Meegan Howlett

The Interleukin-6 Family Cytokine Interleukin-11 Regulates Homeostatic Epithelial Cell Turnover and Promotes Gastric Tumor Development

BACKGROUND & AIMS Gastric cancer is the second most common cause of cancer-related mortality worldwide, mainly as a result of late-stage detection. Interleukin (IL)-11 is a multifunctional cytokine reported to be up-regulated in human gastric cancer. METHODS We investigated the importance of IL-11 in gastric cancer progression by examining its role in a variety of mouse gastric tumor models, as well as in nonneoplastic and tumor tissues taken from gastric cancer patients. We then determined the transcriptional and translational outcomes of IL-11 overexpression in normal gastric mucosa and identified a novel gene signature important early in the progression toward gastric tumorigenesis. RESULTS IL-11 was up-regulated significantly in 4 diverse mouse models of gastric pathology as well as in human biopsy specimens adjacent to and within gastric cancer. Removal of IL-11 co-receptor alpha significantly reduced HKbeta-/- mouse fundic hyperplasia and ablated gp130(757F/F) mouse tumorigenesis. Exogenous IL-11 but not IL-6 activated oncogenic signal transducer and activator of transcription-3, and altered expression of novel proliferative and cytoprotective genes RegIII-beta, RegIII-gamma, gremlin-1, clusterin, and growth arrest specific-1 in wild-type gastric mucosa, a gene signature common in gp130(757F/F) and HKbeta-/- tumors as well as nonneoplastic mucosa of gastric cancer patients. One week of chronic IL-11 administration in wild-type mice sustained the gene signature, causing pretumorigenic changes in both antrum and fundus. CONCLUSIONS Increased gastric IL-11 alters expression of proliferative and cytoprotective genes and promotes pretumorigenic cellular changes.

Inhibition of the JAK2/STAT3 Pathway Reduces Gastric Cancer Growth In Vitro and In Vivo

Signal Transducer and Activator of Transcription-3 (STAT3) is constitutively activated in many cancers where it promotes growth, inflammation, angiogenesis and inhibits apoptosis. We have shown that STAT3 is constitutively activated in human gastric cancer, and that chronic IL-11-driven STAT3 transcriptional activity induces gastric tumourigenesis in the gp130757FF mouse model of gastric cancer development. Here we show that treatment of human AGS gastric cancer cells with the Janus Kinase (JAK) inhibitor WP1066 dose-, and time-dependently inhibits STAT3 phosphorylation, in conjunction with reduced JAK2 phosphorylation, reduced proliferation and increased apoptosis. In addition, application of intraperitoneal WP1066 for 2 weeks, reduced gastric tumour volume by 50% in the gp130757FF mouse coincident with reduced JAK2 and STAT3 activation compared with vehicle-treated, littermate controls. Gastric tumours from WP1066- treated mice had reduced polymorphonuclear inflammation, coincident with inhibition of numerous proinflammatory cytokines including IL-11, IL-6 and IL-1β, as well as the growth factors Reg1 and amphiregulin. These results show that WP1066 can block proliferation, reduce inflammation and induce apoptosis in gastric tumour cells by inhibiting STAT3 phosphorylation, and that many cytokines and growth factors that promote gastric tumour growth are regulated by STAT3-dependent mechanisms. WP1066 may form the basis for future therapeutics against gastric cancer.

