The role of aberrant GP130 signalling in the development and progression of gastric cancer

Abstract

© 2011 Dr. Stefan ThiemGastric cancer (GC) is one of the leading causes of cancer-related deaths. There is no cure, and current treatments are not particularly successful. Therefore, there is an urgent need to develop novel therapies. Our laboratory has previously characterized the gp130Y757F/Y757F mouse (referred to as gp130FF) as a robust model for inflammation-associated gastric tumourigenesis akin to human intestinal-type GC. In this model, a knock-in mutation triggers spontaneous gastric tumour formation driven by interleukin- (IL) 11-mediated hyper-activation of the latent transcription factor signal transducer and activator of transcription 3 (Stat3). This mediator of cell proliferation, survival and angiogenesis is hyper-activated in many types of solid epithelial cancers and also plays a key role in cancer-associated chronic inflammation. The aim of this study was to explore the molecular mechanisms underlying inflammation-associated gastric tumourigenesis in gp130FF mice in three different projects by: 1. identifying Stat3 target genes in gp130FF tumours through microarray and chromatin immunoprecipitation/high throughput sequencing (ChIP-Seq) analysis; 2. examining the role of phosphoinositide 3-kinase (PI3K) signalling and the therapeutic potential of inhibiting the associated mammalian target of rapamycin (mTor) pathway; and 3. generating a novel preclinical mouse model using bacterial artificial chromosome (BAC) transgenesis to confer inducible Cre-recombinase-mediated gene (in-)activation to the gastric epithelium. The combined use of ChIP-Seq, microarray and quantitative PCR analysis revealed genomic Stat3 binding sites in gp130FF tumour cells and associated genes that respond to the pro-inflammatory cytokines IL6 and/or IL11. Careful analysis identified candidate genes that play important roles in oncogenesis, including c-myc. Genetic deletion of c-myc in gp130FF mice resulted in a significantly reduced tumour burden demonstrating that c-myc function is crucial for Stat3-mediated tumour development. Thus, this approach may provide a basis for the identification of novel therapeutic targets for the treatment of Stat3-dependent cancers. The PI3K and mTor signalling pathways promote tumour growth, survival and vascularization and are commonly deregulated in human cancer. In this study, it was demonstrated that mTor complex 1 (mTorc1) and Stat3 are strongly co-activated in human and gp130FF gastric tumours. Furthermore, it was shown that stimulation of mutant gp130 can induce PI3K and mTorc1 signalling, independently of Stat3. Importantly, therapeutic treatment of gp130FF mice with the rapamycin analogue RAD001 (everolimus) suppressed mTorc1 activity and reduced tumour number and burden through inhibition of proliferation and tumour vascularization. Moreover, prophylactic treatment of gp130FF mice alleviated tumour formation. When RAD001 treatment was stopped, mTorc1 activity was re-activated and tumour growth resumed. These findings suggest that inflammation-associated tumour development and progression in gp130FF mice are critically dependent on both Stat3 and mTorc1 activity. Therefore, inhibition of mTOR activity alone or in combination with STAT3 and/or PI3K inhibition may have therapeutic potential. A novel gastric-specific and inducible Cre expressing mouse line, Tg(Tff1/2-CreERT2; Tff3-Luc), was generated and analysed. These mice express the CreERT2 recombinase as well as the firefly luciferase as part of a BAC containing the cis-regulatory elements of the three trefoil factor (Tff) genes. The Tff1 and Tff2 loci confer CreERT2 expression to normal and (on the gp130FF background) neoplastic epithelial cells of the glandular stomach, thus allowing conditional mutagenesis specifically in these cells. The development of the Tg(Tff1/2-CreERT2; Tff3-Luc) mouse provides a novel and powerful tool to study gene functions in the gastric epithelium and identify molecular mechanisms leading to tumour formation. Moreover, intestinal-specific expression of the Tff3-driven luciferase allows in vivo monitoring of intestinal homeostasis and mucosal damage. In order to establish a preclinical model of GC progression, this strain was further used to generate novel compound mutant mice. These are based on the activation of oncogenic mutations associated with invasive human gastric adenocarcinomas and metastasis