Published OnlineFirst February 2, 2016; DOI: 10.1158/0008-5472.CAN-15-3089 Cancer Molecular and Cellular Pathobiology Research Stomach-Specific Activation of Oncogenic KRAS and STAT3-Dependent Inflammation Cooperatively Promote Gastric Tumorigenesis in a Preclinical Model Stefan Thiem1, Moritz F. Eissmann1, Joachim Elzer1, Anna Jonas2, Tracy L. Putoczki3, Ashleigh Poh1,3, Paul Nguyen1,3, Adele Preaudet3, Dustin Flanagan4, Elizabeth Vincan4, Paul Waring5, Michael Buchert1, Andrew Jarnicki1,6, and Matthias Ernst1 Abstract About 5% to 10% of human gastric tumors harbor oncogenic terized by excessive STAT3 activity. To assess the contribution of mutations in the KRAS pathway, but their presence alone is often STAT3 to the spontaneously developing gastric adenomas in insufficient for inducing gastric tumorigenesis, suggesting a gp130F/F mice, which carry a knockin mutation in the Il6 requirement for additional mutagenic events or microenviron- signal transducer (Il6st), we generated Tg(Tff1-CreERT2); fl fl mental stimuli, including inflammation. Assessing the contribu- Stat3 / ;gp130F/F mice that also harbor a conditional Stat3 knock- tion of such events in preclinical mouse models requires Cre out allele and found that tamoxifen administration conferred a recombinase–mediated conditional gene expression in stem or significant reduction in their tumor burden. Conversely, excessive þ progenitor cells of normal and transformed gastric epithelium. Kras activity in Tg(Tff1-CreERT2);KrasLSL-G12D/ ;gp130F/F mice We therefore constructed a bacterial artificial chromosome con- promoted more extensive gastric inflammation, metaplastic taining transgene (Tg), comprising the regulatory elements of the transformation, and tumorigenesis than observed in Tg(Tff1- þ trefoil factor 1 (Tff1) gene and the tamoxifen-inducible Cre CreERT2);KrasLSL-G12D/ mice. Collectively, our findings demon- recombinase (CreERT2)–coding sequence. The resulting Tg strate that advanced gastric tumorigenesis requires oncogenic (Tff1-CreERT2) mice were crossed with mice harboring condi- KRAS or BRAF in concert with aberrant STAT3 activation in tional oncogenic mutations in Kras or Braf. The administration of epithelial precursor cells of the glandular stomach, providing a þ tamoxifen to the resulting adult Tg(Tff1-CreERT2);KrasLSL-G12D/ new conditional model of gastric cancer in which to investigate þ and Tg(Tff1-CreERT2);BrafLSL-V600E/ mice resulted in gastric candidate therapeutic targets and treatment strategies. Cancer Res; metaplasia, inflammation, and adenoma development, charac- 76(8); 1–11. Ó2016 AACR. Introduction ities (1). The most prevalent form of gastric cancer is intestinal- type gastric adenocarcinoma, which progresses from superficial Despite a recent decline in mortality and incidence, gastric gastritis through stages of chronic gastritis, atrophic gastritis, cancer still accounts for one of the largest cancer-related mortal- metaplastic transformation, and dysplasia to invasive adenocar- cinoma (2). Although chronic infection with Helicobacter pylori (H. pylori) remains the main risk factor for intestinal-type gastric 1Cancer and Inflammation, Olivia Newton-John Cancer Research Insti- tute and School of Cancer Medicine La Trobe University, Heidelberg, cancer, others include high-salt diet and viral infections (2). Australia. 2Department of Multiple Sclerosis, The Florey Institute of Tumor initiation and progression is a multistep process that Neuroscience and Mental Health, Melbourne, Australia. 3Inflammation requires the acquisition of genetic alterations and growth-pro- Division, The Walter & Eliza Hall Institute of Medical Research and Department of Medical Biology University of Melbourne, Melbourne, moting conditions in the microenvironment. The most common Australia. 4Department of Anatomy and Neuroscience, University of activating mutations in gastric cancer driver genes affect KRAS, Melbourne, Melbourne, Australia. 5Department of Pathology, Univer- PI3K, and the receptor tyrosine kinases FGFR2, ERBB2, EGFR, 6 sity of Melbourne, Melbourne, Australia. School of Biomedical MET, and related signaling molecules (3). Meanwhile, chronic Sciences and Pharmacy, University of Newcastle, Newcastle, Australia. inflammation of the gastric mucosa, triggered by bacterial infec- Note: Supplementary data for this article are available at Cancer Research tion or other environmental factors, can initiate premalignant Online (http://cancerres.aacrjournals.org/). metaplastic transformation, which often results in the loss of acid- S. Thiem and M.F. Eissmann contributed equally to this article. producing parietal cells (4). Accordingly, the excessive inflamma- Corresponding Author: Matthias Ernst, Olivia Newton-John