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IL11 is a member of the IL6 family of cytokines and signals through its cognate receptor subunits, IL11RA and glycoprotein 130 (GP130), to elicit biological responses via the JAK/STAT signaling pathway. IL11 contributes to cancer progression by promoting the survival and proliferation of cancer cells, but the potential immunomodulatory properties of IL11 signaling during tumor development have thus far remained unexplored. Here, we have characterized a role for IL11 in regulating CD4+ T cell–mediated antitumor responses. Absence of IL11 signaling impaired tumor growth in a sporadic mouse model of colon cancer and syngeneic allograft models of colon cancer. Adoptive bone marrow transfer experiments and in vivo depletion studies demonstrated that the tumor-promoting activity of IL11 was mediated through its suppressive effect on host CD4+ T cells in the tumor microenvironment. Indeed, when compared with Il11ra-proficient CD4+ T cells associated with MC38 tumors, their Il11ra-deficient counterparts displayed elevated expression of mRNA encoding the antitumor mediators IFNγ and TNFα. Likewise, IL11 potently suppressed the production of proinflammatory cytokines (IFNγ, TNFα, IL6, and IL12p70) by CD4+ T cells in vitro, which we corroborated by RNAscope analysis of human colorectal cancers, where IL11RAhigh tumors showed less IFNG and CD4 expression than IL11RAlow tumors. Therefore, our results ascribe a tumor cell–extrinsic immunomodulatory role to IL11 during colon cancer development that could be amenable to an anticytokine-based therapy.See related Spotlight by van der Burg, p. 724.

Increased HCK activity in BMDMs supports tumor cell invasion in vitro.

Persistent activation of the latent transcription factor STAT3 is observed in gastric tumor epithelial and immune cells and is associated with a poor patient prognosis. Although targeting STAT3-activating upstream kinases offers therapeutically viable targets with limited specificity, direct inhibition of STAT3 remains challenging. Here we provide functional evidence that myeloid-specific hematopoietic cell kinase (HCK) activity can drive STAT3-dependent epithelial tumor growth in mice and is associated with alternative macrophage activation alongside matrix remodeling and tumor cell invasion. Accordingly, genetic reduction of HCK expression in bone marrow–derived cells or systemic pharmacologic inhibition of HCK activity suppresses alternative macrophage polarization and epithelial STAT3 activation, and impairs tumor growth. These data validate HCK as a molecular target for the treatment of human solid tumors harboring excessive STAT3 activity.

Constitutive HCK activity in bone marrow-derived cells enhances STAT3 activation in tumors and tumor-associated macrophages.

Increased expression of genes associated with alternative macrophage polarization confers a poorer overall survival in human intestinal-type gastric cancer patients.

Supplementary Figure 1. Gating strategy and representative flow cytometric plots of MC38 tumors from WT and Il11ra-/- hosts. Supplementary Figure 2. IL-11 has no effect on tumor-infiltrating immune cells in MC38 and CT26 tumors from WT and Il11ra-/- hosts. Supplementary Figure 3. IL-11 has no effect on PMA/Iono-induced IFNγ and TNFα production by IL-11RA-deficient CD4+ T cells. Supplementary Figure 4. Single cell profiling of IL11RA and IFNG expression in intra-tumoral CD4+ T cells from colon cancer patients.

Supplementary Table 1

Excessive HCK activity in bone marrow-derived cells promotes STAT3-dependent gastric tumor development and invasion.

Fig S2. MC38 tumor growth is enhanced in St2-deficient hosts.

Suppl Table 1. Oligonucleotide Sequences

Additional supportive data is provided as Fig S1 (Generation and validation of Tg(Tff1-CreERT2)mice), Fig. S2 (Tff1-CreERT2 transgene, endogenous Tff1, and Lgr5 expression in the stomach), Fig. S3 (Tff1-CreERT2-mediated I2-galactosidase reporter activity in various organs), Fig. S4 (KrasG12D-induced gastric tumorigenesis is associated with aberrant mucous production, ectopic expression of intestinal genes and immune cell infiltration), Fig. S5 (Gastric-specific activation of oncogenic BrafV600E triggers tumorigenesis associated with increased Erk and Stat3 phosphorylation), Fig. S6 (Tff1-CreERT2 activity in established gastric tumors of gp130F/F mice), Fig. S7 (Co-activation of Kras and gp130 signalling induces intestinalization associated with severe atrophy) and Table S1 (Incidence and onset of gastric tumor formation).

