Targeting STAT3 in gastric cancer

1. Introduction

2. Constitutive STAT3 activation

is associated with both cancer

initiation and progression

3. Regulation of STAT3

signalling and its relevance to

gastric neoplasia

4. STAT3 in murine models of

gastric cancer

5. Upstream regulators of

STAT3 and their association

with H. pylori inflammation

6. H. pylori, STAT3 and IL-17

7. STAT3 and CagA

8. STAT3 and VacA

9. STAT3-activated genes that

promote the gastric cancer

phenotype

10. Development of IL-11Ra/gp130 antagonists and

STAT3 inhibitors as

therapeutic targets

11. Expert opinion

Review

Targeting STAT3 in gastric cancerAndrew S Giraud†, Trevelyan R Menheniott & Louise M Judd†Murdoch Childrens Research Institute, Royal Childrens Hospital, Parkville, Australia

Introduction: STAT3 is a key transcription factor for many regulatory factors

that modulate gene transcription. Particularly important are cytokines and

growth factors that maintain homeostasis by regulating immunocytes,

stromal and epithelial cells. Dysregulation of STAT3 by constitutive activation

plays an important role in the initiation of inflammation and cellular trans-

formation in numerous cancers, especially of epithelial origin. This review

focuses on STAT3 drive in gastric cancer initiation and progression, with

emphasis on its activation by cytokines, and how targeting the primary drivers

or gastric STAT3 therapeutically may prevent or slow stomach cancer

development.

Areas covered: This review will discuss the mechanics of STAT3 signalling, how

constitutive STAT3 activation promotes gastric tumourigenesis in both human

adenocarcinomas and mouse models, the nature of the upstream regulators

of STAT3, and their association with chronic Helicobacter pylori infection,

STAT3-activated genes that promote transformation and progression, and

finally the development and use of STAT3 and upstream cytokine inhibitors

as therapeutics.

Expert opinion: Chronic STAT3 activation is a key event in gastric cancer

induction and progression. Specific targeting of stomach epithelial STAT3 or

blocking IL-11Ra/gp130 and/or EGFR signal transduction in chronic gastric

inflammation and metaplasia may be therapeutically effective in preventing

gastric carcinogenesis.

Keywords: gastric cancer, IL-11, inhibitors, STAT3

Expert Opin. Ther. Targets [Early Online]

1. Introduction

Gastric cancer is a significant cause of morbidity and mortality worldwide, andhas the second highest death rate of all cancers. It was recently estimated thatnearly 1,000,000 new cases of gastric cancer are diagnosed worldwide each year [1]

(website: http://globocan.iarc.fr). Human gastric cancer is initiated after chronicinfection with the bacterium Helicobacter pylori. Induction of the most commonform, intestinal-type adenocarcinoma, is preceded by chronic gastritis (inflamma-tion), metaplasia and dysplasia in susceptible individuals. This stepwise progressionis known as the Correa paradigm after its originator [2]. The prognosis for treatmentis poor, largely because H. pylori infection is widespread and often asymptomaticfrom childhood, and so patients typically present after metastasis has occurred. Inaddition, antibiotic-based eradication is not globally practiced or available, andthe prospect of a vaccine in the near future is unlikely. Thus, chronic infectionwith H. pylori and persistent inflammation predisposes susceptible individuals todisease, so alternative therapeutic options based on blocking chronic inflammatoryand oncogenic signalling pathways, as well as new approaches to early cancerdetection, need to be addressed.

10.1517/14728222.2012.709238 © 2012 Informa UK, Ltd. ISSN 1472-8222 889All rights reserved: reproduction in whole or in part not permitted

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2. Constitutive STAT3 activation is associatedwith both cancer initiation and progression

Constitutively activated STAT3 is associated with numerouscancers including prostate [3], head and neck [4], breast [5],and colon [6]. When constitutively activated by phosphoryla-tion, STAT3 can transform terminally differentiated andmitotically quiescent cells into proliferating cancer cells [7].Few functional mutations in STAT3 have been reported [8],rather persistent activation is due to overexpression of upstreamligands resulting in chronic JAK2 activity. STAT3 may initiatetumourigenesis by activating transcription of genes involved inproliferation/transformation, cell cycle regulation, inhibition ofprogrammed cell death, and angiogenesis. Once a tumour isinitiated, persistent STAT3 activation can promote invasivegrowth and metastasis.

3. Regulation of STAT3 signalling and itsrelevance to gastric neoplasia

STAT3 is a key transcription factor for many cytokines andgrowth factors that promote gene transcription. Thus EGFreceptor ligands such as HB-EGF, TGFa and amphiregulin,as well as HGF and FGF, all of which are all expressed inthe stomach, can activate STAT3 by phosphorylation afterbinding their respective receptors [9]. Of particular importancein gastric neoplasia, and the focus of this review, are theinflammation-associated cytokines, including members ofthe IL-6 family which signal by binding their cognatea-receptor, which in turn binds to and initiates dimerisationof the signal transducing unit (b receptor or gp130) at theplasma membrane (Figures 1A, 2). The predominantIL-6 family ligands expressed in the stomach are IL-6 andIL-11. Increased expression of IL-11 is associated with chronicH. pylori-associated inflammation [10-12] and gastric cancer [13]

