R

EPRODUCTIONREVIEW

The emerging role of CXC chemokines in epithelialovarian cancer

Adam Rainczuk, Jyothsna Rao, Jessica Gathercole and Andrew N Stephens

Prince Henry’s Institute, Monash Medical Centre, Clayton, Victoria 3168, Australia

Correspondence should be addressed to A N Stephens; Email: andrew.stephens@princehenrys.org

A Rainczuk and J Rao contributed equally to this work

Abstract

In recent years, chemokines have generated intense investigations due to their involvement in both physiological and pathological

processes of inflammation, particularly in ovarian biology. The physiological process of ovulation in the normal ovary involves various

chemokines that mediate the healing of the ruptured endometrium. It is now being reported that many of these chemokines are also

associated with the cancer of the ovary. Chronic inflammation underlies the progression of ovarian cancer; therefore, it raises the

possibility that chemokines are involved in the inflammatory process and mediate immune responses that may favour or inhibit tumour

progression. Ovarian cancer is a gynaecological cancer responsible for highest rate of mortality in women. Although there have been

several investigations and advances in surgery and chemotherapy, the survival rate for this disease remains low. This is mainly because of a

lack of specific symptoms and biomarkers for detection. In this review, we have discussed the emerging role of the CXC chemokines in

epithelial ovarian cancer (EOC). The CXC group of chemokines is gaining importance in the field of ovarian cancer for being angiostatic

and angiogenic in function. While there have been several studies on the angiogenesis function, emerging research shows that ELRK CXC

chemokines, CXCL9 and CXCL10, are angiostatic. Importantly, the angiostatic chemokines can inhibit the progression of EOC. Given

that there are currently no biomarkers or specific therapeutic targets for the disease, these chemokines are emerging as promising targets

for therapy.

Reproduction (2012) 144 303–317

Introduction

As key mediators of the inflammatory response,chemokines represent an area of intense interest andstudy. Their influence on the chemotactic recruitment ofleukocytes towards sites of inflammation is wellestablished and involves multiple cell and tissue types(Charo & Ransohoff 2006). Apart from their establishedroles in chemotactic cell recruitment, however, there is agrowing body of evidence suggesting that interferencewith their established functions may promote tumourdevelopment and metastasis (Mukaida & Baba 2012). Inparticular, a complex chemokine-signalling networkmay influence the development and progression ofepithelial ovarian cancers (EOCs). The study of chemo-kines is therefore becoming important in furthering ourunderstanding of this disease.

An overview of the CXC chemokines and their functions

Chemokines, comprising pairs of ligands and theirassociated receptors, are a superfamily of heparin-binding cytokine molecules with molecular weightsbetween 8 and 10 kDa (Mukaida & Baba 2012). They are

q 2012 Society for Reproduction and Fertility

ISSN 1470–1626 (paper) 1741–7899 (online)

important mediators of the formation of new bloodvessels by neovascularisation, involved in the angio-genic sprouting of vessels in both normal (e.g. embry-ogenesis and menstruation) and pathological states(Keeley et al. 2008). This role is achieved not solelythrough the promotion of angiogenesis but via a complexinterplay between angiogenic and angiostatic signallingto influence the behaviour of the vascular endothelium(Kiefer & Siekmann 2011). Chemokine ligands exert theireffects through their cognate G-protein-coupledreceptors, a group of seven transmembrane-spanningproteins expressed on the cell surface and belonging tothe class A rhodopsin-like family (Kiefer & Siekmann2011; Fig. 1).

On the basis of their primary structure, chemokinesare classified as either C, CC, CXC or CX3C where ‘X’represents a non-conserved amino acid substitution(Fig. 1). With the exception of the ‘C’ subgroup, allchemokines contain a common four-cysteine residuemotif linked by disulphide bonds in conserved positions,one between the first and the third and one between thesecond and the fourth cysteine, to form triple-strandedb-sheet structures (Fernandez & Lolis 2002). The

DOI: 10.1530/REP-12-0153

Online version via www.reproduction-online.org

Figure 1 Chemokine ligands and receptor:intramolecular disulphide bonds form betweencysteine C1–C3 and C2–C4 pairs, leading to theformation of the characteristic triple-strandedb-sheet structures (with the exception of the ‘C’chemokine subgroup, which has only one C–Cpair). CXC chemokines are further groupedaccording to the presence of the ‘ELR’ Glu-Leu-Arg motif. Chemokines bind to their cognateG-protein-coupled receptors (GPCRs), comprisingseven transmembrane-spanning domains andbelonging to the class A rhodopsin-like family.

