Interleukin-33 (IL-33) is an IL-1 family cytokine known to promote T-helper (Th) type 2 immune responses that are often deregulated in gastric cancer (GC). IL-33 is overexpressed in human gastric tumours suggesting a role in driving GC progression although a causal link has not been proven. Here, we investigated the impact of IL-33 genetic deficiency in the well-characterized gp130F/F mouse model of GC. Expression of IL-33 (and it's cognate receptor, ST2) was increased in human and mouse GC progression. IL-33 deficient gp130F/F/Il33-/- mice had reduced gastric tumour growth and reduced recruitment of pro-tumorigenic myeloid cells including key mast cell subsets and type-2 (M2) macrophages. Cell sorting of gastric tumours revealed that IL-33 chiefly localized to gastric (tumour) epithelial cells and was absent from tumour-infiltrating immune cells (except modest IL-33 enrichment within CD11b+ CX3CR1+CD64+MHCII+ macrophages). By contrast, ST2 was absent from gastric epithelial cells and localized exclusively within the (non-macrophage) immune cell fraction together with mast cell markers, Mcpt1 and Mcpt2. Collectively, we show that IL-33 is required for gastric tumour growth and provide evidence of a likely mechanism by which gastric epithelial-derived IL-33 drives mobilization of tumour-promoting inflammatory myeloid cells.

Gastrokines (GKNs) are anti-inflammatory proteins secreted by gastric epithelial (surface mucous and pit) cells, with their aberrant loss of expression causally linked to premalignant inflammation and gastric cancer (GC). Transcriptional mechanisms accounting for GKN expression loss have not been elucidated. Using human clinical cohorts, mouse transgenics, bioinformatics, and transfection/reporter assays, we report a novel mechanism of GKN gene transcriptional regulation and its impairment in GC. GKN1/GKN2 loss is highly coordinated, with both genes showing parallel downregulation during human and mouse GC development, suggesting joint transcriptional control. In BAC transgenic studies, we defined a 152-kb genomic region surrounding the human GKN1/GKN2 genes sufficient to direct their tissue- and lineage-restricted expression. A screen of the 152-kb region for candidate regulatory elements identified a DNase I hypersensitive site (CR2) located 4 kb upstream of the GKN1 gene. CR2 showed overlapping enrichment of enhancer-related histone marks (H3K27Ac), a consensus binding site (GRE) for the glucocorticoid receptor (GR), strong GR occupancy in ChIP-seq data sets and, critically, exhibited dexamethasone-sensitive enhancer activity in reporter assays. Strikingly, GR showed progressive expression loss, paralleling that of GKN1/2, in human and mouse GC, suggesting desensitized glucocorticoid signaling as a mechanism underlying GKN loss. Finally, mouse adrenalectomy studies revealed a critical role for endogenous glucocorticoids in sustaining correct expression (and anti-inflammatory restraint) of GKNs in vivo. Together, these data link the coordinate expression of GKNs to a glucocorticoid-responsive and likely shared transcriptional enhancer mechanism, with its compromised activation contributing to dual GKN loss during GC progression. NEW & NOTEWORTHY Gastrokine 2 (GKN2) is an anti-inflammatory protein produced by the gastric epithelium. GKN2 expression is progressively lost during gastric cancer (GC), which is believed to play a casual role in GC development. Here, we use bacterial artificial chromosome transgenic studies to identify a glucocorticoid-responsive enhancer element that likely governs expression of GKN1/GKN2, which, via parallel expression loss of the anti-inflammatory glucocorticoid receptor, reveals a novel mechanism to explain the loss of GKN2 during GC pathogenesis.

