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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.
Gastric cancer is the fourth most commonly diagnosed cancer and the second most common cause of cancer-related death worldwide. 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.[2-4]
Gastric adenocarcinoma is divided into intestinal and diffuse types using the Lauren classification system, or differentiated and undifferentiated types using the Nakamura classification system. 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 pathways.[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.
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. It is suggested that phenotypic marker expression in gastric carcinoma is associated with clinicopathological variables such as cancer survival,[16-18] 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. 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 α1,4-linked N-acetylglucosamine residues (αGlcNAc) largely attached to a MUC6 scaffold.[21, 22] Previously, we used expression cloning to isolate a human A4GNT cDNA encoding α1,4-N-acetylglucosaminyltransferase (α4GnT), the enzyme responsible for αGlcNAc biosynthesis. We also showed that in vitro αGlcNAc suppresses growth and motility of Helicobacter pylori (H. pylori). Recently, we generated A4gnt-deficient mice to assess the role of αGlcNAc in vivo. Surprisingly, A4gnt null mice developed gastric adenocarcinoma through a hyperplasia–dysplasia–carcinoma sequence in the absence of H. pylori infection. These findings indicate that αGlcNAc loss triggers gastric tumorigenesis.
We also previously reported that gland mucous cells expressing MUC6 in non-neoplastic gastric mucosa also express αGlcNAc. By contrast, αGlcNAc expression is reduced in differentiated-type,[25-27] but not in undifferentiated-type, gastric adenocarcinoma. These findings, coupled with our observations in A4gnt-deficient mice, suggest that αGlcNAc loss is associated with tumorigenesis of differentiated-type but not undifferentiated-type adenocarcinoma. However, to date, the clinicopathological significance of αGlcNAc loss in human gastric cancer remains unclear.
In the present study, we examined expression of mucin phenotypic markers and αGlcNAc in 214 gastric carcinomas by immunohistochemical staining in order to assess the clinicopathological significance of mucin expression and further investigate how αGlcNAc loss is associated with tumor progression.
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In the present study, we showed that αGlcNAc loss in MUC6-positive gastric carcinoma cells was significantly correlated with depth of invasion, stage, and venous invasion in differentiated-type but not undifferentiated-type adenocarcinoma. More importantly, αGlcNAc loss was associated with significantly poorer survival in patients with the MUC6-positive differentiated-type adenocarcinoma. These results suggest that αGlcNAc 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 αGlcNAc) develop differentiated-type but not undifferentiated-type adenocarcinoma. 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, and Ohta et al. 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, and Verbeke et al. showed that CXC chemokines including CXCL1/CXCL5 facilitate tumor progression. Nakayama et al. 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. 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 αGlcNAc loss is related to gastric cancer progression in inflammation-related pathways. It remains to be determined how αGlcNAc 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.[16-20] 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 differentiated-type adenocarcinoma (see Table 1). These results concur with the report of Zheng et al. 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, αGlcNAc 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 αGlcNAc 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 αGlcNAc loss in MUC6-positive cancer cells in progression of differentiated-type gastric adenocarcinoma.
In conclusion, the present study indicates that αGlcNAc 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 αGlcNAc may predict progression and prognosis of patients with these types of tumors.