and this cytokine, unlike IL-6, is absolutely required for neo-plastic initiation in animal models of gastric tumourigene-sis [14,15]. Ligand-induced formation of the gp130 receptorcomplex results in phosphorylation of critical intracellular

tyrosine residues by JAK kinases, and triggers two majorsignalling cascades: the SHP2/ras/erk/AP-1 and STAT3 path-ways, which independently activate transcription of targetgenes. Under normal circumstances, these signalling pathwaysare in homeostatic balance, however when imbalanced andbiased towards the STAT3 cascade, the outcome is inflamma-tion, hyperplasia and eventually neoplasia. This has also beenshown to occur in human gastric adenocarcinoma [16,17] wherehigh levels of activated STAT3 are already present in pre-neoplastic atrophic gastritis, particularly in epithelial cellsassociated with CagA+ H. pylori infection [17]. The early acti-vation of STAT3 suggests that it is associated with initiationof infectious pathology, particularly chronic inflammationwhich ultimately contributes to transformation. Togetherthese data indicate that selective inhibition of STAT3 activa-tion may be therapeutically useful in blocking gastric andpotentially other gut tumour growth.

4. STAT3 in murine models of gastric cancer

Murine models of gastric cancer can be broadly divided into twocategories: inflammatory and non-inflammatory. The inflam-matory models typically require infection with H. pylori [18-20]

or H. felis [21], particularly on the background of other geneticalterations including gastrin overexpression [22-24] or loss ofheterozygosity of tumour suppressor genes. There are alsomodels that involve genetic alterations and chronic inflamma-tion in the absence of Helicobacter infection includinggp1307575FF mutant [14,25-28], MHC Class II mutant, IL-1boverexpression [29] and COX2-2/PGES1 overexpression [30-32].

Non-inflammatory models include those involving muta-tions in key genes required for gastric function: gastrin [33,34],H/K-ATPase subunits [35-37], ion transporters [38-42] and trefoilfactor genes [43]. Many of these models have hypergastrinemiaas a key component although both the gp130757FF [25] andthe gastrin null mouse [44] develop stomach tumour phenotypesin the absence of gastrin. Other non-inflammatory models arethose involving mutations in gastric regulatory genes including:mutations in TGFb and its signalling pathways, and mutationsin cell proliferation pathways, for example p53 and p27. Thefinal examples of non-inflammatory models are those inducedby chemical carcinogenesis including administration of MNUor MNNG, as well as mutations in AhR. It is pertinent tonote that for while the initiating mutation or insult would tra-ditionally be considered non-inflammatory, the ensuing cancerthat develops almost always involves an inflammatory response,and modulating this would be expected to reduce the tumourburden. STAT3 activation is a common feature of bothnon-inflammatory and inflammatory models, although itsexpression is not obligatory for their development.

Interest in the activation of STAT3 in murine models ofgastric cancer grew with the description of the gp1307575FF

mutant mouse. This mouse was generated by engineering aknock-in mutation of the critical tyrosine residue at position757 of gp130, the common co-receptor for the IL-6 family of

Article highlights.

. Chronic STAT3 activation is oncogenic in the stomach.

. IL-11/STAT3 drive is absolutely required for gastrictumourigenesis in the gp130757FF mouse, and isassociated with many mouse models of gastriccarcinogenesis.

. Persistent CagA-positive H. pylori infection activatesSTAT3 in mice and humans.

. STAT3 mediates Th-17 lineage specification andIL-17 effector function via H. pylori-dependentIL-23 signalling

. Targeting STAT3 activation therapeutically maycontribute to preventing gastric cancer development.

This box summarises key points contained in the article.

A. S. Giraud et al.

890 Expert Opin. Ther. Targets [Early Online]

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cytokines. This mutation prevents SHP-2 (and SOCS3)docking after ligand binding, thus blocking signal transductionvia ras/ERK/AP-1 signalling cascade [45]. SOCS3 normallyfunctions in feedback inhibition of the alternate STAT3 signal-ling cascade emanating from gp130 [25,45]. In the absence ofSOCS3 feedback inhibition in the gp1307575FF mutant,increased oncogenic signalling by STAT3 dimers is observed.As a result, mice develop rapid gastric tumourigenesis in thedistal stomach showing several histopathological features typi-cally observed in human gastric adenocarcinoma (Figure 2B).These features include gastritis, atrophy, mucous metaplasiaand SPEM, dysplasia and submucosal invasion, consistentwith the Correa paradigm [2], but without metastasis. In thismodel, increased activation of STAT3 homodimers is crucial

to tumour development [27,46]. Complete absence of STAT3in mice results in embryonic lethality, but gp130 757FF micewith genetic STAT3 haploinsufficiency develop significantlysmaller gastric tumours (Figure 2B-D) [27,46].

