Seminars in Immunology 26 (2014) 29–37

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Review

Epithelial gp130/Stat3 functions: An intestinal signaling node in health and disease Matthias Ernst a,b,∗ , Stefan Thiem a,b , Paul M. Nguyen a,b , Moritz Eissmann a,b , Tracy L. Putoczki a,b a b

The Walter and Eliza Hall Institute for Medical Research, Melbourne, Australia Department of Medical Biology, University of Melbourne, Australia

a r t i c l e

i n f o

Keywords: Intestinal epithelial cells Epithelial Stat3 activity Gastric cancer Colon cancer Interleukin-11

a b s t r a c t 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. Crown Copyright © 2014 Published by Elsevier Ltd. All rights reserved.

1. Introduction The past decade has revealed the existence of phylogenetically conserved signaling networks that provide effective wound healing. In the gastrointestinal tract, for instance, a localized immune cell response to microbiota exposure triggers spatially confined and rapid wound healing to re-establish the epithelial barrier function. During this process myeloid cells act as a critical rheostat linking the degree of wounding to an appropriate repair response. Accordingly, exposure of neoplastic epithelium to this response provides a growth-promoting environment for the emerging tumors. Remarkably many of these interactions, mediated by cytokines, converge on the Signal Transducer and Activation of Transcription (Stat)-3 in epithelial cells and those comprising the tumor microenvironment. Phylogenetic diversification among the Stat proteins appears to have selected Stat3 as a signaling node for the host to counteract against the effects of extracellular pathogens. Therefore, Stat3 mediates a hepatic acute phase response, induces the release of

∗ Corresponding author at: The Walter and Eliza Hall Institute for Medical Research, Melbourne, Australia. Tel.: +61 3 9345 2555. E-mail address: [email protected] (M. Ernst).

antimicrobial proteins, promotes the survival of epithelial cells at mucosal surfaces to prevent pathogen entry and facilitates class switching and production of opsonising and carbohydratetargeting antibodies. Here, we highlight recent insights in to the cytokine networks regulating Stat3 in the gastrointestinal tract and review some of the emerging strategies to therapeutically exploit these findings.

2. The transcription factor Stat3 The Stat3 protein exists in two alternatively spliced isoforms, comprising the full-length Stat3␣ and the carboxyl-terminally truncated Stat3␤. Stat3␣-deficient mice die immediately after birth due to respiratory distress, whereas Stat3ˇKO mice have a normal life expectancy [1]. While Stat3␣ predominantly regulates the responses to IL6 family cytokines, Stat3␤ seems to act as a stabilizer of the ␣ isoform. Although Stat3␤ lacks the transactivation domain, it can regulate transcription, in particular when associated with c-Jun [2]. Like most eukaryotic transcription factors, Stat3 interacts with histone acetyltransferases and transcriptional co-activators including CBP/p300, SP1, c-Fos and c-Jun [3,4]. Stat3 also interacts with

1044-5323/$ – see front matter. Crown Copyright © 2014 Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.smim.2013.12.006

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Fig. 1. Schematic representation of Stat3 activation in response to IL10/IL22 and IL6/IL11 cytokines. The gp130 receptor depicts a gp130-homodimeric complex formed in response to binding of two ligand-occupied IL6R␣ or IL11R␣ subunits. Receptor regions, including phosphorylation (blue) on tyrosine residues (Y) are indicated with the membrane proximal Y757 in murine gp130 providing a docking site for Shp2 and Socs3. Several mechanisms, including those requiring protein kinase C, have been suggested to mediated phosphorylation (red) of a C-terminal serine residue in Stat3, which in its unphosphorylated form interacts with NF-␬B. Activation of the canonical pathways results in nuclear translocation of phosphorylated Stat3 homodimers, which activate transcription of genes conferring cancer hallmark activities, as well as mucosal integrity and negative feedback control.

transcription factors that play a role during inflammatory processes such as the NF-␬B subunits p65 and p50, Stat1, Stat6, hypoxia inducible factor (HIF) 1␣, as well as ␤-catenin which links Wntsignaling to the regulation of the gastrointestinal epithelial stem cell compartment. Fully activated Stat3, which is phosphorylated on tyrosine (Y) residue 705 and serine 727 can form homodimers as well as Stat1/3 heterodimers [5], although it is unclear whether the two kinds of dimers result in activation of different target genes (Fig. 1). Tyrosine phosphorylated Stat3 homodimers selectively bind to Sp1 and Foxo1 [4], while unphosphorylated Stat3 monomers complex with p65 and p50 [6]. Indeed, expression of many NF-␬B target genes in tumor cells and associated immune cells is dependent on Stat3 DNA-binding, thereby modulating NF␬B-mediated gene expression in a tumor-promoting manner [7]. 2.1. Stat3 and intestinal homeostasis Starting at the glandular part of the stomach and extending all the way to the rectum, a single cell layer of epithelium comprises >250 m2 absorptive surface in humans and prevents aberrant immune responses by separating the immune system from trillions of luminal microorganisms. Goblet cells, a specialized intestinal epithelial cell (IEC) type, produce various mucins that account for the major component mucus, which further separates the luminal microflora bacteria from the epithelium. The intestinal epithelium is arranged into villi and crypts, and stem cells at the bottom of the crypt continuously supply progenies into the transient amplifying

