Sage Journals: Discover world-class research

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.

Keywords

ppGalNAc-T6 gastric mucosa intestinal metaplasia gastric carcinoma

Mucins are O-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 X ² 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.

Table 1

Summary of data on the expression level of ppGalNAc-T6 and clinico-pathologic features in 76 gastric carcinoma cases

			Expression of ppGalNAc-T6 a
Category	Number of cases (%)	Positive cases (%)	Low (%)	High (%)	p
Age (years)
<40	1 (1.3)	1 (100.0)	1 (100.0)	0 (0.0)	0.40
40–65	40 (52.6)	32 (80.0)	21 (65.6)	11 (34.4)
>65	35 (46.1)	27 (77.1)	14 (51.9)	13 (48.1)
Laurén's classification
Diffuse	21 (27.6)	18 (85.7)	12 (66.7)	6 (33.3)	0.20
Intestinal	36 (47.4)	29 (80.6)	19 (65.5)	10 (34.5)
Unclassified	19 (25.0)	13 (68.4)	5 (38.5)	8 (61.5)
Ming's classification b
Expanding	28 (37.3)	21 (75.0)	11 (52.4)	10 (47.6)	0.42
Infiltrative	47 (62.7)	38 (80.9)	24 (63.2)	14 (36.8)
Survival
Short survival	67 (88.2)	53 (79.1)	32 (60.4)	21 (39.6)	0.87
Long survival	9 (11.8)	7 (77.8)	4 (57.1)	3 (42.9)
Lymphatic invasion c
Absent	4 (5.4)	3 (75.0)	3 (100.0)	0 (0.0)	0.14
Present	70 (94.6)	55 (78.6)	31 (56.4)	24 (43.6)
Nodal metastasis c
Absent	15 (20.2)	11 (73.3)	7 (63.6)	4 (36.4)	0.71
Present	59 (79.7)	47 (79.7)	27 (57.4)	20 (42.6)
Venous invasion
Absent	33 (43.4)	25 (75.8)	19 (76.0)	6 (24.0)	0.03
Present	43 (56.6)	35 (81.4)	17 (48.6)	18 (51.4)
Staging d
Early	15 (20.8)	11 (73.3)	8 (72.7)	3 (27.3)	0.32
Advance	57 (79.2)	46 (80.7)	26 (56.5)	20 (43.5)
Tumor localization c
Cardia	18 (24.3)	11 (61.1)	8 (72.7)	3 (27.3)	0.50
Body	27 (36.5)	23 (85.2)	12 (52.2)	11 (47.8)
Antrum	29 (39.2)	24 (82.8)	15 (62.5)	9 (37.5)
Total	76 (100.0)	60 (78.9)	36 (60.0)	24 (40.0)

Expression of ppGalNAc-T6 was scored as Low expression (<25% of positive staining cells) and High expression (>25% of positive staining cells).

In one case, the growth pattern was not classified according to Ming's classification because both infiltrative and expanding areas were observed.

In two cases, there was no information regarding lymphatic invasion, nodal metastasis, and tumor localization.

Staging as early (IA and IB) and advance (II-IV) was done based on the pTNM as described in Materials and Methods. In four cases, the pTNM classification was not available.

ppGalNAc-T6, UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactos-aminyltransferase-6.

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).

Table 2

Expression level of ppGalNAc-T6 according to the presence of venous invasion and localization of the tumor in the stomach

		Expression of ppGalNAc-T6 b
Tumor localization a	Number of cases (%)	Low (%)	High (%)	p
Cardia (n = 11)
Absent venous invasion	3 (27.3)	2 (66.7)	1 (33.3)	0.99
Present venous invasion	8 (72.7)	6 (75.0)	2 (25.0)
Body (n = 23)
Absent venous invasion	9 (39.1)	8 (88.9)	1 (11.1)	0.01
Present venous invasion	14 (60.9)	4 (28.6)	10 (71.4)
Antrum (n = 24)
Absent venous invasion	13 (54.2)	9 (69.2)	4 (30.8)	0.67
Present venous invasion	11 (45.8)	6 (54.5)	5 (45.5)

In two cases, there was no information regarding tumor localization.

