K15 promoter-driven enforced expression of NKIRAS exhibits tumor suppressive activity against the development of DMBA/TPA-induced skin tumors.

NKIRAS1 and NKIRAS2 (also called as κB-Ras) were identified as members of the atypical RAS family that suppress the transcription factor NF-κB. However, their function in carcinogenesis is still controversial. To clarify how NKIRAS acts on cellular transformation, we generated transgenic mice in which NKIRAS2 was forcibly expressed using a cytokeratin 15 (K15) promoter, which is mainly activated in follicle bulge cells. The ectopic expression of NKIRAS2 was mainly detected in follicle bulges of transgenic mice with NKIRAS2 but not in wild type mice. K15 promoter-driven expression of NKIRAS2 failed to affect the development of epidermis, which was evaluated using the expression of K10, K14, K15 and filaggrin. However, K15 promoter-driven expression of NKIRAS2 effectively suppressed the development of skin tumors induced by treatment with 7,12-dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol 13-acetate (TPA). This observation suggested that NKIRAS seemed to function as a tumor suppressor in follicle bulges. However, in the case of oncogenic HRAS-driven cellular transformation of murine fibroblasts, knockdown of NKIRAS2 expression drastically suppressed HRAS-mutant-provoked cellular transformation, suggesting that NKIRAS2 was required for the cellular transformation of murine fibroblasts. Furthermore, moderate enforced expression of NKIRAS2 augmented oncogenic HRAS-provoked cellular transformation, whereas an excess NKIRAS2 expression converted its functional role into a tumor suppressive phenotype, suggesting that NKIRAS seemed to exhibit a biphasic bell-shaped enhancing effect on HRAS-mutant-provoked oncogenic activity. Taken together, the functional role of NKIRAS in carcinogenesis is most likely determined by not only cellular context but also its expression level.

Cyclooxygenase 2 in gastric carcinoma is expressed in doublecortin- and CaM kinase-like-1-positive tuft cells.

BACKGROUND/AIMS: Doublecortin and CaM kinase-like-1 (DCAMKL1) is a marker of stem cells expressed predominantly in the crypt base in the intestine. However, DCAMKL1-positive cells have been shown to be differentiated tuft cells rather than quiescent progenitors. Tuft cells are the only epithelial cells that express cyclooxygenase 2 (COX-2) in the normal intestinal epithelium. We previously generated Cdx2-transgenic mice as model mice for intestinal metaplasia and gastric carcinoma. In the current study, we investigated the association between COX-2 and DCAMKL1 in gastric carcinoma. METHODS: We examined the association between COX-2 and DCAMKL1 expression in gastric carcinomas in clinical samples (early gastric well-differentiated adenocarcinoma) and Cdx2-transgenic mice; and the DCAMKL1-transgenic mouse stomach using immunohistochemistry and quantitative real-time polymerase chain reaction. RESULTS: The COX-2-expressing cells were scattered, not diffusely expressed, in gastric carcinomas from humans and Cdx2-transgenic mice. DCAMKL1-positive cells were also scattered in the gastric carcinomas, indicating that tuft cells could still be present in gastric carcinoma. COX-2 was expressed in DCAMKL1-positive tuft cells in Cdx2- and DCAMKL1-transgenic mouse stomachs, whereas the Sox9 transcription factor was ubiquitously expressed in gastric carcinomas, including COX-2-positive cells. CONCLUSIONS: COX-2 is expressed in DCAMKL1-expressing quiescent tuft cells in gastric carcinoma.

SOX9 Is Highly Expressed in Nonampullary Duodenal Adenoma and Adenocarcinoma in Humans.

BACKGROUND/AIMS: SOX9 is a marker for stem cells in the intestine, and overexpression of SOX9 is found in gastric and colon cancer; however, the expression of SOX9 in nonampullary duodenal adenoma and adenocarcinoma has not yet been evaluated. This study aimed to investigate SOX9 expression in nonampullary duodenal adenoma and adenocarcinoma by immunohistochemistry. METHODS: We evaluated SOX9 expression in 43 clinical samples (nonampullary duodenal adenoma in 22 lesions and nonampullary duodenal adenocarcinoma in 21 lesions) resected under endoscopic mucosal resection or endoscopic submucosal dissection. RESULTS: SOX9 was expressed in part of the base of the normal duodenal mucosa surrounding adenomas and adenocarcinomas. In contrast, SOX9-positive cells were found in more than half of the crypts from the bottom part of the crypt in all of the 43 samples. Moreover, in 15 adenoma samples (68.2%) and 19 carcinoma samples (90.5%), SOX9 was expressed in more than three-quarters of the crypts from the bottom part of the crypt. CONCLUSIONS: SOX9 is overexpressed in nonampullary duodenal adenoma and adenocarcinoma in humans.