progenitor cell compartment. In turn, this provides replacement of the four terminally differentiated IEC cell types that are continuously shed at the tip of the villi and mouth of the crypt in the small intestine and colon, respectively. The longer-lived Paneth cells provide the only IEC type migrating in opposite direction toward the bottom of the crypt to provide the stem cell niche in the small intestine and to secrete lysozymes and ␤-defensins with anti-microbial activity. Stat3 expression is uniform along the crypt-villus axis and is required to maintain barrier integrity. Stat3 stimulates expression of the antimicrobial proteins RegIII-␤ and RegIII-␥, produced by Paneth cells [8]. Likewise, Stat3 induces lipocalin-2 and ␤-defensins to buffer the epithelium against an inappropriate innate immune response elicited by commensal bacteria. Some of the mucin genes are direct targets for Stat3, including Muc-1, which is prominently expressed by IECs [9] and Muc4 as a major constituent of the gastric mucous [10]. In line with Stat3’s capacity to induce the production of large amounts of secreted proteins in IECs, it also stimulates expression of the chaperon protein Hsp70 to prevent an unfolded protein response. The latter is associated with IEC apoptosis and ensuing colitis, and is observed in Hsp70-deficient mice [11] or those carrying Muc2 mutations, which traps this mucin in the endoplasmatic reticulum [12]. The exact role of epithelial Stat3 during continuous epithelial renewal, at least of the small intestine, is less clear. For example, using the Cyp1A1-Cre recombinase to mediate conditional Stat3deficiency in IECs of adult mice results in immediate replacement

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of the Stat3-deficient epithelium by Stat3-proficient cells. Since the latter cells are most likely derived from escaping, non-recombined progenitors, these observations suggest a strong selective pressure to retain epithelial Stat3 expression [13]. Constitutive Villin-Cre mediated Stat3 deficiency, which is induced during embryonic development, results in long-term retention of Stat3-deficient IECs, possibly owing to compensatory activity of Stat1 [14]. 2.2. Stat3 and intestinal wound healing The cellular mechanisms associated with inflammatory bowel disease (IBD) suggest that inappropriate activation of the mucosal immune system plays a prominent role in the development of disease-associated inflammation [15]. A decreased mucus layer in IBD patients, in conjunction with defective IEC functions, contributes to increased epithelial permeability to intestinal microbes. Their subsequent recognition by dendritic cells and other antigen presenting cells triggers a T cell response. Accordingly, Crohn’s disease is associated with recruitment of T-helper (Th)-1 and Th17 cells to the lamina propria at the expense of regulatory T (Treg) cells and associated production of IFN␥ and IL17. In contrast, Ulcerative Colitis displays more of a Th2-cytokine mediated inflammatory response associated with elevated IL4 expression. In humans the STAT3 and JAK genes are among the susceptibility loci for IBD [16]. Consistent with this, wide-spread type I interferonmediated Stat3 gene inactivation in macrophages, IECs and other cell types result in enterocolitis [17]. Meanwhile, Stat3+/− mice, or mice carrying a mutant form of gp130 (gp130Stat ) that is incapable of activating Stat3, show impaired intestinal regeneration in response to ␥-radiation and increased susceptibility to models of IBD, including exposure to the luminal irritant dextran sodium sulphate (DSS) [18]. By contrast, excessive Stat3 activity, resulting either from IEC-specific deletion of the negative regulator Socs3, or in response to IEC-specific, transgenic expression of a constitutively dimerizing gp130 mutant [19] in A33:L-gp130Stat3 mice reduced susceptibility to DSS-induced colitis (Putozcki and Ernst, unpublished observations). A similar protective effect is also observed in gp130Y757F mice that harbor a mutation preventing Socs3 from binding to ligand-occupied gp130 receptors and therefore to terminate Stat3 signaling [18]. These observations suggest that epithelial Stat3 activation facilitates survival, proliferation and possibly cellular migration of IECs. Linking the proliferative capacity of IECs to a “gp130/Stat3 rheostat” appears phylogenetically conserved, as it is also required for regeneration of the fly mid-gut [20]. This mechanism is likely to confine the intestine’s capacity for rapid mucosal regeneration to sites of greatest inflammation, which is further aided by the capacity of the inflamed stroma to convert IECs to stem cells through a NF-␬B dependent mechanism. Accordingly, gp130Stat mice display continuous wounding and ulceration at intestinal sphincters exposed to sustained mechanical trauma [21], and fail to regenerate in response to ␥-radiation (Phesse and Ernst, unpublished observations). 2.3. Epithelial Stat3 links inflamed microenvironment to tumorigenesis A link between inflammation and increased cancer incidence is well established with Ulcerative Colitis and Crohn’s disease patients having increased risks of developing cancers of the small intestine and colon, respectively [22]. Meanwhile, chronic infection with Helicobacter pylori (H. pylori) increases the risk for gastric cancer development. Serum IL6 levels are elevated in animal models of IBD, and they also correlate with increased severity of Crohn’s disease [23]. Likewise, stomachs of H. pylori infected mice show elevated IL11 expression [24], consistent with observations that H. pylori infected gastric cell lines display elevated levels of activated

