Keisuke Oguma

Activated macrophages promote Wnt signalling through tumour necrosis factor-α in gastric tumour cells

The activation of Wnt/β‐catenin signalling has an important function in gastrointestinal tumorigenesis. It has been suggested that the promotion of Wnt/β‐catenin activity beyond the threshold is important for carcinogenesis. We herein investigated the role of macrophages in the promotion of Wnt/β‐catenin activity in gastric tumorigenesis. We found β‐catenin nuclear accumulation in macrophage‐infiltrated dysplastic mucosa of the K19‐Wnt1 mouse stomach. Moreover, macrophage depletion in ApcΔ716 mice resulted in the suppression of intestinal tumorigenesis. These results suggested the role of macrophages in the activation of Wnt/β‐catenin signalling, which thus leads to tumour development. Importantly, the conditioned medium of activated macrophages promoted Wnt/β‐catenin signalling in gastric cancer cells, which was suppressed by the inhibition of tumour necrosis factor (TNF)‐α. Furthermore, treatment with TNF‐α induced glycogen synthase kinase 3β (GSK3β) phosphorylation, which resulted in the stabilization of β‐catenin. We also found that Helicobacter infection in the K19‐Wnt1 mouse stomach caused mucosal macrophage infiltration and nuclear β‐catenin accumulation. These results suggest that macrophage‐derived TNF‐α promotes Wnt/β‐catenin signalling through inhibition of GSK3β, which may contribute to tumour development in the gastric mucosa.

Prostaglandin E2 Signaling and Bacterial Infection Recruit Tumor-Promoting Macrophages to Mouse Gastric Tumors

BACKGROUND & AIMSnHelicobacter pylori infection induces an inflammatory response, which can contribute to gastric tumorigenesis. Induction of cyclooxygenase-2 (COX-2) results in production of prostaglandin E(2) (PGE(2)), which mediates inflammation. We investigated the roles of bacterial infection and PGE(2) signaling in gastric tumorigenesis in mice.nnnMETHODSnWe generated a germfree (GF) colony of K19-Wnt1/C2mE mice (Gan mice); these mice develop gastric cancer. We examined tumor phenotypes, expression of cytokines and chemokines, and recruitment of macrophages. We also investigated PGE(2) signaling through the PGE(2) receptor subtype 4 (EP4) in Gan mice given specific inhibitors.nnnRESULTSnGan mice raised in a specific pathogen-free facility developed large gastric tumors, whereas gastric tumorigenesis was significantly suppressed in GF-Gan mice; reconstitution of commensal flora or infection with Helicobacter felis induced gastric tumor development in these mice. Macrophage infiltration was significantly suppressed in the stomachs of GF-Gan mice. Gan mice given an EP4 inhibitor had decreased expression of cytokines and chemokines. PGE(2) signaling and bacterial infection or stimulation with lipopolysaccharide induced expression of the chemokine C-C motif ligand 2 (CCL2) (which attracts macrophage) in tumor stromal cells or cultured macrophages, respectively. CCL2 inhibition suppressed macrophage infiltration in tumors, and depletion of macrophages from the tumors of Gan mice led to signs of tumor regression. Wnt signaling was suppressed in the tumors of GF-Gan and Gan mice given injections of tumor necrosis factor-α neutralizing antibody.nnnCONCLUSIONSnBacterial infection and PGE(2) signaling are required for gastric tumorigenesis in mice; they cooperate to up-regulate CCL2, which recruits macrophage to gastric tumors. Macrophage-derived tumor necrosis factor-α promotes Wnt signaling in epithelial cells, which contributes to gastric tumorigenesis.