304 A Rainczuk, J Rao and others

nomenclature for chemokines is extended by theaddition of receptor ‘R’ (e.g. CXCR1) and respectiveligand ‘L’ (e.g. CXCL8). CXC chemokines are further sub-grouped according to the presence or absence of a threeamino acid ‘ELR’ Glu-Leu-Arg motif preceding the CXCsequence. Thus, the CXC chemokines are often referredto as ELRC or ELRK (Strieter et al. 2004).

Over the past two decades, many studies haveexamined the dual roles of CXC chemokines in boththe promotion and the inhibition of angiogenesis. Theangiogenic sprouting of new, thin-walled capillarynetworks from pre-existing ones is a critical factor intumour growth and spread (Keeley et al. 2008, Kiefer &Siekmann 2011). As a general rule, ELRC CXCchemokines mediate angiogenesis, typically viainteraction with the CXCR2 receptor (see Table 1 for alisting of CXC chemokines and their cognate receptors)and are also involved in other diverse functions such asstem cell migration, lymphocyte migration and lym-phoid tissue neogenesis (Romagnani et al. 2004, Kiefer &Siekmann 2011). CXCR2 is typically expressed onendothelial cells but can be constitutively expressed byspecific tissues and cells (Table 1; Mukaida & Baba2012). By contrast, the ELRK CXC chemokines typicallypromote angiostasis (rather than angiogenesis), throughtheir common receptor CXCR3 (Mukaida & Baba 2012).Chemokine ligands lacking the ELR motif can promote

Reproduction (2012) 144 303–317

anti-tumour effects by recruiting both innate andadaptive immune effector cells (in the case of CXCL9and CXCL10 that can be secreted from tumour cells forexample) and can also inhibit endothelial cell migrationand proliferation (Keeley et al. 2008, Kiefer & Siekmann2011, Whitworth & Alvarez 2011).

There are, however, two exceptions to the rule. CXCL4is an ELRC chemokine, which also binds to CXCR3.Unlike the other ELRC members, CXCR4 promotesangiostasis similar to the ELRK chemokines. Contrast-ingly, the ELRK chemokine CXCL12 binds to the CXCR4receptor to promote angiogenesis (Kiefer & Siekmann2011). Therefore, both the ELR status and the cognatereceptor for each chemokine must be considered whenevaluating the function (either angiogenic or angiostatic)of the CXC chemokine family. The functions of the CXCchemokines are also extensively regulated through post-translational modification. This is a well-establishedmechanism for regulating their bioactivity and functionand has been reviewed elsewhere (Mortier et al. 2011).

It is now well established that besides leukocytes,fibroblasts and endothelial cells, tumour cells alsoexpress chemokines and their receptors (Balkwill 2004,Romagnani et al. 2004, Kiefer & Siekmann 2011,Mukaida & Baba 2012). Importantly, chemokines aresecreted by both the stromal and the malignant part ofthe cancer tissue, and functionally, the chemokine

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Table

1G

ener

alove

rvie

wof

CX

Cch

emoki

nes

invo

lved

inth

ere

gula

tion

of

angi

oge

nes

isan

dly

mphocy

tere

cruitm

ent.

CXCL

ELR

Previous

nomen

clature

CXCR

Rec

eptor

expression

Function

Gen

eral

origin

Commondisea

seReferen

ces

CX

CL1

CX

CL2

CX

CL3

CG

ROa

/MG

SA-a

,G

ROb/M

GSA

-b,

GR

Og

/MG

SA-g

CX

CR

2Ep

i/en

do

Chem

ota

xis

of

neu

trophil

s.En

doth

elia

lce

llm

igra

tion

and

pro

life

rati

on

Neu

trophils

Mel

anom

a,ga

stri

can

dco

l-ore

ctal

cance

rs,

ova

rian

Cuen

caet

al.

(1992),

Wuyt

set

al.