About 5% to 10% of human gastric tumors harbor oncogenic mutations in the KRAS pathway, but their presence alone is often insufficient for inducing gastric tumorigenesis, suggesting a requirement for additional mutagenic events or microenvironmental stimuli, including inflammation. Assessing the contribution of such events in preclinical mouse models requires Cre recombinase–mediated conditional gene expression in stem or progenitor cells of normal and transformed gastric epithelium. We therefore constructed a bacterial artificial chromosome containing transgene (Tg), comprising the regulatory elements of the trefoil factor 1 (Tff1) gene and the tamoxifen-inducible Cre recombinase (CreERT2)–coding sequence. The resulting Tg(Tff1-CreERT2) mice were crossed with mice harboring conditional oncogenic mutations in Kras or Braf. The administration of tamoxifen to the resulting adult Tg(Tff1-CreERT2);KrasLSL-G12D/+ and Tg(Tff1-CreERT2);BrafLSL-V600E/+ mice resulted in gastric metaplasia, inflammation, and adenoma development, characterized by excessive STAT3 activity. To assess the contribution of STAT3 to the spontaneously developing gastric adenomas in gp130F/F mice, which carry a knockin mutation in the Il6 signal transducer (Il6st), we generated Tg(Tff1-CreERT2);Stat3fl/fl;gp130F/F mice that also harbor a conditional Stat3 knockout allele and found that tamoxifen administration conferred a significant reduction in their tumor burden. Conversely, excessive Kras activity in Tg(Tff1-CreERT2);KrasLSL-G12D/+;gp130F/F mice promoted more extensive gastric inflammation, metaplastic transformation, and tumorigenesis than observed in Tg(Tff1-CreERT2);KrasLSL-G12D/+ mice. Collectively, our findings demonstrate that advanced gastric tumorigenesis requires oncogenic KRAS or BRAF in concert with aberrant STAT3 activation in epithelial precursor cells of the glandular stomach, providing a new conditional model of gastric cancer in which to investigate candidate therapeutic targets and treatment strategies. Cancer Res; 76(8); 2277–87. ©2016 AACR.

Supplementary Figure Legends

IL11 is a member of the IL6 family of cytokines and signals through its cognate receptor subunits, IL11RA and glycoprotein 130 (GP130), to elicit biological responses via the JAK/STAT signaling pathway. IL11 contributes to cancer progression by promoting the survival and proliferation of cancer cells, but the potential immunomodulatory properties of IL11 signaling during tumor development have thus far remained unexplored. Here, we have characterized a role for IL11 in regulating CD4+ T cell–mediated antitumor responses. Absence of IL11 signaling impaired tumor growth in a sporadic mouse model of colon cancer and syngeneic allograft models of colon cancer. Adoptive bone marrow transfer experiments and in vivo depletion studies demonstrated that the tumor-promoting activity of IL11 was mediated through its suppressive effect on host CD4+ T cells in the tumor microenvironment. Indeed, when compared with Il11ra-proficient CD4+ T cells associated with MC38 tumors, their Il11ra-deficient counterparts displayed elevated expression of mRNA encoding the antitumor mediators IFNγ and TNFα. Likewise, IL11 potently suppressed the production of proinflammatory cytokines (IFNγ, TNFα, IL6, and IL12p70) by CD4+ T cells in vitro, which we corroborated by RNAscope analysis of human colorectal cancers, where IL11RAhigh tumors showed less IFNG and CD4 expression than IL11RAlow tumors. Therefore, our results ascribe a tumor cell–extrinsic immunomodulatory role to IL11 during colon cancer development that could be amenable to an anticytokine-based therapy. See related Spotlight by van der Burg, p. 724.

Persistent activation of the latent transcription factor STAT3 is observed in gastric tumor epithelial and immune cells and is associated with a poor patient prognosis. Although targeting STAT3-activating upstream kinases offers therapeutically viable targets with limited specificity, direct inhibition of STAT3 remains challenging. Here we provide functional evidence that myeloid-specific hematopoietic cell kinase (HCK) activity can drive STAT3-dependent epithelial tumor growth in mice and is associated with alternative macrophage activation alongside matrix remodeling and tumor cell invasion. Accordingly, genetic reduction of HCK expression in bone marrow–derived cells or systemic pharmacologic inhibition of HCK activity suppresses alternative macrophage polarization and epithelial STAT3 activation, and impairs tumor growth. These data validate HCK as a molecular target for the treatment of human solid tumors harboring excessive STAT3 activity.

Gastric cancer (GC) is the 3rd leading cause of cancer mortality worldwide, therefore providing novel diagnostic and treatment options is crucial for at risk groups. The serine/threonine kinase doublecortin-like kinase 1 (DCLK1) is a proposed driver of GC with frequent amplification and somatic missense mutations yet the molecular mechanism how DCLK1 mediates tumorigenesis is poorly understood. We report how DCLK1 expression orchestrates complementary cancer cell intrinsic and extrinsic processes leading to a comprehensive pro-invasive and pro-metastatic reprogramming of cancer cells and tumor stroma in a DCLK1 kinase-dependent manner. Mechanistically, we identify the chemokine CXCL12 as a key promoter of the pro-tumorigenic properties downstream of DCLK1. Importantly, inhibition of the DCLK1 kinase domain reverses the pro-tumorigenic and pro-metastatic phenotype. Together, this study establishes DCLK1 as a promising, targetable master regulator of GC.TeaserDCLK1 is a druggable cancer driver of GC

Although gastric cancer is a leading cause of cancer-related deaths, systemic treatment strategies remain scarce. Here we explore a metabolite-triggered circuit between epithelial tuft cells and innate lymphoid type 2 cells (ILC2) that is evolutionarily optimized for intestinal remodeling in response to helminth infection. We demonstrate that tuft cell-derived interleukin 25 (IL25) acts as an alarmin on ILC2s to induce the release of IL13 as a growth factor for tuft cells, and propose that this model drives early metaplastic remodeling and gastric tumor formation. Genetic ablation of tuft cells, ILC2s or antibody-mediated neutralization of IL13 or IL25 reduces the growth of established tumors. Thus, the tuft cell/ILC2 axis provides an opportunity to therapeutically inhibit preneoplastic lesions and early-stage gastric cancer through repurposing of antibody-mediated therapies.One-Sentence SummaryTuft cells and type 2 innate lymphoid cells offer a new therapeutic target in gastric disease.

