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Critical Reviews in Oncology/Hematology xxx (2013) xxx–xxx

Interleukin-6: An angiogenic target in solid tumours

Kathryn Middleton a, Joanna Jones a, Zarnie Lwin a, Jermaine I.G. Coward a,b,c,∗a Mater Adult Hospital, Department of Medical Oncology, Raymond Terrace, Brisbane, QLD 4101, Australia

b Inflammation & Cancer Therapeutics Group, Mater Research, Level 4, Translational Research Institute, 37 Kent Street,Woolloongabba, Brisbane, QLD 4102, Australia

c School of Medicine, University of Queensland, St Lucia, Brisbane, QLD 4072, Australia

Accepted 13 August 2013

ontents

. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 001.1. IL-6 and angiogenic processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

. IL-6 and angiogenesis in solid tumours . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.1. Colorectal cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.2. Gastric cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.3. Pancreatic and hepatocellular carcinomas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.4. Cervical cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.5. Ovarian cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.6. Melanoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.7. Prostate cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.8. Renal cell carcinoma (RCC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.9. Glioblastoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Funding source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Reviewers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Biographies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

bstract

During the past decade, incorporating anti-angiogenic agents into the therapeutic management of a myriad of malignancies has in certainases made a significant impact on survival. However, the development of resistance to these drugs is inevitable and swift disease progressionn their cessation often ensues. Hence, there is a drive to devise strategies that aim to enhance response to anti-angiogenic therapies byombining them with other targeted agents that facilitate evasion from resistance. The pleiotropic cytokine, interleukin-6 (IL-6), exerts pro-

ngiogenic effects in the tumour microenvironment of several solid malignancies and there is emerging evidence that reveals significantelationships between IL-6 signalling and treatment failure with antibodies directed against vascular endothelial growth factor (VEGF). This eview summarises the role of IL-6 in pivotal angiogenic processes and preclinical/clinical research to support the future introduction of

Please cite this article in press as: Middleton K, et al. Interleukin-6: An anhttp://dx.doi.org/10.1016/j.critrevonc.2013.08.004

nti-IL-6 therapies to be utilised either in combination with other anti-angecome refractory to these approaches.

2013 Elsevier Ireland Ltd. All rights reserved.

eywords: IL-6; Angiogenesis; VEGF; STAT3; HIF-1�; Bevacizumab; Sunitinib;

∗ Corresponding author at: Inflammation & Cancer Therapeutics Group, Mater Roongabba, Brisbane, QLD 4102, Australia. Tel.: +61 7 3163 1584; fax: +61 7 3163

E-mail addresses: jcoward@mmri.mater.org.au, jim.coward@gmail.com (J.I.G

040-8428/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.critrevonc.2013.08.004

giogenic target in solid tumours. Crit Rev Oncol/Hematol (2013),

iogenic drugs or as a salvage therapy for patients with diseases that

Siltuximab

esearch, Level 4, Translational Research Institute, 37 Kent Street, Wool- 2550.. Coward).

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. Introduction

Over 40 years since Folkman’s seminal work on tumourngiogenesis [1], the era of targeted therapy has witnessed theuccessful incorporation of anti-angiogenic agents into treat-ent algorithms for a plethora of malignancies. Although

heir impact on survival in numerous tumour types has beenignificant, several issues relating to deleterious side effects,evelopment of resistance, establishment of optimal dura-ion of treatment and predictive biomarkers have yet to bedequately addressed. Consequently, there is an urge to dis-over alternative angiogenic targets which serve as a solutiono these problems and ultimately enhance response to the

yriad of therapeutic agents that inhibit angiogenesis. Oneuch factor is the pleiotropic cytokine, interleukin-6 (IL-6);

potent pro-angiogenic mediator that is omnipresent in thenflammatory microenvironment of most solid tumours [2].

IL-6 has a broad spectrum of biological activity relating toegulation of inflammation, cell proliferation, immunomod-lation, haematopoiesis and tumourigenesis. Human IL-6onsists of 184 amino acids and was initially identified as anntigen-nonspecific B-cell differentiation factor that induced-cell production of immunoglobulins. IL-6 acts through the

ormation of a high-affinity complex with a receptor thatonsists of an 80-kDa IL-6 binding glycoprotein gp80 (�-hain, IL-6R�) and the 130-kDa signal transducer gp130�-chain). Both gp80 and gp130 exist in transmembranousnd soluble (sgp80 and sgp130) forms. The transmembraneomain of gp80 consists of a short intracytoplasmic regionhat associates with gp130 as a consequence of IL-6 binding.his results in gp130 homodimerisation and signal trans-uction that characterises classic signalling; the predominantode through which IL-6 orchestrates its homeostatic func-

