Transgenic expression of Helicobacter pylori CagA induces gastrointestinal and hematopoietic neoplasms in mouse

Synthesis and Analysis of the Humanized cagA Gene.

Due to the structural polymorphism in the EPIYA-repeat region, individual CagA species show differential degrees of SHP-2-binding activity, which influences the magnitude of CagA virulence (8). We reported that a CagA species possessing two repeated EPIYA-D segments (ABDD CagA) exhibits the greatest ability to bind and activate SHP-2 (26). Accordingly, we chose a cagA gene encoding ABDD CagA as the transgene in mice. The H. pylori-derived cagA gene is characterized by A/T-rich sequences, which could induce rapid gene silencing after integration into mammalian genomes [supporting information (SI) Fig. 5]. Also, cagA contains multiple ATTTA sequences that act as mRNA degradation motifs in mammalian cells (27). To avoid these potential problems, we chemically synthesized a DNA fragment encoding the entire ORF (3,729 base pairs) of ABDD CagA while converting bacterial cagA codons to those more commonly used in human genes, which significantly reduced the A/T contents and eliminated the ATTTA sequence (SI Fig. 5B). The synthesized DNA was then 3′-tagged with a sequence encoding the hemagglutinin (HA) to generate the humanized cagA gene (cagA^Hs). Expectedly, transfection of a cagA^Hs expression vector gave rise to higher levels of CagA expression and greater magnitude for induction of cells with the hummingbird phenotype than did a bacterial cagA vector in AGS human gastric epithelial cells (SI Fig. 6).

Generation of Transgenic Mice Bearing Humanized cagA Gene.

To express CagA systemically in mice, cagA^Hs was connected downstream of the chicken β-actin and globin fusion promoter (CAG promoter) (28). The cagA^Hs DNA was also connected downstream of the β subunit gene promoter of mouse H⁺/K⁺-ATPase (HK promoter) (29), with the expectation of predominant expression of CagA in the stomach (SI Fig. 7A). After injection of the transgenic constructs into fertilized mouse eggs, three lines (B-10, D-10, and E-01) of the CAG promoter-driven cagA^Hs (CAG-cagA^Hs) transgenic mice and three lines (A-21, F-11, and D-01) of the HK promoter-driven cagA^Hs (HK-cagA^Hs) transgenic mice were established (SI Fig. 7B). These cagA^Hs mice were indistinguishable from the wild-type littermates in behavior and weight when they were born, and they developed normally. No overt difference was found between cagA^Hs heterozygous and homozygous mice.

In CAG-cagA^Hs mice, cagA^Hs mRNAs were detected in various organs and tissues with high levels of expression in the stomach, ileum, colon, brain, lung, thymus, and testis (Fig. 1A and SI Fig. 8A). In HK-cagA^Hs mice, cagA^Hs mRNAs were predominantly expressed in the stomach. However, significant amounts of cagA^Hs transcripts were also present in other tissues such as the lung, intestine, thymus, spleen, and testis, indicating leaky transcription from the transgenic HK promoter (Fig. 1A and SI Fig. 8 A and B). Expression of the CagA protein was confirmed in tissue extracts prepared from the stomachs of 4-week-old CAG-cagA^Hs and HK-cagA^Hs transgenic mice (Fig. 1B), although the levels were much lower than those of gastric epithelial cells transfected with CagA-expression vector or in vitro infected with cagA-positive H. pylori (data not shown). Analysis of embryonic fibroblasts from CAG-cagA^Hs mice confirmed tyrosine phosphorylation of CagA and complex formation of CagA with SHP-2 (Fig. 1C).

Gastrointestinal Abnormalities in Wild-type CagA Transgenic Mice.

At 12 weeks of age, mice were killed and autopsies revealed a broad thickening of gastric mucosa, primarily at the body of the stomach, in both the CAG-cagA^Hs and HK-cagA^Hs mice, although the penetrance of this mucosal change was incomplete (Fig. 1D and SI Fig. 9). This may be attributable to the differences in CagA levels among individual transgenic mice even though they are genetically identical. Histologically, the change was due to epithelial hyperplasia of the glandular stomach, in which the proliferative zone in the gastric gland was markedly expanded, and was concomitantly associated with increased numbers of parietal, chief, and endocrine cells, which were all derived from precursors present in the proliferative zone (Fig. 1D). In the hyperplastic lesion, CagA expression was concomitantly associated with the hyperactivation of the Erk MAP kinase, a downstream effector of CagA-deregulated SHP-2 (Fig. 1E) (15).