Expression of the cytokine IL-11 is elevated in human Helicobacter pylori infection and progressively increases with worsening gastric pathology. Additionally, IL-11 is required for tumor development in STAT3-dependent murine models of gastric cancer (GC) and, when administered acutely, causes resolving atrophic gastritis. However, it is unclear whether locally elevated IL-11 ligand expression can, in isolation from oncogenic gp130-JAK-STAT pathway mutations, initiate GC pathogenesis. Here we developed a transgenic mouse model of stomach-specific (keratin 19 promoter) IL-11 ligand overexpression. Keratin 19 promoter-IL-11 transgenic ( K19-IL11Tg) mice showed specific IL-11 overexpression in gastric corpus and antrum but not elsewhere in the gastrointestinal tract or in other tissues. K19-IL11Tg mice developed spontaneous premalignant disease of the gastric epithelium, progressing from atrophic gastritis to TFF2-positive metaplasia and severe epithelial hyperplasia, including adenoma-like lesions in a subset of older (1 yr old) animals. Although locally advanced, the hyperplastic lesions remained noninvasive. H. pylori infection in K19-IL11Tg mice accelerated some aspects of the premalignant phenotype. Finally, K19-IL11Tg mice had splenomegaly in association with elevated serum IL-11, with spleens showing an expanded myeloid compartment. Our results provide direct in vivo functional evidence that stomach-specific overexpression of IL-11, in isolation from germline gp130-JAK-STAT3 genetic drivers, is sufficient for premalignant progression. These findings have important functional implications for human GC, in which frequent IL-11 overexpression occurs in the reported absence of somatic mutations in gp130 signaling components. NEW & NOTEWORTHY We provide direct in vivo functional evidence that stomach-specific overexpression of the cytokine IL-11, in isolation from gp130-JAK-STAT3 pathway mutations, can trigger spontaneous atrophic gastritis progressing to locally advanced epithelial hyperplasia (but not dysplasia or carcinoma), which does not require, but may be accelerated by, concomitant Helicobacter pylori infection.

Chronic mucosal inflammation is associated with a greater risk of gastric cancer (GC) and, therefore, requires tight control by suppressive counter mechanisms. Gastrokine-2 (GKN2) belongs to a family of secreted proteins expressed within normal gastric mucosal cells. GKN2 expression is frequently lost during GC progression, suggesting an inhibitory role; however, a causal link remains unsubstantiated. Here, we developed Gkn2 knockout and transgenic overexpressing mice to investigate the functional impact of GKN2 loss in GC pathogenesis. In mouse models of GC, decreased GKN2 expression correlated with gastric pathology that paralleled human GC progression. At baseline, Gkn2 knockout mice exhibited defective gastric epithelial differentiation but not malignant progression. Conversely, Gkn2 knockout in the IL-11/STAT3-dependent gp130F/F GC model caused tumorigenesis of the proximal stomach. Additionally, gastric immunopathology was accelerated in Helicobacter pylori-infected Gkn2 knockout mice and was associated with augmented T helper cell type 1 (Th1) but not Th17 immunity. Heightened Th1 responses in Gkn2 knockout mice were linked to deregulated mucosal innate immunity and impaired myeloid-derived suppressor cell activation. Finally, transgenic overexpression of human gastrokines (GKNs) attenuated gastric tumor growth in gp130F/F mice. Together, these results reveal an antiinflammatory role for GKN2, provide in vivo evidence that links GKN2 loss to GC pathogenesis, and suggest GKN restoration as a strategy to restrain GC progression.