ions [3]. Both sgp80 and sgp130 are formed either byleavage from the cell membrane by transmembrane met-lloproteinases or translated from alternate mRNA splicing4–7]. Whilst gp80 expression is restricted to certain cellypes (monocytes, T cells, B cells, neutrophils, hepatocytesnd tumour cells) [7], gp130 expression is ubiquitous. How-ver, as with viral IL-6, human IL-6 signalling transductionan remain in cells lacking transmembrane gp80 by forming

complex with sgp80 and membrane bound gp130 to ini-iate downstream events. This is known as trans-signallingnd is critically involved in inflammatory diseases (e.g.nflammatory bowel disease and rheumatoid arthritis) ands the principal mode for IL-6 tumour promoting activity;hich is particularly evident in colorectal cancer. [3,8,9].rans-signalling is tightly modulated by sgp130 which caneutralise IL-6-sgp80 complexes, and sgp80 that enhanceshe antagonistic activity of sgp130 [10]. Although previouslyhought not to impede classic signalling, recent reports con-rm that sgp130 can indeed inhibit this pathway in addition to

rans-signalling [11]. Gp130 behaves promiscuously in that

Please cite this article in press as: Middleton K, et al. Interleukin-6: An anhttp://dx.doi.org/10.1016/j.critrevonc.2013.08.004

t acts as a common signal transducer for other cytokineslong with IL-6, namely IL-11, IL-27, ciliary neurotrophicactor (CNTF), cardiotropin-1 (CT-1), oncostatin M (OSM),

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logy/Hematology xxx (2013) xxx–xxx

eurotrophin-1 and leukaemia inhibitory factor (LIF) [3,12];ach of which have defined physiological roles. This group ofytokines are collectively known as the IL-6 cytokine super-amily [13] and all, with the exclusion of LIF and OSM,nteract with their specific binding receptor leading to gp130eterodimerisation. Intracellular signalling is then initiatedhrough activation of gp130 associated cytoplasmic tyrosineinases, namely the Janus-activated kinases 1 and 2 (JAK1nd JAK2) which phosphorylate signal transducers and acti-ators of transcription (STAT) proteins, Ras/MEK/ERK andI3K/Akt [14]. These downstream IL-6 signalling pathwaysfficiently facilitate tumour proliferation, migration [2,15],urvival [2,16] and chemoresistance [2] which all contributeo poor outcomes in patients with a broad spectrum of malig-ancies [2,17]. Through these pathways and in particularTAT3, IL-6 provides a fertile environment for angiogenicrocesses to flourish through the induction of factors that areurrently well recognised targets for a host of anti-angiogenicherapies.

.1. IL-6 and angiogenic processes

The phenomena of tumour neo-angiogenesis (charac-erised by vessel sprouting and incorporation of bone-marrowerived endothelial precursors) and co-opting of existinglood vasculature is paramount to the growth of tumourseyond 100–200 �m. This is governed by the balance ofro- and anti-angiogenic factors and the weighting of theseetermine the ‘angiogenic switch’ state [18]. It follows that

preponderance of pro-angiogenic molecules over anti-ngiogenic molecules will turn on this switch and signalsuch as genetic mutations alongside hypoxia and inflam-ation within the tumour microenvironment can assist this

rocess [19–21]. Subsequently, tumours can develop theirwn vasculature through expansion of existing blood ves-els characterised by endothelial tip sprouting and insertionf interstitial tissue columns into the lumen of these vesselsi.e. intussusceptions) [20,22]. The prominent feature of thisprouting phase is tumour vessel dilatation, increased perme-bility and leaking due to the effects of vascular endothelialrowth factor (VEGF). VEGF, a 45 kDa glycoprotein, washe first vascular-specific growth factor to be characterisednd is widely accepted to be the essential driver for vasculo-enesis [23]. It consists of a family of five structurally relatedolecules; namely VEGF-A,-B,-C, -D and placental growth

actor (PlGF) and signals through three receptor tyrosineinases namely VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1)nd VEGFR-3 (Flt-4) [24]. Most of the aforementionedroperties of VEGF are mediated through VEGFR-2 andonversely VEGFR-1 exhibits antagonistic effects by blunt-ng signalling through VEGFR-2 [23]. Furthermore, althoughEGFR-1 has a higher affinity for VEGF than VEGFR-2,

giogenic target in solid tumours. Crit Rev Oncol/Hematol (2013),

t only possesses a weak capacity for signal transduction24,25]. Interestingly, there are additional co-receptors thatxhibit a high affinity for particular VEGF isoforms; namelyhe neuropilins, which include neuropilin-1 (NRP-1) and

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europilin-2 (NRP-2). NRP-1 is able to bind VEGF165 (theredominant exon splice variant of VEGF) and present thisigand to VEGFR2 which results in both increased sig-al transduction through VEGFR2 and enhanced VEGF165elated chemotaxis [23,26].

