the site of O-glycan attachment (Wandall et al. 1997). This enzymatic specificity leads to different functions depending on the cell type and organ (Bennett et al. 1996; Hagen et al. 1997; Sutherlin et al. 1997; Mandel et al. 1999; Brooks et al. 2007; Rajpert-De Meyts et al. 2007). Altered expression of ppGalNAc-Ts could be one of the mechanisms that explain the changes in mucin O-glycosylation during malignant transformation (Hanisch et al. 2001). Variations of the ppGalNAc-Ts expression pattern have been described in oral squamous cell carcinoma, where decreased expression of ppGalNAc-T1 and increased expression of ppGalNAc-T2 and ppGalNAc-T3 have been reported compared with the expression pattern in normal oral mucosa (Mandel et al. 1999). Higher expression of ppGalNAc-T1, ppGalNAc-T2, and ppGalNAc-T3 has also been described in colorectal carcinoma compared with the normal colonic epithelium (Kohsaki et al. 2000). Different levels of expression of ppGalNAc-T3 were detected in patients with colorectal (Shibao et al. 2002), lung (Gu et al. 2004), pancreatic (Yamamoto et al. 2004), gastric (Ishikawa et al. 2004), gallbladder (Miyahara et al. 2004), prostate (Landers et al. 2005), and extrahepatic bile duct (Inoue et al. 2007) carcinomas, and it was identified as an independent factor of prognosis. ppGalNAc-T6, which exhibits a high sequence homology to ppGalNAc-T3, has been recently described to be expressed in most ductal breast carcinomas but not in normal breast epithelium with a significant association with T1 tumor stage (Berois et al. 2006). ppGalNAc-T3 and −T6 were also described in association with breast malignant cell lines (Brooks et al. 2007). Based on these studies ppGalNAc-T6 could be considered an interesting marker for the glycosylation modifications in malignancy with implication in molecular diagnosis (Freire et al. 2006).
In this study, we characterized the expression of ppGalNAc-T6 in normal gastric mucosa, intestinal metaplasia, and gastric carcinoma. We show that ppGalNAc-T6 is expressed in gastric mucosa and changes its expression during gastric carcinogenesis. Expression of ppGalNAc-T6 was found to be associated with a clinico-pathologic characteristic of gastric carcinoma.
Materials and Methods
Tissue Samples and Histological Classification
The study was performed using surgical specimens of gastric carcinomas and adjacent mucosa from patients operated at Hospital S. João, Porto, Portugal. The use of retrospective samples when informed consent cannot be obtained is authorized for research studies by Portuguese Law. Analysis of the expression of ppGalNAc-T6 (MAb T6.3) was performed in 76 tissue samples, fixed in 10% formalin, and embedded in paraffin. Serial sections were cut and used for conventional histo-pathological diagnosis. Carcinomas were classified according to Laurén (1965). The growth pattern was classified according to Ming (1977). Age, patient's survival, presence of lymphatic invasion, nodal metastasis and venous invasion, and tumor localization were also recorded in every case.
The pathological staging was achieved using the unified 1987 TNM system for gastric carcinoma (Pinto-De-Sousa et al. 2001). We evaluated MUC5AC, MUC6, and MUC2 mucin expression in 72, 44, and 55 cases, respectively. MUC5AC, MUC6, and MUC2 were detected using monoclonal antibodies CLH2 (Reis et al. 1997), CLH5 (Reis et al. 2000), and PMH1 (Reis et al. 1998b), respectively.