In gp130757FF mutant mice, STAT3 activation and tumourdevelopment does not occur without gp130 activation.IL-11 is a gastric gp130 ligand and is absolutely required forgp130757FF tumourigenesis, since gp130757FF mutant micethat also lack the IL-11 co-receptor, IL-11Ra, neither haveincreased STAT3 activation, nor do they develop gastrictumours [14,15]. gp1307575FF mice housed under specifiedpathogen-free (SPF) conditions and treated with antibiotics(metronidazole and ciprofloxacin) also exhibit restrictedSTAT3 activation and significantly reduced tumour

gp130757F/F STAT3WT/WT

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Figure 1. A. IL-11 signalling in the gp130757FF mouse model of gastric tumourigenesis. A knockin mutation (F for Y) at position

757 of gp130 prevents SHP2 and SOCS3 docking, thereby preventing MAP kinase signalling and activation of the tumour

suppressor trefoil factor 1 gene (tff1), as well as chronic activation of STAT3 respectively. B -- D. Antral tumours in gp130757FF

mice are much larger than those with haploinsufficiency for STAT3, macroscopically [B] in terms of surface area [C] and

microscopically in terms of cross-sectional area [D]. Reproduced from [27,28] with permission from Elsevier.

Targeting STAT3 in gastric cancer

Expert Opin. Ther. Targets [Early Online] 891

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growth [27]. Therefore STAT3 activation is crucial for tumourdevelopment in gp1307575FF mice, requiring both binding ofthe gp130 receptor by IL-11 and a gastric microbial presenceto initiate tumourigenesis.Increased activation of STAT3 and expression of IL-11 is

also observed in other models of gastric cancer that are not reli-ant on a mutation in gp130. Gastrin-deficient mice developgastric tumours in association with atrophy, inflammationand metaplasia by 12 months of age. These tumours exhibitelevated STAT3 activation driven by interferon (IFN)-g-biasedinflammation [44]. On the other hand, in the absence of aninflammatory stimulus, both activated STAT3 and tumouri-genesis are significantly reduced in gastrin-deficient mice. Iron-ically, activation of gp130 and subsequently STAT3 also occursin mice with hypergastrinaemia. Genetic ablation of the gastricH+K+ATPase b-subunit in mice leads to achlorhydria, hyper-gastrinaemia and tumour formation with co-incident IL-11expression and STAT3 drive [14]. Additionally, micewith experimental autoimmune gastritis exhibit elevatedIL-11 expression and STAT3 activation [37,47]. Finally, trans-genic mice that overexpress COX2-2/PGES1, K19-C2mEand K19-Wnt1C2mE develop fundic tumours with high levelsof IL-11 expression and STAT3 [14]. So although the moleculartriggers for gastric tumourigenesis are numerous and varied,most mouse models assessed to date are unified by an intrinsicmechanism of STAT3-driven inflammation. In keeping withthis, we have now established that intraperitoneal adminis-tration of high doses of IL-11 for 7 days, without any othermitigating factors, is sufficient to cause hyperactivation ofSTAT3 and injury to the gastric mucosa including gastritisand profound fundic atrophy [48].

5. Upstream regulators of STAT3 and theirassociation with H. pylori inflammation

H. pylori is a stomach-restricted bacterium that exerts a pro-found influence on the development of intestinal-type gastricadenocarcinoma. Significant advances have been made inunderstanding the molecular mechanisms by which H. pyloritriggers, but largely evades, host mucosal immunity. Thehost response to H. pylori is mediated by a highly evolvedmolecular dialogue between the bacterium, gastric mucosalcells and immune cells, involving an array of cytokines, theirreceptors and intracellular transduction cascades. Currentliterature indicates that pro-inflammatory cytokines producedby both infiltrating immune, as well as epithelial cells directlyinitiate and maintain a chronic inflammatory state, ulti-mately leading to gastric cancer in susceptible individuals.The widely described oncogenic role of STAT3 in severalorgan systems naturally poses the question of whetherSTAT3 activity is pivotal in the immune response to H. pyloriinfection and indeed whether it may mediate some of theoncogenic effects of the bacterium.STAT3 exerts a strong functional influence in both the

gastric mucosal and immune compartments [14,25-27,45] and,

by controlling specific intracellular signalling events, is well-positioned to dictate inflammatory and oncogenic outcomesin the context of chronic mucosal colonisation by H. pylori.With the potential of STAT3 as a target for pharmacotherapynow recognised, research efforts should prioritise STAT3inhibition at the formative stages of gastric cancer (H. pylorigastritis) as a strategy to prevent the emergence of increasinglysevere (and mostly irreversible) pathologies of atrophicgastritis, metaplasia and adenocarcinoma respectively. Herewe consider the role of STAT3 in the host immune responseto H. pylori infection from both the mucosal and immuno-logical perspectives, as well as delineating STAT3-dependentprocesses amenable to drug targeting.

We previously reported STAT3 hyperactivation inH. pylori-dependent gastritis [17] and on that basis argued fora functional role for STAT3 in the earliest stages of gastriccancer development. We observed nuclear localisation ofphosphorylated STAT3 in mucosal surface and pit epithelialcells of infected, but not uninfected individuals, suggestingthe active engagement of target gene promoters. This suppo-sition was supported by molecular studies showing increasedmRNA for the STAT3 transcriptional target, SOCS3. Ourfindings are also supported by functional studies ingp130757FF mice, as elucidated above.

