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Loss of gastric gland mucin-specific O-glycan is associated with progression of differentiated-type adenocarcinoma of the stomach

Gastric gland mucin secreted from the lower portion of the gastric mucosa contains unique O-linked oligosaccharides having terminal α1,4-linked N-acetylglucosamine (αGlcNAc) residues largely attached to a MUC6 scaffold. Previously, we generated A4gnt-deficient mice, which totally lack αGlcNAc, and showed that αGlcNAc functions as a tumor suppressor for gastric cancer. Here, to determine the clinicopathological significance of αGlcNAc in gastric carcinomas, we examined immunohistochemical expression of αGlcNAc and mucin phenotypic markers including MUC5AC, MUC6, MUC2, and CD10 in 214 gastric adenocarcinomas and compared those expression patterns with clinicopathological parameters and cancer-specific survival. The αGlcNAc loss was evaluated in MUC6-positive gastric carcinoma. Thirty-three (61.1%) of 54 differentiated-type gastric adenocarcinomas exhibiting MUC6 in cancer cells lacked αGlcNAc expression. Loss of αGlcNAc was significantly correlated with depth of invasion, stage, and venous invasion by differentiated-type adenocarcinoma. Loss of αGlcNAc was also significantly associated with poorer patient prognosis in MUC6-positive differentiated-type adenocarcinoma. By contrast, no significant correlation between αGlcNAc loss and any clinicopathologic variable was observed in undifferentiated-type adenocarcinoma. Expression of MUC6 was also significantly correlated with several clinicopathological variables in differentiated-type adenocarcinoma. However, unlike the case with αGlcNAc, its expression showed no correlation with cancer-specific survival in patients. In undifferentiated-type adenocarcinoma, we observed no significant correlation between mucin phenotypic marker expression, including MUC6, and any clinicopathologic variable. These results together indicate that loss of αGlcNAc in MUC6-positive cancer cells is associated with progression and poor prognosis in differentiated, but not undifferentiated, types of gastric adenocarcinoma.

G astric cancer is the fourth most commonly diagnosed cancer and the second most common cause of cancerrelated death worldwide. (1) Despite improvements in surgery, radiotherapy, and chemotherapy, survival rates for advanced gastric cancer are poor. Some patients with gastric cancer, even those with the same TNM stage, have different prognoses and treatment responses. Therefore, we need to understand the biology of gastric cancer better to develop more effective treatment. Recent molecular studies have identified multiple factors that modulate tumor progression, invasion, and metastasis formation. (2)(3)(4) Gastric adenocarcinoma is divided into intestinal and diffuse types using the Lauren classification system, (5) or differentiated and undifferentiated types using the Nakamura classification system. (6) Both classification systems are based on morphological characteristics relevant largely to gland formation and histogenetic background, and these two types of tumor, that is, intestinal or differentiated types and diffuse or undifferentiated types, are known to emerge from different genetic path-ways. (6,7) Although various histological types of tumors can be distinguished using standard H&E staining, recent advances in immunohistochemical methods using gastric and small intestinal cell markers have enabled classification of gastric cancer based on different mucin phenotypes. (8) The MUC6 glycoprotein is expressed in gastric gland mucous cells, such as mucous neck and pyloric gland cells in the lower layer of the mucosa, whereas the MUC5AC glycoprotein is expressed in surface mucous cells in the upper layer of the mucosa. (9,10) Both MUC6 and MUC5AC are commonly used to identify gastric phenotype of tumors. In contrast, MUC2, a marker of intestinal goblet cells, (11,12) and CD10, a marker of intestinal absorptive cells, (13,14) are used to identify intestinal phenotypes. (15) It is suggested that phenotypic marker expression in gastric carcinoma is associated with clinicopathological variables such as cancer survival, (16)(17)(18) and several groups report that MUC5AC and MUC2 are useful clinically to predict malignancy outcomes. (16,17) Others report that downregulation of MUC6 but not of MUC5AC or MUC2 correlates with gastric carcinoma progression. (18) Still others have shown that CD10-positive gastric carcinomas tend to invade blood vessels. (19,20) Thus, analysis of phenotypic mucin markers represents a promising approach to predicting gastric cancer progression.