Fig. 2. Gastric tumorigenesis in mice with impaired Socs3 activity. (A) Representative whole-mount images of stomachs, opened along the outer curvature and pinned out with the lumen facing the viewer, reveal distinct adenomatous tumors in the glandular part (antrum, outlined by white dotted line). Socs3del/del mice have been aged for 52-weeks following gastric epithelium-specific Socs3 ablation using the tamoxifen-inducible transgenic Tff1:CreERT2 driver (Thiem and Ernst, unpublished observations). Tumor burden in 12-week old homozygous gp130Y757F mice (gp130F/F ) is shown for comparison. Scale bar = 1 cm. (B) Incidence and age of gastric tumor onset in gp130Y757F and tamoxifen-induced transgenic Tff1:CreERT2 x Socs3del/del mice.

Stat3. Accordingly, the risk of developing gastric cancer is reduced in populations treated for H. pylori infection, while administration of immunosuppressive and non-steroidal anti-inflammatory drugs can reduce the recurrence of adenomas in IBD patients [25]. The proven efficacy of these anti-inflammatory agents in highrisk individuals highlights the importance of inflammation as a tumor promoter. The protective benefit of aspirin in healthy individuals suggests that tissue inflammation may also be an important contributor to tumor initiation in sporadic colorectal cancer [25]. Both of these situations can be replicated in animal models with excessive Stat3 activity. For instance, constitutive activation of endogenous Stat3 in A33:L-gp130Stat3 transgenic mice sensitized IECs to colonic tumor formation following administration of the somatic mutagen azoxymethane (AOM; Putoczki and Ernst, unpublished observations), which induces missense mutations in exon 3 of ␤-catenin and constitutive activation of the canonical Wnt pathway [14]. Likewise, expression of the constitutive active Stat3C mutant in mouse embryo fibroblasts induced premalignant features and these cells subsequently formed tumors once they acquired additional driver mutations [26]. Excessive Stat3 activity in response to gp130-family cytokines in gp130Y757F mice results in gastric tumor formation at less than 5 weeks of age [27] (Fig. 2). The latter was reconciled by the observation that the tyrosine to phenylalanine substitution mutation in gp130 of these mice not only prevented Socs3 bindings, but also that of Shp2 and hence impaired Shp2-Ras-Erk pathway activation and associated expression of the gastric tumor suppressor gene Tff1 [27]. Therefore, we find that in the presence of wild-type gp130 excessive Stat3 activation in Socs3-depeleted gastric epithelium only infrequently triggers tumorigenesis and only after a prolonged latency period of several months, indicative of a requirement for additional mutations. These observations argue that, although excessive Stat3 activity may serve as the first of several oncogenic mutations, additional genetic events are required for tumor formation.

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In classical two-step tumor initiation/promotion models, where administration of a somatic mutagen is followed by induction of an inflammatory response, Stat3 promotes tumor burden in the skin and liver [25]. Likewise, in the colitis-associated colon (CAC) cancer model, excessive Stat3 activity in Socs3-deficient IECs results in more and larger tubular adenomas, and this is also observed in CAC-challenged gp130Y757F mice. It has been suggested that sphingosine kinase 1, and its product sphingosine-1-phosphate, further exacerbate CAC tumor development by inducing Stat3 phosphorylation directly, as well as indirectly through an NF␬B/IL6-dependent mechanism [28]. By contrast, CAC challenge of mice with Stat3-deficient IECs only results in few superficial adenomas [14]. Excessive global Stat3 activation in gp130Y757F mice or IEC-specific activation in A33:L-gp130Stat3 mice also increased intestinal tumor burden, when crossed to the ApcMin background, where loss-of-heterozygosity of the Apc tumor suppressor gene results in aberrant activation of the Wnt signaling cascade and tumor formation (Putoczki, Phesse and Ernst, unpublished). Surprisingly, IEC-specific Stat3-deficiency in ApcMin mice only initially reduced tumor burden, with the emerging tumors eventually displaying a more aggressive phenotype [29], which may arise from a compensatory (over-) engagement of Stat1 in the IECs of these mice.

2.4. Stat3 and the Hallmarks of cancer Persistent Stat3 activation is a unifying feature of a majority of epithelial malignancies, and transcriptionally active pY-Stat3 is commonly elevated in colon adenocarcinomas and correlates with pathological staging and tissue invasion [30]. Likewise, elevated pY-Stat3 expression is associated with reduced survival of gastric cancer patients [25]. These observations, alongside those cited in the previous sections, warrant assessment of Stat3 in the context on the “hallmark” and “enabling” characteristics of cancer cells [31].