6. H. pylori, STAT3 and IL-17

Aside from well-described mucosal homeostatic roles, it isincreasingly apparent that STAT3 activity in immune cellsmay also influence the outcome of H. pylori-related disease.H. pylori infection in humans is associated with a dramaticaccumulation of gastric mucosal mononuclear cells includingCD4+ T cells of the T helper (Th)-1 and Th17-type line-ages [49,50]. Th-1 cell recruitment and associated cytokinerelease are well-described components of the host immuneresponse to H. pylori. On the other hand, increased mucosalprevalence of Th-17 cells, together with elevated expressionof their lineage-defining cytokine, interleukin (IL)-17, haveonly recently gained recognition as factors mediatingH. pylori-specific immunity [49,50]. IL-17 collectively refers toa family of six members (IL-17A-F; reviewed in [51]). None-theless, current evidence argues strongly that IL-17 familycytokines play a central role in H. pylori-related gastritis andmay influence subsequent oncogenic progression [52,53]

although which members participate in the host response toH. pylori is unclear.

STAT3 plays an essential role in Th-17 lineage specifica-tion by activating expression of the critical transcriptionfactor, RORgt which in turn promotes IL-6-dependent,Th-17 polarisation of naive CD4-positive cells. Disruptionof (IL-6-dependent) STAT3 signalling blocks Th-17 differen-tiation without altering the abundance of Th-17 inhibitoryregulatory T cells (Tregs) [54].

Besides playing a key role in the specification of Th-17 fatevia induction of RORgt, STAT3 may also be required for the

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maintenance of Th-17 effector functions such as IL-17secretion. Caruso and colleagues, in efforts to elucidate themolecular basis of IL-17 production by Th-17 cells duringH. pylori infection, demonstrated a pivotal role for STAT3,acting in concert with the pro-inflammatory cytokineIL-23 [55]. They showed that exogenous IL-23 treatmentdirectly stimulated STAT3 phosphorylation and IL-17 secre-tion in acutely isolated gastric lamina propria mononuclearcells (LPMC), while pharmacological inhibition of STAT3blocked IL-23-dependent IL-17 release. Furthermore, IL-23immunoneutralisation in LPMC cultures derived fromH. pylori-infected patients suppressed both STAT3 activationand IL-17 expression [55]. Therefore, STAT3 positivelyregulates IL-17 transcription in infiltrating Th-17 cells bytransducing H. pylori-related IL-23 signals in infected gastricmucosal tissue.

Elevated IL-23 expression is well-documented in H. pyloriinfection [49] although the cellular basis of this response hasnot yet been formally substantiated in vivo. Nevertheless, thefindings of two recent in vitro studies implicate dendritic cells(DCs) as a likely mucosal source of H. pylori-relatedIL-23 [56,57]. Furthermore, H. pylori-primed DCs are able totrigger IL-17 production in autologous CD4+ T cells [57].Together these studies suggest an attractive model in whichH. pylori-dependent IL-23 release from DCs, acting viaSTAT3, leads to increased mucosal Th-17 cell prevalenceand pro-inflammatory IL-17 production (Figure 2).

IL-17 family cytokines do not exert direct chemotacticactivity but indirectly direct specific cell-mediated immuneresponses by stimulating the release of unrelated pro-inflammatory molecules such as IL-6, a canonical STAT3activator, and a range of chemokines. The process is evidentduring H. pylori infection, where IL-17 has been shown topromote secretion of the chemokine IL-8, by signalling viathe ERK 1/2 MAP kinase pathway [50]. Therefore,IL-17 may indirectly drive cell-mediated immunity (in partvia IL-8) which is a potent chemoattractant for neutrophils.Speculatively, future therapeutic targeting of STAT3 may,by uncoupling the IL-23/STAT3/IL-17 regulatory cascade,provide an effective strategy to subdue IL-17 activity (anddownstream pro-inflammatory actions) and potentiallyimprove the prognosis after chronic H. pylori infection.

The immune responses of immunised and challenged miceinclude T-lymphocyte recruitment and the production ofIL-17 [52]. As a consequence, neutrophil-attracting chemo-kines are released, and the bacterial load is considerablyreduced. Besides regulating gastric inflammation, IL-17 mayalso participate in immune remodelling after immunisation.During infection, Tregs restrain IL-17-specific inflammatoryresponses thus acting in favour of bacterial persistence [52].However a recently published study shows that IL-17 parti-cipates in reducing H. pylori colonisation levels in micefollowing immunisation and challenge [58]. Because immuni-sation is generally thought to produce Helicobacter-specificmemory T-helper cells that potentially alter the ratio between

Th-17 and Treg responses, these findings [58] suggest thatIL-17-mediated inflammatory reactions overwhelm the Tregresponse leading to bacterial clearance. Thus augmentationof STAT3 signalling, rather than inhibition of it, might bepredicted to improve the outcomes of immunisation againstH. pylori by strengthening IL-17-mediated bacterial clearance.