Cancer Research tory response of the gastric epithelium of gp130F/F mice, a vali- Institute and School of Cancer Medicine at La Trobe University, 145 Studley Rd, dated preclinical model for early stage intestinal-type gastric Heidelberg, Victoria 3084, Australia. Phone: 613-9496-9775; Fax: 613-9496- cancer in humans, is associated with intestinal metaplasia and 5334; E-mail: [email protected] inevitably triggers adenoma formation in the most distal part of doi: 10.1158/0008-5472.CAN-15-3089 the stomach, referred to as antrum (5). Meanwhile, aberrant Ó2016 American Association for Cancer Research. activation of Kras induces hyperplasia in many epithelial tissues www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst February 2, 2016; DOI: 10.1158/0008-5472.CAN-15-3089 Thiem et al. and, in the gastric epithelium, is associated with some hallmarks ATG initiation codon in the Tff1 locus. We injected the resulting of spasmolytic peptide–expressing metaplasia (SPEM) and pseu- BAC transgene in the pronucleus of CBB6F1-derived one-cell dointestinal metaplasia (6). However, oncogenic Kras activation embryos and genotyped transgenic founder mice and their off- in isolation is usually insufficient to trigger the formation of spring by PCR (refer to Supplementary Methods). gastric adenomas (7, 8). Thus, akin to the paradigms for the intestinal epithelium, gastric tumorigenesis may also depend on Tamoxifen administration the sequential acquisition of mutations within the precancerous Tamoxifen (Sigma T5648) was dissolved in 100% ethanol metaplastic epithelium or require a permissive tumor microen- (Sigma) and diluted 1:10 in sunflower oil (Sigma). Mice were fl vironment characterized by subclinical in ammation. injected intraperitoneally (100 mL) for 5 consecutive days with The formation of solid tumors is widely believed to depend on tamoxifen (1 mg/20 g body weight; twice daily) or vehicle, unless cancer stem cells, which, at least in the case of the intestinal indicated otherwise. The tamoxifen-containing emulsions were epithelium, express similar markers as their nontransformed prepared freshly every 3 days. tissue stem cells. Accordingly, the gastrointestinal stem cell marker locus Lgr5 can confer Cre recombinase–mediated cellular trans- b formation at the base of the glands of the pylorus and corpus -Gal staining region of the stomach (9). However, the higher abundance of Stomachs and intestines were opened longitudinally, washed Lgr5-positive stem cells in the intestinal epithelium results in three times in PBS by vigorous shaking, and pinned out on fi lethal intestinal tumorigenesis, preventing the analysis of gastric silicone-coated plates. Tissues were pre xed in 4% paraformal- fl fl tumor formation in corresponding Lgr5CreERT2;Apc / mice, which dehyde (PFA; Sigma) for one hour at 4 C, washed three times with b harbor conditional null alleles of the tumor suppressor gene Apc -Gal Wash Buffer (20 minutes/room temperature), and incu- fi bated in b-Gal Staining Solution (refer to Supplementary Meth- (9). Meanwhile, Cre expression that is largely con ned to com- mitted gastric epithelial progenitors in Atp4b-Cre or Tg(Tff2- ods) at 37 C overnight. Photographs were taken and the organs fi CreERT2) mice is insufficient to reproducibly and reliably trans- were xed in 2% PFA (Sigma) at 4 C overnight, transferred to 70% fi duce tumorigenesis, and neither of these Cre-drivers have been ethanol, paraf n sectioned, and counterstained with Nuclear Fast tested for their capacity to confer recombinase activity within Red. established tumors (10, 11). We therefore exploited the regulatory sequences of the stomach-specific trefoil factor 1 (Tff1) gene, YFP detection which is primarily expressed in the epithelium of the glandular Following stomach dissection, specimens were washed in PBS, stomach within the corpus and antrum, and to a lesser extent also fixed in 4% PFA solution (Electron Microscopy Sciences) over- in the neoplastic counterpart in gastric cancer (12, 13). Impor- night, incubated in 30% and 50% sucrose/PBS (w/v) solution, tantly, Tff1 deficiency can give rise to gastric tumorigenesis in mice embedded in OCT (Tissue-Tek Sakura), and snap frozen on dry through a cell-intrinsic mechanism (14), arguing that Tff1-expres- ice. Frozen stomach sections (8 mm) were counterstained with sing gastric epithelium can provide the cell of origin for gastric DAPI before mounting. YFP (shown as green) and DAPI (shown cancer. We therefore exploited bacterial artificial chromosome as blue) were imaged using a confocal microscope (Zeiss LSM (BAC) transgenesis to confer tamoxifen-inducible CreERT2 activ- 780). ity in the glandular stomach of corresponding transgenic Tg(Tff1- CreERT2) mice.