Deletion of IQGAP1 promotes Helicobacter pylori-induced gastric dysplasia in mice and acquisition of cancer stem cell properties in vitro

Helicobacter pylori infection is responsible for gastric carcinogenesis but host factors are also implicated. IQGAP1, a scaffolding protein of the adherens junctions interacting with E-cadherin, regulates cellular plasticity and proliferation. In mice, IQGAP1 deficiency leads to gastric hyperplasia. The aim of this study was to elucidate the consequences of IQGAP1 deletion on H. pylori-induced gastric carcinogenesis. Transgenic mice deleted for iqgap1 and WT littermates were infected with Helicobacter sp., and histopathological analyses of the gastric mucosa were performed. IQGAP1 and E-cadherin expression was evaluated in gastric tissues and in gastric epithelial cell lines in response to H. pylori infection. The consequences of IQGAP1 deletion on gastric epithelial cell behaviour and on the acquisition of cancer stem cell (CSC)-like properties were evaluated. After one year of infection, iqgap1+/- mice developed more preneoplastic lesions and up to 8 times more gastro-intestinal neoplasia (GIN) than WT littermates. H. pylori infection induced IQGAP1 and E-cadherin delocalization from cell-cell junctions. In vitro, knock-down of IQGAP1 favoured the acquisition of a mesenchymal phenotype and CSC-like properties induced by H. pylori infection. Our results indicate that alterations in IQGAP1 signalling promote the emergence of CSCs and gastric adenocarcinoma development in the context of an H. pylori infection.


Bacterial culture
The mouse adapted H. pylori strains SS1 and HPARE and H. felis strain ATTC 49179 were used for mice experiments. H. pylori strain 7.13 was kindly provided by R. Peek (Vanderbilt University, Nashville, TN, USA). All strains were cultured as previously described [4,12]. For coculture, H. pylori strains were grown at 37°C for 24 h, resuspended in PBS and adjusted to an OD 600 nm = 1 (corresponding to 2x10 8 CFU/ml) in PBS before infection. The homogenates from gastric tissues were seeded on selective blood agar media and incubated under microaerobic conditions at 37°C for 3 to 5 days for the culture of H. pylori. The Helicobacter positive status was confirmed by PCR amplification of specific genes as previously described [12].

Quantification scoring and statistical analysis
Scoring criteria for quantification of inflammation, mucosal height, oxyntic atrophy and dysplasia were determined as previously described [12]. Relative quantification of the expression of CD44 in the gastric epithelium was determined in a blind lecture using a scale from 0 to 4 as described previsouly [4] with the following criteria: 0: no staining, 1: <5% of positive epithelial cells; 2: 5 to <25% of positive cells; 3: 25 to <50% of positive cells; 4: ≥50% of positive epithelial cells. For Zeb1 which is discretely diffusely expressed in most of epithelial cells, scores with the same scale were determined but for the quantification of cells with a strong nuclear staining. For Snail, which is detected in almost all epithelial cells, scores with the same scale were determined but for the quantification of cells with a strong staining.

Invasion assay
After cell recovery, 50,000 cells per condition were placed in the upper side of a 8 μm pore size Transwell insert in 24-well culture plates with medium containing 5% FBS. For invasion assays, inserts were previously coated with 0.05 mg/mL of rat type I collagen (BD Biosciences) for 40 min at 37°C. After 18 h of incubation with cells at 37°C, the Transwell inserts were fixed in cold methanol and processed for HES staining, as previously described [4]. Cells having migrated through the lower side of the inserts were counted on five different randomly chosen fields per insert under light microscopy using a x 20 objective.

Tumorsphere assay
After cell recovery, 500 cells were plated on non-adherent 96-well culture plates (coated with 10% polyHEMA (Sigma) solution in absolute ethanol and dried overnight at 56°C). After plating, cells were incubated for 5 days at 37°C in a serum-free medium consisting of DMEM-F12 Glutamax supplemented with 20 ng/ml of EGF, 10 ng/ml of basic-FGF, 1:100 N2-supplement 100X, 0.3% glucose, and 50 μg/ml of vancomycin (all from Invitrogen and Sigma). The number of spheroids (tumorspheres) per well was counted under light microscopy using a x 20 objective.

RNA extraction and quantitative RT-PCR
Total cellular RNAs were extracted using Trizol reagent (Invitrogen) and quantified by their absorbance at 260 nm. Retrotranscription was performed with 1 μg of total RNAs using the Quantitect Reverse Transcription (RT) kit (Qiagen), according to the manufacturer's recommendations. Quantitative PCR was performed using specific primers at 0.3 μM and Maxima SYBR Green qPCR master mix (Fermentas, Courtaboeuf, France).

Statistical analysis
Quantification values represent the means of three or more independent experiments, each performed in triplicate or more ± standard deviation (SD). Statistical analyses were performed using the non-parametric Kruskal-Wallis, for mice scoring results, and Mann Whitney tests on SPSS16.0F software (SPSS Inc., Chicago, IL, USA).