(1999)

and

Eck

etal

.(2

003)

CX

CL4

CPF4

CX

CR

3B

Endo

Anti

-angi

oge

nic

,in

hib

its

tum

our

vasc

ula

risa

tion

Thym

us,

nat

ura

lki

ller

cell

,m

onocy

tes

Mel

anom

a,ova

rian

cance

rG

ott

lieb

etal

.(1

988),

Lasa

gni

etal

.(2

003),

Stru

yfet

al.

(2007),

Cal

lese

net

al.

(2010)

and

Ha

etal

.(2

011)

CX

CL5

CEN

A-7

8C

XC

R2

Epi/

endo

Angi

oge

nes

is,

pro

mote

stu

mour

grow

than

dm

etas

tasi

s

Neu

trophils

Lung,

pan

crea

tic,

ova

rian

cance

rsW

isle

zet

al.

(2004),

Furu

yaet

al.(

2007,2

011),

Fric

ket

al.

(2008)

and

Liet

al.

(2011)

CX

CL6

CG

ranulo

cyte

chem

o-

tact

icpro

tein

2C

XC

R1,

CX

CR

2Ep

i/en

do

Angi

oge

nes

isN

eutr

ophils

Gas

troin

test

inal

tum

our

Gij

sber

set

al.

(2005)

CX

CL7

CN

AP-2

,PB

PC

XC

R2

Epi/

endo

Med

iate

san

gioge

nes

isvi

aM

APK

and

mTO

Rpat

hw

ay

Neu

trophils,

pla

tele

ts,

monocy

tes

Pancr

eati

cca

nce

rsM

atsu

bar

aet

al.

(2011)

CX

CL8

CIL

8C

XC

R1,

CX

CR

2Ep

i/en

do

Neu

trophil

chem

oat

trac

-ta

nta

nd

activa

ting

fact

or.

Angi

oge

nic

agen

t

Neu

trophils

Gas

tric

carc

inom

a,ova

rian

cance

rEc

ket

al.

(2003)

and

Yig

itet

al.

(2011)

CX

CL9

–M

onoki

ne

induce

dby

IFN

-g(M

IG)

CX

CR

3En

do

Att

ract

tum

our-

infilt

rating

lym

phocy

tes,

inhib

iten

doth

elia

lce

llm

igra

tion

and

pro

life

r-at

ion

Thym

us,

nat

ura

lki

ller

cell

,w

ides

pre

adG

astr

ic,

colo

rect

alca

nce

rsM

ush

aet

al.

(2005)

and

Ohta

ni

etal

.(2

009)

CX

CL1

0–

IFN

-g-i

nduci

ble

pro

tein

10

(IP10)

CX

CR

3En

do

Pro

mote

anti

-tum

our

effe

cts

by

attr

acting

T-ly

mphocy

tes

Thym

us,

nat

ura

lki

ller

cell

,w

ides

pre

adG

astr

ic,

colo

rect

al,

endo-

met

rial

,ova

rian

cance

rsM

ush

aet

al.

(2005),

Son

etal

.(2

007),

Ohta

ni

etal

.(2

009)

and

Smir

nova

etal

.(2

012)

CX

CL1

1–

IFN

-induci

ble

T-ce

lla

chem

oat

trac

tant

(I-T

AC

)

CX

CR

3,

CX

CR

7En

do/e

pi

Inhib

iten

doth

elia

lce

llm

igra

tion

and

pro

life

r-at

ion

Thym

us,

nat

ura

lki

ller

cell

Cle

arce

llova

rian

cance

rsFu

ruya

etal

.(2

011)

CX

CL1

2–

SDF-

1a

/bC

XC

R4,

CX

CR

7En

do/e

pi

Neo

vasc

ula

risa

tion

duri

ng

foet

alorg

anoge

nes

is.

Tum

our

cell

mig

rati

on

and

pro

life

rati

on

Wid

espre

adB

reas

t,co

lore

ctal

carc

inom

a,en

dom

etri

al,

ova

rian

can-

cers

Jian

get

al.

(2006),

Aki

shim

a-Fu

kasa

wa

etal

.(2

009),

Gel

min

iet

al.

(2009)

and

Has

san

etal

.(2

009)

CX

CL1

3C

BLC

/BC

A-1

CX

CR

5–

Mig

rati

on

of

T-ce

lls

Bce

lls

Hum

anlu

ng

cance

rde

Chai

sem

arti

net

al.

