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- Materials and Methods
K19-C2mE transgenic (Tg) mice, simultaneously expressing cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase-1 (mPGES-1) in the gastric mucosa under the cytokeratin 19 gene promoter, were here treated with N-methyl-N-nitrosourea (MNU) and inoculated with Helicobacter pylori (H. pylori) to investigate gastric carcinogenesis. Wild-type (WT) and Tg mice undergoing MNU treatment frequently developed tumors in the pyloric region (100% and 94.7%, respectively); multiplicity in Tg was higher than that in WT (P < 0.05) with H. pylori infection. Larger pyloric tumors were more frequently observed in Tg than in WT (P < 0.05). In addition, Tg developed fundic tumors, where WT did not. No gastric tumors were observed without MNU treatment. Transcripts of TNF-α, iNOS, IL-1β, and CXCL14 were up-regulated with H. pylori infection in both genotypes and were also increased more in Tg than in WT within H. pylori-inoculated animals. Immunohistochemical analysis demonstrated significantly greater β-catenin accumulation in pyloric tumors, compared with those in the fundus (P < 0.01) with mutations of exon 3; 18.2% and 31.6% in MNU-alone and MNU + H. pylori-treated WT, whereas 21.4% and 62.5% was observed in the Tg, respectively; the latter significantly higher (P < 0.05), suggesting the role of H. pylori in Wnt activation. In conclusion, K19-C2mE mice promoted gastric cancer in both fundic and pyloric regions. Furthermore β-catenin activation may play the important role of pyloric carcinogenesis especially in H. pylori-infected Tg. Induction of various inflammatory cytokines in addition to overexpression of COX-2/mPGES-1 could be risk factors of gastric carcinogenesis and may serve as a better gastric carcinogenesis model. (Cancer Sci 2008; 99: 2356–2364)
There is a large body of evidence that Helicobacter pylori (H. pylori) infection is involved in development of chronic gastritis, peptic ulceration, and gastric cancer.(1,2) Recent reports have revealed that H. pylori infection induces cyclooxygenase-2 (COX-2) expression and microsomal prostaglandin E synthase-1 (mPGES-1), enzymes responsible for synthesizing prostaglandin E2 (PGE2) in gastric mucosa.(3–5) In the stomach, prostaglandins are major molecules for maintaining the gastric mucosa.(6) PGE2 plays distinct roles in tumor growth and metastasis in several cancers.(7)
Oshima et al. recently constructed transgenic mice (K19-C2mE) that simultaneously overexpress COX-2 and mPGES-1 in the gastric mucosa under the influence of the cytokeratin 19 gene promoter.(4) The transgenic (Tg) mice develop inflammation-associated hyperplastic lesions in the proximal glandular stomach, similar to those found in the Helicobacter-infected stomach.(4) Furthermore, K19-Wnt1/C2mE Tg mice, simultaneously expressing Wnt1 as well as COX-2/mPGES-1, develop dysplastic gastric tumors,(8) indicating that COX-2/mPGES-1 and Wnt pathway activation might be involved in mouse gastric neoplasia.
The Wnt/β-catenin signaling pathway plays important roles in cell–cell adhesion and cell cycle regulation and its alternation is implicated in genesis of many cancers. Abnormal nuclear accumulation of β-catenin due to mutation of the β-catenin gene stimulates the expression of β-catenin/Tcf target genes, such as c-myc, c-jun, and cyclin D1.(9,10) Mutations of β-catenin gene exon 3, where serine and threonine residues are physiologically phosphorylated by glycogen synthase kinase (GSK)-3β, prevent degeneration by APC/GSK-3β/Axin complex. With human gastric cancers, nuclear accumulation of β-catenin has been estimated to occur in 12–37% of cases,(11–15) with mutations in exon 3 reported in a few to over 20%.(12,16,17) However, the degree of involvement of Wnt pathway alteration in the development of mouse gastric cancers remains unclear.
In the present study, Tg mice were treated with a stomach carcinogen, N-methyl-N-nitrosourea (MNU), and inoculated with H. pylori to investigate the influence of COX-2/mPGES-1 expression and H. pylori infection on mouse gastric carcinogenesis. Furthermore, we analyzed the frequency of β-catenin activation and gene mutations to assess involvement of the Wnt pathway.