The influence of hypoxia on VEGF induction is particu-arly highlighted by the intimate relationship between the Vonippel Lindau (VHL) tumour suppressor gene and hypoxia

nducible factor-1� (HIF-1�). Under normoxic conditions,HL induces the hydroxylation and subsequent ubiquitinediated degradation of HIF-1� which in turn modulates

he expression of VEGF mRNA [27,28]. Conversely, underypoxic conditions, this process is reversed and hence pro-otes angiogenesis. More specifically, in malignancies such

s renal cell carcinomas characterised by VHL mutations,IF-1� is constitutively activated [29] and aberrant angio-enesis is a prominent feature.

As inflammation is one of several conditions that resultsn HIF-1� and VEGF upregulation, it stands to reasonhat considerable interplay exists between these factors andnflammatory mediators within the tumour microenviron-ent. Amongst the plethora of such mediators, IL-6 in

articular exhibits an intimacy with HIF-1� and VEGF whichs predominantly driven by STAT3 signalling [30]. For exam-le, using chromatin immunoprecipitation assays, Niu et al.onfirmed that STAT3 binds directly to the VEGF promoternd an activated STAT3 mutant (STAT3C) could effectivelypregulate VEGF and tumour angiogenesis [31]. Moreover,reatment of various epithelial cell lines with IL-6 for 6–48 han significantly induce VEGF mRNA to a level comparableo the effects of hypoxia or cobalt chloride, an activator ofypoxia-induced genes [32]. Furthermore close correlationsave been identified between IL-6 and hypoxia inducible fac-ors (both HIF-1� and HIF-2�), which suggest an alternativeink between this pleiotropic cytokine and VEGF regulation33].

IL-6 also has notable effects on other critical angio-enic processes such as promoting endothelial progenitorell migration and proliferation [34,35], regulation of basicbroblast growth factor (bFGF) [36], stimulation of vascularmooth muscle cell (VSMC) migration [37] and inductionf platelet derived growth factor (PDGF) mediated VSMCroliferation [38]. However, the relationship between IL-

signalling and its influence on PDGF-mediated pericyteecruitment warrants further investigation. IL-6 also has theotential to manipulate factors responsible for either inhib-ting or stabilising angiogenic switching. Loganadane et al.ave previously demonstrated that secretion of the endoge-ous angiogenesis inhibitor thrombospondin-1 (TSP-1) intohe subendothelial matrix can be modulated by IL-6; how-ver, this effect was predominantly influenced by increasedell density [39]. Although, the angiostatic factor, endostatin

Please cite this article in press as: Middleton K, et al. Interleukin-6: An anhttp://dx.doi.org/10.1016/j.critrevonc.2013.08.004

a 20 kDa carboxy-terminal fragment of collagen XVIII) haseen shown to effectively reduce IL-6 in human umbilicalein endothelial cells (HUVECs) [40], further studies areequired to verify the extent of its regulation by IL-6.

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logy/Hematology xxx (2013) xxx–xxx 3

IL-6 has also been implicated in other angiogenicignalling pathways. Activation of the evolutionary con-erved Notch signalling pathway by its ligands jagged-1 andelta-like 4 ligand (DLL4) regulate numerous aspects of the

umour angiogenesis spectrum including endothelial tip cellprouting and vascular maturation [41]. Sansone et al. notedhat IL-6 can stimulate Notch3 dependent upregulation ofagged-1 (Fig. 1) and this promoted growth of breast cancerells and maintained their aggressive phenotype [42]. Moreecently, targeting IL-6 has been shown to impede jagged-1xpression alongside decreased tumour vasculature in ovar-an cancer in vivo models [43].

With such compelling evidence highlighting the angio-enicity of IL-6, it would intuitively appear to be an attractivearget in numerous tumour types; especially in view of theecent success witnessed with other anti-angiogenic regimens44–48]. This review will summarise the evidence linking thisleiotropic cytokine with angiogenic processes (Fig. 1) in aost of solid malignancies and point towards the possibleuture therapeutic implications alongside other novel agentsnd as a salvage strategy for patients developing resistanceo these therapies.