(2011)

CX

CL1

4C

BR

AK

/bole

kine

Unkn

ow

nU

nkn

ow

nM

igra

tion

of

T-ce

lls,

che-

moat

trac

tion

of

NK

cell

sin

epit

hel

ium

of

the

endom

etri

um

Monocy

tes

Endom

etri

alca

nce

rva

nder

Hors

tet

al.

(2012)

CX

CL1

6C

SR-P

SOX

OX

;C

XC

LG16;

SR-P

SOX

CX

CR

6Ep

iSo

luble

CX

CL1

6en

han

ces

pro

life

rati

on

and

mig

ration.

Tran

s-m

embra

ne

CX

CL1

6in

hib

its

pro

life

rati

on

Act

ivat

edT-

cells

Gli

om

a,co

lore

ctal

carc

i-nom

a,re

nal

cance

r,pro

s-ta

teca

nce

r,bre

ast

cance

r

Ludw

iget

al.(2

005),

Hojo

etal

.(2

007),

Dar

ash-Y

ahan

aet

al.

(2009),

Gutw

ein

etal

.(2

009)

and

Den

get

al.

(2010)

Epi,

epit

hel

ial

cell

expre

ssio

n;

Endo,

endoth

elia

lce

llex

pre

ssio

n.

CXC chemokines in epithelial ovarian cancer 305

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306 A Rainczuk, J Rao and others

ligand and receptor pairs can exert a direct effect ontumour proliferation and survival (Erreni et al. 2009,Ha et al. 2011, Boimel et al. 2012). The chemokinesfrom stromal cells may influence the survival ofmalignant cells by binding to the functional receptorsacquired on the cancerous cells, enhancing metastasis ina chemokine-rich environment (Mantovani et al. 2010,Balkwill 2012).

This review will discuss the emerging roles of theCXC chemokines in the pathogenesis of EOC, in thecontext of their inflammatory, angiogenic and metastaticproperties.

The role of CXC chemokines in normal ovarianphysiology

The surface epithelium of the ovary is a natural extensionof the peritoneal lining and is directly exposed to anymetabolic and environmental stress present in theperitoneal cavity (Maccio & Madeddu 2012). Regularcycling and release of ova from the ovary causes theovarian epithelium to undergo regular cycles of wound-ing and repair throughout a female’s reproductive life.This process is generally considered to induce aninflammatory response and triggers (Murdoch et al.2010) the secretion of a large number of chemokines,cytokines, enzymes and growth factors (Murdoch &McDonnel 2002).

A model of CXC chemokine-coordinated regulation ofangiogenesis and inflammation after wounding of anormal epithelial cell layer is summarised in Fig. 2(Griffioen & Molema 2000, Spinetti et al. 2001,Romagnani et al. 2004), and a more detailed review ofwound repair involving chemokines can be found inRomagnani et al. (2004). At the wound site, plateletsrelease growth factors including vascular endothelialgrowth factor (VEGF), platelet-derived growth factor andvarious cytokines including CXCL7, CXCL1, CXCL5(which initiate neutrophil recruitment) and CXCL4(to assist in blood-clot formation) (Griffioen & Molema2000, Spinetti et al. 2001, Romagnani et al. 2004).Wounded epithelial cells then express CXCL8, inducingan inflammatory response and resulting in angiogenesisand the sprouting of new blood vessels with high CXCR2expression (Romagnani et al. 2004). The epithelial layeralso produces angiostatic CXCL9, -10 and -11, arrestingthe migration of proliferating CXCR3C endothelial cellsand leading to lymphocyte recruitment during the periodof healing (Romagnani et al. 2004). CXCL8 is alsoinvolved in re-epithelialisation of the wound site (notshown in Fig. 2; Romagnani et al. 2004). Other non-CXCchemokines are also involved in wound healing; forexample, CCL2 expression during wound healing alsorecruits CCR2C macrophages and monocytes to theinflamed ovarian epithelium (Fader et al. 2010). The roleof non-CXC chemokines in this process is reviewedelsewhere (Charo & Ransohoff 2006).