Inflammation-induced repression of tumor suppressor miR-7 in gastric tumor cells

Inflammation has an important role in cancer development through various mechanisms. It has been shown that dysregulation of microRNAs (miRNAs) that function as oncogenes or tumor suppressors contributes to tumorigenesis. However, the relationship between inflammation and cancer-related miRNA expression in tumorigenesis has not yet been fully understood. Using K19-C2mE and Gan mouse models that develop gastritis and gastritis-associated tumors, respectively, we found that 21 miRNAs were upregulated, and that 29 miRNAs were downregulated in gastric tumors in an inflammation-dependent manner. Among these miRNAs, the expression of miR-7, a possible tumor suppressor, significantly decreased in both gastritis and gastric tumors. Moreover, the expression of miR-7 in human gastric cancer was inversely correlated with the levels of interleukin-1β and tumor necrosis factor-α, suggesting that miR-7 downregulation is related to the severity of inflammatory responses. In the normal mouse stomach, miR-7 expression was at a basal level in undifferentiated gastric epithelial cells, and was induced during differentiation. Moreover, transfection of a miR-7 precursor into gastric cancer cells suppressed cell proliferation and soft agar colony formation. These results suggest that suppression of miR-7 expression is important for maintaining the undifferentiated status of gastric epithelial cells, and thus contributes to gastric tumorigenesis. Although epigenetic changes were not found in the CpG islands around miR-7-1 of gastritis and gastric tumor cells, we found that activated macrophage-derived small molecule(s) (<3u2009kDa) are responsible for miR-7 repression in gastric cancer cells. Furthermore, the miR-7 expression level significantly decreased in the inflamed gastric mucosa of Helicobacter-infected mice, whereas it increased in the stomach of germfree K19-C2mE and Gan mice wherein inflammatory responses were suppressed. Taken together, these results indicate that downregulation of tumor suppressor miR-7 is a novel mechanism by which the inflammatory response promotes gastric tumorigenesis.

Induction and Down-regulation of Sox17 and Its Possible Roles During the Course of Gastrointestinal Tumorigenesis

BACKGROUND & AIMSnThe activation of Wnt/beta-catenin signaling causes the development of gastric and colon cancers. Sox17 represses Wnt/beta-catenin signaling and is down-regulated in colon cancer. This study was designed to elucidate the role of Sox17 during the course of gastrointestinal tumorigenesis.nnnMETHODSnSox17 expression was examined in gastrointestinal tumors of mouse models and humans. The roles of Sox17 in gastric tumorigenesis were examined by cell culture experiments and by construction of Sox17 transgenic mice.nnnRESULTSnSox17 was induced in K19-Wnt1/C2mE mouse gastric tumors and K19-Wnt1 preneoplastic lesions, where Wnt/beta-catenin signaling was activated. Consistently, Wnt activation induced Sox17 expression in gastric cancer cells. In contrast, Sox17 was rarely detected by immunohistochemistry in gastric and colon cancers, whereas strong nuclear staining of Sox17 was found in >70% of benign gastric and intestinal tumors. Treatment with a demethylating agent induced Sox17 expression in gastric cancer cells, thus indicating the down-regulation of Sox17 by methylation. Moreover, transfection of Sox17 in gastric cancer cells suppressed both the Wnt activity and colony formation efficiency. Finally, transgenic expression of Sox17 suppressed dysplastic tumor development in K19-Wnt1/C2mE mouse stomach.nnnCONCLUSIONSnSox17 plays a tumor suppressor role through suppression of Wnt signaling. However, Sox17 is induced by Wnt activation in the early stage of gastrointestinal tumorigenesis, and Sox17 is down-regulated by methylation during malignant progression. It is therefore conceivable that Sox17 protects benign tumors from malignant progression at an early stage of tumorigenesis, and down-regulation of Sox17 contributes to malignant progression through promotion of Wnt activity.

