the role of interleukin-6 in gynecological malignancies

8/13/2019 The role of Interleukin-6 in gynecological malignancies

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Mini review

The role of interleukin-6 in gynaecological malignancies

Jermaine I.G. Coward a ,*, Hagen Kulbe ba Cancer Therapeutics Group, Mater Medical Research Institute, Level 3 Aubigny Place, Mater Hospitals, Brisbane, QLD 4101, Australiab Barts Cancer Institute, Centre for Cancer and Inammation, Charterhouse Square, London EC1 6BQ, United Kingdom

1. Introduction

Collectively, gynaecological malignancies (i.e. epithelial ovarian(EOC), endometrial and cervical cancers) represent one of the mostcommon causes of female cancer death worldwide. Althoughadvanced presentations of these diseases are amenable to surgeryand exhibit modest sensitivity to chemotherapy, resistance invari-ably occurs and most patients eventually die within 5 years. Fromresearch into the biology of gynaecological cancers (GC) during thepast few decades, inammation has emerged as a chief orchestratorof processes that underpin disease progression and ultimately poorprognosis. Cancer-related inammation is particularly exempliedby the role of cytokines and chemokines in both the pathogenesisand progression of GC. Indeed, inammatory aetiological factorshave been well established in these tumour types, for example;

(a) Unopposed oestrogens have profound inammatory effects onthe endometrium that can result in the initiation andpropagation of neoplastic activity through upregulation of cytokines including interleukin (IL)-1, IL-6, tumour necrosisfactor (TNF)-a and matrix metalloproteases (MMPs) [1,2] .These processes are mediated through NF-k B signalling inendometrial adenocarcinoma [3] .

(b) Ovulation is characterised by local release of inammatorymediators including the cytokines IL-1b , IL-6, TNF-a , GM-CSF[4] , platelet activating factor, prostaglandins, histamine andbradykinin [5] . These substances may inuence the epithelium

in ovulatory tissue by enhancing cell replication and createoxidative stresses that can facilitate the development of malignant cells [5] .

(c) Cervical inammation is associated with high-grade cervicalneoplasia or invasive cervical cancer [68] . In addition,elevated systemic levels of IL-6, IL-8, TNF-a and CCL3 areassociated with persistent human papilloma virus (HPV)infection [9] .

As inammation prominently features in normal reproductiveprocesses, it is not surprising that aberrant inammatorysignalling is associated with chronic diseases and malignanciesin the lower female genital tract [10,11] . It follows that GC appearto share inammatory pathway signatures within their tumourmicroenvironments that are integral to processes that expeditedisease progression (Table 1). However, despite the evidence tosupport the importance of these networks in disease recurrence,there is a paucity of translational clinical trials that focus onmanipulating these pathways in order to prolong survival.Although some studies have shown modest increments inprogression free survival (PFS) [1219] , the impact on overallsurvival (OS) has been negligible due to the development of resistance. Furthermore, these limited benets are in some partdue to targeting single molecules that consequently inducecompensatory pathways leading to relapse. Within the pro-inammatory cytokine milieu, IL-6 is emerging to be a pivotalmediator of tumourigenesis (Fig. 1) and subsequently a potentialtherapeutic target. Moreover, the concept of manipulatinginammatory mediators in cancer therapeutics is particularlysignicant in light of the recent ndings from the Cancer GenomeAtlas project in ovarian cancer [20] . The genomic analysis

Cytokine & Growth Factor Reviews 23 (2012) 333342

A R T I C L E I N F O

Article history:Available online 30 September 2012

Keywords:Interleukin-6Tumour microenvironmentEpithelial ovarian cancerEndometrial cancerCervical cancer

A B S T R A C T

There are many parallels between gynaecological cancers in relation to cytokine networks within theirrespectivetumour microenvironmentsandevidence to support interleukin-6 (IL-6) being an appropriatetherapeutic target in these diseases. This article provides an overview on IL-6 biology including updateson novel discoveries in IL-6 signalling and then focuses on the role of IL-6 in processes such as cellproliferation, migration, angiogenesis, evasion of tumour immunity and chemoresistance and presentsdata relating to the abrogation of these processes with anti-IL-6 targeted therapy in preclinical andclinical studies. The overall aim will be to highlight the necessity for further translational studiesconcentrating on combinations of anti-IL-6/IL-6R therapies with other novel targets in an attempt tosignicantly improve overall survival in patients with gynaecological cancers.

2012 Elsevier Ltd. All rights reserved.

* Corresponding author.E-mail address: [email protected] (Jermaine I.G. Coward).

Contents lists available at SciVerse ScienceDirect

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1359-6101/$ see front matter 2012 Elsevier Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.cytogfr.2012.08.005
http://dx.doi.org/10.1016/j.cytogfr.2012.08.005http://dx.doi.org/10.1016/j.cytogfr.2012.08.005http://dx.doi.org/10.1016/j.cytogfr.2012.08.005http://dx.doi.org/10.1016/j.cytogfr.2012.08.005http://dx.doi.org/10.1016/j.cytogfr.2012.08.005http://dx.doi.org/10.1016/j.cytogfr.2012.08.005http://dx.doi.org/10.1016/j.cytogfr.2012.08.005http://dx.doi.org/10.1016/j.cytogfr.2012.08.005http://dx.doi.org/10.1016/j.cytogfr.2012.08.005http://dx.doi.org/10.1016/j.cytogfr.2012.08.005http://dx.doi.org/10.1016/j.cytogfr.2012.08.005http://dx.doi.org/10.1016/j.cytogfr.2012.08.005http://dx.doi.org/10.1016/j.cytogfr.2012.08.005http://dx.doi.org/10.1016/j.cytogfr.2012.08.005http://dx.doi.org/10.1016/j.cytogfr.2012.08.005mailto:[email protected]:[email protected]://www.sciencedirect.com/science/journal/13596101http://www.sciencedirect.com/science/journal/13596101http://www.sciencedirect.com/science/journal/13596101http://dx.doi.org/10.1016/j.cytogfr.2012.08.005http://dx.doi.org/10.1016/j.cytogfr.2012.08.005http://www.sciencedirect.com/science/journal/13596101mailto:[email protected]://dx.doi.org/10.1016/j.cytogfr.2012.08.005


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conrmed that high grade serous subtypes (HGSC) consistedpredominantly of TP53 mutations (96%) with a low frequency of additional targetable oncogenes. However, Notch signalling, apathway downstream of TNF-a and IL-6 [14,21] , was amplied/mutated in 11% of HGSC cases [20] . Hence targeting theinammatory microenvironment in this particular disease couldprove be benecial in improving clinical outcome.