. IL-6 and angiogenesis in solid tumours

.1. Colorectal cancer

Over the past few years, there has been increasing evi-ence linking IL-6 to the development and progressionf colorectal cancers [49]. Elevated expression in serumnd tumour cells have been reported alongside correlationsith advancing stage and poor survival [50–52]. Further-ore, in colitis associated cancer (CAC) in vivo models,

L-6 can enhance proliferation of tumour initiating cells,ncrease tumour burden at late stages of disease and pro-ect both normal and malignant intestinal epithelial cellsrom apoptosis in a STAT3 dependent fashion [53]. Sig-ificantly, reduction in tumour size and number has alsoeen observed in CAC models with IL-6 ablation via sta-le knockdown or IL-6R monoclonal antibodies [53,54].ith respect to the influence of IL-6 on angiogenesis in this

isease, the evidence unsurprisingly leans towards its rela-ionship with VEGF. Eldesoky et al. measured preoperativeerum VEGF and IL-6 in 35 colorectal cancer patients and 30ontrols. Colorectal cancer patients had significantly higherL-6 and VEGF levels and were elevated further in thoseith advanced pathological tumour stage and metastatic dis-

ase [55]. Interestingly, in the quest to define biomarkersredicting response to anti-angiogenic therapy in CRC, IL-

is also emerging as a potential candidate. A Phase IItudy with bevacizumab combined with chemoradiation in

giogenic target in solid tumours. Crit Rev Oncol/Hematol (2013),

ectal cancer patients noted that elevated IL-6 and circu-ating endothelial cells levels were associated with poorerutcomes [56].

Please cite this article in press as: Middleton K, et al. Interleukin-6: An angiogenic target in solid tumours. Crit Rev Oncol/Hematol (2013),http://dx.doi.org/10.1016/j.critrevonc.2013.08.004

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Fig. 1. IL-6 and tumour angiogenesis. IL-6 exerts pro-angiogenic activity predominantly through STAT3 signalling leading to VEGF transcription, endothelialcell proliferation and migration. IL-6 may potentially orchestrate resistance to anti-angiogenic agents through induction of CXCL12 and influencing thephenotyping of immune cells which also secrete numerous angiogenic factors.

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.2. Gastric cancer

The aetiological origins of gastric cancer (GC) are clearlyinked to Helicobacter pylori infestation and chronic gastritisnd inevitably the GC microenvironment is rich in inflam-atory mediators promoting disease progression [57,58].pecifically, elevated IL-6 expression relates to poor surgicalutcome, increased invasive and metastatic potential in GC59–62]. As with colorectal cancer, the correlations betweenncreased IL-6 and VEGF in GC compared to healthy subjectslso contribute to its pathogenesis [58]. Additionally, gas-ric cancer cells can produce significant amounts of VEGFith increasing dose and duration of IL-6 stimulation via

AK-STAT signalling [63]. In parallel with this, IL-6 inducesEGF to promote HUVEC cell proliferation and tube for-ation in vitro and Matrigel plug vascularisation in vivo

63].

.3. Pancreatic and hepatocellular carcinomas

As with a host of malignancies, circulating levels of IL-6re increased in pancreatic cancer patients and higher lev-ls have been shown to correlate with worse survival [64].n vitro, pancreatic cell lines can express higher levels of IL-

than normal human pancreatic duct epithelium and therere various reports of IL-6 induced expression of phospho-ylated STAT3 (pSTAT3), HIF-1�, VEGF and the VEGFR-2o-receptor, NRP-1 in these cells [65,66]. Furthermore, moreecently, the hypoxia induced aggressive phenotypic nature ofancreatic cancer has been partly attributed to both VEGF andL-6 [67]. However, in view of the avascular nature of pancre-tic cancer and lack of clinical efficacy in targeting VEGF-Aith bevacizumab, it is unlikely that any potential benefits

xerted by anti-IL-6 therapeutics will be as a consequence ofmeliorating angiogenesis in this unrelenting tumour type.