2.4.1. Sustained proliferation, evasion of growth suppression and resistance to cell death Stat3 regulates the transcription of survival genes, including the anti-apoptotic Bcl-XL , Bcl-2, Bcl-w, Mcl-1 proteins of the intrinsic apoptosis pathway alongside the survival and proliferation marker Survivin [32]. Stat3 also directly promotes cell cycle progression and proliferation through transcriptional induction of c-Myc, CyclinD1, CyclinB1, and Cdc2, alongside the induction of c-Jun and c-Fos [27]. Accordingly, inhibition of Stat3 activation in the intestinal epithelium or in human cancer cell lines results in cell cycle arrest at the G2/M transition and is associated with histone H3 phosphorylation-associated mitotic arrest. In cooperation with c-Jun, Stat3 can also inhibit the extrinsic apoptosis pathways through transcriptional repression of the Fas death receptor [33].

2.4.2. Sustained angiogenesis, invasion and metastasis Stat3 regulates the transcription of factors that coordinate angiogenesis, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor and HIF1␣ [25]. Hence, Stat3 and VEGF expression often correlate with vascular density in gastric cancer samples. Excessive Stat3 activity promotes metastasis of colon cancer cells to the liver, melanoma cells to the brain and breast tumor metastasis to the lung and this is associated with the capacity Stat3 to induces expression of the tissue-degrading metalloproteinases Mmp-2 and Mmp-9 [25]. In addition, Twist, Snail, Cten and other regulators of the epithelial-to-mesenchymal transition are also activated by Stat3 [25,32], most likely owing to the phylogenic role of Stat3 as a regulator of wound-healing.

2.4.3. Reprogramming of cellular metabolism Recent reports suggest a non-transcriptional effect by serine phosphorylated Stat3 in mitochondria to augment the electron transport chain that underpins ATP synthesis [25]. However, mitochondrial Stat3 can also stimulate the glycolysis pathway, thereby promoting the “Warburg effect” in cancer cells [34]. In part this is attributed to the capacity of Stat3 to increase enzymatic activities in the glycolytic pathway through transcriptional activation and stabilization of HIF1␣, and simultaneous suppression of mitochondrial gene functions [34]. 2.4.4. Avoid immune destruction Stat3 promotes the secretion of IL10 and VEGF from tumor cells, thereby curbing the Th1 anti-tumor response [35]. Stat3 also inhibits the maturation and activation of dendritic cells, which favors the polarization and activation of tumor associated macrophages and results in reduced cytotoxic activity of neutrophils and natural killer cells [35]. Socs3-deficiency in macrophages, and associated excessive Stat3 activation, favors a M2-like, “wound-healing” phenotype, while excessive IFN␥/Stat1 activity in Socs2-deficient macrophages promotes polarization toward a phagocytic M1-like phenotype [36]. Furthermore, the physical contact between tumor and antigen presenting cells directly activates Stat3 and triggers a tolerogenic dendritic cell phenotype [37]. Accordingly, myeloid-specific Stat3 ablation enhanced anti-tumor immunity. Meanwhile excessive, Stat3-dependent IL23 production by tumor-associated myeloid cells helps stabilizing the phenotype of Th17 cells, which secrete large amounts of IL17A to stimulate tumor angiogenesis by acting on cancer-associated fibroblasts and endothelial cells. 2.4.5. Maintenance of cancer stem cells Based on its capacity to maintain pluripotency of embryonic stem cells, the gp130/Stat3 signaling cascade has also been implicated in regulating cancer stem cells. The dynamic equilibrium between non-stem cancer cells and the inducible formation of breast cancer stem cells, for instance, is dependent on IL6/Stat3 signaling [38]. Likewise, gp130/Stat3 signaling maintains glioma stem cells [39] although the Ezh2 methyltransferase also enhances Stat3 activation in these cells [40]. Meanwhile tumor-initiating ALDH+ /CD133+ human colon cancer cells expressed higher levels of pY-Stat3 than non-stem cell populations [41]. Furthermore, the stem cell factors Klf4 and Klf5 are transcriptionally regulated by Stat3, and at least the latter is required for the maintenance of intestinal stem cells. 3. Stat3 regulating cytokines In most cancers persistent Stat3 activation occurs in the absence of Stat3 gene amplification, with somatic mutations that confer constitutive Stat3 dimerization being relatively rare. More frequently, constitutive Stat3 activation results from somatic mutations in upstream receptors (i.e. gp130, EGFR) and cytoplasmic tyrosine kinases (incl. Jak, Src, Abl) [25]. Likewise, genetic or epigenetic impairment mutations in negative regulators, such as Socs3, PTPRT, PTPRD, CD45 [25] have been described and result in excessive, albeit ligand-dependent, Stat3 activation. IL6-dependent gp130 signaling in dysplastic colon cancer cells, for instance, is associated with Dnmt1-mediated Socs3 hypermethylation and gene silencing [42]. However, excessive Stat3 activity is predominantly the consequence of an oversupply of autocrine and/or paracrine activating cytokines secreted by tumor cells and/or those residing in the tumor microenvironment. In the context of communicating Stat3-depenent responses in gastrointestinal cancers, the most important regulators are cytokines of the IL6 and IL10 families,