Cell lineage dynamics in the process leading to intestinal metaplasia.

BACKGROUND: Gene expression in the early stage of the transition to intestinal metaplasia in human gastric mucosa has not been determined. In this study, we investigated the temporal relationship between cell lineage changes and intestine-specific gene expression in the process leading to intestinal metaplasia, using Cdx2-transgenic mice. METHODS: Cellular phenotypes were analyzed by immunohistochemistry and were compared with the gene expression profiles of cell lineage markers by real-time polymerase chain reaction. RESULTS: Up to postnatal day (PD) 20, the gastric mucosae of Cdx2-transgenic mice were histologically similar to those of their normal littermates. However, at approximately PD 20, we observed the sporadic appearance of glands in which all the epithelial cells expressed Cdx2 (Cdx2-diffuse positive glands). In the Cdx2-diffuse positive glands, parietal cells had disappeared, the proliferating zone had moved from the isthmus to the base, and absorptive cells and goblet cells were recognized. In contrast, the surrounding mucosa retained the phenotype of the gastric gland in which only some of the epithelial cells expressed Cdx2. During PDs 30 and 40, the entire fundic mucosa changed to transdifferentiated mucosa that was a composite of intestinal metaplasia and spasmolytic polypeptide-expressing metaplasia. An increase in the expression of intestine-specific genes, with a reciprocal decrease in gastric-specific gene expression, began much earlier than the emergence of Cdx2-diffuse positive glands. CONCLUSIONS: A dramatic increase in intestine-specific gene expression precedes the morphological appearance of intestinal metaplasia and spasmolytic polypeptide-expressing metaplasia.

Sox2 expression is maintained while gastric phenotype is completely lost in Cdx2-induced intestinal metaplastic mucosa.

Sox2 is closely related to the gastric phenotype. Sox2 plays a pivotal role in gastric epithelial differentiation in the adult. Sox2 expression is reduced in Helicobacter pylori-associated intestinal metaplastic change of the gastric epithelium. The gastric mucosa is replaced by intestinal metaplastic mucosa in the stomach of caudal type homeobox 2 (Cdx2)-transgenic mice. The aim of this study was to use Cdx2-transgenic mice to investigate: (i) Sox2 expression in the intestinal metaplastic mucosa of the Cdx2-transgenic mouse stomach; and (ii) the relationship between Sox2 and Cdx2. Quantitative real-time PCR was performed to determine Sox2, Cdx2, Muc5Ac, and alkaline phosphatase mRNA expression levels and single- or double-label immunohistochemistry was used to evaluate the localization of Sox2, Cdx2, gastric mucin and alkaline phosphatase activity. We determined that Sox2 mRNA in the intestinal metaplastic mucosa of the Cdx2-transgenic mouse stomach was expressed 3.5-fold compared to the normal mouse stomach. Immunohistochemical analysis showed that the same cells in the intestinal metaplastic mucosa expressed both Cdx2 and Sox2. Gastric mucin was not expressed while alkaline phosphatase activity was recognized in the intestinal metaplastic mucosa in spite of the Sox2 expression. Cdx2 increased the transcriptional activity of the Sox2 gene, and Sox2 increased the transcriptional activity of the Muc5Ac gene, which was reduced by cotransfecion of Cdx2 together with Sox2 in the human gastric carcinoma cell line AGS. In conclusion, Sox2 expression is maintained while gastric phenotype is completely lost in the intestinal metaplastic mucosa of Cdx2-transgenic mouse stomach.

Monocyte chemoattractant protein-1 is generated via TGF-beta by myofibroblasts in gastric intestinal metaplasia and carcinoma without H. pylori infection.