This review will summarise the role of IL-6 in GC along with thepreclinical and clinical research to support the rationale fortargeting this cytokine in conjunction with established cytotoxicregimes and/or other novel therapies in future treatment algo-rithms.

2. Interleukin-6 signalling

IL-6 is a pleiotropic cytokine with a broad spectrum of biological activity relating to regulation of inammation, celldifferentiation, cell proliferation, immunomodulation, haemato-poiesis and oncogenesis. Human IL-6 consists of 184 amino acidsand is produced by multiple host and tumour cells in the tumourmicroenvironment. It was initially cloned in 1986 and identied asan antigen-nonspecic B-cell differentiation factor that induced B-cell production of immunoglobulins. IL-6 acts through theformation of a high-afnity complex with its receptor that consistsof an 80-kDa IL-6 binding glycoprotein gp80 (a -chain, IL-6R a ) andthe 130-kDa signal transducer gp130 (b -chain). Both gp80 andgp130 exist in transmembrane and soluble forms (sgp80 andsgp130). The transmembrane domain of gp80 consists of a short

Table 1Cancer-related inammation in gynaecological cancers.

Gene signatures conrm IL-6 upregulation [14,112,113] Upregulation of JAK-STAT signalling pathway that results in the

transcription of anti-apoptotic genes such as bcl-xl, survivin, mcl-1 [36,39]PTEN and KRAS mutations associated with activation of inammatory

signalling and subsequent upregulation of inammatory mediators resultingin tumourogenesis [114121]

Poor prognosis correlating with elevated circulating concentrations of IL-6[52,62,65] and increased inltration with tumour associated macrophages

[100,122,123] Increased metastatic potential associated with increased expression of the

chemokine receptor CXCR4 and its ligand CXCL12 (also known as stromalcell derived factor-1 (SDF-1)) [82,124,125]

Fig. 1. IL-6 function and the GC tumour microenvironment IL-6 can exhibit pleiotropy within the GC microenvironment primarily through pSTAT3 signalling and interactionwith other cytokine and chemokines. IL-6 contributes to GC with aggressive phenotypes by promoting tumour cell proliferation, angiogenesis and platinum resistancealongside subverting tumour immunity by enhancing dendritic cell tolerogenicity and phenotypic skewing of M1 macrophages to TAM.

J.I.G. Coward, H. Kulbe / Cytokine & Growth Factor Reviews 23 (2012) 333342334


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intracytoplasmic region that associates with gp130 as a conse-quence of IL-6 binding. This leads to gp130 homodimerisation andsignal transduction that characterises classic signalling; thepredominant mode through which IL-6 orchestrates its homeo-static functions [22] (Fig. 2). Both sgp80 and sgp130 are formedeither by cleavage from the cell membrane by transmembranemetalloproteinases (e.g. ADAM17) or translated from alternatemRNA splicing [2326] . Whilst gp80 expression is limited tocertain cell types (hepatocytes, monocytes, T cells, B cells,neutrophils and malignant cells) [26] , gp130 expression isubiquitous. However, IL-6 can still exert inuences on cells lackingtransmembrane gp80 by forming a complex with sgp80 andmembrane bound gp130 to initiate downstream events. This isknown as trans-signalling and is critically involved in inammatorydiseases (e.g. rheumatoid arthritis, peritonitis, asthma and inam-matory bowel disease) and malignancies such as colorectal cancerand AIDS-associated Kaposis sarcoma [22,27,28] . Trans-signallingcan be regulated by sgp130 which is able to neutralise IL-6-sgp80complexes and sgp80 can enhance the antagonistic activity of sgp130 [29] . Although previously thought not to inuence classicsignalling effects, a recent study conrms that sgp130 can indeed

inhibit this pathway in addition to trans-signalling [30] . Consideringthat most pathophysiological conditions are characterised by molarexcesses of sgp80 over IL-6 in serum, Garbers et al. demonstratedthat sgp130 can indirectly block classic signalling by trapping IL-6-sgp80 complexes which subsequently eliminate free IL-6 molecules(Fig. 2) [30] . Gp130 behaves promiscuously in that it acts as acommon signal transducer for other cytokines along with IL-6,namely IL-11, IL-27, ciliary neurotrophic factor (CNTF), cardiotropin-1 (CT-1), oncostatin M (OSM), neurotrophin-1 and leukaemiainhibitory factor (LIF) [22,31] ; each of which have denedphysiological roles. This group of cytokines are collectively knownas the IL-6 cytokine superfamily [32] and all (with the exception of OSM and LIF that engage directly with gp130) interact with theirrespective binding receptor leading to gp130 hetrodimerisation.Intracellular signalling is then initiated through activation of gp130associated cytoplasmic tyrosine kinases, namely the Janus-activatedkinases 1 and 2 (JAK1 and JAK2) which phosphorylate signaltransducers and activators of transcription (STAT) proteins, Ras/MAPK and PI3K/Akt [33] .

The association between IL-6 and cancer revolves around gp130activation of IL-6 signalling pathways that ultimately lead to

Fig. 2. IL-6 signalling A. The classic mode of IL-6 signalling involves IL-6 complexing with membrane bound IL-6R. Trans-signalling is mediated via IL-6-sgp80 complexes. Bothmodes involve association with membrane bound gp130 to induce downstream signalling. B. sgp130 abrogates both classic signalling and trans-signalling by preferentiallybinding to IL-6-sgp80 complexes.