Ets homologous factor (EHF) is a member of the epithelial-specific Ets (ESE) family of transcription factors. To investigate its role in development and epithelial homeostasis, we generated a series of novel mouse strains in which the Ets DNA-binding domain of Ehf was deleted in all tissues (Ehf−/−) or specifically in the gut epithelium. Ehf−/− mice were born at the expected Mendelian ratio, but showed reduced body weight gain, and developed a series of pathologies requiring most Ehf−/− mice to reach an ethical endpoint before reaching 1 year of age. These included papillomas in the facial skin, abscesses in the preputial glands (males) or vulvae (females), and corneal ulcers. Ehf−/−mice also displayed increased susceptibility to experimentally induced colitis, which was confirmed in intestinal-specific Ehf knockout mice. Gut-specific Ehf deletion also impaired goblet cell differentiation, induced extensive transcriptional reprogramming in the colonic epithelium and enhanced Apc-initiated adenoma development. The Ets DNA-binding domain of EHF is therefore essential for postnatal homeostasis of the epidermis and colonic epithelium, and its loss promotes colonic tumour development.

Helicobacter pylori (H pylori) infection is the main risk factor for gastric cancer. The role of fibroblast growth factor receptors (FGRFs) in H pylori-mediated gastric tumorigenesis remains largely unknown. This study investigated the molecular and mechanistic links between H pylori, inflammation, and FGFR4 in gastric cancer.Cell lines, human and mouse gastric tissue samples, and gastric organoids models were implemented. Infection with H pylori was performed using in vitro and in vivo models. Western blot, real-time quantitative reverse-transcription polymerase chain reaction, flow cytometry, immunofluorescence, immunohistochemistry, chromatin immunoprecipitation, and luciferase reporter assays were used for molecular, mechanistic, and functional studies.Analysis of FGFR family members using The Cancer Genome Atlas data, followed by validation, indicated that FGFR4 messenger (m)RNA was the most significantly overexpressed member in human gastric cancer tissue samples (P.001). We also detected high levels of Fgfr4 mRNA and protein in gastric dysplasia and adenocarcinoma lesions in mouse models. Infection with J166, 7.13, and PMSS1 cytotoxin-associated gene A (CagA)+ H pylori strains induced FGFR4 mRNA and protein expression in in vitro and in vivo models. This was associated with a concordant activation of signal transducer and activator of transcription 3 (STAT3). Analysis of the FGFR4 promoter suggested several putative binding sites for STAT3. Using chromatin immunoprecipitation assay and an FGFR-promoter luciferase reporter containing putative STAT3 binding sites and their mutants, we confirmed a direct functional binding of STAT3 on the FGFR4 promoter. Mechanistically, we also discovered a feedforward activation loop between FGFR4 and STAT3 where the fibroblast growth factor 19-FGFR4 axis played an essential role in activating STAT3 in a steroid receptor coactivator-dependent manner. Functionally, we found that FGFR4 protected against H pylori-induced DNA damage and cell death.Our findings demonstrated a link between infection, inflammation, and FGFR4 activation, where a feedforward activation loop between FGFR4 and STAT3 is established via steroid receptor coactivator in response to H pylori infection. Given the relevance of FGFR4 to the etiology and biology of gastric cancer, we propose FGFR4 as a druggable molecular vulnerability that can be tested in patients with gastric cancer.

BackgroundEts homologous factor (EHF) is a member of the epithelial-specific Ets (ESE) transcription factors. EHF is specifically expressed in epithelial tissues, however its role in development and epithelial homeostasis is largely uncharacterized.MethodsWe generated a novel mouse strain in which the Ets DNA binding domain (exon 8) of Ehf was flanked by loxP sites (EhfLox/Lox). To inactivate Ehf in the whole body, EhfLox/Lox mice were crossed to CMVCre mice, which were then bred out to generate germline Ehf null (Ehf−/−) mice. To inactivate Ehf specifically in the intestinal epithelium, EhfLox/Lox mice were bred to tamoxifen-inducible VillinCre-ERT2 mice. EhfLox/Lox mice were also crossed to tamoxifen-inducible Cdx2CreERT2; ApcLox/+ mice to determine the impact of Ehf deletion on Apc-initiated colon cancer development.ResultsTranscripts encoding the Ets binding domain of EHF were effectively deleted in all tissues in Ehf−/− mice. Ehf−/− mice were born at the expected Mendelian ratio, but showed reduced body weight gain and developed a series of pathologies during their lifespan that led the majority of Ehf−/− mice to reach an ethical endpoint within one year of age. Most prominent of these were the development of papillomas in the chin, and abscesses in the preputial glands (males) or vulvae (females) which showed evidence of Staphylococcus and Proteus infection. Consistent with the development of papillomas, the epidermis of Ehf−/− mice showed evidence of mild hyperplasia. A subset of Ehf−/− mice also developed cataracts and corneal ulcers. EHF is highly expressed in the colonic epithelium and Ehf−/− mice displayed increased susceptibility to dextran sodium sulphate-induced colitis. This phenotype was confirmed in intestinal-specific Ehf knockout mice, and histopathological analyses revealed reduced numbers of goblet cells and extensive transcriptional reprogramming in the colonic epithelium. Finally, colon-specific deletion of Ehf enhanced Apc-initiated adenoma development, unveiling a novel, tumour suppressive role for EHF in colorectal cancer.ConclusionThe Ets DNA-binding domain of EHF is essential for post-natal homeostasis of the epidermis and colonic epithelium, and functions as a tumour suppressor in the colon.