Expression of ppGalNAc-T6 was scored as low expression (<25% of positive staining cells) and high expression (>25% of positive staining cells).

ppGalNAc-T6, UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactos-aminyltransferase-6.

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.

Table 3

Summary of data on the coexpression level of mucins (MUC5AC, MUC6, and MUC2) and ppGalNAc-T6 in gastric carcinoma cases

			Expression of ppGalNAc-T6 a
Mucins	Number of cases (%)	Positive cases (%)	Low (%)	High (%)	p
MUC5AC b
Negative	22 (30.6)	17 (77.3)	6 (35.3)	11 (64.7)	0.02
Positive	50 (69.4)	39 (78.0)	27 (69.2)	12 (30.8)
MUC6 c
Negative	28 (63.6)	21 (75.0)	13 (61.9)	8 (38.1)	0.51
Positive	16 (36.4)	12 (75.0)	6 (50.0)	6 (50.0)
MUC2 d
Negative	32 (58.2)	24 (54.5)	13 (54.2)	11 (45.8)	0.70
Positive	23 (41.8)	20 (45.4)	12 (60.0)	8 (40.0)

Expression of ppGalNAc-T6 was scored as low expression (<25% of positive staining cells) and high expression (>25% of positive staining cells).

MUC5AC mucin expression was evaluated in 72 gastric carcinoma cases.

MUC6 mucin expression was evaluated in 44 gastric carcinoma cases.

MUC2 mucin expression was evaluated in 55 gastric carcinoma cases.

ppGalNAc-T6, UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactos-aminyltransferase-6.

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.

Footnotes

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.

References

Bennett

Hassan

Clausen

(1996) cDNA cloning and expression of a novel human UDP-N-acetyl-alpha-D-galactosamine. Polypeptide N-acetylgalactosaminyltransferase, Gal-NAc-T3. J Biol Chem 271:17006–17012

Bennett

Hassan

Hollingsworth

Clausen

(1999a) A novel human UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase, GalNAc-T7, with specificity for partial GalNAc-glycosylated acceptor substrates. FEBS Lett 460:226–230

Bennett

Hassan

Mandel

Hollingsworth

Akisawa

Ikematsu

Merkx

. (1999b) Cloning and characterization of a close homologue of human UDP-N-acetyl-alpha-Dgalactosamine: polypeptide N-acetylgalactosaminyltransferase-T3, designated GalNAc-T6. Evidence for genetic but not functional redundancy. J Biol Chem 274:25362–25370

Bennett

Hassan

Mandel

Mirgorodskaya

Roepstorff

Burchell

Taylor-Papadimitriou

. (1998) Cloning of a human UDP-N-acetyl-alpha-D-Galactosamine:polypeptide N-acetylgalactosaminyltransferase that complements other GalNAc-transferases in complete O-glycosylation of the MUC1 tandem repeat. J Biol Chem 273:30472–30481

Berois

Mazal

Ubillos

Trajtenberg

Nicolas

Sastre-Garau

Magdelenar

. (2006) UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase-6 as a new immunohistochemical breast cancer marker. J Histochem Cytochem 54:317–328

Brooks

Carter

Bennett

Clausen

Mandel

(2007) Immunolocalisation of members of the polypeptide N-acetylgalactosaminyl transferase (ppGalNAc-T) family is consistent with biologically relevant altered cell surface glycosylation in breast cancer. Acta Histochem 109:273–284

Cheng

Tachibana

Iwasaki

Kameyama

Zhang

Kubota

Hiruma

. (2004) Characterization of a novel human UDP-GalNAc transferase, pp-GalNAc-T15. FEBS Lett 566:17–24

Clausen

Bennett

(1996) A family of UDP-GalNAc: polypeptide N-acetylgalactosaminyl-transferases control the initiation of mucin-type O-linked glycosylation. Glycobiology 6:635–646

David

Nesland

Clausen

Carneiro

Sobrinho-Simões

(1992) Simple mucin-type carbohydrate antigens (Tn, sialosyl-Tn and T) in gastric mucosa, carcinomas and metastases. APMIS Suppl 27:162–172

10.