J.I.G. Coward, H. Kulbe / Cytokine & Growth Factor Reviews 23 (2012) 333342 335


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evasion of apoptosis which appears to be principally mediatedthrough the aforementioned JAK/STAT pathway. Within normaleukaryotic cells, STATs, particularly STAT3, are involved in thetransduction of signals from the cell surface to its nucleus andactivation of gene transcription. This results in the inhibition of apoptosis during inammation and cell survival in suboptimalconditions. However, dysregulation of such processes may alsopromote the development of a number of malignancies andconstitutive STAT signalling is prominent in GC [3439] . JAK2mediated phosphorylation of STAT3 occurs at a single tyrosineresidue (Tyr705) [40] which is situated in the Src homologue (SH2)domain and binding between these residues results in theformation of stable STAT dimer complexes that contain an exposednuclear translocation signal. The complex is then able totranslocate to the nucleus where enhancer elements in thepromoter and enhancer regions of target genes are recognisedleading to the initiation of transcription [41] . Induced genesinclude acute phase proteins (CRP, haptoglobin and brinogen),the anti-apoptotic bcl-2 family members (e.g. bcl-2, b-1, bcl-x L ,XIAP and mcl-1), alongside myc and cyclin D1 that both enhancecellular proliferation [34] . IL-6 also modulates STAT3 signalling viaa negative feedback loop through induction of suppressor of cytokine signalling 3 (SOCS3) proteins that bind to the phosphor-ylated tyrosine residue, Tyr759, on the gp130 receptor [42] .Conversely, recent research in an ulcerative colitis relatedcolorectal cancer model has demonstrated that IL-6 can induceDNA methyltransferase gene 1 (DNMT1) mediated SOCS3 promot-er methylation that consequently results in aberrant STAT3signalling and increased malignant potential [43] . Furthermore,this pathway blunts anti-tumour immunity by tolerising dendriticcells and augments angiogenesis by upregulation of VEGF [44,45] .These effects, which are complemented by other oncogenic eventsmanifesting through parallel Ras/MAPK and PI3/Akt signallingpathways, fuel the malignant repertoire of IL-6 within the tumourmicroenvironment and hence make this key cytokine an attractivetherapeutic target in GC.

3. IL-6 as an autocrine growth factor in GC

A number of in vitro and in vivo studies have reported increasedIL-6 expression in GC. In EOC, elevated levels of IL-6 have beenidentied in ovarian tumour cell cultures with cells displayingintracellular IL-6 by immunoperoxidase staining [46] . Theseconcentrations also appear to be considerably higher in subtypeswith poor prognosis. This was shown clearly by Anglesio et al.,who demonstrated a panel of ovarian clear cell cancer (OCCC) lines(namely TOV21G, RMG1, HAC2, JOHCS5, JOHCS7 and JOHCS9)secreting IL-6 in concentrations several logs higher than other EOCsubtypes [47] . Watson et al. have also demonstrated constitutiveproduction of IL-6 in EOC cell lines such as CAOV-3, OVCAR-3 andSKOV-3 and its activity appears to be upregulated by IL-1b , TNF-a

and

IFN-g [46] .

This

group

also

discovered

that

inhibition

of

IL-6gene expression using IL-6 anti-sense oligonucleotides could resultin up to 85% inhibition of cellular proliferation [48] ; however thiseffect was not reversed with the addition of exogenous IL-6.Recently, we have shown bi-weekly intraperitoneal (i.p.) injectionsof the anti-IL-6 monoclonal antibody (mAb), siltuximab (CentocorOrtho Biotech Inc.) into mice xenografted with either IGROV-1 orTOV21G cells, diminished tumour colonisation analysed bybioluminescence imaging and signicantly decreased cellularproliferation as measured by Ki67 expression on tumour sectionsafter 4 weeks of treatment [14] .

For endometrial cancer the evidence to support existing IL-6autocrine networks in this disease requires further exploration. Inthe normal endometrium, IL-6 promotes proliferation, differentia-

tion

and

invasion

of

trophoblast

cells

[49] .

Furthermore,

IL-6

modulates the expression of growth hormone (GH) and its receptor[50] ; which is also responsible for endometrial cell proliferation. Inview of this, a retrospective study by Slater et al. demonstratedthat compared to normal uterine cells, GH and IL-6 are increasedby 3.8 and 4.4-fold in endometrial adenocarcinoma respectively[51] . They concluded that these two growth factors may act inconcert to enhance endometrial cancer progression [51] . Belloneet al. [52] identied detectable levels of IL-6 from the supernatantsof endometrioid (EC; Type I) and uterine serous papillary cell lines(UPSC; Type II) with the latter poorer prognostic subtype secretingsignicantly higher concentrations of IL-6. In addition, endome-trial cell proliferation enhanced through estrogenic G protein-coupled receptor 30 (GPR30) signalling via the MAPK/ERK pathwaycan result in increased IL-6 production [2] . Although these studiessuggest a tenuous association between IL-6 and endometrial cellgrowth, more focused experiments are required to rmly establishits autocrine function in this disease. In contrast, such effects havebeen established in cervical cancer cell lines in vitro . IL-6 is secretedin variable quantities from numerous epithelial cervical neoplasticcells cultured on plastic and cell proliferation is inhibited with theaddition of anti-IL-6 neutralising agents [5355] ; an effect that canbe rescued with recombinant human IL-6 (rhIL-6) [54] . Predict-ably, targeting IL-6 with anti-IL-6/IL-6R mAb also impedes thetranscription of pro-survival genes and this effect appears to bespecic to certain signalling pathways within GCs. Wei et al.elegantly demonstrated that the anti-apoptotic effects of IL-6through mcl-1 upregulation occurs preferentially via PI3/Aktsignalling [56] ; whereas in EOC, the expression of the bcl2 genefamily is predominantly inuenced by STAT3 activity [57] .