With respect to hepatocellular carcinoma (HCC), IL-6 isignificantly linked to both its pathogenesis and poor prog-osis [68,69]. In addition, IL-6 knockdown in HCC in vivoodels can abrogate cell proliferation, migration and inva-

ion [70]. Although its pro-tumorigenic activity appears toe principally mediated through STAT3 signalling [71], itspecific influence on angiogenesis in HCC requires furtherxploration. Nevertheless, higher levels of IL-6 alongsideoluble c-Kit and CXCL12 (stromal derived factor-1�; SDF-�) were documented by Zhu et al. throughout a coursef sunitinib (VEGFR2, c-kit and PDGFR tyrosine kinasenhibitor) treatment in HCC patients who rapidly progressed72]; another example of the potential role of IL-6 in theevelopment of anti-angiogenic refractory disease.

.4. Cervical cancer

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IL-6 is certainly omnipresent in the inflammatory milieu ofynaecological cancers and is central to a host of tumorigenicrocesses in this group of diseases [73]. Again, within thepectre of angiogenesis, its role is mediated through VEGF

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nduction. In FIGO stage IB-IIA cervical cancer patients,ei et al. reported consistently higher levels of IL-6 andEGF in cancerous tissues than in adjacent non-malignant

issues in early-stage cervical cancer patients [74]. Further-ore, they confirmed that the addition of recombinant human

L-6 induced VEGF in a time- and dose-dependent man-er in vitro. Moreover, impeding autocrine IL-6 signallingith anti-IL-6 or anti-IL-6R antibodies reduced the expres-

ion of VEGF at the transcriptional level [74]. In parallelith this observation, an in vivo model using a Matrigel plug

ssay has shown that IL-6 increases angiogenic activity viapregulation of VEGF in a STAT3 dependent manner (Fig. 1)75].

.5. Ovarian cancer

The pleiotropic nature of IL-6 is lucidly exemplifiedn ovarian cancer by its extensive influence on cell sur-ival, migration and chemoresistance through JAK/STATignalling and the observation that elevated circulating levelsre associated with poor prognosis [73]. It also facilitates theevelopment and progression of malignant ascites by enhanc-ng tumour endothelial cell migration; a process mediatedhrough trans-signalling [76]. Similarly, Nilsson et al. haveemonstrated that this process is directed by STAT3 in vitrond elegantly demonstrated significant increases in microvas-ular density in vivo as a consequence of exogenous IL-6timulation [77].

Conversely, by using IL-6 producing intraperitonealGROV-1 and TOV21G xenograft models, Coward et al. havehown that a high affinity anti-IL-6 monoclonal antibody,iltuximab, can significantly reduce neovascularisation ononfocal imaging of tumour sections and decrease expres-ion of jagged-1 [43]; also known to have angiogenic effectsn advanced epithelial ovarian cancer [78]. Furthermore, in

phase II clinical trial of siltuximab in 18 patients withlatinum resistant ovarian cancer, VEGF and IL-8 plasmaoncentrations decreased markedly in platinum resistantatients treated with siltuximab monotherapy for 6 months43]. The authors also concluded that IL-6 co-regulates TNF-, IL-1�, CCL2, CXCL12 and VEGF, resulting in paracrineromotion of angiogenesis within the tumour microenviron-ent [43]. IL-6 may also pose as an attractive angiogenic

arget in sub-types which are inherently chemoresistant.nglesio et al. reported upregulation of IL-6-STAT3-HIF-1�

ignalling in ovarian clear cell carcinomas and some modestesponse to the anti-angiogenic agent, sunitinib [33]. The pro-ngiogenic repertoire of IL-6 also extends to its influence onumour immunity, where it enables skewing of macrophageso the M2 (i.e. tumour associated macrophage; TAM) pheno-ype which themselves secrete a host of angiogenic mediatorsincluding VEGF, PDGF, bFGF and IL-8) and, in conjunction

giogenic target in solid tumours. Crit Rev Oncol/Hematol (2013),

ith TGF-�1, drives T cell differentiation towards the Th17ineage; a subset of immune cells also critical to angiogenesisFig. 1) [79–82].

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.6. Melanoma

IL-6 transcription and downstream signalling have sig-ificant roles in melanoma progression, especially in theontext of angiogenesis. Karst et al. demonstrated that theF-�B p50 subunit strongly induces IL-6 upregulation inelanoma cells at both transcriptional and translational lev-

ls and consequently enhanced the growth of endothelial cellsn vitro [57]. In addition, knockdown of p50 expression usingentiviral-based shRNA abrogated cellular IL-6 expression,ndothelial cell growth and blood vessel formation [57]. Thenduction of angiogenesis by IL-6 also relates to the relation-hip between the NF-�B p65 subunit and the integrin-linkedinase (ILK); whose overexpression strongly correlates withelanoma progression, invasion and the poor overall sur-