Gastric gland mucin secreted from the lower portion of the gastric mucosa contains unique O-linked oligosaccharides (O-glycan) exhibiting terminal a1,4-linked N-acetylglucosamine residues (aGlcNAc) largely attached to a MUC6 scaffold. (21,22) Previously, we used expression cloning to isolate a human A4GNT cDNA encoding a1,4-N-acetylglucosaminyltransferase (a4GnT), the enzyme responsible for aGlcNAc biosynthesis. (23) We also showed that in vitro aGlcNAc suppresses growth and motility of Helicobacter pylori (H. pylori). (24) Recently, we generated A4gnt-deficient mice to assess the role of aGlcNAc in vivo. (25) Surprisingly, A4gnt null mice developed gastric adenocarcinoma through a hyperplasia-dysplasiacarcinoma sequence in the absence of H. pylori infection. These findings indicate that aGlcNAc loss triggers gastric tumorigenesis.
We also previously reported that gland mucous cells expressing MUC6 in non-neoplastic gastric mucosa also express aGlcNAc. (22) By contrast, aGlcNAc expression is reduced in differentiated-type, (25)(26)(27) but not in undifferentiated-type, gastric adenocarcinoma. (26) These findings, coupled with our observations in A4gnt-deficient mice, suggest that aGlcNAc loss is associated with tumorigenesis of differentiated-type but not undifferentiated-type adenocarcinoma. However, to date, the clinicopathological significance of aGlcNAc loss in human gastric cancer remains unclear.
In the present study, we examined expression of mucin phenotypic markers and aGlcNAc in 214 gastric carcinomas by immunohistochemical staining in order to assess the clinicopathological significance of mucin expression and further investigate how aGlcNAc loss is associated with tumor progression.

Materials and Methods
Patients. Our series consisted of 214 patients who had undergone gastrectomy for gastric cancer between 2002 and 2005 at Aizawa Hospital, Matsumoto, Japan. The patients included 150 men and 64 women with an age range of 40-90 years. No preoperative radiotherapy and ⁄ or chemotherapy had been given. The Ethical Committees of Shinshu University School of Medicine (Matsumoto, Japan) and Aizawa Hospital approved the protocol and use of human materials in this study.
Histopathology. Tissue specimens were fixed in 10% buffered formalin and embedded in paraffin. Hematoxylin-eosin and immunohistochemical stainings were carried out on 4-lm serial sections. Tumors were classified as differentiated or undifferentiated types according to the classification of Nakamura et al. (6) Pathological diagnosis, tumor invasion depth, lymph node metastasis, distant metastasis, and stage were determined according to the Japanese Classification of Gastric Carcinoma, 14th edition. (28) Immunohistochemistry. Expression of MUC5AC, MUC6, MUC2, CD10, and aGlcNAc was analyzed by immunohistochemistry. Primary antibodies used included anti-MUC5AC (CLH2) (Novocastra, Newcastle upon Tyne, UK) diluted 1 ⁄ 100, anti-MUC6 (CLH5) (Novocastra) diluted 1 ⁄ 200, anti-MUC2 (Ccp58) (Novocastra) diluted 1 ⁄ 200, anti-CD10 (56C6) (Novocastra) diluted 1 ⁄ 100, and anti-aGlcNAc (HIK1083) (Kanto Chemical, Tokyo, Japan) diluted 1 ⁄ 20. Before immunostaining, antigen retrieval was carried out by microwaving tissue sections in 10 mM Tris-HCl buffer (pH 8.0) containing 1 mM EDTA for 30 min for anti-MUC5AC, anti-MUC6, anti-MUC2, and anti-CD10 antibodies. The secondary antibody was anti-mouse Dako EnVision+ System-HRP Labeled Polymer (Dako North America, Carpinteria, CA, USA). Peroxidase activity was visualized using a diaminobenzidine-H 2 O 2 solution. Controls were undertaken by omitting the primary antibody, and no specific staining was seen. Tissue specimens containing >5% of positively stained carcinoma cells out of the total number of carcinoma cells on the slide were defined as positive, and others were classified as negative according to the criteria of Machida et al. (29) Statistical analysis. Categorical data were compared using the v 2 -test. When the expected number in any cell was less than five, Fisher's exact test was used. Age and tumor size were compared using the Mann-Whitney U-test. Cancer-specific survival curves were estimated by the Kaplan-Meier method, (30) and the difference between the curves was evaluated by a log-rank test. (31) P-values < 0.05 were considered significant. Statistical analysis was carried out using the SPSS software package (version 21) (IBM, Armonk, NY, USA).