4. Serum and peritoneal uid IL-6 concentrations

Several studies have focused on analysing IL-6 levels in bloodand ascites from patients with GCs. In healthy human subjects, IL-6concentrations found in blood greater than 10 pg/ml are consid-ered to be abnormally elevated [58,59] and in patients with newly

diagnosed EOC, these levels portend signicantly worse survival(median OS: IL-6 < 10 pg/ml: 5.99 yrs, IL-6 > 10 pg/ml: 3.38 yrs; p < 0.001) [60] . Additionally, there is evidence to suggest arelationship between serum IL-6, tumour burden, disease stageand prognosis in EOC. Berek et al. published data illustrating thathigh IL-6 levels correlated with bulky disease, elevated CA-125,disease progression and ultimately poorer prognosis [61] . Similar-ly, the Tempfer study conrmed that elevated serum IL-6measured prior to treatment (mean 55.6 pg/ml) correlatedstatistically signicantly with FIGO stage, poorer PFS and OS[62] . There also appears to be inter-subtype variability in IL-6concentrations [47,63] (Table 2). For example, analysis of serumfrom the Australian Ovarian Cancer Study (AOCS) dataset, hasconrmed patients with OCCC have signicantly higher concen-

trations

of

IL-6

than

patients

with

HGSC.

These

elevated

levelswere associated with poor PFS and OS in OCCC; and, unlike theHGSC cohort, there was no correlation with increasing tumourstage [47] . However, IL-6 has yet to be validated as a tumourmarker in EOC and in combination with CA-125 only marginallyincreases overall sensitivity compared to CA-125 alone [63] .

The in vitro results from the Bellone study were also reected inthe analysis of serum from patients with endometrial cancer. Notonly were IL-6 levels signicantly higher relative to healthycontrols ( p < 0.01), but these concentrations were particularlyelevated in UPSC as compared to EC ( p < 0.03) [52] . Theseobservations conict with an earlier study that showed normallevels of IL-6 throughout all stages of endometrial cancer; howeverall of these patients had type I disease only [64] . Hence, as with

EOC,

IL-6

appears

to

be

associated

with

subtypes

exhibiting

poor



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prognosis and may serve as a more appropriate therapeutic targetin UPSC [52] .

Similarly elevated levels of IL-6 are also found in serum frompatients with cervical cancer. Takano et al. reported that amongst66 patients with squamous cell carcinoma (SCC) approximately33% had IL-6 levels > 20 pg/ml (mean 83.4 pg/ml) [55] . Thisconcentration was substantially higher than in 4 patients withadenocarcinoma (14.1 pg/ml) and 8 healthy controls (4.4 pg/ml).This pattern was also seen when cells from these subtypes werecultured in vitro and IL-6 concentrations correlated with the extentof SCC differentiation [55] . In addition, for both subtypes,increasing IL-6 levels appear to correlate with advancing stagesof disease [65] .

In comparison with plasma or serum, IL-6 concentrations inascitic uid are substantially higher; predominantly due to therelative abundance of inammatory cells such as tumour

associated

macrophages

(TAM),

neutrophils,

lymphocytes

andmesothelial cells which are all rich sources of IL-6 [66,67] . Theselevels have also been attributed to the surge in IL-6 production byEOC cells followed by rapid clearance into the vasculature whereIL-6 becomes either protein bound or degraded [68,69] . Plante etal. reported that ascitic IL-6 levels can correlate with increasingvolume of ascites ( p < 0.0001) and tumour size found at surgery( p = 0.05) but found no relationship with survival or tumour gradeand stage [69] . However, a recent study has associated poor PFSwith high ascitic levels of IL-6 in patients newly diagnosed withEOC [70] . The subtype variability observed in serum IL-6concentrations in EOC also applies to peritoneal uid. This wasrst intimated by Kutteh et al. who identied that papillary caseshad a tendency to secrete higher levels than non-papillary

histotypes

[71] .

A

subsequent

study

by

Kryczek

et

al.

observed

substantially higher concentrations in patients with serous andmucinous tumours compared with endometrioid and undifferen-

tiated carcinomas and also noted an inverse correlation betweenIL-6 and p53 staining on EOC cytospins [72] . In conjunction with IL-6, elevated levels of sIL-6R are also evident in ascites. In EOCxenograft models, IL-6 trans-signalling through ERK activationfacilitates the development of ascites by enhancing endothelial cellhyperpermeability and transendothelial migration of ovariancancer cells [73] . Furthermore, ascites formation can be reducedby blocking trans-signalling using the ERK inhibitor PD98059 [73] .

Punnonen et al. studied the peritoneal uid cytokine prolesfrom GC patients and discovered that IL-6 levels were elevatedamongst all GCs and these concentrations were signicantly higherin patients with benign ovarian tumours and EOC than in patientswith endometrial or cervical cancer. Interestingly, amongst themyriad of cytokines and growth factors measured (i.e. IL-2, IL-4, IL-

10,

IFN-g ,

TNF-a ,

M-CSF,

G-CSF

and

GM-CSF),

IL-6

was

the

solitarycytokine that exhibited signicant differences amongst thedifferent GCs [66] . The reasons for this observation have yet tobe determined. Contrary to the Plante study, which included thesame selection of EOC subtypes, there was no correlation betweenIL-6 levels and volume of ascites; so it remains to be ascertainedwhether ascitic IL-6 concentrations could be utilised as a reliablemarker for tumour response in patients receiving treatment forpredominantly ascitic disease.

5. Cell migration and invasion

During inammation, IL-6 certainly employs a central role withintegrin induction and migration of monocytes [74] ; a phenome-

non

also

commonplace

within

tumorigenic

processes

in

a

variety

Table 2Serum and ascitic/peritoneal uid IL-6 concentrations in GC.

GC type Subtype Specimen Mean IL-6 pg/ml (unless indicated)(range SEM)

Reference

Cervical cancer Squamous cell Serum 83.4 [55]Adenocarcinoma Serum 14.1

Squamous cell & adenocarcinoma Serum Stage I: 21.6 9.8 [65]Stage II: 32.8 8.9Stage III: 27.5 5.7Stage IV: 42.4 3.6

Squamous cell & adenocarcinoma Peritoneal uid 1222 546 [66]

Endometrial cancer Endometrioid Serum 20.43 (2.8682.13) [52]Endometrioid Serum < 10.0 (all stages) [64]Uterine serous papillary Serum 125.7 (16.3500.1) [52]Adenocarcinoma, carcinosarcoma& stromal sarcoma

Peritoneal uid 1977 616 [66]

Ovarian cancer Unspecied Serum 0.26 U/ml 0.04 [61]Serous, mucinous, clear cell andundifferentiated