Ferreira

Marcos

David

Nakayama

Reis

(2006) Terminal alpha1,4-linked N-acetylglucosamine in Helicobacter pylori-associated intestinal metaplasia of the human stomach and gastric carcinoma cell lines. J Histochem Cytochem 54:585–591

11.

Freire

Berois

Sóñora

Varangot

Barrios

Osinaga

(2006) UDP-N-acetyl-D-galactosamine:polypeptide N-acetyl-galactosaminyltransferase 6 (ppGalNAc-T6) mRNA as a potential new marker for detection of bone marrow-disseminated breast cancer cells. Int J Cancer 119:1383–1388

12.

Oyama

Osaki

Takenoyama

Izumi

Sugio

. (2004) Low expression of polypeptide GalNAc N-acetyl-galactosaminyl transferase-3 in lung adenocarcinoma: impact on poor prognosis and early recurrence. Br J Cancer 90:436–442

13.

Guo

Zhang

Cheng

Iwasaki

Wang

Kubota

Tachibana

. (2002) Molecular cloning and characterization of a novel member of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family, pp-GalNAc-T12. FEBS Lett 524:211–218

14.

Hagen

Ten Hagen

Beres

Balys

VanWuyckhuyse

Tabak

(1997) cDNA cloning and expression of a novel UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase. J Biol Chem 272:13843–13848

15.

Hanisch

Reis

Clausen

Paulsen

(2001) Evidence for glycosylation-dependent activities of polypeptide N-acetylgalactosaminyltransferases rGalNAc-T2 and −T4 on mucin glycopeptides. Glycobiology 11:731–740

16.

Hassan

Bennett

Mandel

Hollingsworth

Clausen

(2000a) Control of mucin-type-O-glycosylation: O-glycan occupancy is directed by substrate specificities of polypeptide GalNAc-transferases. In Ernst

Hart

Sinay

, eds. Carbohydrates in Chemistry and Biology, A Comprehensive Handbook. New York, Wiley-VCH, 273–292

17.

Hassan

Reis

Bennett

Mirgorodskaya

Roepstorff

Hollingsworth

Burchell

. (2000b) The lectin domain of UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactos-aminyltransferase-T4 directs its glycopeptide specificities. J Biol Chem 275:38197–38205

18.

Hollingsworth

Swanson

(2004) Mucins in cancer: protection and control of the cell surface. Nat Rev Cancer 4:45–60

19.

Homa

Hollander

Lehman

Thomsen

Elhammer

(1993) Isolation and expression of a cDNA clone encoding a bovine UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase. J Biol Chem 268:12609–12616

20.

Hsu

Faine

Fanger

(1981) A comparative study of the peroxidase-antiperoxidase method and the avidin-biotin complex method for studying polypeptide hormones with radioimmunoassay antibodies. Am J Clin Pathol 75:734–738

21.

Inoue

Eguchi

Oda

Nishiyama

Fujii

Izumi

Kohno

. (2007) Expression of GalNAc-T3 and its relationships with clinicopathological factors in 61 extrahepatic bile duct carcinomas analyzed using stepwise sections - special reference to its association with lymph node metastases-. Mod Pathol 20:267–276

22.

Ishikawa

Kitayama

Nariko

Kohno

Nagawa

(2004) The expression pattern of UDP-N-acetyl-alpha-d-galactosamine: polypeptide N-acetylgalactosaminyl transferase-3 in early gastric carcinoma. J Surg Oncol 86:28–33

23.

Kohsaki

Nishimori

Nakayama

Miyazaki

Enzan

Nomoto

Hollingsworth

. (2000) Expression of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase isozymes T1 and T2 in human colorectal cancer. J Gastroenterol 35:840–848

24.