Cytokine signalling via gp130 in gastric cancer

Cytokine signalling pathways that depend on gp130 are dysregulated in several epithelial cancers including gastric cancer. It has been established that blockade of SHP2 activation of MAPK signalling results in hyperactivation of STAT3 resulting in increased cell proliferation, angiogenesis, inflammation and inhibition of both immunocyte and epithelial cell apoptosis. Additionally, key genes regulated downstream of gp130 via MAPK activation such as the stomach-specific tumor suppressor gene tff1 are suppressed, contributing to the oncogenic outcome. The main cytokine driver of gp130 signalling in the stomach is IL-11, with IL-6 having little activity in the antral stomach in which most pathology initiates. IL-11 is up-regulated in both mouse and human gastric cancer and in pre-neoplastic mucosa. A characteristic gene signature specifically associated with IL-11 drive has been observed, although the prognostic value of the signature has not yet been assessed. Infection of human or mouse stomach with Helicobacter pylori, especially that expressing the CagA cytotoxin, produces constitutive MAPK activation, but also activated STAT3 and increases IL-11 expression. The possibility of designing and utilising small molecule inhibitors of either IL-11 or STAT3 activation may be worthwhile in developing new cancer therapeutics.

IL-11 is a parietal cell cytokine that induces atrophic gastritis

Background and Aims IL-is important in gastric damage, mucosal repair and gastric cancer progression. We analysed IL-11 expression in H.pylori infected mouse stomach, the site of gastric IL-11 expression in mice and humans, and the effect of exogenous IL-11 on gastric mucosal homeostasis. Methods IL-11 protein was localised in mouse and human stomach. The impact of chronic, exogenous IL-11 on normal mouse stomach was examined histologically and transcriptionally by microarray, confirmed by mRNA and protein analysis. Functional impact of IL-11 on gastric acid secretion was determined. Results In mice infected with H.pylori, IL-11 was increased in fundic mucosa with temporal expression similar to IL-1b. IL-11 protein was localised predominantly to parietal cells in mouse and human stomach. Application of exogenous IL-11 to resulted in fundic parietal and chief cell loss, hyperplasia, mucous cell metaplasia and inflammation. Coincident with cellular changes were an increased gastric pH, altered parietal cell ultrastructure and altered gene expression, particularly genes involved in immune response and ion transport which could result in compromised acid secretion. We confirmed that a single dose of IL-11 effectively ablated the gastric response to histamine. Conclusions IL-11 is a parietal cell cytokine that blocks gastric acid secretion, likely via reducing expression of parietal cell ion transport genes, CCKb and histamine H2 receptors. IL-11 expression is increased in H. pylori infected mouse stomach and treatment of wild type mice with IL-11 induced changes in the gastric fundic mucosa reminiscent of chronic atrophic gastritis, a precursor to gastric cancer.

Insights into the mechanisms of gastric adaptation to aspirin-induced injury : a role for regenerating protein but not trefoil peptides

The phenomenon of reduced gastric mucosal injury despite repeated doses of a damaging agent is termed adaptation. Adaptation to nonsteroidal anti-inflammatory drug-induced injury has been clearly demonstrated in both humans and experimental animals; however, the precise mechanisms remain unclear. We hypothesized that mediators of adaptation might be the regenerating protein (RegI) and the trefoil peptides TFF1 and TFF2, because these proteins play pivotal roles in gastric mucosal protection and repair. The gene expression and the protein levels of these proteins were measured and compared in normal, aspirin-injured, and aspirin-adapted rat stomachs. TFF gene and protein expression levels were similar in all three groups, whereas RegI gene expression and protein levels in adapted stomach were increased. A time course analysis of RegI expression during the onset and offset of adaptation showed that mucosal RegI increased during the development of adaptation, was maintained during subsequent aspirin dosing, and returned to baseline levels once dosing had ceased and adaptation was lost—indicative of a causal role in the adaptation process. Colocalization of increased RegI with gastric epithelial areas showing increased proliferation also suggests that RegI may be an important mediator of the resolution of mucosal injury that is characteristic of gastric adaptation to aspirin.