Serum Stage I: 16.1 (0316.3)Stage II: 44.7 (0877.9)Stage IIIIV: 285.3 (66.02869.0)

[62]

Serous, endometrioid, mucinous,

clear cell and undifferentiated

Serum 10.0 (< 11221) [69]

Serous Serum 7.0 (0.3660) [63]Mucinous 1.5 (0.455)Endometrioid 4.4 (0.420)Undifferentiated 4.5 (0.426)Unspecied 7.7 (0.455)

Serous, endometrioid, mucinous,clear cell and undifferentiated

Ascites 49,612 (< 1680,330) [69]

Serous cystadenocarcinoma,endometrioid, mucinous andgranulosa cell

Ascites 5572 1266 [66]

Serous, mucinous, endometrioid,mixed and unspecied

Ascites 6419 1409 [70]



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of malignancies [7577] . The inuence of IL-6 on ovarian cancercell motility is also mediated through STAT3 signalling. Aspreviously mentioned, Silver et al. correlated constitutive pSTAT3production in ovarian cancer cell lines with phenotypicallyaggressive tumours [37] . By using confocal micrographs of SKOV3and OVCAR3 cells plated on bronectin, this group detectedlocalisation of pSTAT3 in focal adhesions, structures that enhancecell migration by transducing extracellular signals into changes inthe cytoskeleton and hence potentially facilitate invasion of malignant cells. Furthermore, this localisation occurred whenthese cells were stimulated with IL-6 in vitro [37] . More recently,Colombiere et al. have shown with wound healing assays that IL-6can induce OVCA 433 cell migration and this can be reversed with aneutralising anti-IL-6R antibody [78] . In addition, in vitro studieshave conrmed that IL-6 can enhance EOC cell line invasion andattachment to matrigel [79,80] and targeting IL-6 can effectivelysuppress MMP-9 secretion [81] . Interestingly, although inamma-tory mediators related to IL-6 such as CXCL12 [82] , TNF-a [83] andPGF2 a [84] are known to promote endometrial cancer cellmigration and invasion, the direct role of IL-6 in these processesfor this disease has yet to be conrmed. Similarly, although non-signicant trends have been reported between increasing intra-tumoural IL-6 expression and extent of loco-regional invasion andmetastasis in cervical cancer [85] , further research is requiredrelating to this issue.

6. IL-6 and angiogenesis

There is ample evidence supporting proangiogenic activity of IL-6 in various tumour types [76,86,87] . With respect to thisreview, it seems tting that the pivotal work implicating IL-6 as apotent inducer of VEGF was established in a cervical carcinomamodel [85,88] . Wei et al. demonstrated that rhIL-6 can induceVEGF in a time- and dose-dependent manner in C33A cells in vitroand at a transcriptional level this effect can be substantiallyrepressed with either anti-IL-6 or anti-IL-6R treatment [85] . Thisgroup also highlighted that IL-6 upregulation of VEGF transcription

is mediated by STAT-3 [88] ; a signalling pathway notorious forpromoting tumour angiogenesis through its constitutive activation[44] . Subsequently, using matrigel plugs containing C33A cellsoverexpressing IL-6, Su et al. have shown that inhibition of IL-6R with S7 peptide can effectively impede angiogenesis and tumourgrowth in vivo [89] .

Within normal ovarian tissue, IL-6 is involved in the formation of new blood vessels that accompany ovarian folliculogenesis [90] . Inrelation to ovarian cancer and angiogenesis there are a few studiesemerging that specically focus on the role of IL-6 in this process.Using IHC analysis, Nilsson et al. showed that IL-6R is expressed onendothelial cells in human ovarian cancer specimens and IL-6induced pSTAT3 in ovarian and mesenteric endothelial cells.Furthermore, through STAT3 activation, IL-6 promoted endothelial

cell

migration

in

vitro

[91] .

This

study

also

reported

potentangiogenic effects associated with IL-6 in vivo . Gel foam spongespreviously incubated with IL-6, bFGF or PBS, were implanted intothe peritoneum of BALB/c mice for two weeks and thenimmunostained with CD31 and VEGFR-1 antibodies to estimatemicrovascular density. The vessel density with IL-6 treated sectionswas equivalent to bFGF, but signicantly greater than PBS( p < 0.0001) [91] . More recently, using IL-6 producing intraperito-neal IGROV-1 and TOV21G xenograft models, we have shown thatsiltuximab can signicantly reduce microvascular density onconfocal imaging of tumour sections and reduce expression of jagged-1; also known to have angiogenic effects in advanced EOC[14,21] . In addition, VEGF and IL-8 plasma concentrations decreasemarkedly in platinum resistant patients treated with siltuximab

monotherapy

for 6

months

[14] .

In

response

to

hypoxia,

IL-6-STAT3

mediated angiogenesis leads to transcription of HIF-1 a along withVEGF [92] . Anglesio et al. have recently reported overexpression of the IL-6-STAT3-HIF1 a pathway in OCCC tumours compared toHGSC and evidence of response in patients treated with the anti-angiogenic agent; sunitinib [47] .

Another mode of IL-6 induced angiogenesis in EOC could occurthrough its ability to polarize macrophages in ascites to theimmunosuppressive M2 phenotype, i.e. TAM [93] . TAM secreteproangiogenic factors such as TGF-b , PDGF, VEGF, FGF andnumerous chemokines including CXCL1 and IL-8 (CXCL8) thatpromote the angiogenic switch [94] . In conjunction with inhibitingangiogenesis in our aforementioned EOC xenograft models,siltuximab also inhibited macrophage inltration in tumoursections as evidenced by signicant decreases in F4/80 IHCstaining [14] . Equally, from an immunogenic perspective, IL-6with TGF-b could also inuence angiogenic effects by mediatingthe preferential differentiation of CD4+ Th cells to Th17 subsets asopposed to FoxP3 + regulatory T cells [95,96] . Within the EOCtumour microenvironment, IL-6 promotes expansion of Th17 cells[97] that secrete a host of cytokines including IL-17 which issignicantly associated with increased microvascular density inEOC tissue sections [98] . Although TAM, IL-8, VEGF and FGF arewell documented to have proangiogenic activity within endome-trial cancers [99,100] , further studies are required in verifywhether IL-6 per se has a profound inuence on angiogenicprocesses in this disease.