At 48 weeks of age, several CAG-cagA^Hs and HK-cagA^Hs mice had developed polyps in the glandular stomach. These polyps were mostly hyperplastic polyps, consisting of surface epithelial cells with limited atypical cellularity without nuclear pseudostratification. By 72 weeks of age, 15 of 184 CAG-cagA^Hs/line B-10 mice, 8 of 35 CAG-cagA^Hs/line D-10 mice and 5 of 98 HK-cagA^Hs/line A-21 mice had developed hyperplastic polyps (Fig. 2A and SI Table 1). Furthermore, a small number of cagA^Hs mice at 72 weeks of age had developed adenocarcinomas, two in the stomach and four in the small intestine (five in CAG-cagA^Hs/line B-10 and 1 in HK-cagA^Hs/line F-11) (Fig. 2 B and C and SI Table 1). These carcinomas consisted of irregular branching glands lined by atypical columnar cells that showed plump nuclei with large nucleoli and high mitotic index (SI Fig. 10A). The high proliferating activity of the tumors was confirmed by Ki-67 immunostaining (Fig. 2 C and D). In several cases, neoplastic glands had penetrated the lumina muscularis mucosa (SI Fig. 10B). Immunostaining of the tumor specimens demonstrated intense nuclear positivity of p53 and β-catenin in intestinal adenocarcinomas, suggesting mutations in the β-catenin and/or Apc and p53 genes in the neoplastic lesions but not in gastric adenocarcinomas (Fig. 2C and data not shown). These findings indicate the requirement of cell type-specific secondary events for the development of gastrointestinal neoplasms. No gastrointestinal polyps or tumors were found in the wild-type littermate mice by 72 weeks after birth with continual observation (n = 100).

It has been generally believed that atrophic gastritis and intestinal metaplasia, a transdifferentiation of gastric epithelial cells to an intestinal phenotype, are precancerous gastric mucosal changes, from which intestinal-type gastric adenocarcinoma arises (30). We therefore performed histological examination of gastric mucosa from 72-week-old cagA^Hs mice but did not find any signs of pathological abnormalities (data not shown). From these observations, we concluded that CagA has the ability to induce epithelial hyperplasia, polyps, and adenocarcinomas in the stomach, without causing overt inflammation or intestinal metaplasia. We also note that no overt histopathological abnormalities were found in the intestinal tract of cagA^Hs mice without tumors.

Hematopoietic Abnormalities in Wild-Type CagA Transgenic Mice.

In addition to the gastrointestinal abnormalities, some of the cagA^Hs mice had developed hematopoietic malignancies by 72 weeks of age (16 of 152 in CAG-cagA^Hs/line B-10; 1 of 77 in HK-cagA^Hs/line A-21; and 1 of 38 in HK-cagA^Hs/line F-11) (SI Table 1). It should be noted here that CagA was also expressed in the spleen of HK-cagA^Hs mice (SI Fig. 8B). The leukemic mice showed marked splenomegaly, and immunohistochemical analyses of the spleens revealed that they were of myeloid (5 cases), B cell (12 cases), or T cell (1 case) origin (Fig. 3 A–C). Mild leukocytosis (granulocytosis) was also found in the peripheral blood of 72-week-old cagA^Hs mice without hematological malignancies (Fig. 3D). No age-matched wild-type littermate mice developed hematological abnormality (n = 100).

The observed spectrum of hematological malignancies in CagA transgenic mice was similar to that evoked by gain-of-function SHP-2 mutations (13). We thus suspected that CagA-deregulated SHP-2 is also involved in the transformation of hematopoietic cells as well. Indeed, in spleen cells from cagA^Hs mice, the Erk MAP kinase was again hyperactivated (Fig. 3E). Because SHP-2 potentiates cellular responses to interleukin (IL)-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) (31), we performed a colony assay to evaluate activation status of SHP-2. Bone marrow cells from cagA^Hs mice showed hypersensitivity to IL-3 and GM-CSF and also exhibited spontaneous colony formation in the absence of cytokines (Fig. 3F). These observations are consistent with the idea that SHP-2 is deregulated in cagA^Hs mice and suggest that CagA-deregulated SHP-2 plays a key role in the abnormal proliferation of multiple hematopoietic cells.

Role of Tyrosine Phosphorylation in the in Vivo Pathogenic Activity of CagA.

Tyrosine-phosphorylated CagA specifically binds to and deregulates SHP-2. To explore the role of CagA tyrosine phosphorylation in the development of gastrointestinal and hematopoietic lesions observed in cagA^Hs mice, we next generated a PR-cagA^Hs gene encoding a phosphorylation-resistant ABDD CagA (PR-CagA), which cannot bind SHP-2, from cagA^Hs (SI Fig. 11 A and B). Transgenic constructs were made by connecting the PR-cagA^Hs DNA fragment downstream of the CAG or HK promoter (SI Fig. 11C). After injection of the transgenic constructs into fertilized mouse eggs, three lines each for the CAG promoter-driven PR-cagA^Hs (CAG-PR-cagA^Hs) and HK promoter-driven PR-cagA^Hs (HK-PR-cagA^Hs) transgenic mice were established (Fig. 4A). Systemic expression of PR-cagA^Hs in CAG-PR-cagA^Hs mice was confirmed by RT-PCR analysis (SI Fig. 11D). Again, expression of PR-cagA^Hs in HK-PR-cagA^Hs mice was not specific to the stomach.