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- Materials and Methods
In the present study, K19-C2mE Tg mice developed gastric tumors not only in the pyloric mucosa but also the fundic region, whereas WT mice developed tumors only in pyloric areas with the carcinogen treatment but irrelevant to H. pylori infection. Furthermore, Tg mice possessed larger tumors even without H. pylori infection. On the other hand, Tg mice showed increased tumor multiplicity compared with the corresponding WT mice only with H. pylori infection. These findings indicated that transgenes in combination with H. pylori infection and subsequent inflammatory response should play important roles in promotion of gastric carcinogenesis in various ways in this mouse model.
Immunohistochemical analysis here demonstrated that tumor cells in the fundic region more markedly express COX-2 than those in the pyloric region. COX-2 expression was predominantly observed in foveolar epithelial cells in K19-C2mE Tg mice. Thus fundic tumors might be derived from foveolar epithelial cells and be more significantly affected by COX-2 expression compared with pyloric tumors. In human gastric neoplasia, proximal gastric tumors are suggested to be specific subtypes of gastric carcinoma based on histological and genetic research.(21,22) K19-C2mE Tg mice may serve as a new animal model for proximal gastric carcinogenesis.
In the present study, H. pylori infection did not promote gastric carcinogenesis in the fundic region, and influence could not be evaluated in the pyloric region in terms of cancer incidence. However, there is abundant evidence from rodent gastric cancer models that H. pylori infection promotes gastric cancers induced by stomach carcinogens, MNU and N-methyl-N′-nitroso-N-nitrosoguanidine (MNNG),(23–27) although not without exceptions.(28) H. pylori infection induced gastritis and caused hyperplasia of the gastric mucosa in the current mouse system, but heterotopic proliferating glands arising with long-term infection of H. pylori and considered as high-grade inflammation in the Mongolian gerbil,(29,30) were not observed here. Such lesions are reversible and are considered as regenerative lesions due to excessive cell proliferation. The observations indicate that the influence of H. pylori infection may depend on the animal species with clear differences between the mouse and Mongolian gerbil. In addition, host immune responses or H. pylori virulence factors may affect gastritis and gastric carcinogenesis.(31,32) By the fact of increased multiplicity of pyloric tumors in H. pylori-infected Tg mice, overexpression of COX-2 and mPGES-1 may serve as a better mouse model for mimicking human cases.
For further analysis of factors promoting gastric carcinogenesis, we investigated the expression of inflammatory cytokines in gastric mucosa. In the present study, expression of those such as TNF-α, iNOS, IL-1β, and CXCL14 were significantly increased in Tg mice with H. pylori infection and MNU treatment. Among them, CXCL14 has been known to selectively attract monocytes, where PGE2 up-regulates their responsiveness.(33) The combination of these factors may contribute to the participation of macrophages in increased tumorigenesis in Tg mice with H. pylori infection plus MNU treatment. We previously demonstrated that the severity of chronic gastritis, characterized by high-level expression of IL-1β, TNF-α, COX-2, and iNOS was concerned with glandular gastric carcinogenesis in H. pylori-infected Mongolian gerbils.(34) Thus, in Tg mice with H. pylori infection a higher level of inflammatory cytokines may be induced that eventually promotes gastric carcinogenesis.