7. IL-6 and chemoresistance

IL-6 behaves as a prominent mediator of chemoresistance in avariety of malignancies [101] . In EOC, Scambia et al. were the rstgroup to report the association of elevated serum IL-6 concentra-tions ( 6 pg/ml) with poor response to chemotherapy [63] . Sincethen, Wang et al. revealed that both exogenous and endogenousIL-6 increased platinum and paclitaxel resistance in non-IL-6producing A2760 cell lines and sensitivity to these drugs could beretrieved with IL-6 antisense in IL-6 over-expressing SKOV-3 cells

[102] . This group also showed that IL-6 specically promoted suchcytotoxic resistance through upregulation of multidrug resistancegenes MDR-1 and GSTpi, transcription of anti-apoptotic proteins(bcl-xL and XIAP) and signalling through PI3/Akt and Ras/MEK/ERKsignalling pathways [102] . Similar studies conducted by Duan etal. showed that IL-6 is preferentially overexpressed in certainpaclitaxel-resistant EOC cell lines compared with chemo-na vecounterparts [103] . Additionally, using paired resistant andsensitive human ovarian cancer cell lines, they demonstrated thatSTAT3 was overexpressed in some paclitaxel resistant lines andcorrelated high levels of IL-6 with increased STAT3 expression inthe chemoresistant cells [103] . They concluded that chemoresis-tance could be reversed by STAT3 knockdown using siRNA, andfound that apoptosis was enhanced when resistant lines were

treated

with

a

STAT3

inhibitor

(AG490),

paclitaxel

or

a

combina-tion of both drugs [103] . Using siltuximab, on multidrug resistantEOC cell lines, Guo et al. have recently recapitulated these ndingsin vitro but witnessed no signicant effect on paclitaxel resistanttumour growth with SKOV-3 TR xenograft mouse models [57] .

Although fewer reports explicitly focus upon the role of IL-6 andchemoresistance in endometrial and cervical cancers; the path-ways that have been implicated in this process appear to beinextricably linked with IL-6. For example, the cytoprotectiveredox sensitive transcription factor, nuclear factor erythroid-2-related factor 2 (Nrf2), is not only a potent inducer of the IL-6promoter, but is upregulated in a myriad of tumour types [104] .Nrf2 is highly expressed in SPEC-2 (Type II EC) cell lines, confersresistance to platinum and paclitaxel and this resistance can be

reversed

by

Nrf2

siRNA

[105] .

Nrf2

expression

also

increases

with



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advancing stages of cervical cancer and Nrf2 knockdown withshRNA in CaSki cells can reverse multidrug resistance [106] .Moreover, with respect to IL-6 signalling pathways, isoforms of Akt(Akt2 and Akt3) are involved in mediating cisplatin resistance inKLE endometrial cancer cell lines [107] . Furthermore, IL-6 over-expressed in C33A cervical cancer cells can diminish doxorubicinand cisplatin related apoptosis in a PI3/Akt dependent fashion [56] .

8. Translational anti -IL-6/anti-IL-6R studies in GC

With evidence supporting the signicant inuence of IL-6 innumerous cancers, this cytokine is emerging as an attractivetherapeutic target in the clinical setting. Several clinical trialsusing anti-IL-6/IL-6R therapy have been conducted in a range atumour types with diverse results [101] . These studies haveprimarily been conducted in patients with poor prognosis diseaseand clinical benet (stable disease or better) has been achievedwith durable response in select cases. The encouraging outcomefrom these trials has provided a sound rationale for developingfurther studies with anti-IL-6(R) therapy in GC; however, at thetime of writing, only one such study has been reported. Weconducted an open label non-randomised phase II clinical trialinvestigatingthe effects of siltuximab monotherapy in 20 patients(age 4175) with advanced platinum resistant EOC [14] . All, butone patient with OCCC, had tissue biopsy conrmed HGSC andnearly half of the cohort had an ECOG performance status of 2.Each had radiological evidence of disease progression at the pointof study recruitment and received between 2 and 6 prior lines of platinum-based treatment. The primary endpoint was responserate as assessed by combined RECIST and CA125 criteria. Onepatient of eighteen evaluable had a partial response, whilst sevenothers had periods of disease stabilization. None of the seriousadverse events reported in the study were attributable tosiltuximab and it was generally well tolerated. In 4 patientstreated for 6 months, there was a signicant decline in plasmalevels of IL-6-regulated CCL2, CXCL12 and VEGF. Alongside this,sgp130 receptor concentrations increased 2.5-fold (baseline:

260.4 10 3 pg/ml; 6-month: 662.4 10 3 pg/ml); a differencethatwas statistically signicant ( p < 0.05) [108] . This implies thatlong term response to si ltuximab in these patients could beinuenced by inhibition of IL-6 trans-signalling. In addition, geneexpression levels of factors that were reduced by siltuximabtreatment in the patients signicantly correlated with high IL-6pathway gene expression and macrophage markers in microarrayanalyses of ovarian cancer biopsies [14] . The mechanisms of action underlying these phenomena are currentlynot clear, but itserves as an intriguing avenue for further exploration in order toenhance the efcacy of anti-IL-6 therapies in future combinatorialtrials. Together with the in vitro and in vivo studies previouslymentioned, we concluded that IL-6 stimulates inammatorycytokine production, tumour angiogenesis and the TAM inltrate

in

ovarian cancer

and both

paracrine and

autocrine actions

can

beinhibited by a neutralising anti-IL-6 antibody [14] .Subsequent to this research, Stone et al. have noted an

important relationship between IL-6 and the development of paraneoplastic thrombocytosis in ovarian cancer [60] . Targetingeither IL-6 or platelets with siltuximab and an anti-plateletantibody (APA; directed against mouse glycoprotein 1b a ) respec-tively, impeded tumour development and reduced circulatingplatelets in EOC xenograft mouse models. With siltuximab, thiseffect was particularly enhanced in combination with paclitaxel;however APA signicantly inhibited tumour cell proliferation,microvascular density, pericyte coverage and endothelial apopto-sis [60] . Whether the anti-neoplastic/anti-angiogenic effectsexerted with APA are an indirect consequence of IL-6 inhibition

requires

further

investigation.