Helicobacter pylori (H. pylori) stimulates secretion of monocyte chemoattractant protein 1 (MCP-1) from gastric mucosa. Monocyte chemoattractant protein-1 (MCP-1) expression and macrophage infiltration are recognized in human gastric carcinoma. We have previously generated Cdx2-transgenic mice as model mice for intestinal metaplasia. Both chronic H. pylori-associated gastritis and Cdx2-transgenic mouse stomach develop intestinal metaplasia and finally gastric carcinoma. In this study we have directed our attention to MCP-1 expression in the intestinal metaplastic mucosa and the gastric carcinoma of Cdx2-transgenic mouse stomach. Quantitative real-time PCR was performed to determine MCP-1 and transforming growth factor-beta1 (TGF-beta1) mRNA expression levels and single- or double-label immunohistochemistry was used to evaluate the localization of MCP-1, TGF-beta type I receptor, and alpha-smooth muscle actin (alphaSMA). We determined that MCP-1 mRNA dramatically increased in the intestinal metaplastic mucosa and the gastric carcinoma of Cdx2-transgenic mouse stomach, compared with normal mouse stomach. Both MCP-1 and TGF-beta type I receptor were co-expressed in the alphaSMA-positive myofibroblasts of intestinal metaplastic mucosa and gastric carcinoma. Exogenous application of TGF-beta1 increased MCP-1 mRNA expression levels in the intestinal metaplastic tissue. Furthermore, TGF-beta1 was overexpressed and macrophage was strongly infiltrated in the gastric carcinoma. In conclusion, MCP-1 expression, which was stimulated by TGF-beta1, was recognized in the TGF-beta type I receptor-expressing myofibroblasts of the intestinal metaplastic mucosa and the gastric carcinoma of Cdx2-transgenic mouse stomach. The present results suggest that intestinal metaplasia and gastric carcinoma themselves induce MCP-1 expression independently of H. pylori infection.

Direct repression of Sonic Hedgehog expression in the stomach by Cdx2 leads to intestinal transformation.

Shh (Sonic Hedgehog) is a morphogen involved in gastric fundic gland differentiation in the adult. Shh expression is reduced in Helicobacter pylori-associated intestinal metaplastic change of the gastric epithelium and mice that lack Shh show intestinal transformation of the gastric mucosa. Similarly, in the stomach of Cdx2 (caudal-type homeobox 2)-transgenic mice, the gastric mucosa is replaced by intestinal metaplastic mucosa. The aim of the present study was to use Cdx2-transgenic mice to investigate: (i) Shh expression in the intestinal metaplastic mucosa of the Cdx2-transgenic mouse stomach; and (ii) the relationship between Shh and Cdx2. We determined that Shh mRNA levels were dramatically reduced in the intestinal metaplastic mucosa of the Cdx2-transgenic mouse stomach compared with the normal (wild-type) mouse stomach. This was not due to hypermethylation of the Shh promoter, but instead we showed that Cdx2 directly bound to the TATA box region of the Shh promoter. Cdx2 also down-regulated transcription of the Shh gene in the human gastric carcinoma cell lines AGS, MKN45 and MKN74. In conclusion, Cdx2 reduced Shh expression by binding to the unmethylated Shh promoter in the intestinal metaplastic mucosa of Cdx2-transgenic mouse stomach.

Transgenic Cdx2 induces endogenous Cdx1 in intestinal metaplasia of Cdx2-transgenic mouse stomach.

Cdx1 and Cdx2, which are transcription factors regulating normal intestinal development, have been studied as potential key molecules in the pathogenesis of the precancerous intestinal metaplasia of the human stomach. However, the regulation of Cdx1 expression in the intestinal metaplasia is poorly understood. Cdx2-expressing gastric mucosa of Cdx2-transgenic mouse stomach was replaced by intestinal metaplastic mucosa. The aim of this study was to investigate the following: (a) Cdx1 expression in the intestinal metaplastic mucosa of the Cdx2-transgenic mouse stomach; and (b) the relationship between Cdx1 and Cdx2. A mouse model of intestinal metaplasia, the Cdx2-transgenic mouse, was used to investigate Cdx1 gene expression by RT-PCR. DNA methylation profile analysis was performed by bisulfite sequencing, and the interaction of Cdx2 with the Cdx1 promoter was examined by chromatin immunoprecipitation assay, electrophoretic mobility shift assay, and luciferase reporter assays. Cdx2 mRNA was expressed in the Cdx2-transgenic mouse stomach. However, endogenous Cdx2 mRNA was not expressed in the intestinal metaplasia of the Cdx2-transgenic mouse stomach. On the other hand, endogenous Cdx1 mRNA and protein were expressed in the intestinal metaplasia of the Cdx2-transgenic mouse stomach. The Cdx1 promoter was unmethylated in the intestinal metaplasia of the Cdx2-transgenic mouse stomach. Chromatin immunoprecipitation assay and electrophoretic mobility shift assay showed that Cdx2 was bound to the Cdx1 promoter region in the intestinal metaplasia and the normal intestine. Cdx2 upregulated and siRNA-Cdx2 downregulated the transcriptional activity of the Cdx1 gene in the human gastric carcinoma cell lines AGS, MKN45, and MKN74. In conclusion, transgenic Cdx2 induced endogenous Cdx1 through the binding of Cdx2 to the unmethylated Cdx1 promoter region in the intestinal metaplasia of the Cdx2-transgenic mouse stomach.