Landers

Burger

Tebay

Purdie

Scells

Samaratunga

Lavin

. (2005) Use of multiple biomarkers for a molecular diagnosis of prostate cancer. Int J Cancer 114:950–956

25.

Laurén

(1965) The two histological main types of gastric carcinoma: diffuse and so-called intestinal-type carcinoma. Acta Pathol Microbiol Scand 64:31–49

26.

Lesuffleur

Zweibaum

Real

(1994) Mucins in normal and neoplastic human gastrointestinal tissues. Crit Rev Oncol Hematol 17:153–180

27.

Mandel

Hassan

Therkildsen

Rygaard

Jakobsen

Juhl

Dabelsteen

. (1999) Expression of polypeptide GalNAc-transferases in stratified epithelia and squamous cell carcinomas: immunohistological evaluation using monoclonal antibodies to three members of the GalNAc-transferase family. Glycobiology 9:43–52

28.

Marcos

Cruz

Silva

Almeida

David

Mandel

Clausen

. (2003) Polypeptide GalNAc-transferases, ST6GalNAc-transferase I, and ST3Gal-transferase I expression in gastric carcinoma cell lines. J Histochem Cytochem 51:761–771

29.

Mesquita

Raquel

Nuno

Reis

Silva

Serpa

Van Seuningen

. (2006) Metaplasia: a transdifferentiation process that facilitates cancer development: the model of gastric intestinal metaplasia. Crit Rev Oncog 12:3–26

30.

Ming

S-C

(1977) Gastric carcinoma. A pathobiological classification. Cancer 39:2475–2485

31.

Miyahara

Shoda

Kawamoto

Furukawa

Ueda

Todoroki

Tanaka

. (2004) Expression of UDP-N-acetyl-alpha-D-galactosamine-polypeptide N-acetylgalactosaminyltransferase isozyme 3 in the subserosal layer correlates with postsurgical survival of pathological tumor stage 2 carcinoma of the gallbladder. Clin Cancer Res 10:2090–2099

32.

Pinto-De-Sousa

David

Seixas

Pimenta

(2001) Clinicopathologic profiles and prognosis of gastric carcinomas from the cardia, fundus/body and antrum. Dig Surg 18:102–110

33.

Rajpert-De Meyts

Poll

Goukasian

Jeanneau

Herlihy

Bennett

Skakkebaek

. (2007) Changes in the profile of simple mucin-type O-glycans and polypeptide GalNAc-transferases in human testis and testicular neoplasms are associated with germ cell maturation and tumour differentiation. Virchows Arch 451:805–814

34.

Reis

David

Carvalho

Mandel

de Bolós

Mirgorodskaya

Clausen

. (2000) Immunohistochemical study of the expression of MUC6 mucin and co-expression of other secreted mucins (MUC5AC and MUC2) in human gastric carcinomas. J Histochem Cytochem 48:377–388

35.

Reis

David

Correa

Carneiro

de Bolós

Garcia

Mandel

. (1999) Intestinal metaplasia of human stomach displays distinct patterns of mucin (MUC1, MUC2, MUC5AC, and MUC6) expression. Cancer Res 59:1003–1007

36.

Reis

David

Nielsen

Clausen

Mirgorodskaya

Roepstorff

Sobrinho-Simoes

(1997) Immunohistochemical study of MUC5AC expression in human gastric carcinomas using a novel monoclonal antibody. Int J Cancer 74:112–121

37.

Reis

David

Seixas

Burchell

Sobrinho-Simoes

(1998a) Expression of fully and under-glycosylated forms of MUC1 mucin in gastric carcinoma. Int J Cancer 79:402–410

38.

Reis

Sørensen

Mandel

David

Mirgorodskaya

Roepstorff

Kihlberg

. (1998b) Development and characterization of an antibody directed to an alpha-N-acetyl-D-galactosamine glycosylated MUC2 peptide. Glycoconj J 15:51–62

39.

Schwientek

Bennett

Flores

Thacker

Hollmann

Reis

Behrens

. (2002) Functional conservation of subfamilies of putative UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases in Drosophila, Caenorhabditis elegans, and mammals. One subfamily composed of l(2)35Aa is essential in Drosophila. J Biol Chem 277:22623–22638

40.