IHC
IHC evaluation of ppGalNAc-T6 was performed by the avidin-biotin-complex staining method (Hsu et al. 1981). All the formalin-fixed, paraffin-embedded samples were deparaffinated, rehydrated, and treated with 0.3% hydrogen peroxide in methanol for 30 min to block endogenous peroxidase. Sections were incubated with normal rabbit serum (DakoCytomation; Glostrup, Denmark) diluted 1:5 in PBS containing 10% of BSA for 20 min. After that, they were incubated overnight at 4C with the monoclonal antibody T6.3 for ppGalNAc-T6, diluted 1:400 in PBS containing 5% of BSA (Berois et al. 2006). The slides were washed in PBS and incubated for 30 min with biotinylated rabbit anti-mouse secondary antibody (DakoCytomation) diluted 1:200 in PBS containing 5% of BSA. Samples were washed with PBS and incubated with avidin-biotin peroxidase complex for 30 min (Vectastain Elite ABS kit; Burlingame, CA). Sections were stained with 3,3′-diaminobenzidine tetrahydrochloride (Sigma; St. Louis, MO) in a buffer containing 0.1% hydrogen peroxide, counterstained with Mayer's hematoxylin, dehydrated, and mounted. Negative controls were performed replacing primary antibody with PBS.
IHC for mucin expression and classification of intestinal metaplasia was performed as previously described (Reis et al. 2000).
Scoring of the Immunostaining and Statistical Analysis
A semiquantitative approach was used to score the immunostaining. Samples were scored as follows: low expression (<25% of positive staining cells) and high expression (>25% of positive staining cells) for IHC.
Statistical analysis was performed using the X2 test with Yates correction using Statview 5.0 software. Fisher's test was applied whenever appropriate. Differences were considered statistically significant at p<0.05. Logistic regression was performed using Statview 5.0 software.
Results
Expression of ppGalNAc-T6 in Normal Gastric Mucosa
Normal gastric mucosa showed expression of ppGal-NAc-T6 in 25/36 (69%) cases. The expression was localized in the superficial foveolar epithelium and glandular cells of both antrum and body regions of the normal gastric mucosa (Figures 1A, 1B, and 2A). The staining pattern observed for ppGalNAc-T6 was always perinuclear and/or supranuclear characterizing a Golgi staining pattern.
Expression of ppGalNAc-T6 in Intestinal Metaplasia
Intestinal metaplasia showed expression of ppGalNAc-T6 in 14/27 (52%) cases in both goblet and columnar cells of metaplastic glands. The staining pattern observed for ppGalNAc-T6 was always perinuclear and/or supranuclear characterizing a Golgi staining pattern (Figures 1D and 1E). Expression of ppGaNAc-T6 was observed in both complete and incomplete types of intestinal metaplasia (Figure 2).
Expression of ppGalNAc-T6 in Gastric Carcinomas
Evaluation of ppGalNAc-T6 using IHC in paraffin sections of a series of 76 gastric carcinomas showed expression of this enzyme in 60/76 (79%) of the cases. ppGalNAc-T6 expression was observed in 29/36 (81%) of intestinal type carcinomas, in 18/21 (86%) of diffuse type carcinomas, and in 13/19 (68%) of unclassified carcinomas, according to Laurén's classification (Table 1; Figures 1G and 1H). The immunostaining pattern of ppGalNAc-T6 was either perinuclear or diffuse cytoplasmic in gastric carcinoma cells.
A significant association was observed between the levels of expression of ppGalNAc-T6 and venous invasion (Table 1). Most cases with low levels of ppGal-NAc-T6 expression showed absence of venous invasion (p<0.03). In addition, analysis of contingency split by tumor localization showed that venous invasion was more associated with ppGalNAc-T6 in tumors localized in the body region of the stomach (Table 2). Further logistic regression showed no deterministic relation in which venous invasion was solely explained by ppGalNAc-T6.
Figure 1 IHC detection of UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase-6 (ppGalNAc-T6) evaluated in normal gastric mucosa (A–C), intestinal metaplasia (D–F), and gastric carcinoma (G–I). Normal gastric mucosa showing ppGalNAc-T6 expression in the foveolar and glandular epithelial cells with a supranuclear staining (A,B). Normal gastric mucosa without expression of ppGalNAc-T6 (C). Intestinal metaplasia showing expression of ppGalNAc-T6 in columnar and goblet cells of metaplastic glands with a supranuclear staining pattern (D,E) and intestinal metaplasia with glands lacking expression of ppGalNAc-T6 (F). Gastric carcinoma of the diffuse (G), intestinal (H), and unclassified (I) histological types showing expression of ppGalNAc-T6. Bars: A,D,F = 25 μm; B,C,E,G–I = 12.5 μm.