Reproduction (2012) 144 303–317

Chemokines and cytokines also modulate ovarianfunction via their involvement in follicular developmentand ovulation and the formation, function and death ofthe corpus luteum (Auersperg et al. 1998). CXCL8, forexample, is significantly elevated in normal folliculo-genesis, suggesting a role in mediating neutrophilattraction (Ness & Modugno 2006). Moreover, anti-CXCL8 antiserum can suppress hCG-induced ovulationand neutrophil activity in women undergoing IVF (Ness& Modugno 2006). CXCL8 levels also correlate withcollagenases, which are crucial to the remodelling ofthe cervical matrix (Auersperg et al. 1998). CXCL1,another neutrophil attractant, is present in periovulatoryfollicular fluid and is expressed by ovarian stromalcells. On completion of ovulation, the rupturedepithelium undergoes repair to form the corpus luteum,characterised by significantly increased infiltration ofleukocytes and modulated by pro-angiogenic factors inthe follicular fluid such as CXCL8 and CCL2 (Merritt &Cramer 2011).

Recent studies have shown that CXCL12 is expressedin the cells of the normal ovarian surface epithelium andthe epithelium of the fallopian tube (Machelon et al.2011). Interestingly, CXCL12 is absent in the follicles andthe oocytes (Machelon et al. 2011, Merritt & Cramer2011). Constitutive expression of CXCL10 has also beenreported in normal ovary, with cyclic increases observedduring ovulation (Wong et al. 2002, Zhou et al. 2004).While the precise roles played by these chemokines inthe physiology of the ovary are still unclear, theirpresence firmly establishes their importance in normalovarian function.

Epithelial ovarian cancer

EOCs account for between 80 and 95% of all ovariantumours diagnosed and exhibit the highest mortality rateof any gynaecological tumour type (Roett & Evans 2009).EOCs are commonly classified according to histopatho-logical appearance as clear cell, endometrioid, mucinousor serous; amongst these, serous epithelial tumoursare the most commonly diagnosed tumour type (Kobelet al. 2008). However, wide varieties of less commonovarian neoplasms exist and can include mixed, undiffer-entiated or Brenner-type tumours (Kaku et al. 2003,Arnogiannaki et al. 2011). While traditionally sub-grouped according to FIGO stage and grade, a growingbody of evidence now suggests that low- and high-gradetumours have different aetiologies arising from unrelated,underlying molecular pathologies (Gilks et al. 2008,Kobel et al. 2008). Low-grade tumours typically exhibitmicropapillary structures and originate from borderlinetumours. These tumours are believed to progress throughbenign and borderline stages to malignancy and aregenerally associated with good prognosis. They also havewell-defined mutational pathways and often expressmutated KRAS or BRAF (Cho 2009). By contrast, a

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Figure 2 An overview of CXC chemokine involvement in normal wound repair (expanded details found in Romagnani et al. (2004)). (A) Generationof blood clot and neutrophil recruitment: (A1) growth factors, cytokines and chemokines are released by clotted platelets, while CXCL4, -5 and -7initiate neutrophil recruitment to the wound site; (A2) epithelial cells at the wound site express CXCL8, while endothelial cells express CXCL1; (A3)neutrophils expressing CXCR2 migrate into the wound site along the CXCL1/CXCL8 chemotactic gradient. (B) Recruitment of lymphocytes andmonocytes: (B1) epithelial cells continue to express CXCL8, promoting the growth of new blood cells. Epithelial cells also express CXCL9, -10 and-11 to limit and regulate endothelial cell proliferation and new blood vessel growth; (B2) CXCL9 and -10 recruit lymphocytes during wound healing;(B3) ongoing CXCL1 expression by endothelial cells promotes continued neutrophil recruitment, while CXCL9, -10 and -11 regulate blood vesselgrowth and the migration of proliferating endothelial cells; (B4) CXCL4 helps promote and maintain blood clotting.

CXC chemokines in epithelial ovarian cancer 307

significantly large number of TP53 gene mutations havebeen found in high-grade serous tumours (Milner et al.1993), and they also have a poorly defined mutationalpathway with mutated TP53 expression found in up to80% of cases (Vang et al. 2009). They appear to arisespontaneously, metastasise early in their progression andare associated with poor prognosis (Cho 2009, Cho &Shih Ie 2009, Roett & Evans 2009).