ival of melanoma patients [83]. Wani et al. demonstratedhat ILK enhances IL-6 gene transcription by supporting theinding of NF-�B p65 to the IL-6 promoter [83]. Further-ore both STAT3 and VEGF levels were increased in ILK

verexpressing melanoma cells and this was associated withnhanced tube forming capacity of endothelial cells in vitrond microvessel formation in vivo. [83]. Interestingly, theseffects diminished with IL-6siRNA and resulted in declin-ng VEGF levels, suggesting that the angiogenic effects areeliant on IL-6 signalling through STAT3 [83]. Li et al. havelso confirmed an additional link to establish the importancef the NF-�B-IL-6 axis in angiogenesis within the melanomaumour microenvironment. They discovered overexpressionf the breast cancer metastasis suppressor 1 (BRMS1) genenhibited endothelial cell growth and tube formation abil-ty in vitro along with decreased microvasculature in vivo byuppressing NF-�B activity and IL-6 expression [84]. Subse-uently, they also confirmed BRMS1 knockdown increasedL-6 expression and promoted these processes; indicatinghat the inhibitory effects of BRMS1 on IL-6 expression areependent on NF-�B [84].

.7. Prostate cancer

Amongst the solid tumours highlighted in this review, theargest body of research highlighting the significance of IL-6n tumourigenesis relates to prostate cancer. Several reportsave consistently confirmed its role as an autocrine growthnd survival factor, mediator of chemoresistance, metasta-is and upregulator of androgen receptors [85–90]. Withespect to angiogenesis, IL-6 has a further pivotal role. Inormone resistant prostate cancer cells, Wu et al. observedhat IL-6 inhibition impeded recruitment of myeloid deriveduppressor cells (MDSCs) in tumour- bearing mice, result-ng in attenuated angiogenesis and abrogated tumour growth91]. Furthermore, Jemaa et al. recently confirmed crossalk between IL-6 and bFGF through MAPK signalling and

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ostulated that IL-6 could potentially enhance angiogenesishrough this relationship [92]. Wang et al. have also shownhat the angiogenic properties of the chemokine CXCL12nd its receptor, CXCR4 are mediated through IL-6 via ERK

cape

logy/Hematology xxx (2013) xxx–xxx

ctivation (Fig. 1) [93]. In addition, they confirmed that angio-tatin levels inversely correlated with CXCR4 expression93]; however, the influence of IL-6 on this phenomenonas yet to be determined. To date, two Phase I/II trials havenvestigated the efficacy of combining chemotherapy withiltuximab in castrate resistant prostate cancer [94,95]. How-ver, the conflicting results from these studies highlight theeed to further investigate the optimal scheduling of anti-IL-6herapy in this disease.

.8. Renal cell carcinoma (RCC)

As previously mentioned, VHL mutations and consequentpregulation of the angiogenic mediators HIF-1� and VEGFre synonymous with RCC pathogenesis. In turn, the inter-lay of IL-6 with these factors certainly contributes to itsole in orchestrating metastatic spread and consequent poorrognosis and survival [96,97]. Furthermore, prior to the cur-ent sunitinib era where immunotherapy with either IL-2 ornterferon-� (IFN-�) was standard treatment, IL-6 also con-ributed to poor outcomes with this management [98,99].his latter observation fuelled the development of a Phase

/II study with siltuximab which resulted in disease stabilisa-ion in more than 50% of patients who had relapsed afterumerous lines of immunotherapy [100]. In parallel withmmunotherapy failure, a recent small study in 85 patientsith advanced RCC has also implicated IL-6 in the develop-ent of resistance to sunitinib [101]. Porta et al. performed

erum cytokine assays for angiogenic factors (IL-6, hep-tocyte growth factor (HGF) and bFGF) and specificallyxcluded VEGF analysis. Significant increases (>1.5 timesigher than baseline) in HGF, and in particular, IL-6 andFGF preceded progression on sunitinib in approximately0–44% of these patients [101].

.9. Glioblastoma

The functional role of IL-6 in glioblastoma (GBM) devel-pment was demonstrated by Weissenberger et al. who notedailure of GBM development with IL-6 ablation in trans-enic mice expressing the src oncogene in astrocytes [102].n vitro, IL-6 also promotes GBM cell invasion and angio-enesis by enhancing vascular endothelial cell migration viaTAT3 signalling [103]; a pathway also confirmed as a directffector for the EGFRvIII mutant protein which has beeninked to poor long term survival [104].