The Interleukin (IL-)1 family IL33 is best known for eliciting type 2 immune responses by stimulating mast cells (MCs), regulatory T-cells (Tregs), innate lymphoid cells (ILCs) and other immune cells. MCs and IL33 provide critical control of immunological and epithelial homeostasis in the gastrointestinal (GI) tract. Meanwhile, the role of MCs in solid malignancies appears tissue-specific with both pro and anti-tumorigenic activities. Likewise, IL33 signaling significantly shapes immune responses in the tumor microenvironment, but these effects remain often dichotomous when assessed in experimental models of cancer. Thus, the balance between tumor suppressing and tumor promoting activities of IL33 are highly context dependent, and most likely dictated by the mixture of cell types responding to IL33. Adding to this complexity is the promiscuous nature by which MCs respond to cytokines other than IL33 and release chemotactic factors that recruit immune cells into the tumor microenvironment. In this review, we integrate the outcomes of recent studies on the role of MCs and IL33 in cancer with our own observations in the GI tract. We propose a working model where the most abundant IL33 responsive immune cell type is likely to dictate an overall tumor-supporting or tumor suppressing outcome in vivo. We discuss how these opposing responses affect the therapeutic potential of targeting MC and IL33, and highlight the caveats and challenges facing our ability to effectively harness MCs and IL33 biology for anti-cancer immunotherapy.

IL11 is a member of the IL6 family of cytokines and signals through its cognate receptor subunits, IL11RA and glycoprotein 130 (GP130), to elicit biological responses via the JAK/STAT signaling pathway. IL11 contributes to cancer progression by promoting the survival and proliferation of cancer cells, but the potential immunomodulatory properties of IL11 signaling during tumor development have thus far remained unexplored. Here, we have characterized a role for IL11 in regulating CD4

The contribution of mast cells in the microenvironment of solid malignancies remains controversial. Here we functionally assess the impact of tumor-adjacent, submucosal mast cell accumulation in murine and human intestinal-type gastric cancer. We find that genetic ablation or therapeutic inactivation of mast cells suppresses accumulation of tumor-associated macrophages, reduces tumor cell proliferation and angiogenesis, and diminishes tumor burden. Mast cells are activated by interleukin (IL)-33, an alarmin produced by the tumor epithelium in response to the inflammatory cytokine IL-11, which is required for the growth of gastric cancers in mice. Accordingly, ablation of the cognate IL-33 receptor St2 limits tumor growth, and reduces mast cell-dependent production and release of the macrophage-attracting factors Csf2, Ccl3, and Il6. Conversely, genetic or therapeutic macrophage depletion reduces tumor burden without affecting mast cell abundance. Therefore, tumor-derived IL-33 sustains a mast cell and macrophage-dependent signaling cascade that is amenable for the treatment of gastric cancer., Mast cells within the tumor microenvironment have controversial roles. Here, the authors show, using genetic mouse models, that in gastric cancer, mast cells at the periphery of the tumors are activated via cancer cell produced-IL33 and promote tumorigenesis by recruiting macrophages within the tumors.