Despite significantly higher levels of PR-CagA expression than those of wild-type CagA in transgenic mice (Fig. 4 B and C), PR-cagA^Hs mice neither showed gastric epithelial hyperplasia or leukocytosis nor developed neoplasms (except one case of gastric hyperplastic polyp in a HK-PR-cagA^Hs mouse) (Fig. 4 D and E, SI Fig. 11E, and SI Table 1). Furthermore, bone marrow cells from PR-cagA^Hs mice did not show hypersensitivity to IL-3 or GM-CSF (Fig. 4F). These observations indicate that tyrosine-phosphorylated CagA, which enables CagA to bind SHP-2, plays an important role in the abnormal proliferation of gastric epithelial and hematopoietic cells and subsequent development of neoplasms.

Transgenic Mice.

Chemically synthesized cagA^Hs DNA was subcloned into pCAGGS (28) or pHKATP vector (29). The CAG-cagA^Hs or HK-cagA^Hs fragment consisting of the promoter, cagA^Hs and polyA cassettes derived from β-globin gene was excised from the resulting plasmid by restriction enzyme digestion, and then injected directly into fertilized eggs of C57BL/6J mice. Transgenic mice were identified by PCR analysis of tail DNAs, using cagA^Hs-specific primers (cagA^Hs-forward 5′-CACAATAACGCTCTGTCATCAGTGCTG-3′, cagA^Hs-reverse 5′-TCAACGTATAAGACACTTCCCCATTGC-3′). PCR amplification was performed at 94°C for 5 min, 94°C for 30 seconds, 62°C for 30 seconds, 72°C for 30 seconds (30 cycles), and 72°C for 7 min. All of the animal experiments were carried out according to the protocol approved by the Ethics Committee for Animal Experiments at Hokkaido University.

RT-PCR and Quantitative RT-PCR.

Total RNA was extracted from mouse tissues with the use of TRIzol Reagent (Invitrogen). Quantitative RT-PCR was performed by using SYBR Green fluorescence detection system (Qiagen) with ABI PRISM 7700 sequencer (Applied Biosystems). The following primer pairs were used for PCR amplification: cagA^Hs-forward: 5′-AAGCTGCTTCTGCGATTAACCG-3′, cagA^Hs-reverse: 5′-GGAGTCTTTCAGTTCGTC-3′, GAPDH-forward: 5′-ACCACAGTCCATGCCATCAC-3′, and GAPDH-reverse: 5′-TCCACCACCCTGTTGCTGTA-3′.

Immunoprecipitation and Immunoblotting.

Protein extracts prepared from tissues or cells were subjected to immunoprecipitation or immunoblotting with an anti-HA antibody (3F10; Roche) as described in ref. 9.

Histopathological Analyses.

Tissue specimens were fixed in formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). Thickness of gastric mucosa was evaluated by measuring three distinct points of the glandular stomach for each mouse. Cancer diagnosis was made based on Ki-67 labeling index, nuclear atypia, and mitosis counts by two independent pathologists (S.T. and H.S.). For immunohistochemical staining, sections were deparaffinized, rehydrated, and then incubated with anti-PCNA (PC10; Dako), anti-Ki-67 (Dako), anti-B220 (RA3–6B2), anti-p53 (CM5; Novocastra), anti-CD3 (Dako), or anti-β-catenin antibody (BD Bioscience). Sections were then washed and incubated with appropriate secondary antibodies. Reacted antibodies were detected by the peroxydase reaction, using diaminobenzidine as substrate. Chief cells were identified by H&E staining. Parietal cells and endocrine cells were identified by immunostaining with anti-H⁺/K⁺-ATPase α-subunit antibody (Chemicon) and anti-chromogranin A antibody (Dako), respectively. Peripheral blood smears were stained with May–Giemsa solution.

Flow Cytometry.

Cells were washed in PBS containing 0.5% BSA and 0.05% NaN₃. Fc receptor-mediated binding was blocked by preincubation of cells with supernatants of 2.4G2 hybridoma. Fluorescence isothiocyanate (FITC)-anti-Gr-1 and phycoerythrin (PE)-anti-CD11b (Mac-1) antibodies were purchased from BD Bioscience. Flow cytometric analysis was performed by using FACSCalibur (Becton–Dickinson) and analyzed with CellQuest software.

Colony Assay.

Methylcellulose cell culture was performed in 35-mm culture dishes as described in ref. 40.

Statistical Analysis.

Statistical analysis was performed by using Student's t test, the χ² test, or Fisher's exact test.