Cytokine signalling by gp130 regulates gastric mucosal healing after ulceration and, indirectly, antral tumour progression†

The cytokines IL‐6 and IL‐11, which signal via the receptor gp130, have been implicated in various gut pathologies, including inflammation and wound healing. We used mouse cytokine signalling mutants to evaluate the role of gp130 pathways in gastric ulceration and healing and the effect of spatially remote fundic ulceration on antral tumour progression, since compromised wound healing may impact tumourigenesis. Glacial acetic acid applied to the serosal surface of stomachs from wild‐type, gp130757FF, IL‐6−/− and IL‐11 receptor (R)α−/− mice was used to induce discrete haemostasis/necrosis and resultant mucosal ulceration. Wound pathology and mRNA expression of key cytokine target genes were examined 2 and 14 weeks after ulcer induction. The outcome of fundic ulceration on antral tumour development in gp130757FF mice was also examined. Chemical haemostasis in gp1307575FF mice produces more severe gastric ulcers than in wild‐type mice. Lack of IL‐6 produces more severe ulceration, while loss of IL‐11Rα less severe ulcers, suggesting a role for IL‐11 in ulcer induction. Increased expression of ulcer‐associated IL‐11 and its established mitogenic target genes RegI, IIIβ and IIIγ paralleled severe ulceration in gp130757FF mice. In this model, coincident with fundic ulceration, antral tumour development was inhibited and correlated with decreased RegI, IIIβ and IIIγ and reduced MMP9 and 13 expression. IL‐11‐driven transcription via gp130 contributes to the gastric mucosal response to ulceration. Fundic mucosal ulceration modulates antral growth factor and metalloproteinase gene expression, thereby contributing to restricted tumour growth. Copyright

Chronic IL-11 Promotes Proliferation, Bmp1 Signalling and Activation of the Mucosal Alarmin IL-33 in the Colon

Background and AimsIL-33 is an IL-1 related cytokine and like IL-1a has been suggested to amplify the immune response during tissue injury. IL-33 is expressed in endothelium and mucosal epithelium and is a ligand for the receptor ST2. Recently IL-33 has been shown to be a crucial amplifier of immunity that can modulate the response in a number of models of innate-type mucosal immunity. Significantly IL-33 is involved in the development of DSS induced colitis. Additionally we have demonstrated that IL-11, a member of the IL-6 family of cytokines, is also important in DSS induced colitis and others have demonstrated an antiinflammatory role for IL-11 in intestinal inflammation. Here we examine the effects of IL11 on the normal colon and establish that IL-11 is upstream of Bmp1 and IL-33, and that it regulates mucosal proliferation via Reg3β in a STAT3-dependent manner. MethodsNormal mice were treated with 5 ug of recombinant human IL-11 (rhIL-11) every 6 hours and culled either 6 hours, 24 hours of 5 days after the initial dose. Colons were removed for mRNA and protein analysis. ResultsIL-11 is a ligand for gp130 and should initiate both the Ras/MAPK/ERK and STAT1/STAT3 signalling pathways. Chronic IL-11 treatment of normal mice activates STAT3 (Tyr705) but not ERK1/2 (Thr202/Tyr204), and acute IL-11 caused a drop in ERK1/2 activation. This is a very similar to the signalling outcome downstream of IL-11 in the gastric mucosa, where signalling emanating from gp130 activation by IL-11 is skewed towards STAT3. Expression of SOCS3, a bona fide STAT3 responsive gene, is also increased in the colon following IL-11 treatment. In the gastric antrum IL-11 treatment is pro-proliferative. The same is true of the colonic response to IL-11 with a 1.6 fold increase in the expression of the proliferation marker PCNA by immunoblot. IL-11 also caused an increase in the expression of the pro-proliferative gene Reg3β and the mRNAs involved in BMP signalling Bmp1 and Bmp1rb. However, most significantly IL-11 treatment caused a large and significant increase in the expression of IL-33 and other mediators of the innate immune response in the absence of any significant damage to the mucosa. ConclusionsColonic tissue responds to IL-11 largely by activation of STAT3 signalling. There are three major outcomes resulting from this signalling; 1) increased proliferation and expression of pro-proliferative genes, 2) increased expression of components of BMP signalling and 3) activation of IL-33 and the innate immune system.