Cytokine-mediated inflammation is a driver of gastric and colonic tumorigenesis. IL1-family cytokine Interleukin 33 (IL33) regulates inflammatory responses and antibodies targeting IL33 or its receptor ST2 are in clinical trials against various inflammatory and allergy diseases. Only recently, a role of IL33 in cancer started to emerge. Depending on the cancer stage or type IL33 can provoke either pro- or anti-tumoral responses (1). To evaluate the potential of targeting IL33 signalling to abrogate gastric and colonic cancer growth, we employ gp130Y757F/ Y757F (FF) gastric cancer mice and the 6xAOM sporadic colon cancer model as well subcutaneously engrafted MC38 colon cancer models. IL33 signalling inhibition was achieved via genetic deficiency (St2-/-), while recombinant IL33 was administered to hyper activate the IL33-signalling pathway. We found, that IL33 cytokine and ST2 receptor are overexpressed in human gastric cancers and high ST2 expression predicts poor survival for gastric cancer patients. In accordance, IL33 and ST2 are highly elevated in our FF mouse tumors. Deficiency of IL33 signalling (ST2-/-) diminishes gastric tumour growth, and is associated with a decrease in tumour-adjacent pro-tumoral mast cells and tumour-infiltrating macrophages (TAM) as well as reduced angiogenesis. Furthermore, ST2-deficiency potentiates the anti-tumor effects of oxaliplatin chemotherapy in the FF mouse model. Mechanistically, we show that tumour-produced IL33 can activate mast cells, which in turn recruit pro-tumoral and pro-angiogenic macrophages to the tumour through release of chemo-attractants like Ccl2, Ccl3 and Ccl7 (2). In the colon cancer setting, ST2-deficiency led to increased tumour burden in 6xAOM sporadic colorectal cancer. Reciprocal bone marrow chimera experiments indicated that the radio-resistant non-hematopoietic cell compartment contributes to the increased tumour growth. Indeed, we found St2 expression in the mesenchymal cells of the tumor microenvironment. Loss of IL33 signalling in the non-haematopoietic radio-resistant compartment coincided with a strong reduction of an IFNy gene expression signature. Importantly, IL33 cytokine administration reduced colon cancer allograft growth associated with tumour-infiltrating IFNy-positive T cells (3). Our data demonstrates the opposing roles of IL33 signalling in the growth of gastric and colonic cancers and identifies cellular mediators of these divergent tumor responses. Further studies are warranted to stratify IL33-signalling sensitive GI cancers and to verify the potential of anti-IL33/ST2 antibodies against gastric cancer and activating IL33 cytokine administration in colon cancers as novel therapy strategies. 1 Eissmann M, et al. Front Immunol 2020 Jul; 11:1389. 2 Eissmann M, et al. Nat Commun 2019 Jun; 21;10(1):2735. 3 Eissmann M, et al. Cancer Immunol Res 2018 April; 6(4):409-421. This abstract is also being presented as PO017. Citation Format: Moritz Eissmann, Christine Dijkstra, Matthias Ernst. IL33 cytokine signalling in gastrointestinal cancers - a therapy target? [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PR015.

Temporal and spatial regulation of genes mediated by tissue-specific promoters and conditional gene expression systems provide a powerful tool to study gene function in health, disease, and during development. Although transgenic mice expressing the Cre recombinase in the gastric epithelium have been reported, there is a lack of models that allow inducible and reversible gene modification in the stomach. Here, we exploited the gastrointestinal epithelium-specific expression pattern of the three trefoil factor (Tff) genes and bacterial artificial chromosome transgenesis to generate a novel mouse strain that expresses the CreERT2 recombinase and the reverse tetracycline transactivator (rtTA). The Tg(Tff1-CreERT2;Tff2-rtTA;Tff3-Luc) strain confers tamoxifen-inducible irreversible somatic recombination and allows simultaneous doxycycline-dependent reversible gene activation in the gastric epithelium of developing and adult mice. This strain also confers luciferase activity to the intestinal epithelium to enable in vivo bioluminescence imaging. Using fluorescent reporters as conditional alleles, we show Tff1-CreERT2 and Tff2-rtTA transgene activity in a partially overlapping subset of long-term regenerating gastric stem/progenitor cells. Therefore, the Tg(Tff1-CreERT2;Tff2-rtTA;Tff3-Luc) strain can confer intermittent transgene expression to gastric epithelial cells that have undergone previous gene modification, and may be suitable to genetically model therapeutic intervention during development, tumorigenesis, and other genetically tractable diseases. Birth Defects Research (Part A) 106:626-635, 2016. © 2016 Wiley Periodicals, Inc.

About 5% to 10% of human gastric tumors harbor oncogenic mutations in the KRAS pathway, but their presence alone is often insufficient for inducing gastric tumorigenesis, suggesting a requirement for additional mutagenic events or microenvironmental stimuli, including inflammation. Assessing the contribution of such events in preclinical mouse models requires Cre recombinase–mediated conditional gene expression in stem or progenitor cells of normal and transformed gastric epithelium. We therefore constructed a bacterial artificial chromosome containing transgene (Tg), comprising the regulatory elements of the trefoil factor 1 (Tff1) gene and the tamoxifen-inducible Cre recombinase (CreERT2)–coding sequence. The resulting Tg(Tff1-CreERT2) mice were crossed with mice harboring conditional oncogenic mutations in Kras or Braf. The administration of tamoxifen to the resulting adult Tg(Tff1-CreERT2);KrasLSL-G12D/+ and Tg(Tff1-CreERT2);BrafLSL-V600E/+ mice resulted in gastric metaplasia, inflammation, and adenoma development, characterized by excessive STAT3 activity. To assess the contribution of STAT3 to the spontaneously developing gastric adenomas in gp130F/F mice, which carry a knockin mutation in the Il6 signal transducer (Il6st), we generated Tg(Tff1-CreERT2);Stat3fl/fl;gp130F/F mice that also harbor a conditional Stat3 knockout allele and found that tamoxifen administration conferred a significant reduction in their tumor burden. Conversely, excessive Kras activity in Tg(Tff1-CreERT2);KrasLSL-G12D/+;gp130F/F mice promoted more extensive gastric inflammation, metaplastic transformation, and tumorigenesis than observed in Tg(Tff1-CreERT2);KrasLSL-G12D/+ mice. Collectively, our findings demonstrate that advanced gastric tumorigenesis requires oncogenic KRAS or BRAF in concert with aberrant STAT3 activation in epithelial precursor cells of the glandular stomach, providing a new conditional model of gastric cancer in which to investigate candidate therapeutic targets and treatment strategies. Cancer Res; 76(8); 2277–87. ©2016 AACR.