7. STAT3 and CagA

In the past decade, no other aspect of H. pylori biology hasreceived more attention than the cytotoxin-associated antigenA (CagA) [59,60]. CagA is a high molecular weight(120 -- 145 kDa) protein and principal cytotoxin of H. pyloriencoded by the cagA virulence gene. The cagA gene is locatedwithin the cag pathogenicity island (cagPAI), a 40 kb genomicDNA region containing 27 -- 31 virulence genes, some ofwhich encode components of a bacterial type IV secretionsystem (T4SS) that mediate the delivery of CagA into gastricepithelial cells [59,61]. A substantial body of evidence showsthat CagA-positive H. pylori strains exhibit greater virulencethan their CagA-negative counterparts, and carry an increasedrisk of gastric adenocarcinoma [62]. Functional evidence tosupport these studies comes from mouse transgenic experi-ments in which CagA overexpression led to uniformovergrowth of the gastric epithelium and low frequency,late-onset focal tumourigenesis, without significant inductionof gastritis or atrophy [63]. CagA therefore deregulates gastricepithelial cell homeostasis autonomously, but additionalfactors such as secondary somatic mutations, or inflammatorystimuli are evidently required for fully penetrant oncogenesis.The concept is supported by in vitro experiments showingthat CagA mediates oncogenic transformation of primarygastric epithelial cells but only in combination with otherimmortalising agents [64]. The functions of CagA are bestsummarised as follows: it has a demonstrated capacity to altercell polarity and growth kinetics, but its intrinsic transformingeffects are limited (when uncomplemented) and may contri-bute to oncogenic progression only in the subset ofH. pylori-infected individuals with pre-existing genetic orinflammatory susceptibility.

CagA is translocated from the bacterium to gastric epithelialcells via a bacterial type-IV secretion system [65]. Once interna-lised, CagA localises to the inner surface of the plasma mem-brane and is tyrosine phosphorylated at specific C-terminalGlu-Pro-Ile-Tyr-Ala (EPIYA) repeat motifs by src and c-Ablkinases [66,67]. Translocated CagA has been shown to interactwith several intracellular signal transduction cascades associatedwith cell growth and motility [66,68-73]. Current literature iden-tifies Src-homology protein tyrosine phosphatase (SHP)2 as themost widely described intracellular target of CagA [59]. Phos-phorylated CagA specifically binds and activates SHP2, leadingto inappropriate signalling through the SHP2-(Ras)-ERK(MAP-kinase) cascade, deregulated epithelial cell polarityand increased motility; the ‘hummingbird phenotype’ [68,74].Additionally, RNA interference (RNAi)-mediated knockdown



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of SHP2 prevents CagA-dependent ERK activation and deve-lopment of the hummingbird phenotype [74]. Therefore,SHP2 is a pivotal mediator of CagA function in gastricepithelial cells.The association of SHP2 with gastric gp130 signalling [45],

together with the necessity of correctly regulated gp130/JAK/STAT3 pathway activation for the maintenance of gastricmucosal homeostasis [25,45] prompted us to investigate the func-tional impact of CagA on STAT3. We observed significantlyhigher STAT3 activation levels in CagA-positive comparedto CagA-negative, H. pylori-infected human gastric mucosalbiopsies [17], providing empirical support for the perceivedlink between STAT3 and CagA, and have substantiatedthis by demonstrating that transfected CagA triggers STAT3phosphorylation in gastric epithelial cells [73].EPIYA-motif phosphorylation is a major determinant of

CagA-mediated signalling and target gene transcription withgeneral consensus that CagA functions predominantly, thoughnot exclusively, in the phosphorylated state [66,68-73,75,76]. Ourrecently published work shows that phosphorylation of tyrosineresidues within the CagA EPIYA repeat motifs is necessary foractivation of STAT3 since mutation of these tyrosines to serines(EPISA) abolished CagA-dependent STAT3 activation [73].

Two other groups have also demonstrated a link betweenCagA and STAT3 activation [76,77], however, all three studiesdisagree on the mechanism of STAT3 activation. As discussedabove, we have shown a requirement for CagA tyrosine phos-phorylation [73] whereas the other two studies report thatSTAT3 activation occurs either independently of CagA tyro-sine phosphorylation [76] or exclusively by unphosphorylatedCagA [77].

These discrepancies are not easily reconciled but may beexplained by the contrasting cell types and different experi-mental approaches used. One of the groups performed adetailed investigation of CagA-dependent STAT3 activationin non-gastric, laryngeal carcinoma-derived HEp-2 cells [76],whereas the second group assessed STAT3 responses in gastricAGS cells following infection with different H. pylori strainsincluding an internally deleted phosphorylation defectiveCagA mutant lacking an EPIYA motif. [77]. Our studies basedin gastric cell lines used inducible transgenes carrying eitherwild-type CagA or an amino acid substitution mutant inwhich the (EPIYA) tyrosine residues had been replaced withserines (EPISA) [73]. This approach allowed specific abolitionof EPIYA tyrosine phosphorylation without compromisingthe plasma membrane tethering of CagA [78].

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Figure 2. The H. pylori cytotoxin CagA activates signalling pathways downstream of gp130 to increase proliferation, motility

and cell polarity as well as synthesis of bactericidal Reg3g in an IL-11/STAT3-dependent fashion. In addition, CagA causes

IL-23 release from mucosal dendritic cells which activates STAT3 in CD4+ T cells and targets such as RORgt which can alter

Th17 differentiation and neutrophil recruitment. Reproduced from [27] with permission from Elsevier.