BACKGROUND & AIMS: During aging, physiological changes in the stomach result in more tenuous gastric tissue that is less capable of repairing injury, leading to increased susceptibility to chronic ulceration. Spasmolytic polypeptide/trefoil factor 2-expressing metaplasia (SPEM) is known to emerge after parietal cell loss and during Helicobacter pylori infection, however, its role in gastric ulcer repair is unknown. Therefore, we sought to investigate if SPEM plays a role in epithelial regeneration.METHODS: Acetic acid ulcers were induced in young (2-3 mo) and aged (18-24 mo) C57BL/6 mice to determine the quality of ulcer repair with advancing age. Yellow chameleon 3.0 mice were used to generate yellow fluorescent protein-expressing organoids for transplantation. Yellow fluorescent protein-positive gastric organoids were transplanted into the submucosa and lumen of the stomach immediately after ulcer induction. Gastric tissue was collected and analyzed to determine the engraftment of organoid-derived cells within the regenerating epithelium.RESULTS: Wound healing in young mice coincided with the emergence of SPEM within the ulcerated region, a response that was absent in the aged stomach. Although aged mice showed less metaplasia surrounding the ulcerated tissue, organoid-transplanted aged mice showed regenerated gastric glands containing organoid-derived cells. Organoid transplantation in the aged mice led to the emergence of SPEM and gastric regeneration.CONCLUSIONS: These data show the development of SPEM during gastric repair in response to injury that is absent in the aged stomach. In addition, gastric organoids in an injury/transplantation mouse model promoted gastric regeneration.

STAT3 regulates the expansion of myeloid-derived suppressor cells (MDSCs) during inflammation, infection and cancer. Hyperactivation of STAT3 in gp130 757F/F mice is associated with protection from experimental colitis. This study determined mechanisms for this protection and compared this to mice with myeloid-specific STAT3-deficiency (LysMcre/STAT3 flox ; gp130 757F/F LysMcre/STAT3 flox ). Acute and chronic colitis was induced and colons were removed for histological, mRNA and protein analysis. Cell populations from spleen, mesenteric lymph node and colon were analyzed for different myeloid cell populations using flow cytometry. Functions of MDSCs and LPS-stimulated peritoneal macrophages were further characterized by in vitro and in vivo assays. Here we show that the resistance to experimental colitis in gp130 757F/F mice is via myeloid-cell specific STAT3 activation, MDSC expansion and increased production of suppressive and protective cytokines.

IL-1 is key driver of gastric tumorigenesis and is a downstream target of IL-11 signaling. Recently, IL-1 cytokines, particularly IL-1 beta, have been flagged as therapeutic targets for gastric cancer treatment. Here, we assess the requirement for IL-1 signaling in gastric tumorigenesis. gp130(757FF) xIL-1RT1(-/-) mice were generated to determine the pathological consequence of ablated IL-1 signaling in the IL-11 dependent gp130(757FF) mouse model of gastric tumorigenesis. Gastric lesions in gp130(757FF) xIL-1RT1(-/-) mice were increased in incidence and size compared to gp130(757FF) mice. Proximal gastric lesions originated from the cardiac region and were associated with elevated STAT3 activation, loss of specialized gastric cells and a modulated immune response including increased expression of TNF-alpha and MDSC associated genes. Administration of IL-11 to IL-1RT1(-/-) mice showed similar changes to gp130(757FF) xIL-1RT1(-/-) mice. Spleens from IL-11 treated wildtype mice showed an enrichment of MDSC and gp130(757FF) xIL-1RT1(-/-) mice had increased MDSCs in the stomach compared to gp130(757FF) mice. Furthermore, crossing TNF-alpha(-/-) to gp130(757FF) mice resulted in reduced lesion size. We conclude that IL-1 signaling antagonizes IL-11/STAT3 mediated pathology and the genetic deletion of IL-1RT1 results in increased tumor burden. We provide evidence that a likely mechanism is due to IL-11/STAT3 dependent enrichment of MDSCs.

STAT3 imparts a profound influence on both the epithelial and immune components of the gastric mucosa, and through regulation of key intracellular signal transduction events, is well placed to control inflammatory and oncogenic outcomes in the context of Helicobacter (H.) pylori infection. Here we review the roles of STAT3 in the host immune response to H. pylori infection, from both gastric mucosal and systemic perspectives, as well as alluding more specifically to STAT3-dependent mechanisms that might be exploited as drug targets.