Correlation between clinicopathologic findings and mucin expression in differentiated-type adenocarcinoma. Expression of MUC6 was significantly correlated with depth of invasion, lymph node metastasis, stage, lymphatic invasion, venous invasion, and tumor size (Table 1). In addition, MUC2 expression was significantly correlated with venous invasion. However, no significant correlation was seen between MUC5AC or CD10 expression and any variable analyzed. Also, expression of individual mucin phenotypic markers was not associated with 5-year cancer-specific survival rates in patients with differentiated-type adenocarcinoma (Fig. 2).
Correlation between clinicopathologic findings and mucin expression in undifferentiated-type adenocarcinoma. In undifferentiated-type adenocarcinoma, no significant correlations were observed between mucin marker expression and any clinicopathologic variable analyzed (Table 2). Mucin marker expression was also not associated with 5-year cancer-specific survival in patients with undifferentiated-type adenocarcinoma (Fig. 3).
Expression of aGlcNAc in MUC6-positive gastric cancer. As aGlcNAc is attached to MUC6 in the normal gastric mucosa, (22) we tested whether the glycan could be eliminated from gastric cancer cells expressing MUC6 using immunohistochemistry. Fifty-five of 102 (53.9%) MUC6-positive gastric cancers, including differentiated-type and undifferentiated-type adenocarcinomas, lacked aGlcNAc expression (Table 3). In addition, aGlcNAc expression in MUC6-positive adenocarcinomas was not significantly correlated with differentiated or undifferentiated tumor types (P = 0.12). However, in the case of signet ring cell carcinoma, a subtype of undifferentiated-type adenocarcinoma, only 6 (26.1%) of 23 patients lacked aGlcNAc expression, compared with differentiated-type adenocarcinoma (P = 0.0049).
Expression of aGlcNAc in gastric cancer was heterogeneous, irrespective of the histological types. As shown previously, (25,32) and further confirmed here, aGlcNAc-positive cancer cells tended to be located in the lower layer of the gastric mucosa rather than in the upper layer, regardless of differentiated or undifferentiated types (Fig. 4a,c). Once the cancer cells invaded beyond the muscularis mucosae, aGlcNAc-positive cancer cells were irregularly distributed throughout carcinoma tissues, and the expression levels of aGlcNAc in the invasive region tended to mirror the expression levels of the glycan in the intramucosal cancer region within the same tumor (Fig. 4b,c).  (Table 3). Notably, aGlcNAc expression was inversely correlated with depth of invasion, stage, and venous invasion. More importantly, analysis of 5-year cancer-specific survival rates of patients with MUC6-positive cancer cells revealed that individuals with aGlcNAc-negative tumors had a significantly poorer outcome than those showing aGlcNAc-positive tumors (P = 0.048) (Fig. 5a).
Correlation between clinicopathological variables and aGlcNAc expression in undifferentiated-type adenocarcinoma showing MUC6-positive cancer cells. As shown in Table 3      did not express aGlcNAc. In undifferentiated-type adenocarcinoma, no significant correlation was found between aGlcNAc expression and any variable examined, and aGlcNAc status in tumor cells had no significant effect on 5-year cancer-specific survival rates in patients (P = 0.549) (Fig. 5b).