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Homeostasis

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Colitis

Tumor

pY-Stat3

Commensal bacteria

pY-Stat3

IL11

Mucous barrier

pY-Stat3 pY-Stat3 pY-Stat3 pY-Stat3 pY-Stat3

Dendritic cells

IL17 IL22

IL6 IL23 Th17

pY-Stat3 pY-Stat3

IL6 IL11 IL17 IL22

IL12 IL23

Macrophages

IL6

Fig. 3. Schematic representation of the major cytokines affecting Stat3 signaling within the intestinal epithelium in health and disease. During intestinal homeostasis, a continuously renewing single layer of epithelial cells and its associated layer of mucus provides a barrier between the intestinal microflora and the resident immune cells in the submucosa. When the integrity of the barrier is compromised, dendritic cells trigger an immune response through production of IL12/IL23 and other mediators, which in turn recruit and/or activate macrophages and lymphoid (incl Th17) effector cells. Among the cytokines produced by these cells, those that activate epithelial Stat3 mediated survival and proliferation to promote a wound-healing response. However in mutated and/or neoplastic transformed IECs, these signals also stimulate cancer hallmark capabilities and promote the growth of gastrointestinal tumors. Intestinal wound-healing and tumorigenesis critically depends on IEC- and hematopoietic cells-derived IL11 and both processes are severely impaired in ILR␣1KO mice (see text for details).

whose activity in turn is required for differentiation and polarization of other IL17/IL23/IL22 producing lymphoid cell types (Fig. 3). In addition, Stat3 is also activated by the IL17/IL23 cytokine families, tumor necrosis factor ␣, platelet-derived growth factor, granulocyte colony stimulating factor, and integrins [25]. 3.1. IL6 family cytokines The IL6 cytokine family is defined by the shared use of the transmembrane receptor gp130, also referred to as IL6st or CD130, and comprises IL6, IL11, IL27, IL31, leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor (CNTF), cardiotrophin-1, cardiotrophin-like cytokine, and neuropoietin (NP). Upon receptor engagement, IL6 and IL11 mediate gp130 homodimerization, while all other ligands (except IL31), induce heterodimerization of gp130 with one of the related trans-membrane molecules LIFR, OSMR or WSX-1/T [43] (Fig. 1). Meanwhile, IL31 induces formation of an OSMR/IL31R␣ receptor heterodimer. Upon gp130 dimerization and Jak-mediated phosphorylation of specific Y residues, these receptor ␤-chains mediate Y-phosphorylation and activation of Stat1/Stat3, the Shp2/Ras/Erk and the PI3K/Akt signaling cascade as well as Stat3-mediated induction of the negative regulators Socs3. The latter requires binding to the membrane proximal pY residue in gp130 to limit the extent and duration of gp130-dependent signaling [44]. 3.1.1. IL6 and IL11 specific receptor subunits Owing to the wide expression of gp130, IL6 family cytokines are not only pleiotropic in activity, but also somewhat redundant in their function, where tissue-specific activity is governed by the more restricted expression of the additional receptor components that enable binding of the ligands to gp130. In the case, of IL6, IL11 and CNTF these components comprise membraneassociated receptor ␣-subunits. Accordingly, IL6KO and IL6R␣KO

mice display impaired acute phase response upon immunological challenge and mature IL6KO mice have reduced numbers of T-lymphocytes and thymocytes and fewer T-cell progenitors and bone marrow stem cells [45]. In mice, the IL6R␣ is relatively broadly expressed, including lung, liver, spleen, pancreas, stomach and IECs and in many immune cells types [46]. A soluble form of IL6R␣ occurs in serum and can confer IL6 responsiveness to cells devoid of membrane-associated IL6R␣, including T cells, neural cells, smooth muscle cells, endothelial cells and epithelial cells in a process known as “trans-signaling”. Meanwhile the IL11R␣ exists in two isoforms, IL11R␣1 and IL11R␣2, with the latter lacking an intracellular domain [46]. In mice, low levels of IL11R␣1 expression occurs in the lung, thymus, spleen, kidney, IECs, bone marrow and uterus, and IL11R␣1-deficiency in pregnant females prevents formation of a functional decidua [46]. Meanwhile, IL11R␣2 expression is restricted to the thymus, testis and lymph nodes [46]. Although a soluble version of the IL11R␣ receptors does not occur naturally, engineered soluble IL11R1␣ can bind to IL11 and activate gp130 signaling. IL11 signaling plays an important role in thrombopoiesis, embryogenesis, immunomodulation, IEC homeostasis, mucosal protection, hematopoiesis, macrophage and osteoclast differentiation and promotion of stem cell development [46]. IL11 protects the clonogenic stem cells in murine small-intestinal crypts from impairment of their reproductive capacity after ␥-radiation [47]. Although many of these biological activities of IL11 are also shared with IL6 and converge at the level of Stat1/3 activation, the full extent by which IL6 and IL11 display unique biological activities is still unknown. 3.1.2. IL11 and colitis In serum of healthy individuals IL11 remains undetectable, but is abundantly present in serum of mice during viral-induced inflammation [46]. Recently, subepithelial myofibroblasts have