Meeting abstractIntestinal monocytes/macrophages sustain the intestinal immune homeostasis and might be an attractive therapeutic target for the management of inflammatory bowel disease (IBD). Granulocyte macrophage colony-stimulating factor (GM-CSF) exerts beneficial effects in intestinal inflammation and promotes STAT3-mediated expansion of myeloid-derived suppressor cells (MDSCs). We explored whether GM-CSF mediates its beneficial effects in IBD via myeloid regulatory cells (Mreg).Here we show that GM-CSF i) provokes non-classical monocyte activation; ii) drives monocytes towards an anti-inflammatory phenotype; iii) enhances innate immune functions; iv) primes monocyte responses to secondary microbial stimuli; and v) accelerates epithelial healing via monocytes. GM-CSF-activated monocytes (GMaM) show therapeutic activity in T cell-induced colitis in Rag1-/- mice with increased production of IL-4, IL-10, IL-13 and decreased production of IFNγ in LPMCs. Confirming this finding, GMaM attract T cells and shape their differentiation towards Th2 cells in vitro. In addition, GMaM induce regulatory (Foxp3+) T cells (Treg) in vitro and adoptive transfer of GMaM in chronic DSS-induced colitis ameliorates disease in vivo with accelerated gut homing of GMaM and induction of colonic Treg. Myeloid-cell specific STAT3 activation protects gp130757F/F mice from colitis via MDSC expansion and increased production of suppressive and protective cytokines. LysMcre/STAT3flox mice with myeloid-specific STAT3-deficiency show opposite effects and are not protected from colitis. Additionally, MDSCs of gp130757F/F mice produce significantly more IL-4, IL-10 and IL-13.In summary, beneficial effects of GM-CSF in IBD may possibly be mediated through reprogramming of monocytes and MDSCs via enhanced innate immune functions as well as regulation of adaptive immunity. Our findings support the exploration of stimulating rather than suppressive therapies for patients with IBD and underpin that Mreg might become a promising novel cell-based therapeutic option.

Objectives The mucin MUC1, best known for providing an epithelial barrier, is an important protective host factor in both humans and mice during Helicobacter pylori pathogenesis. This study aimed to identify the long-term consequences of MUC1 deficiency on H. pylori pathogenesis and the mechanism by which MUC1 protects against H. pylori gastritis.Design Wildtype and Muc1(-/-) mice were infected for up to 9 months, and the gastric pathology, immunological response and epigenetic changes assessed. The effects of MUC1 on the inflammasome, a potent inflammatory pathway, were examined in macrophages and H. pylori-infected mice deficient in both MUC1 and inflammasome components.Results Muc1(-/-) mice began to die 6 months after challenge, indicating Muc1 deficiency made H. pylori a lethal infection. Surprisingly, chimaeric mouse infections revealed MUC1 expression by haematopoietic-derived immune cells limits H. pylori-induced gastritis. Gastritis in infected Muc1(-/-) mice was associated with elevated interleukin (IL)-1 beta and epigenetic changes in their gastric mucosa similar to those in transgenic mice overexpressing gastric IL-1 beta, implicating MUC1 regulation of an inflammasome. In support of this, infected Muc1(-/-) Casp1(-/-) mice did not develop severe gastritis. Further, MUC1 regulated Nlrp3 expression via an nuclear factor (NF)-kappa B-dependent pathway and reduced NF-kappa B pathway activation via inhibition of IRAK4 phosphorylation. The importance of this regulation was proven using Muc1(-/-) Nlrp3(-/-) mice, which did not develop severe gastritis.Conclusions MUC1 is an important, previously unidentified negative regulator of the NLRP3 inflammasome. H. pylori activation of the NLRP3 inflammasome is normally tightly regulated by MUC1, and loss of this critical regulation results in the development of severe pathology.