Figure 2 IHC of ppGalNAc-T6 and mucins MUC5AC and MUC2, in normal gastric mucosa (A–C), complete intestinal metaplasia (D–F), and incomplete intestinal metaplasia (G–I). Foveolar epithelium of a normal gastric mucosa showing expression of ppGalNAc-T6 (A, arrowheads), MUC5AC (B, arrowheads), and lacking expression of MUC2 (C). Complete type of intestinal metaplasia expressing ppGalNAc-T6 (D, arrowheads), MUC2 (F, arrowheads), and lacking expression of MUC5AC (E, arrows). Incomplete type of intestinal metaplasia expressing ppGalNAc-T6 (G, arrowheads), MUC5AC (H, arrowheads), and MUC2 (I, arrowheads). Bar = 12.5 μm.
The expression of ppGalNAc-T6 was not associated with other clinico-pathological characteristics of gastric carcinomas.
Coexpression of Mucins (MUC5AC, MUC6, and MUC2) and ppGalNAc-T6 in Gastric Carcinomas
Analysis of the coexpression of mucins (MUC5AC, MUC6, and MUC2) and ppGalNAc-T6 in gastric carcinoma is shown in Table 3. The expression of MUC5AC was inversely associated with the levels of expression of ppGalNAc-T6. Most cases positive for MUC5AC showed low expression of ppGalNAc-T6, whereas most cases negative for MUC5AC showed high expression of the enzyme (Table 3). No association was observed between the expression of ppGalNAc-T6 and the expression of mucins MUC6 and MUC2 (Table 3).
Discussion
Alterations in mucin-type O-glycans are associated with malignant transformation. This study evaluated the pattern of expression of ppGalNAc-T6, a key enzyme involved in O-glycan biosynthesis in normal, metaplastic, and neoplastic gastric tissues.
Our results showed that ppGalNAc-T6 was expressed both in the foveolar epithelium and glands of both antrum and body regions of the normal gastric mucosa.
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Table 1 Summary of data on the expression level of ppGalNAc-T6 and clinico-pathologic features in 76 gastric carcinoma cases
In intestinal metaplasia, a precursor lesion of gastric carcinoma, we observed a slightly lower percentage of positive cases. Intestinal metaplasia shows a highly regulated pattern of expression of genes, reproducing the differentiation characteristics of intestinal cells. This is the case of mucins such as MUC2 (Reis et al. 1999) and a modified pattern of mucin-type O-glycans such as Sialyl-Tn. The percentage of cases expressing ppGalNAc-T6 observed in intestinal metaplasia may reflect the differentiation characteristics of these cells and may explain the modified pattern of mucin-type O-glycans observed in intestinal metaplasia (David et al. 1992; Ferreira et al. 2006; Mesquita et al. 2006).
Studies in vitro have shown that ppGalNAc-Ts display site specificity and different kinetic properties toward O-glycosylation sites in mucins, including the MUC1 tandem repeat (Wandall et al. 1997; Hassan et al. 2000b). These characteristics of ppGalNAc-Ts suggest that the members of this family of enzymes have different functions and that the repertoire of ppGalNAc-Ts expressed in a given cell may determine the pattern of O-glycan attachment, especially the density of glycosylation of the MUC1 tandem repeat (Bennett et al. 1998; Hassan et al. 2000b).
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Table 2 Expression level of ppGalNAc-T6 according to the presence of venous invasion and localization of the tumor in the stomach
This study is, to the best of our knowledge, the first evaluation of the expression of ppGalNAc-T6 in gastric carcinomas. This evaluation of ppGalNAc-T6 of gastric carcinomas was possible because of the capability of MAb T6.3 to recognize formalin-fixed paraffin-embedded sections (Berois et al. 2006). The evaluation of ppGalNAc-T6 in 76 gastric carcinomas showed expression of this enzyme in 79% of the cases. Our results showed that the expression of ppGalNAc-T6 in gastric carcinomas was associated with the presence of venous invasion. In addition, our results also showed a particular association of venous invasion with ppGalNAc-T6 expression in tumors localized in the body region of the stomach.