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Balancing inflammation, T-cell recruitment,angiogenesis and angiostasis: the role of chemokinesin ovarian cancer development and progression

Inflammation and the ovarian surface epithelium

Similar to other tumour types, chronic inflammation isan important condition underlying the growth andprogression of ovarian tumours (Ness & Cottreau 1999,

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308 A Rainczuk, J Rao and others

Ness et al. 2000, Fleming et al. 2006, Son et al. 2007,Coffelt & Scandurro 2008, Maccio & Madeddu 2012). Askey mediators of inflammation, chemokines play twomajor roles: i) they are responsible for the recruitmentand activation of immune cells into sites of malignancy(and possibly areas of pre-neoplastic growth) and ii) theymediate pro- and anti-angiogenic effects, processesrequired for the vascularisation and progression oftumours. In addition, the multiple biological effectsexerted by CXC chemokines mean that each of theseprocesses is inextricably linked. Therefore, it is ofparamount importance to identify molecules that areinvolved and their contribution towards anti-tumouractivity and in preventing recurrence – or, conversely,how their activity may promote tumour growth andmetastasis.

Although an accepted view is that a majority ofovarian cancer cases originate from the ovarian surfaceepithelium, emerging evidence suggests the involvementof the fimbriae of fallopian tubes. Crum et al. showedthat fallopian tubes and ovaries from women with BRCAmutations often contained precursor lesions in thefimbriae of the tube (Medeiros et al. 2006, Crum et al.2007). By contrast, Clarke et al. have demonstrated anassociation between BRCA and inflammation in serousovarian carcinomas. BRCA mutations (or epigenetic loss)were associated with favourable prognosis via recruit-ment of intraepithelial CD8C (and to a lesser extentCD3C) tumour-infiltrating lymphocytes (TILs). This novelassociation between intraepithelial T-cell infiltration andBRCA mutation in serous EOC has also been observedwith certain breast cancer types and can lead to afavourable patient prognosis (Clarke et al. 2009).Chronic inflammation from diseases such as hepatitis,human papilloma virus in the cervix and ulcerativecolitis are well known to cause cancer of the tube(Demopoulos et al. 2001). Similarly, it has beensuggested that proliferation and hyperplasia in thefallopian tube are associated with lesions that may becarcinogenic in nature (Moore & Enterline 1975).Compelling evidence for this hypothesis was recentlypresented by Kim et al. (2012) who demonstrated, usinga Dicer–Pten knockout mouse, that serous epithelialtumours can arise in the fallopian tube and rapidlymetastasise to the ovary. Mice in this study exhibited a100% mortality rate from tumours that bore a strikingresemblance to clinical disease (Kim et al. 2012).

Retrograde menstruation via the fallopian tube is acommon condition, where the flow of endometrial fluidbathes the tubes with inflammatory molecules such asCXCL8 (interleukin-8 (IL8)), tumour necrosis factor alpha(TNF-a) and granulocyte–macrophage colony-stimulat-ing factor (GM–CSF). Each of these are elevated in ovariancarcinomas (Strandell et al. 2004). It is well establishedthat a combination of tubal ligation and hysterectomy isprotective against ovarian cancer (Green et al. 1997,Seidman et al. 2002). Together, these findings strongly

Reproduction (2012) 144 303–317

support the notion that inflammation in the fallopiantube, the ovarian epithelium or both are contributingfactors in the development of ovarian cancer.

The inflammation-mediated release of chemokines,cytokines and growth factors can also promote migrationand proliferation of leukocytes, epithelial and endo-thelial cells (Fig. 2; Mukaida & Baba 2012). At amolecular level, there is evidence that pro-inflammatorycytokines such as IL1, IL6 and TNF-a are linked to a poorprognosis in EOC patients (Clendenen et al. 2011,Maccio & Madeddu 2012). Oxidative stress, cell necrosisand rapid cell division are hallmarks of chronicinflammation (Ness & Modugno 2006). Rapid celldivision gives rise to a possibility of replication errorand the need for DNA repair, such as at the key regulatorysite TP53, increasing the risk of mutagenesis. This isfurther supported by the high rate of TP53 mutationcommonly observed in high-grade serous EOC (Merritt &Cramer 2011).