The stem cells that maintain and repair the postnatal skeleton remain undefined. One model suggests that perisinusoidal mesenchymal stem cells (MSCs) give rise to osteoblasts, chondrocytes, marrow stromal cells, and adipocytes, although the existence of these cells has not been proven through fate-mapping experiments. We demonstrate here that expression of the bone morphogenetic protein (BMP) antagonist gremlin 1 defines a population of osteochondroreticular (OCR) stem cells in the bone marrow. OCR stem cells self-renew and generate osteoblasts, chondrocytes, and reticular marrow stromal cells, but not adipocytes. OCR stem cells are concentrated within the metaphysis of long bones not in the perisinusoidal space and are needed for bone development, bone remodeling, and fracture repair. Grem1 expression also identifies intestinal reticular stem cells (iRSCs) that are cells of origin for the periepithelial intestinal mesenchymal sheath. Grem1 expression identifies distinct connective tissue stem cells in both the bone (OCR stem cells) and the intestine (iRSCs).

Altered DNA methylation is a hallmark of cancer genomes and has been advocated as both a marker and mechanism of the malignant phenotype. In the conventional view, such alterations include global DNA hypomethylation, linked to aberrant expression of oncogenic driver genes and more localised de novo hypermethylation at ‘CpG-island’ promoters, argued to cause inappropriate silencing of tumor suppressor genes. Although paradigms like these have been unquestionably influential, deregulation of the epigenome is not the only source of altered DNA methylation in tumors. Cell lineage and tissue composition have recently emerged as additional determinants of cancer-specific methylation patterns [1, 2]. During differentiation, cell lineages acquire stable and heritable gene expression programs associated with characteristic epigenetic marks, including DNA methylation. Lineage-specific DNA methylation profiles can therefore serve as ‘epigenetic fingerprints’ of cellular identity, suggesting their application as diagnostic biomarkers for diverse cell types. Screening for subliminal lineage signatures concealed within ‘DNA methylomes’ may allow simultaneous detection and enumeration of multiple cell types in complex tissues [3], including cancers [2]. Indeed, methylation signatures reflecting the presence of key prognostic indicators, such as T lymphocytes, have already shown immense promise in predicting clinical outcome of cancers and response to therapy [2]. Gastric cancer (GC), which has one of the highest malignancy-related mortality rates globally, arises through stepwise, inflammation-dependent transformation of gastric epithelial cells. Improved detection of the premalignant inflammatory stages of GC may facilitate earlier intervention and better outcomes [4]. In a recent study by Kurklu et al. [5] we described an aberrant hypomethylation signature at the RUNX3 locus, associated with GC progression, specifically within an upstream alternative promoter (RUNX3 P1). We found that, based on the degree of RUNX3 P1 hypomethylation, premalignant gastric epithelial lesions and tumors could be segregated from disease-free tissues. This novel signature may therefore be diagnostic for premalignant inflammation in individuals who are, by definition, at risk of developing GC. Importantly, our analysis utilised whole tissues/tumors comprised primarily by cancer epithelial cells, but also by non-neoplastic cell types including leukocytes. It had not escaped our attention that RUNX3, a key regulator of haematopoiesis, is highly expressed in several immune cell lineages [6]. We therefore hypothesised that enrichment of RUNX3 P1 hypomethylated alleles in these tissues might reflect leukocyte recruitment to the premalignant epithelium and/or tumor microenvironment. DNA methylation analysis of epithelial and immune cell lineages independently isolated directly from gastric tumors confirmed this expectation; RUNX3 P1 was hypermethylated and repressed in the epithelial cells but, critically, lacked methylation and was transcriptionally active in all the isolated immune cell types tested. Therefore, RUNX3 P1 hypomethylation, as a ‘pan’-marker of haematopoietic lineages, can distinguish progressive changes in cellular composition that accompany GC pathogenesis, namely those due to leukocyte infiltration (Figure ​(Figure11). Figure 1 RUNX3 P1 methylation levels reflect leukocyte infiltration during GC progression A key question not addressed in our study is whether RUNX3 P1 methylation, as a surrogate of leukocyte influx, might classify gastric tumors into useful prognostic categories? As a case in point, lymphocyte-rich GC generally has a better prognosis than GC lacking significant lymphocytic infiltrate [7]. Intra-tumoral lymphocytes often display cytolytic properties positively linked to survival, hence defining specific methylation signatures of cancers that elicit this type of anti-tumor response would be of high translational priority. The value of such an approach has been shown to great effect in breast cancer. Dedeurwaerder et al. described a multi-gene methylation signature that could not only discern the degree of tumor infiltration by T lymphocytes, but was also strongly prognostic for relapse-free survival [2]. They concluded that their signature was likely indicative of an anti-tumor T cell response. While the jury is still out, RUNX3 P1 methylation has links with key mediators of anti-tumor responses that warrant exploration of its use in similar context. For example, RUNX3 P1 hypomethylation is a feature of cytolytic CD8+ T lymphocytes and natural killer cells (NK) [5], correlating with both the high RUNX3 mRNA expression of these two cell types and genetic requirement of RUNX3 for their intrinsic ‘tumor killing’ functions [6]. Nevertheless, we caution against over interpretation of these observations, as RUNX3 P1 hypomethylation also marks other immune lineages that can elicit both pro- and anti-tumor effects. Ultimately, whether RUNX3 P1 methylation can reveal the specific polarity of cancer immune responses, used alone or in combination, awaits formal validation in appropriately powered clinical studies. We are not the first to study RUNX3 methylation as a cancer biomarker. Aberrant hypermethylation of the canonical RUNX3 CpG island promoter (RUNX3 P2) hitherto proposed [8], but since refuted as a driver of GC has been avidly pursued as a biomarker, albeit with limited success (reviewed in [6]). However, our analysis of RUNX3 P1 is unique in the field, providing fresh perspectives on cell lineage as a modifier of cancer methylomes and further highlighting its potential for translation in GC as well as other malignancies in which leukocyte infiltration has diagnostic and prognostic significance.