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Table 3 Summary of data on the coexpression level of mucins (MUC5AC, MUC6, and MUC2) and ppGalNAc-T6 in gastric carcinoma cases
Different levels of ppGalNAc-T3 have been detected in patients with various types of carcinomas (Shibao et al. 2002; Gu et al. 2004; Ishikawa et al. 2004; Miyahara et al. 2004; Yamamoto et al. 2004; Landers et al. 2005; Inoue et al. 2007), and this has been associated with a poor prognosis. ppGalNAc-T6 exhibits a high sequence homology to ppGalNAc-T3, and in vitro studies have shown that both enzymes display similar acceptor substrate specificities (Bennett et al. 1999b). Our results suggest that ppGalNac-T6 may also play a role in the biological characteristics of gastric carcinoma cells, most probably through the variation in mucin O-glycosylation. This is in line with previous studies showing that gastric carcinoma cell lines expressing different set of ppGalNAc-Ts are associated with variable patterns of mucin glycosylation (Marcos et al. 2003). The abnormal expression of ppGalNAc-T6 in gastric carcinomas, and the mucin glycoforms produced by this enzyme, could induce changes in cellular functions including adhesion and invasion. Although a preliminary evaluation of coexpression between ppGalNAc-T6 and simple mucin-type carbohydrate antigens (Tn and sialyl-Tn) in a subseries of gastric carcinomas did not show a direct association (data not shown), we cannot exclude that the expression of ppGalNAc-T6 contributes to a different level of O-glycan site occupancy in the mucin tandem repeats. Our results warrant further study on the relationship between ppGalNAc-T6 expression and alterations in the O-glycan density and expression of carbohydrate antigens in gastric carcinomas (Marcos et al. 2003). This evaluation should also include other glycosyltransferases that can contribute to the synthesis of these carbohydrate antigens. In addition, future functional in vitro studies will clarify the biological role of ppGalNAc-T6 overexpression in gastric carcinoma cells. Furthermore, the expression of the mucin protein core is tightly regulated in gastric mucosa. Our results showed that ppGalNAc-T6 is inversely associated with the expression of mucin MUC5AC. This result may stem from characteristics of differentiation of the cells expressing either protein. No association was observed with the other mucins evaluated. Finally, ppGalNAc-T6 expression was observed in both complete and incomplete intestinal metaplasia cases (Reis et al. 1999).
In summary, we showed that (a) ppGalNAc-T6 is expressed in normal gastric mucosa in both antrum and body regions; (b) in intestinal metaplasia, there is expression of ppGalNAc-T6 in 52% of the cases; (c) in gastric carcinomas, the expression of ppGalNAc-T6 is heterogeneous, with most cases (79%) expressing ppGalNAc-T6; and (d) ppGalNAc-T6 expression in gastric carcinomas is associated with venous invasion. Our results provide evidence that ppGalNAc-T6 is a novel IHC marker associated with venous invasion in gastric carcinomas and contributes to the understanding of the molecular mechanisms that underlie aberrant glycosylation during gastric carcinogenesis and gastric carcinoma.
Acknowledgements
This work was supported by Fundação para a Ciěncia e a Tecnologia (FCT) (PTDC/CTM/65330/2006 and PTDC/CVT/65537/2006); financiado no âmbito Programa Operacional Ciěncia e Inovação 2010 do Quadro Comunitário de Apoio III e comparticipado pelo FEDER; and Association for International Cancer Research (AICR Grant 05–088). J.G. (SFRH/BD/40563/2007), N.T.M. (SFRH/BD/11764/2003), and A.M. (SFRH/BD/36339/2007) acknowledge FCT for financial support.
We thank Leonor David and Ulla Mandel for suggestions, Mário Seixas for statistical analysis, and Nuno Mendes for technical assistance.
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