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8. STAT3 and VacA

The H. pylori vacuolating toxin VacA induces apoptosisof gastric epithelial cells after activation of Bcl-2 familyproteins. Since STAT3 activity is anti-apoptotic, it is notsurprising that VacA mediates STAT3 inhibition as well asreducing expression of Bcl-2 and Bcl-XL in gastric cancercell lines [79]. Whether functional parallels betweenSTAT3 and VacA exist in vivo is unknown, however thestrong and sustained activation of STAT3 in chronicH. pylori infection in both humans and mouse models (seeprevious sections) suggests that the STAT3-inhibitoryactions of VacA are overwhelmed by distinct mechanismsof H. pylori pathogenicity.

9. STAT3-activated genes that promote thegastric cancer phenotype

STAT3 is known to transcriptionally activate numerous targetgenes. We and others have investigated the spectrum of genesregulated by gp130/STAT3 drive that may have particularrelevance to gastric cancer. It has been established that the dis-tal stomach, where most gastric cancers initiate is particularlyresponsive to IL-11 (but is refractory to IL-6), and thatexogenous IL-11 preferentially activates only STAT and notMAPK/Erk signalling pathways [14]. Therefore the simplestway to ascertain which genes are directly activated bySTAT3 in the stomach in the absence of other confoundingpathologies is to administer IL-11 in vivo, and then quantifyeffects on the gastric transcriptome prior to significantmorphological changes to the gastric mucosa. In this contextit is important to understand that the stomach is composedof a number of functionally distinct compartments: theacid/enzyme secreting fundus (body) and the distal antrum.The spectrum of genes induced by IL-11/STAT3 is differentin these two compartments. The early gene changes in theantral stomach driven directly by activation of the IL-11/gp130/STAT3 pathway include: Regenerating-islet derived(Reg) family members, Clusterin, Gremlin-1, gp130,SOCS3, Jak1, TIMP1 and Gas1 [14]. Significantly, the expres-sion pattern for this IL-11-induced gene signature is alsoobserved in mouse genetic models of gastric disease indepen-dently of exogenous IL-11 administration. These modelsinclude gp130757FF mice, K19-C2mE transgenic mice andH+K+ATPase b-subunit-/- knockout mice. Incidentally, agene expression profile bearing remarkable similarity to themurine IL-11-induced gene signature is observed in humangastric biopsies collected from patients with H. pyloriinfection, pre-neoplastic and neoplastic gastric disease [14].

In contrast, the IL-11/STAT3 gene signature in the fundicmucosa is much more diverse and includes: i) proliferative genessuch as the Reg family members, Igfbp4, Gsdmc1, Grem1 andBlm1, ii) immune-related genes, e.g., Serpin a family members,Dmbt1, IL-33, Spp1, MyD88, and iii) signal transduction genes,e.g., Socs3, Jak3 and Jun b [14,28]. Many of these genes are

reported to be associated with human cancers especially theReg family members [11,80], Grem1 [81,82] and Blm1 [83].

Other studies have indirectly investigated STAT3-regulatedgenes by examining the correlation between STAT3 activationand gene expression, particularly in the setting of gastriccancer. Han and colleagues demonstrated a high correlationbetween STAT3 activation and expression of VEGF, c-Myc,survivin, MMP-7, CD44v6 and cyclinD1 [84], many of whichhave been confirmed to be STAT3-responsive in mouse [46].Others have shown that the expression of survivin and alsoBcl-2 is dependent on the activation of STAT3 [16,46,85]. Interms of the inflammatory response to H. pylori, it has beendemonstrated that the levels of IL-6 and IL-8 expression cor-relate with the activation of STAT3 in both the human [17]

and mouse [27]. In summary, a significant number of genesare proposed to be STAT3-responsive in the stomach. Someof these genes have been identified in mouse genetic studiesin which gastric STAT3 is activated in the absence of othersignificant pathogenic changes. Others have been identifiedin comparative mouse models and human studies in whichthere is a correlation between STAT3 activation and geneexpression, but on the background of complex gastric patho-logies. In spite of this complication all of these studies suggestthat, under permissive conditions, chronic activation ofSTAT3 is oncogenic in the stomach.

10. Development of IL-11Ra/gp130antagonists and STAT3 inhibitors astherapeutic targets

There are numerous points in the IL-11/STAT3 signallingpathway that are amenable to inhibitory blockade. Theseinclude antagonism of IL-11 binding to the IL-11Ra, blockadeof JAK kinase activation by de-phosphorylation, inhibition ofSTAT3 phosphorylation, preventing STAT3 dimerisation, pro-moting STAT3 de-phosphorylation, and inhibiting STAT3-mediated gene transcription. As well as endogenous STAT3inhibitors which include suppressor of cytokine signalling3 (SOCS3), protein inhibitor of activated STAT3 (PIAS3),the SHP2 tyrosine phosphatase, GRIM19 and Grb2 [28] thereare now many small molecule inhibitors of STAT3 that specifi-cally target each part of the IL-6/11 signalling cascade describedpreviously. These have been detailed recently in several compre-hensive reviews [86-89] and the most salient in relation to gastriccancer development are summarised below. To date, only a fewSTAT3 inhibitors have entered clinical trials despite the utilityof blocking constitutive STAT3 activation pathways inepithelial cancers.