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been described to produce IL11 in response to Th17 cell-derived IL22 or colon cancer cell-derived TGF␤ [48,49]. Furthermore, serum IL11 levels are elevated in a rat model of IBD, although this has not been reported for serum from IBD patients, which have increased levels of serum IL6 [46]. Administration of recombinant IL11, or gene delivery of IL22 [50], ameliorates colitis in mouse models, while reduced epithelial Stat3 expression in response to either IL6 or IL11R␣1 gene ablation [30], or administration of neutralizing antibodies against IL22 [51], enhanced susceptibility of mice to DSS-induced colitis.

locally confined, boosting mechanism to stimulate IL6R␣-deficient epithelial “stem cells”. In support of this, IL6 and IL11 signaling is functionally redundant for epithelial regeneration following ␥-radiation, and both cytokines were equally potent in conferring partial resistance to DSS-induced colitis (Putoczki, Phesse and Ernst, unpublished). Since IL6 trans-signaling also promotes regeneration in the liver [55], and when concomitantly over-expressed with IL6R␣, induced hepatocellular carcinomas in mice [56], it is likely that IL11 also plays a role in the regenerative capacity of the liver and other organs [57].

3.1.3. IL11 and gastric tumorigenesis We have observed that in the absence of IL11R␣1 that gp130Y757F mice remain free of gastric tumors [52]. Meanwhile, IL6 gene ablation did not affect tumor burden in these mice, but reduced inflammatory cell accumulation in the liver and lung, although in the stomach the presence of these cells correlated with the presence of tumors. All of these pathologies required excessive Stat3 activation and were prevented upon systemic administration of Stat3 antisense oligonucleotides of genetic reduction of Stat3 expression [27,52]. Established tumor burden was also reduced upon Stat3 ablation in the gastric epithelium [53], while monoallelic IL11R␣1 ablation delayed the onset and reduced overall tumor burden [30]. The same effect was observed upon therapeutic administration of the IL11 antagonist IL11 Mutein. This correlated with reduced tumor cell proliferation and Cyclin D1/D2 expression, as well as increased epithelial apoptosis, although the latter coincided with re-expression of the BH3-only protein Bim, rather than with suppression of Bcl2 family members as prototypical Stat3 target genes [30]. Furthermore, Stat3 antisense, Jak-inhibitor or IL11 Mutein treatment also reduced the abundance of tumor infiltrating CD45+ hematopoietic cells and inflammatory cytokines, but tumor burden remained unaffected in chimeric mice reconstituted with IL11R␣1-deficient bone marrow [30]. As CD45+ and EpCam+ epithelial cells account for much of the source of IL11 in these tumors, and IL11 gene expression is induced upon Stat3 activation, these feed forward mechanisms fuel tumor growth once IL11 Mutein or Jak-inhibitor treatment ceased [30,53].

3.1.5. IL27 and intestinal tumorigenesis IL27 confers potent anti-tumor activity in a model of colon carcinoma cells, since it synergizes with IL12 to induce IFN␥ production and proliferation of naive CD8+ T cells. This activity depends on the capacity of IL27 to activate Stat1 rather than Stat3 [58] and is reminiscent of similar observations that Stat1 was also required for IL27 to inhibit the activity of IL17 during chronic inflammation [59].

3.1.4. IL11 and intestinal tumorigenesis In the CAC-model, IL6 gene deletion, or treatment of mice with anti-IL6 antibodies, suppresses tumor burden [5,52] in part by disrupting IL6 trans-signaling, which suppresses apoptosis of infiltrating T cells [23]. The latter, alongside myeloid cells within the lamina propria, are the major source of IL6. Thus, administration of neutralizing IL6R␣ antibodies or soluble gp130Fc suppressed Stat3 activation and IEC proliferation, and reduced tumor incidence [54]. However, we identified a much more prominent role for IL11, which not only promoted inflammation-associated, but also sporadic colon cancer in ApcMin mice or mice challenged repeatedly with AOM [30]. Consistent with a more profound capacity of IL11 to promote colon cancer growth than IL6, administration of a Hyper-IL6 (a fusion protein between IL6 and IL6R␣) but not IL6, increased CAC tumor burden in wild-type mice [5]. These observations suggest that the tumor initiating stem cells, and possibly to some extent their normal counterparts, may have a strong bias toward expression of IL11R␣1 over IL6R␣. Indeed, gene expression analysis of gastric tumors from gp130Y757F mice suggest that IL11 induced genes primarily associate with epithelial functions, while those induced by IL6 are linked to immune functions [13]. This suggests a phylogenetic advantage of IL11 over IL6 to locally maximize epithelial proliferation during intestinal wound-healing and possibly owing to the fact that IL6 is systemically induced by many different inflammatory stimuli. Therefore, we speculate that IL6 trans-signaling, and the capacity of inflammatory cells to mediate shedding of membrane bound IL6R␣, provides an additional,

