Research Article Development of Gastric Tumors in ApcMin/+ Mice by the Activation of the B-Catenin/Tcf Signaling Pathway Hiroyuki Tomita,1,2 Yasuhiro Yamada,1 Takeru Oyama,1 Kazuya Hata,1 Yoshinobu Hirose,1 Akira Hara,1 Takahiro Kunisada,3 Yasuyuki Sugiyama,2 Yosuke Adachi,2 Heinz Linhart,4 and Hideki Mori1 Departments of 1Tumor Pathology, 2Oncologic Surgery, and 3Tissue and Organ Development, Gifu University Graduate School of Medicine, Gifu, Japan and 4Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, Massachusetts Abstract 5q21, is responsible for such phenotypes in FAP (1, 2). Therefore, inactivating APC is considered to initiate the multistep progression Although several lines of evidence suggest the involvement of of colorectal cancer (3). The knowledge about how APC acts as a the Wnt pathway in the development of gastric cancers, the tumor suppressor gene was considerably advanced by the demon- functional significance of the pathway in gastric carcinogen- stration that APC contributes to the degradation of cytosolic esis is still poorly defined. To examine the role of the Apc/B- h-catenin, thereby linking APC to the Wnt/h-catenin pathway as a catenin signaling pathway in the development of gastric Min/+ negative regulator (4–7). The critical event in the Wnt pathway cancers, we investigated the gastric mucosa of the Apc activation is the elevation of the cytoplasmic pool of h-catenin and mouse, which is a murine model for familial adenomatous its resultant transport to the nucleus (5, 8). Consequently, the polyposis, carrying a germ-line mutation at codon 850 of Apc. inactivation of APC leads to a constitutive accumulation of h- We found that aged ApcMin/+ mice spontaneously develop catenin, which triggers an aberrant transcriptional activation of the multiple tumors in the stomach, which are accompanied by h-catenin/Tcf target genes, such as Myc and Cyclin D1 (9, 10). loss of heterozygosity of Apc. Such tumors consisted of Gastric cancer is the fourth most common cancer and the adenomatous glands with strong nuclear accumulation of B- second leading cause of cancer-related death in the world (11). catenin. Even a single adenomatous gland already showed Although several lines of evidence suggest the involvement of the nuclear accumulation of B-catenin, suggesting that Apc/B- Wnt pathway in the development of sporadic gastric cancers, the catenin pathway is an initiating event in gastric tumorigenesis functional significance of the Apc/h-catenin pathway in gastric in ApcMin/+ mice. Myc and cyclin D1 expressions, which are carcinogenesis is still poorly defined in comparison with that in transcriptional targets of B-catenin/Tcf, increased in the colon carcinogenesis. The nuclear accumulation of h-catenin, a adenomatous lesions. Furthermore, B-catenin/Tcf reporter hallmark of Wnt activation, has been found in 17% to 54% of transgenic mice with ApcMin allele showed higher levels of the gastric adenocarcinomas (12–14), and mutations in APC and transcriptional activity of B-catenin/Tcf in the gastric tumors. b-catenin are found in a subset of gastric adenocarcinomas. We also treated ApcMin/+ and wild-type mice with N-methyl-N- However, the incidence of such mutations varies according to nitrosourea (MNU), an alkylating agent that induces adeno- evidence from different studies (0–60% for APC mutations and mas and adenocarcinomas in the stomach. Consequently, 0–27% for b-catenin mutations; refs. 13–18). In addition, FAP MNU-treated ApcMin/+ mice significantly enhanced the tumor patients have been reported to have an increased risk for gastric development in comparison with ApcMin/+ mice or MNU- cancer in Japan but not in Western countries (19–22). Taken treated wild-type mice. Several gastric tumors in MNU-treated together, the functional significance of the Wnt pathway in gastric ApcMin/+ mice showed invasion into the submucosal layer. B carcinogenesis therefore remains controversial. These results indicate that the Apc/ -catenin pathway may ApcMin/+ mouse is a mouse model for FAP and harbors a play an important role in at least subset of gastric carcinomas. dominant mutation at the Apc, the mouse homologue of the ApcMin/+ In addition, mice combined with MNU could be a human APC gene, resulting in the truncation of the gene product at useful short-term model to investigate multistage carcinogen- amino acid 850 (23). Originally, this lineage was established from an esis in the stomach. [Cancer Res 2007;67(9):4079–87] ethylnitrosourea-treated C57BL/6Jmale mouse, and its phenotype is an autosomal dominant trait. Although homozygous ApcMin/Min Introduction mice die as embryos, ApcMin/+ mice develop multiple neoplasias in Familial adenomatous polyposis (FAP) is regarded as an their intestinal tracts within several weeks after birth (23). It is also autosomal dominant disease in which many adenomatous polyps known that most of the intestinal tumors in the mice that are develop in the colon, thereafter progressing to colorectal carcinoma. heterozygous for a mutant allele of Apc have lost the Apc function Genetic linkage studies have