Despite this, anti-IL-6 monotherapy may not completelybrogate GBM invasiveness. IL6 and VEGF are both pro-uced by glioma cells and possibly act in union to facilitateumour growth and survival through angiogenesis, cell pro-iferation and resistance to apoptosis. Data suggest that theombinatorial approach of IL-6 targeting alongside beva-

giogenic target in solid tumours. Crit Rev Oncol/Hematol (2013),

izumab may be more efficient in dampening invasivenessnd growth in malignant cell clusters [105]. Saidi et al. com-ared the effect of inhibiting IL6 and VEGF on U87-derivedxperimental glioma grown on the chick chorio-allantoic

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embrane (CAM) or in the brain of xenografted mice.n vitro, IL-6 knockdown had no effect on proliferation butubstantially enhanced invasion. In the CAM glioma model,L-6 or VEGF knockdown equally reduced growth andascularisation of the tumours, but paradoxically increasednvasion of residual tumour cells. In contrast, combinedL6 and VEGF knockdown showed enhanced reduction ofumour growth, angiogenesis and also significantly impedednvasion. In mice, combining IL-6 knockdown and beva-izumab treatment completely inhibited tumour developmentnd infiltration. Hence these results suggest that a combina-ion of IL6 and VEGF inhibitors could induce a synergisticnti-tumoural effect [105]; a significant finding for the devel-pment of future trials with these agents in GBM and otheralignancies.In line with a selection of aforementioned tumour types,

L-6 has an inferred role in anti-angiogenic resistance inBM. Jin et al. demonstrated that IFN regulatory factor 7erived IL-6 had a pivotal role in maintaining GBM stem cellroperties, increased tumour heterogeneity and formationhrough JAK/STAT activation of the jagged-Notch pathwayFig. 1) [106]. Interestingly, alongside stem cell accumula-ion, myeloid infiltration and mesenchymal phenotype aressociated with anti-VEGF therapy resistance in GBM [107].ost significantly, all such processes can be governed by

L-6 within the tumour microenvironment [78,107,108]. Fur-hermore, recent reports have confirmed correlations betweenncreased glioma cell pSTAT3 expression and patients fail-ng bevacizumab therapy [109]. These findings have beenecapitulated by Jahangiri et al., whose micro-array genexpression analysis comparing GBM primary tumours withevacizumab resistant glioblastoma (BRG) has shown sig-ificant upregulation of c-Met expression in BRG specimens110]. Subsequently, they demonstrated that the increasedypoxia (analysed via immunostaining for hypoxia marker,A9) associated with BRG cells correlated with increasedhosphorylation of STAT3, c-MET and c-MET activatedocal adhesion kinase (FAK) [110]. Moreover, in BRGenograft models, both intrinsic and acquired bevacizumabesistance could be ameliorated with the c-MET inhibitor;L184 (Exelixis) [110].Consequently, attention has now been drawn towards com-

inations with drugs targeting both the JAK/STAT pathwayAZD1480) and VEGF (cedirinib) which may potentiallyvert resistance to angiogenic inhibitors [109].

. Conclusions

Arguably, the introduction of anti-angiogenic agentslongside chemotherapy represents one of the key advancesn oncological practise over the past decade. However, as with

Please cite this article in press as: Middleton K, et al. Interleukin-6: An anhttp://dx.doi.org/10.1016/j.critrevonc.2013.08.004

ll novel medical therapeutics, challenges swiftly emergen developing robust biomarkers to assist appropriate strat-fication of patients most likely to gain significant benefit.

oreover, the inevitable development of resistance to these

oew6

logy/Hematology xxx (2013) xxx–xxx 7

gents also puts the spotlight on creating effective strate-ies to avert processes underpinning this phenomenon. Thiseview outlines considerable evidence to support the rolef IL-6 in both tumour angiogenesis and treatment failureith anti-angiogenic drugs including bevacizumab and suni-

inib; both of which best exemplify the recent success of thislass of targeted agents in the current era of cancer thera-eutics. Although they have brought varying increments inmproved progression free survival in several malignancies,he impact on overall survival for certain tumour types haseen negligible predominantly due to either intrinsic or adap-ive resistance. This is driven by compensatory signallingathways facilitating relapse and hence there is an urge toefresh current treatment algorithms with new strategies torolong response to these therapies.