3.2. IL10/22 family cytokines The IL10/IL22 cytokine family is defined by its shared use of the IL10R2 ␣-receptor and comprises IL10, IL19, IL20, IL22, IL24 IL26, IL28 and IL29. Ligand binding triggers the formation of heterodimeric receptor complexes, which comprise IL22R and IL10R2 in the case of IL22. A soluble IL22R (also referred to IL22 binding protein) acts as a decoy to limit the availability of free ligand to bind to the trans-membrane receptor. Signaling through the IL10R2 heterodimeric receptor complex involves Jaks, although activation of Stat3 can also occur independently of receptor Yphosphorylation [60]. Since the IL10R2 receptor family lacks Socs3 binding sites and associated negative feedback regulation, receptor engagement results in persistent Stat3 activation. Accordingly, at least in macrophages, transient (gp130-mediated) Stat3 activation confers pro-inflammatory effects, while sustained Stat3 activation in response to engagement of the IL10R2 (or the gp130Y757 ) receptors triggers an anti-inflammatory response [25]. IL10 confers broad anti-inflammatory responses, which can be amplified by a feed-forward loop whereby Stat3 transcriptionally stimulates IL10 expression [25]. Accordingly, IL10KO mice, akin those with Stat3-deficient macrophages, show excessive cytokine release and develop colitis. However, IL10 also induces gp130 expression in mast cells [61] to enable their responsiveness to gp130 cytokines and to support gastric tumor growth in gp130Y757F mice (Eissmann and Ernst, unpublished observations). IL22 serum levels are elevated in IBD patients, as well as in animal models of enterocolitis, and correlate positively with disease severity [49]. IL22 producing cells are found in the lamina propria and submucosa of IBD patients and include natural killer cells alongside Th1, Th17 and the newly discovered innate lymphoid cells [61]. IL22 acts as a “directional cytokine”, owing to its immune cell expression and to the IL22R receptor expressed by skin, intestine, liver, colon, lung, kidney and pancreas [8]. IL22 targets IECs to induce proliferation and wound-healing, and through enhanced expression of Muc1, Muc3, Muc13, RegIII-␤ and RegIII-␥ mediate epithelial protection against bacterial pathogens [14,49]. Therefore, ablation of IL22 in knockout mice, or following administration of neutralizing antibodies, increases susceptibility to DSS-induced or citrobacter infection-associated colitis, while ablation of the IL22 binding protein increased tumor burden in the CAC model [62]. 3.3. IL17/IL23 cytokines During prolonged inflammation CD4+ cells become polarized toward distinct subsets that promote different effector activities

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including the Th17 lineage. In vitro polarization of murine naïve CD4+ T cells toward the Th17 lineage requires a combination of T cell antigen receptor stimulation together with IL6, IL21 and TGF␤ [63]. In addition, IL23 is essential for the pathogenic maintenance of the Th17 population, which also requires Stat3-mediated induction of the transcription factors Ror␥T and Ror␣ [64]. Stat3 activation in the tumor microenvironment also shifts the cytokine balance from IL12 to IL23 [65] by inhibiting transcription of the IL12p35 in favor of enhancing IL23p19 transcription as the two subunits, which compete for covalent binding with p40 to form mature IL12 and IL23, respectively. Finally, Stat3 also binds directly to the promoters of the IL17A and IL17F genes [66] as the major cytokine produced by Th17 cells. Th17 cells are most abundant at steady state in gut-associated tissues, particularly the small intestinal lamina propria [63], where they respond to pathogen invasion [67]. IL17A and IL23 gene expression has been linked to human cancers [68,69], with increased numbers of CD4+ Th17 cells correlating with cancer stage. In mice, Th17 lamina propria cells have been associated with enhanced tumor formation in ApcMin mice [70] and thus implicate IL17/IL23 cytokines for mucosal homeostasis.

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5. Conclusions Within the gastrointestinal epithelium, Stat3 provides a central signaling node that ensures intestinal homeostasis as well as an effective wound-healing response. Alongside NF-␬B, Stat3 provides a functional link by which activation of inflammatory cells within the tumor microenvironment triggers epithelial cell survival, growth and an angiogenic response. In turn, gastrointestinal tumors become addicted to persistent Stat3 activity, which arises predominantly from oversupply of cytokines rather than the less frequent acquisition of somatic mutations in the associated signaling cascades. Among the Stat3 activating cytokines and growth factors, IL22 and IL11 appear to play unique and indispensible roles and this can be exploited therapeutically by targeting these cytokines, the associated Jak kinases or Stat3 itself. Conflict of interest statement The authors declare no competing financial interests. Acknowledgments