Gastric cancer (GC) remains the third leading cause of cancer related death worldwide. While inflammation is a well-established driver of gastric tumorigenesis, only a small subset of GC patients responds to immunotherapy. The understanding of the tumor microenvironment, which suppresses anti-tumor immune responses, is a field of great interest. One proposed mechanism of immune escape is silencing tumor antigen expression by DNA methylation, and thereby avoiding immune recognition. DNA methyltransferases (DNMTs) are the enzymes responsible for DNA methylation and hence, for the epigenetic silencing of gene expression of tumor antigens and other immune related molecules. DNMTs are often overexpressed in solid tumors. Epigenetic drugs inhibiting these DNMTs have shown anti-tumor effects in combination with immune checkpoint inhibitors by re-activating the expression of tumor antigens, amongst others. Here we are studying the role of DNMTs in GC mouse models and the possibility of combining DNMT inhibitors with immunotherapy for the treatment of GC. This project utilizes various mouse models of GC. The Gp130Y757F/Y757F (gp130FF) mutant mouse model develops spontaneous inflammation driven gastric adenomas. We established a gp130FF Dnmt3a overexpressing gastric cancer mouse model (gp130FF, A33Dnmt3a) where Dnmt3a overexpression occurs in gastric tumors. In a second mouse model, mutant Kras, Pi3kca and Tp53 expression results in highly advanced invasive gastric carcinoma formation (KPT model). We successfully established gastric cancer organoids of one of these triple mutant tumors (KPT organoids) which can be injected subcutaneously into wild type mice and result in allograft tumor formation. DNMT3A overexpression was detected in human GC specimen and associated with bad overall survival of patients. Gastric adenomas of our (gp130FF, A33Dnmt3a mouse model have a 10-fold elevated Dnmt3a expression. Importantly, tumor specific Dnmt3a overexpression significantly increased gastric tumor burden. In the KPT model, we have identified DNMT3A as being highly expressed in the invasive front of tumors, their liver metastases as well as in the allograft tumors established by the KPT organoids. In this model of advanced gastric cancer, treatment with the DNMT inhibitor decitabine significantly decreased tumor growth and we are currently investigating the efficacy of decitabine in combination with anti PD 1 immunotherapy. Taken together, we provide evidence for a driver function of Dnmt3a in gastric tumorigenesis and gastric tumor growth. In addition, we demonstrate the vulnerability of an aggressive gastric carcinoma model to DNMT inhibition and hence its non-oncogene addiction to DNMTs. Therefore, our findings encourage further studies to investigate the potential of DNMT inhibitors for the treatment of GC. Citation Format: Anne Huber, Christine Dijkstra, Matthias Ernst, Moritz Eissmann. DNA methyltransferases drive gastric cancer growth and present a therapy target for gastric cancer [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-014.

Interleukin 33 (IL33) is an inflammatory cytokine released during necrotic cell death. The epithelium and stroma of the intestine express large amounts of IL33 and its receptor St2. IL33 is therefore continuously released during homeostatic turnover of the intestinal mucosa. Although IL33 can prevent colon cancer associated with inflammatory colitis, the contribution of IL33 signaling to sporadic colon cancer remains unknown. Here, we utilized a mouse model of sporadic colon cancer to investigate the contribution of IL33 signaling to tumorigenesis in the absence of preexisting inflammation. We demonstrated that genetic ablation of St2 enhanced colon tumor development. Conversely, administration of recombinant IL33 reduced growth of colon cancer cell allografts. In reciprocal bone marrow chimeras, the concurrent loss of IL33 signaling within radioresistant nonhematopoietic, and the radiosensitive hematopoietic, compartments was associated with increased tumor burden. We detected St2 expression within the radioresistant mesenchymal cell compartment of the colon whose stimulation with IL33 induced expression of bona fide NF-κB target genes. Mechanistically, we discovered that St2 deficiency within the nonhematopoietic compartment coincided with increased abundance of regulatory T cells and suppression of an IFNγ gene expression signature, whereas IL33 administration triggered IFNγ expression by tumor allograft-infiltrating T cells. The decrease of this IFNγ gene expression signature was associated with more aggressive disease in human colon cancer patients, suggesting that lack of IL33 signaling impaired the generation of a potent IFNγ-mediated antitumor immune response. Collectively, our data reveal that IL33 functions as a tumor suppressor in sporadic colon cancer. Cancer Immunol Res; 6(4); 409–21. ©2018 AACR.