10.1 IL-11Ra/gp130 antagonistsRelatively few IL-11 receptor antagonists have been described,likely because of limited information about the potentialpathogenic actions of this cytokine in the stomach, and itsclinical use (oprelvekin) in promoting megakaryocytopoiesisafter chemotherapy. In addition IL-11 has been proposed as



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a therapeutic agent in chronic inflammatory diseases such asCrohn’s disease [90], lending support to the erroneous ideathat it may have generalised anti-inflammatory actions inthe gut.One of the early antagonists to be evaluated for its therapeu-

tic potential was Madindoline A, a low molecular weight, non-peptide inhibitor of IL-6 and IL-11 based on a fulindolinestructure. Madindoline A was active in the 10 -- 100 µM rangefor osteoclastogenesis and proposed to act by suppression ofgp130 and respective ligand a-receptor dimerisation [91].More recently, a high affinity IL-11-specific antagonistW147A has been described [92]. This tryptophan-modifiedIL-11 analogue competitively disrupts gp130/IL-11Ra multi-mer formation and resultant signalling, with biological activityon human endometrial cells [92]. Since IL-11 and LIF areabsolutely required for blastocyst implantation [93], antago-nists of IL-11 action might also have application as contracep-tives. In response to this, polyethylene glycol (PEG)-ylatedIL-11 antagonist has been synthesised [93] and shown to bindthe IL-11Ra, thereby preventing proper interaction ofIL-11Ra and gp130 [93] and blocking STAT3 activation.Although effective after injection, PEG-ylated IL-11 is toolarge to be commercially synthesised as an IL-11 antagonistand is also unlikely to be sufficiently bioavailable if givenorally. To date, the successful application of PEG-ylatedIL-11 as an anti-cancer agent in the stomach has notbeen reported.

10.2 STAT3 inhibitorsInhibition of STAT3 in cancer progression is desirablebecause STAT3 plays a major role in a broad spectrum oftumours, promoting cell growth, survival, angiogenesis andthe local inflammatory response [89]. Since STAT3 is a tran-scriptional regulatory end-point for numerous and diverseregulatory pathways, it has been traditionally viewed as“undruggable” with respect to inhibition in cancer. However,more recently this view has changed [86-88], in part because ofthe realisation that STAT3 also acts as a signalling mediatorand its activation can be specifically blocked at numerouspoints in the signal transduction pathway. Additionally,because STAT3 activity in many normal tissues is low or tran-sient, but constitutively activated to high levels in cancer, ithas a high therapeutic index as a target for inhibition [94]

(but see section 10.3 below). Recent advances in STAT3inhibitor development have demonstrated that new genera-tion inhibitors not only have direct anti-cancer effects, butcan also synergise with other therapies [94,95] and reducemetastatic success. The main types of inhibitors are smallpeptides or peptidomimetics, small molecules derived fromlibrary or natural compound screens, oligonucleotides, andplatinum-based inhibitors [89]. Although medicinal chemistshave generally concentrated on inhibitors which targetSTAT dimerisation and DNA binding, there are many recentreports in the literature of STAT3 inhibitors which act bytyrosine kinase (especially JAK kinase) inhibition. Although

regarded as sub-optimal [86] likely due to concerns aboutspecificity, JAK kinase inhibitors have been described whichare of low molecular weight, orally bioavailable, stable, effica-cious in reducing tumour volume in vivo, and which haveIC50 values in the same range as STAT3 dimer inhibitors.An example is the caffeic acid derivative, WP1066, which pro-motes glioma cell apoptosis in vivo via STAT3 inhibition [96].Moreover, in unpublished work we have shown WP1066 toeffectively block gastric tumour progression in thegp130757FF mouse when given orally (Louise M. Judd,unpublished), and at similar doses to antisense oligonucleoti-des given intraperitoneally in the same model as reportedpreviously [15].

There are relatively few reports in the literature on the useand outcome of using STAT3 inhibitors for blocking humangastric cancer progression, despite the fact that increasedSTAT3 expression and activation has been shown to be associ-ated with gastric cancer development [17,84,97] and with poorsurvival [98-100]. Exceptions to this are three recent studies, test-ing novel phytochemicals against STAT3 activation in vitro andin vivo in the context of VEGF stimulation [101,102]. Thus, eupa-tilin, a flavone from Artemisia asiatica dose-dependentlyreduced STAT3 phosphorylation and VEGF promoter bindingunder hypoxic conditions in MKN45 gastric cancer cells, andalso inhibited VEGF neovascularisation of xenotransplantedcancer cells and tumour growth [101]. Likewise nitidine chloride,derived from Zanthoxylum nitidum had similar effects onSGC-7901 gastric cancer cells and tumours, against the sametargets and at a comparable dose range (7-10 mg/kg/day) [102].This latter cell line and its cisplatin-resistant derivative havealso been used to demonstrate that the porphyrin compoundDPP which prevents STAT3 dimerisation [103] is effective inreducing cisplatin resistance and inducing apoptosis in aSTAT3-dependent fashion [104]. Finally, the green tea deriva-tive (-)-epigallocatechin-3-gallate inhibited STAT3-mediatedVEGF expression in the gastric cancer cell line AGS [105].