In

correlation

with

our

Phase

II

study data, for all evaluable patients there were signicant andsustained reductions in mean platelet counts after the completionof 3 cycles of siltuximab ( p = 0.009) [60] ; however there were nostatistical differences in these levels between patients attainingclinical benet and those who progressed at this time-point (J.Coward, PhD Thesis, Queen Mary University, London).

In light of the potential additive effect of targeting IL-6 incombination with chemotherapy, a recently initiated feasibilitystudy in the Netherlands is investigating the efcacy of varyingschedules of carboplatin/caelyx or carboplatin/doxorubicin withanti-IL-6R mAb (Tocilizumab; Roche) and Peg-Intron (interferon- a2b) in patients with recurrent EOC. The primary objective is toassess the safety and efcacy of these novel regimens. In view of the immunosuppressive effects of IL-6 signalling, the secondaryoutcomes focus on the inuence of anti-tumour immunity onsurvival by analyzing changes in dendritic cell subsets andassessing T- and B-cell responses to known ovarian cancerantigens such as NY-ESO and p53 (ClinicalTrials.gov identier:NCT01637532).

9. Future directions

Although there have been some signicant improvements inresponse rates with different combinations of chemotherapy overthe past 20 years, it is clear that the therapeutic ceiling is beingreached with this approach in treating GC. Naturally, for thisreason, attention has turned towards targeting mediators of specic processes intrinsic to tumour progression; a strategy thathas had varying degrees of efcacy, both as monotherapy and incombination with cytotoxic agents. Certainly the most convincingapproach in this group of diseases to date has centred uponinhibiting angiogenesis through VEGF blockade with bevacizumab(anti-VEGF-A mAb) with signicant PFS benet evident inadjuvant, maintenance and palliative settings. However, despitethese recent encouraging results, the goal of attaining a distinct OSsurvival has yet to be realised in GC and this consequentlynecessitates the rapid development of further combinatorial

clinical trials with novel targeted agents. This review has providedconsiderable evidence to support the role of IL-6 as a therapeutictarget in various GC subtypes and the aforementioned trials withsiltuximab and bevacizumab have set the foundation for theincorporation of drugs targeting IL-6(R) and associated signallingpathways into the standard pharmacological management of thesediseases.

Although the EOC siltuximab trial was conducted on a smallcohort, the results are striking in that clinical benet was evidentfor monotherapy in a heavily pretreated poor prognostic group.Although the exact mechanisms of action have to yet undergofurther investigation, the anti-angiogenic effects of this drug arecompelling and in view of the recent ICON 7 and OCEANS studies[19,109] , it warrants further investigation as both rst-line and

salvage

treatment

in

conjunction

with

chemotherapy.

This

is

apoint of particular interest as both platinum and taxane drugs canthemselves transiently induce IL-6 in in vitro and in vivo cancermodels [110,111] . Hence, theoretically at least, the introduction of anti-IL-6 blockade both in combination and as maintenancetherapy could avert the development of chemoresistant clonesin addition to suppressing neoangiogenesis. Such trials could alsobe introduced in the neoadjuvant settings, whereby tissueobtained during debulking surgery could provide a wealth of information on the effects previously exerted by targeting IL-6 onthe tumour microenvironment. Moreover, high throughputsystematic analysis for the upregulation of compensatory signal-ling pathways that foster resistance to this therapy could assist intailoring appropriate adjuvant therapies in order to prolong

survival.

Not

only

will

it

be

important

to

expand

translational



8/10


9/10


10/10

ceedings of the National Academy of Sciences of the United States of America2008;105:1550510.

[98] Kato T, Furumoto H, Ogura T, Onishi Y, Irahara M, Yamano S, et al. Expressionof IL-17 mRNA in ovarian cancer. Biochemical and Biophysical ResearchCommunications 2001;282(3):7358.

[99] Fujimoto J. Novel therapeutic strategy for uterine endometrial cancers.International Journal of Clinical Oncology 2008;13:4115.

[100] Hashimoto I, Kodama J, Seki N, Hongo A, Miyagi Y, Yoshinouchi M, et al.Macrophage inltration and angiogenesis in endometrial cancer. AnticancerResearch 2000;20(6C):48536.

[101] Guo Y, Xu F, Lu T, Duan Z, Zhang Z. Interleukin-6 signaling pathway in

targeted

therapy

for

cancer.

Cancer

Treatment

Reviews

2012;38(7):90410.[102] Wang Y, Niu XL, Qu Y, WuJ, Zhu YQ, Sun WJ, et al. Autocrine production of interleukin-6 confers cisplatin and paclitaxel resistance in ovarian cancercells. Cancer Letters 2010;295(1):11023.

[103] Duan Z, Foster R, Bell DA, Mahoney J, Wolak K, Vaidya A, et al. Signaltransducers and activators of transcription 3 pathway activation in drug-resistant ovarian cancer. Clinical Cancer Research 2006;12(17):505563.

[104] Wruck CJ, Streetz K, Pavic G, Gotz ME, Tohidnezhad M, Brandenburg LO, et al.Nrf2 induces interleukin-6 (IL-6) expression via an antioxidant responseelement within the IL-6 promoter. Journal of Biological Chemistry2011;286(6):44939.

[105] Jiang T, Chen N, Zhao F, Wang XJ, Kong B, Zheng W,et al. High levels of Nrf2determine chemoresistance in type II endometrial cancer. Cancer Research2010;70(13):548696.

[106] Ma X, Zhang J, Liu S, Huang Y, Chen B, Wang D. Nrf2 knockdown by shRNAinhibits tumor growth and increases efcacy of chemotherapy in cervicalcancer. Cancer Chemotherapy and Pharmacology 2012;69:48594.

[107] Gagnon V, Mathieu I, Sexton E, Leblanc K, Asselin E. AKT involvement incisplatin chemoresistance of human uterine cancer cells. Gynecologic On-

cology 2004;94:78595.[108] Balkwill FR, Coward J, Kulbe H, Milagre C, Gopinathan G, McNeish IA. IL-6 and

ovarian cancer response. Clinical Cancer Research 2011;17:7838.[109] Aghajanian C, Blank SV, Goff BA, Judson PL, Teneriello MG, Husain A, et al.