Prostaglandin E2, Wnt, and BMP in gastric tumor mouse models

The development of gastric cancer is closely associated with Helicobacter pylori (H. pylori) infection. The expression of cylooxigenase‐2 (COX‐2), a rate‐limiting enzyme for prostaglandin biosynthesis, is induced in H. pylori‐associated chronic gastritis, which thus results in the induction of proinflammatory prostaglandin, PGE2. The COX‐2/PGE2 pathway plays a key role in gastric tumorigenesis. On the other hand, several oncogenic pathways have been shown to trigger gastric tumorigenesis. The activation of Wnt/β‐catenin signaling is found in 30–50% of gastric cancers, thus suggesting that Wnt signaling plays a causal role in gastric cancer development. Mutations in the bone morphogenetic protein (BMP) signaling pathway are responsible for the subset of juvenile polyposis syndrome (JPS) that develops hamartomas in the gastrointestinal tract. BMP suppression appears to contribute to gastric cancer development because gastric cancer risk is increased in JPS. Wnt signaling is important for the maintenance of gastrointestinal stem cells, while BMP promotes epithelial cell differentiation. Accordingly, it is possible that both Wnt activation and BMP suppression can cause gastric tumorigenesis through enhancement of the undifferentiated status of epithelial cells. Recent mouse model studies have indicated that induction of the PGE2 pathway is required for the development of both gastric adenocarcinoma and hamartoma in the Wnt‐activated and BMP‐suppressed gastric mucosa, respectively. This article reviews the involvement of the PGE2, Wnt, and BMP pathways in the development of gastric cancer, and gastric phenotypes that are found in transgenic mouse models of PGE2 induction, Wnt activation, BMP suppression, or a combination of these pathways. (Cancer Sci 2009; 100: 1779–1785)

Activation of epidermal growth factor receptor signaling by the prostaglandin E2 receptor EP4 pathway during gastric tumorigenesis

Cyclooxygenase‐2 (COX‐2) plays an important role in tumorigenesis through prostaglandin E2 (PGE2) biosynthesis. It has been shown by in vitro studies that PGE2 signaling transactivates epidermal growth factor receptor (EGFR) through an intracellular mechanism. However, the mechanisms underlying PGE2‐induced EGFR activation in in vivo tumors are still not fully understood. We previously constructed transgenic mice that develop gastric tumors caused by oncogenic activation and PGE2 pathway induction. Importantly, expression of EGFR ligands, epiregulin, amphiregulin, heparin‐binding EGF‐like growth factor, and betacellulin, as well as a disintegrin and metalloproteinases (ADAMs), ADAM8, ADAM9, ADAM10, and ADAM17 were significantly increased in the mouse gastric tumors in a PGE2 pathway‐dependent manner. These ADAMs can activate EGFR by ectodomain shedding of EGFR ligands. Notably, the extensive induction of EGFR ligands and ADAMs was suppressed by inhibition of the PGE2 receptor EP4. Moreover, EP4 signaling induced expression of amphiregulin and epiregulin in activated macrophages, whereas EP4 pathway was required for basal expression of epiregulin in gastric epithelial cells. In contrast, ADAMs were not induced directly by PGE2 in these cells, suggesting indirect mechanism possibly through PGE2‐associated inflammatory responses. These results suggest that PGE2 signaling through EP4 activates EGFR in gastric tumors through global induction of EGFR ligands and ADAMs in several cell types either by direct or indirect mechanism. Importantly, gastric tumorigenesis of the transgenic mice was significantly suppressed by combination treatment with EGFR and COX‐2 inhibitors. Therefore, it is possible that inhibition of both COX‐2/PGE2 and EGFR pathways represents an effective strategy for preventing gastric cancer. (Cancer Sci 2011; 102: 713–719)

Inflammation, tumor necrosis factor and Wnt promotion in gastric cancer development

Infection-associated chronic inflammation plays an important role in tumorigenesis, and macrophages are a key player in both inflammation and tumorigenesis. Tumor-associated macrophages accelerate tumorigenesis through the enhancement of angiogenesis, remodeling and the suppression of antitumor immunity. Helicobacter pylori infection induces inflammatory responses, which are closely associated with gastric cancer development. Recent studies using mouse models indicate that activated macrophages in the infected and inflamed gastric mucosa express TNF-alpha, which stimulates the surrounding epithelial cells to promote Wnt signaling activity. Such a promotion of Wnt signaling activity beyond the threshold for tumorigenesis may, therefore contribute to gastric cancer development. Accordingly, it is possible that the TNF-alpha-induced promotion of Wnt signaling is a novel protumorigenic mechanism of inflammation in gastric carcinogenesis.