Although gastric cancer is a multifaceted disease that hasproved resistant to standard chemotherapeutic treatment [106]

unless detected early, recent advances have begun to define themain molecular targets, for which focussed therapies might bedeveloped. For instance, a recent comprehensive genomic anal-ysis using SNP arrays to interrogate a panel of more than200 gastric cancers concluded that nearly 40% of those investi-gated could be independently classified according to discretegenomic alterations in one of EGFR (ERBB1), ERBB2,KRAS, FGFR2 or MET genes [107]. Since these receptor orreceptor-associated genes either have intrinsic tyrosine kinaseactivity, or are associated with particular tyrosine kinases whichcontribute to downstream signalling, it was concluded thatthese patients would be likely to respond to tyrosine kinase/RAS directed therapies. Changes in STAT3 copy numberwere not reported in this study, however all identified genegroups utilise STAT3-dependent signalling pathways, sugges-ting that inhibition of activated STAT3 might also havetherapeutic value in these patients along with targeted receptor

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approaches. In support of this, dual use of PI3-kinase andSTAT3 inhibitors have shown utility in inhibiting proliferationin gastric cancer cell lines with KRAS mutations [95], andFGFR2-amplified gastric cancers were sensitive to dovitinib [107]

a multi-kinase inhibitor that also targets STAT3 [108].

10.3 Off-target effects of STAT3 inhibitorsBecause STAT3 is a highly utilised transcription factor inmany tissues, global inhibition of its activity is likely toimpact normal cellular function in both health and disease.Therefore, off-target effects of STAT3 inhibition need to beconsidered when designing inhibitors.

Two potential undesirable actions of global STAT3 inhibi-tion are briefly considered here. The first relates to the obser-vation that STAT3 plays an important role in cardiacprotection and regulation of cardiac inflammation [109]. Theformer is thought to occur by induction of anti-oxidantenzymes as well as by promoting cell survival after inhibitionof key anti-apoptotic mediators [110]. STAT3 appears to bepleiotrophic with respect to regulation of inflammation; itcan mediate pro-inflammatory outcomes but can also inhibitinflammation when activated downstream of cardiacIL-11 [111] and IL-10 [112] in models of myocardial ischaemia.In addition STAT3 is intimately involved in mitochondrialrespiration [113], required for maintenance of energy balancein normal myocardial function [110].

A second area of concern for STAT3 inhibition is inIL-10 signalling. IL-10 is an important anti-inflammatorycytokine, particularly in the gut, where it is expressed by cellsof both the innate and adaptive immune systems, signallingvia the JAK/STAT3 pathway [114]. It is well established thatblockade of IL-10 signalling leads to the development ofinflammatory bowel disease (IBD) in mouse models [115]

and loss of function polymorphisms in IL-10R1 andIL-10R2 increase the chance of IBD development inhumans [116]. These examples highlight the contrasting rolesthat STAT3 plays in normal physiology and tumourigenesis,as well as emphasising the importance of selectively targeting

acute and chronic STAT3 activation in different tissuecompartments by therapeutic STAT3 inhibitors.

11. Expert opinion

STAT3 is a pivotal signalling molecule and transcriptionfactor in gastric mucosal function. Constitutive activation ofSTAT3 by hyperphosphorylation (often accompanied byonly a modest increase in gene expression as quantified bysteady-state mRNA analysis) contributes to pre-neoplasticprogression in the stomach, where it promotes inflammation,cell proliferation, angiogenesis, and inhibits programmed celldeath. Recent data from both animal models and humangastric cancers suggest that a major endogenous driver ofoncogenic STAT3 is the cytokine IL-11. IL-11 has pleiotro-phic actions with respect to inflammation and tumourigene-sis, which are directed in a tissue-specific fashion. Furtherresearch is required to understand the relative roles at eachstage of gastric cancer development of IL-11 and IL-6, bothof which can signal through gp130 along several differentsignal transduction pathways. Selective inhibition of STAT3appears to be a potentially useful therapeutic option inblocking gastric neoplastic progression. Caveats include off-target effects especially in the cardiovascular system, theability to develop selective, orally available antagonists, aswell as establishing whether STAT3 blockade will be effectivein ameliorating metastasis to secondary sites; a commonsituation at first presentation for therapy.

Acknowledgements

Supported by NHMRC (Australia) and the VictorianGovernment’s Operational Infrastructure Support Program.

Declaration of interest

The authors state no conflict of interest and have received nopayment in preparation of this manuscript.



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AffiliationAndrew S Giraud†1,2 PhD,

Trevelyan R Menheniott1 PhD &

Louise M Judd1 PhD†Author for correspondence1Murdoch Childrens Research Institute,

Royal Childrens Hospital,

Parkville, Australia2Research Director (Infection & Immunity),

Murdoch Childrens Research Institute,

Royal Childrens Hospital,

Flemington Rd,

Parkville, 3052, Australia

Tel: +61+3 8341 6446;

E-mail: [email protected]



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Targeting STAT3 in gastric cancer

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