shown that the inactivation of the due to a loss of heterozygosity (LOH; refs. 24, 25). Although mice adenomatous polyposis coli (APC) gene, located on chromosome generated by gene targeting with a mutation at codon 1638 of the Apc have been reported to occasionally develop gastric tumors after 30 weeks of age (26, 27), the relationship between gastric tumorigenesis and the activation of the Apc/h-catenin pathway has Note: Supplementary data for this article are available at Cancer Research Online Min/+ (http://cancerres.aacrjournals.org/). not yet been well characterized. Apc mice combined with À/À Requests for reprints: Yasuhiro Yamada, Department of Tumor Pathology, Gifu Foxl1 mice (28) or Cdx2 transgenic mice (29) have been shown University Graduate School of Medicine, 1-1 Yanagido, Gifu 501-1194, Japan. Phone: 81- to develop gastric tumors. However, previous studies have 58-230-6225; Fax: 81-58-230-6226; E-mail: [email protected]. I2007 American Association for Cancer Research. indicated no evidence of a spontaneous development of gastric Min/+ doi:10.1158/0008-5472.CAN-06-4025 tumors in Apc mice. www.aacrjournals.org 4079 Cancer Res 2007; 67: (9). May 1, 2007 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2007 American Association for Cancer Research. Cancer Research In the present study, we examined the gastric lesions of ApcMin/+ University Hospital, was examined for h-catenin expression. The experi- mice and found, for the first time, that aged ApcMin/+ mice develop ments were carried out according to the protocol approved by the Ethics multiple adenomas in the stomach, which are accompanied by Apc Committee of Gifu University. LOH and the activation of the h-catenin/Tcf signaling pathway. We Immunohistochemistry. The avidin-biotin peroxidase complex (ABC) A also treated ApcMin/+ mice with N-methyl-N-nitrosourea (MNU), an technique was used for immunohistochemical studies. Sections (5 m thick) were cut, deparaffinized, rehydrated in PBS, placed in 10 mmol/L citrate alkylating agent that induces adenomas and adenocarcinomas in the Min/+ buffer (pH 6.0), and heated in a 750-W microwave four times for 6 min. The stomach, to elucidate the mode of gastric carcinogenesis in Apc endogenous peroxidase activity was blocked by incubation for 30 min in mice. Consequently, MNU-treated ApcMin/+ mice were found to 0.3% H2O2. After washing thrice with PBS, the sections were preincubated strongly promote tumor development, and several gastric tumors with normal blocking serum for 20 min at room temperature and then showed invasive adenocarcinomas. These results indicate that the incubated with h-catenin (1:1,000; BD Biosciences PharMingen), c-myc Apc/h-catenin pathway plays a significant role in a subset of gastric (1:200; Santa Cruz Biotechnology), cyclin D1 (1:200; Santa Cruz Biotechnol- carcinogenesis, thus suggesting that this model could be a short- ogy), Ki-67 (1:200; DAKO Corp.), AE1/AE3 (1:100; DAKO), and green term model to investigate multistage carcinogenesis in the stomach. fluorescent protein (GFP) antibody (1:1,500; Molecular Probes) overnight at 4jC. Subsequently, the sections were incubated with biotinylated secondary antibodies (Vectastain ABC kit, Vector Laboratories) for 30 min followed by Materials and Methods incubation with avidin-coupled peroxidase (Vector Laboratories) for 30 min. Animals. This study was approved by the Institutional Ethics Review The sections were developed with 3,3¶-diaminobenzidine (DAB) using Committee for Animal Experiments at Gifu University. ApcMin/+ mice in the DAKO Liquid DAB Substrate-Chromogen System (DAKO) and then C57BL/6Jbackground were obtained from The Jackson Laboratory and counterstained with hematoxylin. For immunofluorescence, FITC- or maintained by breeding ApcMin/+ males to C57BL/6Jfemales. All mice were TRITC-conjugated secondary antibodies (Jackson ImmunoResearch) were maintained under specific pathogen-free conditions with isolated ventila- used and then counterstained with 4¶,6-diamidino-2-phenylindole. No tion cages in an air-conditioned room with a 12-h light/12-h dark cycle. specific staining was observed in the negative control slides prepared They were bred and maintained on a basal diet, CE-2 (CLEA Japan, Inc.), without primary antibody. until the termination of the study. Heterozygous progeny were identified by LOH analysis and laser capture microdissection. In the current study, a PCR analysis of tail DNA using allele-specific primers (24). To examine the DNA for the analysis of the Apc allelic loss was extracted from the cells gastric lesions, ApcMin/+ and age-matched wild-type littermates, 15 to 35 isolated with laser microdissected tissue sections as described previously weeks of age, were used in the present study. It is known that the life span (38, 39). For laser capturing, the slides were put into xylene for 30 min to of ApcMin/+ mice on C57BL/6Jbackground is f20 weeks (23), whereas the dissolve the paraffin that otherwise interfered with laser capture mice in our facility have a better survival as was also observed in several microdissection (LCM).