Discussion
In the present study, we showed that aGlcNAc loss in MUC6positive gastric carcinoma cells was significantly correlated with depth of invasion, stage, and venous invasion in differentiatedtype but not undifferentiated-type adenocarcinoma. More importantly, aGlcNAc loss was associated with significantly poorer survival in patients with the MUC6-positive differentiated-type adenocarcinoma. These results suggest that aGlcNAc loss promotes progression of differentiated-type adenocarcinoma in humans. This conclusion is consistent with our previous study showing that mice deficient in A4gnt in gastric gland mucous cells (which lack aGlcNAc) develop differentiated-type but not undifferentiated-type adenocarcinoma. (25) Microarray and quantitative RT-PCR analysis of the gastric mucosa of those mutant mice revealed upregulation of genes encoding inflammatory chemokine ligands, proinflammatory cytokines, and growth factors, such as Ccl2, Cxcl1, Cxcl5, Il-11, and Hgf. Chemokine ligand 2 (CCL2) attracts tumor-associated macrophages, (33) and Ohta et al. (34) have reported that CCL2 expression in tumor cells is correlated with depth of tumor invasion and increased microvessel density and macrophage infiltration. Those authors conclude that CCL2 produced by human gastric carcinoma cells functions in angiogenesis through macrophage recruitment and activation. The CXC chemokines CXCL1 ⁄ CXCL5 are potent angiogenic factors, (33) and Verbeke et al. (35) showed that CXC chemokines including CXCL1 ⁄ CXCL5 facilitate tumor progression. Nakayama et al. (36) observed that interleukin-11 expression is significantly higher in differentiated compared to undifferentiated types of adenocarcinoma. That group also reported that interleukin-11 functions in gastric carcinoma progression. Furthermore, Mohri et al. (37) suggest that hepatocyte growth factor is an important prognostic determinant in gastric cancer. Thus, all of these factors likely promote tumor-promoting inflammation. Accordingly, our results suggest that aGlcNAc loss is related to gastric cancer progression in inflammation-related pathways. It remains to be determined how aGlcNAc loss in gastric cancer promotes tumor-promoting inflammation in the stomach. It is generally thought that intestinal or differentiated types of adenocarcinoma emerge from gastric mucosa with intestinal metaplasia, whereas diffuse or undifferentiated types of adenocarcinoma arise from ordinary gastric mucosa. However, gastric and intestinal phenotypic markers are widely expressed in gastric cancer, irrespective of histological types. (17,19,38) In the present study, altered expression of phenotypic mucin markers was not significantly correlated with histological type (see Tables 1,2). However, it has been suggested that phenotypic mucin marker expression in tumor cells is associated with clinicopathological findings and tumorigenesis in gastric cancer. (16)(17)(18)(19)(20) Our evaluation of clinicopathological findings and phenotypic mucin marker expression indicates that gastric carcinomas lacking MUC6 expression show deep invasion, frequent lymph node metastasis, high stage, frequent lymphatic and venous invasion, and large tumor size in differentiatedtype adenocarcinoma (see Table 1). These results concur with the report of Zheng et al. (18) showing that MUC6 downregulation correlates with gastric carcinoma progression. Those authors concluded that gastric carcinomas lacking MUC6 expression show aggressive behavior, as mucin loss is an indicator of cellular dedifferentiation or anaplasia. In contrast, other studies indicated no correlation between MUC6 expression and aggressive parameters. (39,40) Notably, in the present study, we found that even when cancer cells express MUC6, aGlcNAc loss in MUC6-positive cancer cells is significantly correlated with depth of invasion, venous invasion, stage, and poorer patient prognosis in the case of differentiated-type adenocarcinoma (see Table 3, Fig. 5a), strongly implying that in humans aGlcNAc acts as a tumor suppressor in this type of cancer. Prospective studies analyzing larger numbers of gastric cancer patients will be of great significance to verify the impact of aGlcNAc loss in MUC6-positive cancer cells in progression of differentiated-type gastric adenocarcinoma.
In conclusion, the present study indicates that aGlcNAc loss in MUC6-positive cancer cells is significantly associated with progression of differentiated-type but not undifferentiated-type adenocarcinoma of the stomach. Thus, immunohistochemistry for not only MUC6 but also for aGlcNAc may predict progression and prognosis of patients with these types of tumors.