Interestingly, there is significant evidence supporting thentegral role of inflammatory pathways in the development ofvasive resistance to anti-angiogenic therapy. Hypoxic con-itions that occur during vessel regression with anti-VEGFherapy can lead to compensatory increases in angiogenic

ediators within tumours and also enhance recruitmentf bone marrow derived-cells (BMDCs) which themselvesotentiate neovascularisation [111]. Such pro-angiogenicMDCs include vascular progenitors (i.e. endothelial andericyte progenitor cells) and vascular modulatory cells112,113]. The latter consist of TAM [114] and CD11b+ cells115,116] which all express a myriad of pro-inflammatoryytokines and growth factors. Recruitment of BMDCs inreas of low oxygen tension is mediated through HIF-1�nd downstream effectors such as CXCL12 and VEGF117–119]. Furthermore, compensatory mechanisms throughL-8 have been shown to maintain angiogenic potential inumours that were otherwise impaired due to the absencef HIF-1� [120]. Indeed, within the tumour microenviron-ent, IL-6 shares close relations with these pathways and

rocesses as highlighted in recent clinical and preclinicaltudies with siltuximab; which has been shown to inhibitacrophage infiltration and angiogenic mediators such asXCL12, jagged-1, IL-8 and VEGF [43]. Therefore it is fea-

ible that targeting IL-6 could impede the development ofesistance to anti-angiogenic agents. Indeed, in view of theyriad of other cytokines within the IL-6 superfamily that

ould also potentially enhance such resistance, therapeuticlockade of IL-6R may prove to be a more intuitive approacho prolong response to VEGF tyrosine kinase inhibitors.

In addition, increased circulating levels of IL-6 may alsoerve as a putative biomarker for poor response to sunitinibnd bevacizumab as reported in HCC, RCC, glioblastomand colorectal cancers [56,72,101,109]. Nevertheless, to date,ost translational studies with anti-IL-6 therapies have been

imited to patients with advanced disease and only mod-st benefits have been apparent [43,94,95,100]. In light

giogenic target in solid tumours. Crit Rev Oncol/Hematol (2013),

f the evidence for the pro-angiogenic role of IL-6, anyfforts to develop further clinical trials targeting this cytokineill need to focus on combinatorial (e.g. anti-IL-6/anti-IL-R with MET inhibition) as opposed to monotherapeutic

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pproaches. Additionally, its efficacy in adjuvant, mainte-ance and chemoresistant settings alongside other novelnti-angiogenic agents will require thorough explorationrior to being established as a viable treatment option.

onflict of interest statement

None to declare.

unding source

Dr. Jermaine Coward is currently funded by the Cancerouncil Queensland and the Mater Foundation, Brisbane,ustralia.

eviewers

Hagen Kulbe, Barts Cancer Institute – a Cancer ResearchK Centre of Excellence, Queen Mary, University ofondon, John Vane Science Centre, Charterhouse Square,ondon, EC1M 6BQ, United Kingdom.

Christudas Morais, Centre for Kidney Disease Research,chool of Medicine, University of Queensland at Princesslexandra Hospital, Brisbane, Queensland 4102, Australia

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taCdTacAMRTtis currently funded by the Cancer Council Queensland and theremit of his research revolves around efficient translation ofnovel bench-side discoveries in cancer related inflammationinto nimble combinatorial clinical trials.

K. Middleton et al. / Critical Reviews

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iographies

Kathryn Middleton is an Advanced Trainee in medicalncology at Mater Adult Hospital, Brisbane, Australia.

Joanna Jones is an Advanced Trainee in medical oncologyt Mater Adult Hospital, Brisbane, Australia.

Zarnie Lwin is a Consultant Medical Oncologist at Materdult Hospital, Brisbane, Australia. She specialises in the

Please cite this article in press as: Middleton K, et al. Interleukin-6: An anhttp://dx.doi.org/10.1016/j.critrevonc.2013.08.004

reatment of thoracic and neurological malignancies.

Jermaine I.G. Coward is a consultant medical oncolo-ist specialising in gynaecological and thoracic cancers at

logy/Hematology xxx (2013) xxx–xxx 11

he Mater Adult Hospital, Brisbane. He was appointed as UK Medical Research Council Clinical Fellow at Bartsancer Institute, Queen Mary, University of London and con-ucted basic research into the role of IL-6 in ovarian cancer.his included the first translational clinical trial of anti-IL-6ntibody therapy in patients with platinum resistant ovarianancer and this work culminated in his Ph.D. award in 2010.fter completing specialist oncology training at the Royalarsden Hospital, London, he was appointed as a Senioresearch Fellow and Leader of the Inflammation & Cancerherapeutics Group at Mater Research currently housed at

he Translational Research Institute, Brisbane, Australia. He

giogenic target in solid tumours. Crit Rev Oncol/Hematol (2013),