BACKGROUND & AIMS: Interleukin (IL)(33) is a recently described alarmin that is highly expressed in the gastric mucosa and potently activates Th2 immunity. It may play a pivotal role during Helicobacter pylori infection. Here, we delineate the role of IL33 in the normal gastric mucosa and in response to gastropathy.METHODS: IL33 expression was evaluated in mice and human biopsy specimens infected with H pylori and in mice after dosing with aspirin. IL33 expression was localized in the gastric mucosa using immunofluorescence. Mice were given 1 or 7 daily doses of recombinant IL33 (1 mu g/dose), and the stomach and the spleen responses were quantified morphologically, by flow cytometry and using quantitative reverse-transcription polymerase chain reaction and immunoblotting.RESULTS: In mice, the IL33 protein was localized to the nucleus of a subpopulation of surface mucus cells, and colocalized with the surface mucus cell markers Ulex Europaeus 1 (UEA1), and Mucin 5AC (Muc5AC). A small proportion of IL33-positive epithelial cells also were Ki-67 positive. IL33 and its receptor Interleukin 1 receptor-like 1 (ST2) were increased 4-fold after acute (1-day) H pylori infection, however, this increase was not apparent after 7 days and IL33 expression was reduced 2-fold after 2 months. Similarly, human biopsy specimens positive for H pylori had a reduced IL33 expression. Chronic IL33 treatment in mice caused systemic activation of innate lymphoid cell 2 and polarization of macrophages to the M2 phenotype. In the stomach, IL33-treated mice developed transmural inflammation and mucous metaplasia that was mediated by Th2/signal transducer and activator of transcription 3 signaling. Rag-1(-/-) mice, lacking mature lymphocytes, were protected from IL33-induced gastric pathology.CONCLUSIONS: IL33 is highly expressed in the gastric mucosa and promotes the activation of T helper 2-cytokine-expressing cells. The loss of IL33 expression after prolonged H pylori infection may be permissive for the T helper 1-biased immune response observed during H pylori infection and subsequent precancerous progression.