A contiguous intestinal epithelial barrier safeguards against aberrant activation of the immune system and therefore requires molecular mechanisms that ensure effective wound-healing responses. During this processes cytokine-producing myeloid cells serve as rheostats that link the degree of wounding and local inflammation to the epithelial repair response. Likewise, intestinal inflammation is an important factor by which the microenvironment promotes tumorigenesis and the progression of established cancers by facilitating neoplastic cell survival and proliferation. Among the cytokines and chemokines orchestrating this process, those comprising the interleukin (IL) IL6, IL10/IL22 and IL17/IL23 families play a prominent role by virtue of converging on the latent Signal Transducer and Activator of Transcription (Stat)-3. Accordingly, aberrant and persistent Stat3 activation is a frequent observation in cancers of the gastrointestinal tract where it promotes "cancer hallmark capabilities" in the malignant epithelium and suppresses the anti-tumor response of innate and adaptive immune cells. Here, we discuss recent insights arising from situations where persistent activation of the gp130/Stat3 signaling cascades result from excessive abundance of IL6 family cytokines. In particular, we highlight novel and unique roles for IL11 in promoting intestinal wound-healing and, in its corrupted form, enabling and facilitating growth of inflammation-associated and sporadic gastrointestinal tumors.

The transcription factor Stat3 plays essential roles in biological processes involving development, immunity and inflammation. Stat3 is known to regulate the expression of a large repertoire of genes which when deregulated, as in the context of tumorigenesis, can lead to the development of some of the well-known hallmarks of cancer, such as resistance to cell death and sustained proliferation. Indeed the aberrant activation of Stat3 has been characterized across a number of different cancers including gastrointestinal cancers, presenting Stat3 and its signalling pathway a promising therapeutic target. Although intrinsic Stat3 signalling in tumor cells is well characterized, the effect(s) of Stat3 signalling among the infiltrating cells of the tumor microenvironment is still poorly understood. With this project we aim to elucidate the role of Stat3 in the non-tumoral compartment, and explore the therapeutic value of Stat3 inhibition for gastric and colon cancer. We generated the CAGsrtTA3;STAT3.1348 (shStat3) mouse that utilizes short-hairpin RNAi technology allowing for conditional and reversible Stat3 reduction. To study the effects of systemic Stat3 inhibition, the shStat3 was crossed with the gp130F/F mutant mouse that spontaneously develops gastric cancer. To assess the effects of Stat3 suppression in the non-tumoral compartment alone, the shStat3 mice were sub-cutaneously injected with MC38 murine colon cancer cells. Tumors and other tissues of these mice were excised and analysed. We found that shStat3-mediated systemic Stat3 suppression resulted in significant tumor reduction in the gp130F/F gastric cancer mice. Similarly, isolated Stat3 reduction in the non-tumoral compartment of the shStat3 mice, restricted the growth of Stat3-proficient MC38 tumor allografts. Immunophenotyping of excised tumors highlighted an increase of a monocytic Ly6C+Ly6G- myeloid population. Our data provides evidence for the therapeutic value of specific Stat3 targeting in gastrointestinal cancers. Interestingly, we have shown that Stat3 suppression in the non-tumoral compartment alone was enough to result in significant tumor reduction, indicating a prominent role of Stat3 in creating a pro-tumorigenic microenvironment. We have also identified a monocytic population as candidate cell type that drives this Stat3 suppression mediated anti-tumor effect. Citation Format: Mariah Grace Alorro, Frederick Masson, Moritz Eissmann, Matthias Ernst. Selective Stat3 inhibition in the tumor microenvironment restricts gastrointestinal tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2365.

Signal transducer and activator of transcription 3 (Stat3) is a transcription factor that has many essential roles during inflammation, development and cancer. Stat3 is therefore an attractive therapeutic target in many diseases. While current Stat3 knockout mouse models led to a better understanding of the role of Stat3, the irreversible nature of Stat3 ablation does not model the effects of transient Stat3 therapeutic inhibition, and does not inform on potential dosage effects of Stat3. Using RNAi technology, we have generated a new mouse model allowing the inducible and reversible silencing of Stat3 in vivo, which mirrors the effects of specific Stat3 therapeutic interference. We showed that upon Doxycycline-mediated activation of the Stat3 short-hairpin RNA, Stat3 expression was efficiently reduced by about 80% in multiple organs and cell types. Moreover, Stat3 reduction was sufficient to reduce tumor burden in a clinically-validated mouse model of gastric cancer. Finally, we demonstrated that Stat3 silencing during embryonic development led to reduced birth rate without leading to complete embryonic lethality, in contrast to full Stat3 ablation. In conclusion, this new mouse model will be invaluable to understand the effects of Stat3 therapeutic interference and Stat3 dosage effects.