OCEANS: a randomized, double-blind, placebo-controlled phase III trial of chemotherapy with or without bevacizumab in patients with platinum-sensitive recurrent epithelial ovarian, primary peritoneal, or fallopian tubecancer. Journal of Clinical Oncology 2012;30(17):203945.

[110] Poth KJ, Guminski AD, Thomas GP, Leo PJ, Jabbar IA, Saunders NA. Cisplatintreatment induces a transient increase in tumorigenic potential associatedwith high interleukin-6 expression in head and neck squamous cell carcino-ma. Molecular Cancer Therapeutics 2010;9:24309.

[111] Wang TH, Chan YH, Chen CW, Kung WH, Lee YS, Wang ST, et al. Paclitaxel(Taxol) upregulates expression of functional interleukin-6 in human ovariancancer cells through multiple signaling pathways. Oncogene 2006;25(35):485766.

[112] Santin AD, Zhan F, Cane S, Bellone S, Palmieri M, Thomas M, et al. Geneexpression ngerprint of uterine serous papillary carcinoma: identicationof novel molecular markers for uterine serous cancer diagnosis and therapy.British Journal of Cancer 2005;92(8):156173.

[113] Tartour E, Gey A, Sastre-Garau X, Pannetier C, Mosseri V, Kourilsky P, et al.Analysis of interleukin 6 gene expression in cervical neoplasia using aquantitative polymerase chain reaction assay: evidence for enhanced inter-leukin 6 gene expression in invasive carcinoma. Cancer Research1994;54(23):62438.

[114] Enomoto T, Inoue M, Perantoni AO, Terakawa N, Tanizawa O, Rice JM. K-rasactivation in neoplasms of the human female reproductive tract. CancerResearch 1990;50:613945.

[115] Tashiro H, Blazes MS, WuR, Cho KR, Bose S, Wang SI, et al. Mutations in PTENare frequent in endometrial carcinoma but rare in other common gyneco-logical malignancies. Cancer Research 1997;57(18):393540.

[116] Daikoku T, Hirota Y, Tranguch S, Joshi AR, DeMayo FJ, Lydon JP, et al.Conditional loss of uterine Pten unfailingly and rapidly induces endometrialcancer in mice. Cancer Research 2008;68(14):561927.

[117] St-Germain ME, Gagnon V, Parent S, Asselin E. Regulation of COX-2 proteinexpression by Akt in endometrial cancer cells is mediated through NF-kappaB/IkappaB pathway. Molecular Cancer 2004;3:7.

[118] Cheung TH, Lo KW,Yim SF, Chan LK, Heung MS, Chan CS, et al. Epigenetic andgenetic alternation of PTEN in cervical neoplasm. Gynecologic Oncology2004;93(3):6217.

[119] Rizvi MM, Alam MS, Ali A, Mehdi SJ, Batra S, Mandal AK. Aberrant promotermethylation and inactivation of PTEN gene in cervical carcinoma from Indianpopulation. Journal of Cancer Research and Clinical Oncology 2011;137:125562.

[120] Pappa KI, Choleza M, Markaki S, Giannikaki E, Kyroudi A, Vlachos G, et al.

Consistent

absence

of

BRAF

mutations

in

cervical

and

endometrial

cancerdespite KRAS mutation status. Gynecologic Oncology 2006;100(3):596600.[121] Shih Ie M, Kurman RJ. Ovarian tumorigenesis: a proposed model based on

morphological molecular genetic analysis. American Journal of Pathology2004;164:15118.

[122] Fujimoto J, Sakaguchi H, Aoki I, Tamaya T. Clinical implications of expressionof interleukin 8 related to angiogenesis in uterine cervical cancers. CancerResearch 2000;60:26325.

[123] Wan T, Liu JH, Zheng LM, Cai MY, Ding T. Prognostic signicance of tumor-associated macrophage inltration in advanced epithelial ovarian carcinoma.Ai Zheng 2009;28:3237.

[124] Zhang JP, Lu WG, Ye F, Chen HZ, Zhou CY, Xie X. Study on CXCR4/SDF-1alphaaxis in lymph node metastasis of cervical squamous cell carcinoma. Interna-tional Journal of Gynecological Cancer 2007;17:47883.

[125] Milliken D, Scotton C, Raju S, Balkwill F, Wilson J. Analysis of chemokines andchemokine receptor expression in ovarian cancer ascites. Clinical CancerResearch 2002;8:110814.

Dr Jermaine Coward is a medical oncology specialist ingynaecological malignancies and is the Cancer Thera-peutics Group Leader at the Mater Medical ResearchInstitute, Brisbane, Australia. He completed his medicaltraining at Imperial College, London in 1998 and becamea member of the Royal College of Physicians in 2002. Hisinterests in oncology began during his tenure as a juniordoctor and he subsequently undertook specialist regis-trar training at Westmead Hospital, Sydney and theRoyal Marsden Hospital, London. He has extensive ex-perience in the development and conduct of Phase IIclinical trials in addition to clinical management of numerous tumour types. In 2006, he was appointed

as a MRC clinical fellow at the Barts Cancer Institute, London and investigatedthe effects of anti-IL-6 in advanced ovarian cancer which involved the successfultranslation of laboratory research into a clinical trial in patients with platinumresistant disease. This research culminated in the award of his PhD from QueenMary, University London in 2010. Since then, he has been involved in a number of international collaborations with a focus on investigating the role of inammatorycytokine networks in gynaecological cancers.

Dr Hagen Kulbe is a postdoctorate research assistant atthe Centre for Cancer and Inammation, Barts CancerInstitute, London. In 2001, he was awarded his PhD inthe eld of tumour immunology and genetics from theMax-Delbrueck-Centre, Berlin. Over the past 5 years hehas published a number of key articles relating to theeffects of targeting inammation in ovarian cancer andis currently involved in several projects focusing oninammatory signalling within tumour microenviron-ments using novel animal models.


the role of interleukin-6 in gynecological malignancies

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