We tested whether the T helper (Th) type 2 (Th2) cell agonist and allergenic ligand IL-33 was associated with eosinophilic esophagitis (EoE) development in a pediatric cohort and whether IL-33 protein could induce disease symptoms in mice. Biopsies from EoE patients or controls were used to measure IL-33 mRNA and protein expression. Increased expression of IL-33 mRNA was found in the esophageal mucosa in EoE. IL-33 protein was detected in cells negative for CD45, mast cells, and epithelial cell markers near blood vessels. Circulating levels of IL-33 were not increased. The time course for IL-33 gene expression was quantified in an established Aspergillus fumigatus allergen mouse model of EoE. Because IL-33 induction was transient in this model and chronicity of IL-33 expression has been demonstrated in humans, naive mice were treated with recombinant IL-33 for 1 wk and esophageal pathology was evaluated. IL-33 application produced changes consistent with phenotypically early EoE, including transmural eosinophilia, mucosal hyperproliferation, and upregulation of eosinophilic genes and chemokines. Th2 cytokines, including IL-13, along with innate lymphoid cell group 2, Th1/17, and M2 macrophage marker genes, were increased after IL-33 application. IL-33-induced eosinophilia was ablated in IL-13 null mice. In addition, IL-33 induced a profound inhibition of the regulatory T cell gene signature. We conclude that IL-33 gene expression is associated with pediatric EoE development and that application of recombinant protein in mice phenocopies the early clinical phase of the human disease in an IL-13-dependent manner. IL-33 inhibition of esophageal regulatory T cell function may induce loss of antigenic tolerance, thereby providing a mechanistic rationale for EoE development.

STAT3 imparts a profound influence on both the epithelial and immune components of the gastric mucosa, and through regulation of key intracellular signal transduction events, is well placed to control inflammatory and oncogenic outcomes in the context of Helicobacter (H.) pylori infection. Here we review the roles of STAT3 in the host immune response to H. pylori infection, from both gastric mucosal and systemic perspectives, as well as alluding more specifically to STAT3-dependent mechanisms that might be exploited as drug targets.

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 gp130(757FF) 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 gp130(757FF) 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.

Runt domain transcription factor 3 (RUNX3) is widely regarded as a tumour-suppressor gene inactivated by DNA hypermethylation of its canonical CpG (cytidine-phosphate-guanidine) island (CGI) promoter in gastric cancer (GC). Absence of RUNX3 expression from normal gastric epithelial cells (GECs), the progenitors to GC, coupled with frequent RUNX3 overexpression in GC progression, challenge this longstanding paradigm. However, epigenetic models to better describe RUNX3 deregulation in GC have not emerged. Here, we identify lineage-specific DNA methylation at an alternate, non-CGI promoter (P1) as a new mechanism of RUNX3 epigenetic control. In normal GECs, P1 was hypermethylated and repressed, whereas in immune lineages P1 was hypomethylated and widely expressed. In human GC development, we detected aberrant P1 hypomethylation signatures associated with the early inflammatory, preneoplastic and tumour stages. Aberrant P1 hypomethylation was fully recapitulated in mouse models of gastric inflammation and tumorigenesis. Cell sorting showed that P1 hypomethylation reflects altered cell-type composition of the gastric epithelium/tumour microenvironment caused by immune cell recruitment, not methylation loss. Finally, via long-term culture of gastric tumour epithelium, we revealed that de novo methylation of the RUNX3 canonical CGI promoter is a bystander effect of oncogenic immortalization and not likely causal in GC pathogenesis as previously argued. We propose a new model of RUNX3 epigenetic control in cancer, based on immune-specific, non-CGI promoter hypomethylation. This novel epigenetic signature may have utility in early detection of GC and possibly other epithelial cancers with premalignant immune involvement.

tumor cells.These findings provide evidence for a novel matrix remodeling process conducted by mesenchymal cells that is substantially more effective than conventional invadopodia, distinct in structural organization, independent of the classical invadopodial machinery, and capable of promoting invasion by pancreatic tumor cells.