Shibao

Izumi

Nakayama

Ohta

Nagata

Nomoto

Matsuo

. (2002) Expression of UDP-N-acetyl-alpha-Dgalactosamine-polypeptide galNAc N-acetylgalactosaminyl transferase-3 in relation to differentiation and prognosis in patients with colorectal carcinoma. Cancer 94:1939–1946

41.

Sutherlin

Nishimori

Caffrey

Bennett

Hassan

Mandel

Mack

. (1997) Expression of three UDP-N-acetylalpha-D-galactosamine:polypeptide GalNAc N-acetylgalactosaminyl transferases in adenocarcinoma cell lines. Cancer Res 57:4744–4748

42.

Ten Hagen

Bedi

Tetaert

Kingsley

Hagen

Balys

Beres

. (2001) Cloning and characterization of a ninth member of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase family, ppGaNTase-T9. J Biol Chem 276:17395–17404

43.

Ten Hagen

Fritz

Tabak

(2003) All in the family: the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases. Glycobiology 13:1R–16R

44.

Ten Hagen

Hagen

Balys

Beres

Van Wuyckhuyse

Tabak

(1998) Cloning and expression of a novel, tissue specifically expressed member of the UDP-Gal-NAc:polypeptide N-acetylgalactosaminyltransferase family. J Biol Chem 273:27749–27754

45.

Ten Hagen

Tetaert

Hagen

Richet

Beres

Gagnon

Balys

. (1999) Characterization of a UDP-Gal-NAc: polypeptide N-acetylgalactosaminyltransferase that displays glycopeptide N-acetylgalactosaminyltransferase activity. J Biol Chem 274:27867–27874

46.

Wandall

Hassan

Mirgorodskaya

Kristensen

Roepstorff

Bennett

Nielsen

. (1997) Substrate specificities of three members of the human UDP-N-acetyl-alpha-Dgalactosamine:polypeptide N-acetylgalactosaminyltransferase family, GalNAc-T1, −T2, and −T3. J Biol Chem 272:23503–23514

47.

Wang

Tachibana

Zhang

Iwasaki

Kameyama

Cheng

Guo

. (2003) Cloning and characterization of a novel UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, pp-GalNAc-T14. Biochem Biophys Res Commun 300:738–744

48.

White

Bennett

Takio

Sorensen

Bonding

Clausen

(1995) Purification and cDNA cloning of a human UDP-Nacetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase. J Biol Chem 270:24156–24165

49.

Yamamoto

Nakamori

Tsujie

Takahashi

Nagano

Dono

Umeshita

. (2004) Expression of uridine diphosphate N-acetyl-alpha-D-galactosamine: polypeptide N-acetylgalactosaminyl transferase 3 in adenocarcinoma of the pancreas. Pathobiology 71:12–18

50.

Zhang

Iwasaki

Wang

Kudo

Kalka

Hennet

Kubota

. (2003) Cloning and characterization of a new human UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase, designated pp-GalNAc-T13, that is specifically expressed in neurons and synthesizes GalNAc alpha-serine/threonine antigen. J Biol Chem 278:573–584

Expression of UDP- N -acetyl-D-galactosamine: Polypeptide N -acetylgalactosaminyltransferase-6 in Gastric Mucosa,Intestinal Metaplasia,and Gastric Carcinoma

Abstract

Keywords

Materials and Methods

Tissue Samples and Histological Classification

IHC

Scoring of the Immunostaining and Statistical Analysis

Results

Expression of ppGalNAc-T6 in Normal Gastric Mucosa

Expression of ppGalNAc-T6 in Intestinal Metaplasia

Expression of ppGalNAc-T6 in Gastric Carcinomas

Coexpression of Mucins (MUC5AC, MUC6, and MUC2) and ppGalNAc-T6 in Gastric Carcinomas

Discussion

Footnotes

Acknowledgements

References