Temporal and spatial regulation of genes mediated by tissue-specific promoters and conditional gene expression systems provide a powerful tool to study gene function in health, disease, and during development. Although transgenic mice expressing the Cre recombinase in the gastric epithelium have been reported, there is a lack of models that allow inducible and reversible gene modification in the stomach. Here, we exploited the gastrointestinal epithelium-specific expression pattern of the three trefoil factor (Tff) genes and bacterial artificial chromosome transgenesis to generate a novel mouse strain that expresses the CreERT2 recombinase and the reverse tetracycline transactivator (rtTA). The Tg(Tff1-CreERT2;Tff2-rtTA;Tff3-Luc) strain confers tamoxifen-inducible irreversible somatic recombination and allows simultaneous doxycycline-dependent reversible gene activation in the gastric epithelium of developing and adult mice. This strain also confers luciferase activity to the intestinal epithelium to enable in vivo bioluminescence imaging. Using fluorescent reporters as conditional alleles, we show Tff1-CreERT2 and Tff2-rtTA transgene activity in a partially overlapping subset of long-term regenerating gastric stem/progenitor cells. Therefore, the Tg(Tff1-CreERT2;Tff2-rtTA;Tff3-Luc) strain can confer intermittent transgene expression to gastric epithelial cells that have undergone previous gene modification, and may be suitable to genetically model therapeutic intervention during development, tumorigenesis, and other genetically tractable diseases. Birth Defects Research (Part A) 106:626-635, 2016. © 2016 Wiley Periodicals, Inc.

Activation of the Wnt/β-catenin pathway occurs in the vast majority of colorectal cancers. However, the outcome of the disease varies markedly from individual to individual, even within the same tumor stage. This heterogeneity is governed to a great extent by the genetic make-up of individual tumors and the combination of oncogenic mutations. In order to express throughout the intestinal epithelium a degradation-resistant β-catenin (Ctnnb1), which lacks the first 131 amino acids, we inserted an epitope-tagged ΔN(1-131)-β-catenin-encoding cDNA as a knock-in transgene into the endogenous gpA33 gene locus in mice. The resulting gpA33ΔN-Bcat mice showed an increase in the constitutive Wnt/β-catenin pathway activation that shifts the cell fate towards the Paneth cell lineage in pre-malignant intestinal epithelium. Furthermore, 19% of all heterozygous and 37% of all homozygous gpA33ΔN-Bcat mice spontaneously developed aberrant crypt foci and adenomatous polyps, at frequencies and latencies akin to those observed in sporadic colon cancer in humans. Consistent with this, the Wnt target genes, MMP7 and Tenascin-C, which are most highly expressed in benign human adenomas and early tumor stages, were upregulated in pre-malignant tissue of gpA33ΔN-Bcat mice, but those Wnt target genes associated with excessive proliferation (i.e. Cdnn1, myc) were not. We also detected diminished expression of membrane-associated α-catenin and increased intestinal permeability in gpA33ΔN-Bcat mice in challenge conditions, providing a potential explanation for the observed mild chronic intestinal inflammation and increased susceptibility to azoxymethane and mutant Apc-dependent tumorigenesis. Collectively, our data indicate that epithelial expression of ΔN(1-131)-β-catenin in the intestine creates an inflammatory microenvironment and co-operates with other mutations in the Wnt/β-catenin pathway to facilitate and promote tumorigenesis., Summary: The gpA33ΔN-Bcat mice provide a model that better mimics some aspects of sporadic colon cancer induction in humans in terms of tumor multiplicity and latency and site-specific (i.e. colon) tumor occurrence that coincides with upregulation of markers for human colorectal cancer progression.

[reaction: see text] As a contribution to bioorganometallic chemistry, an enantioselective synthesis of novel carbocyclic nucleoside analogues with a ferroceno-cyclopentene backbone was developed. Diastereoselective cuprate 1,4-addition or Mukaiyama-Michael addition to a planar-chiral enoate (ethyl (E)-2-[2-methoxycarbonyl-ferrocenyl]-acrylate) allowed for the introduction of different side chains (RCH(2)). Other important steps include a Dieckmann cyclization and the attachment of the nucleobase (NB) in an iron-assisted S(N)1 reaction. Some of the target compounds were shown to exhibit significant apoptosis-inducing activity (LD(50) = 10-20 microM) against tumor cells.

Most colon cancers arise from somatic mutations in the tumor suppressor gene APC (adenomatous polyposis coli), and these mutations cause constitutive activation of the Wnt–to–β-catenin pathway in the intestinal epithelium. Because Wnt–β-catenin signaling is required for homeostasis and regeneration of the adult intestinal epithelium, therapeutic targeting of this pathway is challenging. We found that genetic activation of the cytokine-stimulated pathway mediated by the receptor gp130, the associated Jak (Janus kinase) kinases, and the transcription factor Stat3 (signal transducer and activator of transcription 3) was required for intestinal regeneration in response to irradiation-induced damage in wild-type mice and for tumorigenesis in Apc-mutant mice. Systemic pharmacological or partial genetic inhibition of gp130-Jak-Stat3 signaling suppressed intestinal regeneration, the growth of tumors in Apc-mutant mice, and the growth of colon cancer xenografts. The growth of Apc-mutant tumors depended on gp130-Jak-Stat3 signaling for induction of the polycomb repressor Bmi-1, and the associated repression of genes encoding the cell cycle inhibitors p16 and p21. However, suppression of gp130-Jak-Stat3 signaling did not affect Wnt–β-catenin signaling or homeostasis in the intestine. Thus, these data not only suggest a molecular mechanism for how the gp130-Jak-Stat3 pathway can promote cancer but also provide a rationale for therapeutic inhibition of Jak in colon cancer.