4. Therapeutic implications The concept of Stat3 providing a prime anti-cancer target is reinforced by therapeutically favorable differences in sensitivity between normal and tumor cells to Stat3 signaling. At least in adult mice, a 50–75% systemic reduction of Stat3 triggers enterocolitis, impairs T-cell migration or causes Th1 autoimmunity [71]. Likewise, the Hyper-IgE syndrome associated with dominant-negative mutations in STAT3 in humans only occurs upon reduction of STAT3 activity in CD4 cells by approximately 75% [72]. Encouragingly, many tool compounds designed to interfere with Stat3 activity suppress the growth of breast cancer, myeloma and melanoma cell lines in xenograph models [25]. Meanwhile, chemically modified antisense oligonucleotides are currently undergoing clinical trials [73]. Although extensive redundancy among Stat3 activating cytokines provides a strong rationale to target Stat3 itself, interference with upstream components may increase specificity. Therapeutic inhibition of Jak kinases with small molecules reduces gastric tumor burden in gp130Y757F mice and colonic tumor burden in CAC-challenged wild-type mice [53]. Thus, current advanced clinical trials with Jak inhibitors for myeloproliferative disease should allow for their timely expansion to gastrointestinal cancers. Meanwhile, the notion of cancer cells developing high reliance on non-mutated pathways indicates novel therapeutic opportunities. Growth and maintenance of gastrointestinal tumors that arise in the context of an inflamed microenvironment and show excessive activation of the gp130/Stat3 pathway, for instance, becomes exquisitely sensitive to therapeutic inhibition of the PI3K/mTor pathway [74]. We predict that therapeutic targeting of IL11 signaling may confer a selective effect and therefore prevent thrombocytopenia as an on-target side effect often associated with systemic inhibition of Stat3 or Jak2. In mice we find that peptide-based IL11 Mutein was well tolerated with no adverse effects on platelet numbers [30], despite the capacity of IL11 to stimulate megakaryopoiesis. These findings are also consistent with normal steady-state hematopoiesis in IL11R␣1KO mice and their response to chemoablative stress [46]. Therapeutic interference with IL11 signaling is therefore likely to selectively suppress Stat3-associated cancer hallmark capabilities, which collectively promote progression of gastrointestinal disease in humans.

TLP and ME thank the members of their laboratories for their contributions. Work in the laboratories of TLP and ME is supported by the Ludwig Institute for Cancer Research, the Victorian State Government Operational Infrastructure Support, the IRIISS scheme of the National Health and Medical Research Council Australia (NHMRC), and NHMRC grant #1008614 awarded to TLP, and grants #487922, #433617 and #603122 awarded to ME. ME is an NHMRC Senior Research Fellow. Apologize that due to space limitations not all original research articles could be cited. References [1] Maritano D, Sugrue ML, Tininini S, Dewilde S, Strobl B, Fu X, et al. The STAT3 isoforms alpha and beta have unique and specific functions. Nat Immunol 2004;5:401–9. [2] Schaefer TS, Sanders LK, Park OK, Nathans D. Functional differences between Stat3alpha and Stat3beta. Mol Cell Biol 1997;17:5307–16. [3] Korzus E, Torchia J, Rose DW, Xu L, Kurokawa R, McInerney EM, et al. Transcription factor-specific requirements for coactivators and their acetyltransferase functions. Science 1998;279:703–7. [4] Yang G, Lim CY, Li C, Xiao X, Radda GK, Cao X, et al. FoxO1 inhibits leptin regulation of pro-opiomelanocortin promoter activity by blocking STAT3 interaction with specificity protein 1. J Biol Chem 2009;284:3719–27. [5] Grivennikov S, Karin E, Terzic J, Mucida D, Yu GY, Vallabhapurapu S, et al. IL-6 and Stat3 are required for survival of intestinal epithelial cells and development of colitis-associated cancer. Cancer Cell 2009;15:103–13. [6] Yoshida Y, Kumar A, Koyama Y, Peng H, Arman A, Boch JA, et al. Interleukin 1 activates STAT3/nuclear factor-kappaB cross-talk via a unique TRAF6- and p65-dependent mechanism. J Biol Chem 2004;279:1768–76. [7] Grivennikov SI, Karin M. Dangerous liaisons: STAT3 and NF-kappaB collaboration and crosstalk in cancer. Cytokine Growth Factor Rev 2010;21: 11–9. [8] Wolk K, Kunz S, Witte E, Friedrich M, Asadullah K, Sabat R. IL-22 increases the innate immunity of tissues. Immunity 2004;21:241–54. [9] Li YY, Hsieh LL, Tang RP, Liao SK, Yeh KY. Macrophage-derived interleukin-6 up-regulates MUC1, but down-regulates MUC2 expression in the human colon cancer HT-29 cell line. Cell Immunol 2009;256:19–26. [10] Mejias-Luque R, Linden SK, Garrido M, Tye H, Najdovska M, Jenkins BJ, et al. Inflammation modulates the expression of the intestinal mucins MUC2 and MUC4 in gastric tumors. Oncogene 2010;29:1753–62. [11] Sikora A, Grzesiuk E. Heat shock response in gastrointestinal tract. J Physiol Pharmacol 2007;58(Suppl. 3):43–62. [12] Heazlewood CK, Cook MC, Eri R, Price GR, Tauro SB, Taupin D, et al. Aberrant mucin assembly in mice causes endoplasmic reticulum stress and spontaneous inflammation resembling ulcerative colitis. PLoS Med 2008;5:e54. [13] Matthews JR, Sansom OJ, Clarke AR. Absolute requirement for STAT3 function in small-intestine crypt stem cell survival. Cell Death Differ 2011;18: 1934–43. [14] Bollrath J, Phesse TJ, von Burstin VA, Putoczki T, Bennecke M, Bateman T, et al. Gp130-mediated Stat3 activation in enterocytes regulates cell survival and cell-cycle progression during colitis-associated tumorigenesis. Cancer Cell 2009;15:91–102.

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