The Tg mice feature increased PGE2 synthesis due to over-expressed COX-2/mPGES-1 in gastric mucosa.(4) PGE2 exerts its biological effects by binding to four isoform receptors, EP1, EP2, EP3, and EP4,(35,36) categorized in the family of seven transmembrane G protein coupled rhodopsin-type receptors. Accumulating evidence indicates that PGE2 promotes tumor growth by stimulating EP receptor signaling with subsequent enhancement of cell proliferation, promotion of angiogenesis, and inhibition of apoptosis.(37) Previous reports based on mouse studies demonstrated that EP1, EP2, and EP4 receptors play important roles in colon carcinogenesis.(38–41) Furthermore, expression of EP1, EP2, and EP4 receptors has been found to be elevated in mouse mammary tumors as well as colon cancers.(42) EP3 receptor activation was furthermore suggested to contribute to breast cancer progression.(43) These observations indicate that the expression pattern of EP receptors in cancer cells might determine the potential of PGE2 to drive tumor progression. Although the expression level of EP receptors in gastric cancers was unclear in the present study, EP receptor signaling stimulated by PGE2 might have influenced gastric carcinogenesis. It should be stressed that PGE2 transactivates epidermal growth factor receptor (EGFR) and triggers PI3K/Akt signaling,(44–46) and Ras/MEK/ERKs,(47) pathways in gastric epithelial and colon cancer cells in vitro as well as rat gastric mucosa in vivo.(46)
To analyze the differences between present mouse gastric tumors developed in fundic and pyloric mucosas, we investigated β-catenin activation, which was suggested to play an important role in gastric carcinogenesis. Immunohistochemical analysis here showed that β-catenin activation characterized by its intracellular accumulation was frequently observed in tumors in the pyloric region, in contrast to those in the fundic region, indicating the unnecessariness of the Wnt pathway. Conversely, pyloric tumorigenesis might be promoted by Wnt activation. In a rat model, a type of adenocarcinoma resembling foveolar epithelium showed nuclear accumulation of cyclin D1 without β-catenin activation,(48) whereas β-catenin was accumulated in cytoplasms or nuclei in the majority of less-differentiated adenocarcinomas.(20) Since fundic tumors were histologically classified as being of the foveolar type and pyloric ones were classified as the less differentiated in this experiment, oncogene activation could depend on cell/tissue differentiation or vice versa.(49) Similarly, mucin expression characterized by AB-PAS staining was observed in fundic tumors as in the hyperplastic fundic tumors in the previous report;(4) pyloric tumors, however, lost most of those mucin production.
Several previous studies of gastric carcinogenesis models in rodents such as the mouse, rat, and Mongolian gerbil have indicated that β-catenin activation plays an important role in gastric carcinogenesis.(20,50–52) In the present study, this was more frequently observed than in previous reports (rat, 18.2%; mouse, 12.5%; and Mongolian gerbil, 2.2%).(20,50,51) Such variation in the frequency of β-catenin activation might be caused by differences in experimental design such as the experimental period, chemical carcinogen applied, or type of experimental animal.
In rat and Mongolian gerbil models, mutations of β-catenin gene in exon 3 have been identified in codons 34, 41, and 45 at GSK-3β phosphorylation sites, and are significantly associated with nuclear β-catenin accumulation.(20,51) In human gastric cancers, gene alternations have been found in the same sites including codons 29, 37, 41, and 47 as well as in adjacent sites at codons 28, 32, 34, 36, 38, 39, and 48.(12,13,19,53) In the present experiment, the mutation spectrum was codons 32, 33, 34, 35, 41, and 45, consistent with the previous reports. Furthermore, β-catenin mutations were particularly frequent in Tg mice with H. pylori infection. Tumor multiplicity was also increased in Tg mice with H. pylori infection, indicating the possibility that H. pylori infection in addition to COX-2/mPGES-1 expression might contribute to progression of gastric adenocarcinomas though β-catenin gene alternations at least in part. In humans, stomach cancers with intestinal differentiation markers feature more β-catenin mutations compared to those with gastric markers.(19) Furthermore, intestinal markers may be induced only in stomach tumors in H. pylori-infected gerbils.(54) Thus, alteration of β-catenin could be related to H. pylori infection, although further work is needed to reveal interactions between these two factors.
β-Catenin accumulation without β-catenin gene mutations was detected in some tumors in the present study, indicating involvement of other alterations of Wnt pathway regulatory genes. Indeed, there have been a large number of previous reports suggesting that APC gene mutation,(17,55–61) APC loss of heterozygosity,(59) over-expression of various Wnt ligands, and altered frizzled receptors(62–64) may be involved in β-catenin activation. Furthermore, degradation of E-cadherin and microsatellite instability might also be responsible for β-catenin accumulation. In one rodent model, the APCMin/+ mouse which harbors a nonsense mutation at codon 850 of the APC gene, gastric tumors frequently develop with APC loss of heterozygosity.(50)
In conclusion, the present study indicated that over-expression of COX-2/mPGES-1 promotes gastric carcinogenesis, especially in the fundic region, further showing the K19-C2mE Tg mouse to be a new animal model for proximal gastric carcinogenesis. Furthermore Tg mice developed multiple tumors in the pyloric region with H. pylori infection partly with β-catenin gene mutation and activation. This indicates the risk of multiple or metachronous gastric cancers also in human cases with H. pylori infection and supports the idea to eradicate the bacterium or to suppress inflammatory response for the prevention of secondary malignancies.(65)