Recently, inflammatory cytokine production in thehuman ovarian cancer microenvironment has beendescribed in terms of an autocrine cytokine network.The TNF network includes TNF-a, IL6 and CXCL12(Kulbe et al. 2012); in particular, the CXCL12/CXCR4axis plays a major role in the proliferation and metastasisof ovarian tumour cells (Barbieri et al. 2010). EnhancedTNF network activity has been associated with genesinvolving angiogenesis, inflammation and leukocyteinfiltration. Targeting members of the TNF network viasiRNA or neutralising antibodies reduced experimentalperitoneal ovarian tumour growth, as well as angiogen-esis in human ovarian tumour biopsies and mousexenograft models (Kulbe et al. 2012).

The nuclear factor-kB (NF-kB) pathway is also import-ant in modulating autocrine and paracrine signallingbetween inflammatory markers in the cancer microenvir-onment (Son et al. 2007). Notably, NF-kB regulates theCXCL12/CXCR4 autocrine signalling in ovarian cancercell lines (Miyanishi et al. 2010). Over-expression ofprostaglandins, leading to altered expression of cytokinesand chemokines, increases the invasiveness of ovariantumour cells (and other tumour types) (Aitokallio-Tallberget al. 1988, Subbaramaiah et al. 1997, Ness & Modugno2006). The expression of cytokines and chemokines ininflamed malignancies of the female genital tract may,therefore, lead to dysfunctional autocrine loops, resultingin increased tumour progression and invasion.

Recruitment of T-cells to epithelial ovarian tumours

In the normal anti-tumour immune response,interactions between CXCL9 (MiG), CXCL10 (IP10) andtheir receptor CXCR3 influences the chemoattraction ofnatural killer (NK) cells, activated Th1 cells, gamma-delta T-cells, macrophages and dendritic cells towardstumours (Strieter et al. 2006, Whitworth & Alvarez2011). The importance of CXCL9 and CXCL10 to

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CXC chemokines in epithelial ovarian cancer 309

generate an anti-tumour response is supported by theirroles as mediators of IL12, a potent immunoregulatorycytokine (also being trialled as a therapeutic agent inovarian cancer patients, although with modest results)(Whitworth & Alvarez 2011). Additionally, in aninvestigation into links between coagulation and inflam-mation, it was shown that culture of cells derived fromovarian cancer ascites and peripheral blood mono-nuclear cells (PBMCs) from healthy donors also inducedsignificant up-regulation of inflammatory cytokines(IL1b and IL6), along with CCL2 and CXCL8 (Naldiniet al. 2011). This suggests that the ovarian cancermicroenvironment is inflammatory in nature and candirect PBMCs to increase the release of inflammatorychemokines and cytokines.

Recent gene expression studies identified CXCL10,CXCL11 and their receptor CXCR3 as amplified in asubset of ovarian cancer patients from a sample of 489high-grade, serous epithelial tumours. These studiesdefined this group of tumours as ‘immunoreactive’(CGARN 2011), suggesting an important role for thesechemokines in the tumour microenvironment. A primaryfunction for these chemokine ligands is the trafficking ofleukocytes and the recruitment of activated CD4C Th1cells, CD8CT-cells and NK cells towards sites ofinflammation (Clark-Lewis et al. 2003). High concen-trations of CXCL9 and CXCL10 are also associated withthe presence of TILs, and this is a positive predictor ofpatient outcome with patients exhibiting lower recur-rence, less dissemination and longer survival (Zhanget al. 2003, Sato et al. 2005, Tomsova et al. 2008,Kryczek et al. 2009). By contrast, negative patientoutcomes are associated with tumour infiltration byregulatory T-cells (Treg) that mediate immune tolerance(Curiel et al. 2004, Sato et al. 2005, Kryczek et al. 2009).Sato et al. (2005) demonstrated that high intraepithelial

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CD8C/CD4CT-cell and CD8CT-cell/Treg ratios resultedin improved survival in EOC. Moreover, neitherintraepithelial CD4C or other CD3C TILs alone wereassociated with survival in this study (Sato et al. 2005).Others have more recently shown that longer survival ispositively correlated with both CD8C and CD3C TILs(Hwang et al. 2012). However, CD8C positive TILsshowed a more consistent association with patientsurvival in this meta-analysis (Hwang et al. 2012).

EOC tissue and ascites fluid contain high levels ofCD4C CD25C FOXP3C Treg cells, which are not presentin normal ovarian tissues (Curiel et al. 2004). FOXP3 is amarker of Treg cells, crucial for maintaining immunologichomoeostasis by preventing autoimmunity in a normalimmune environment (Kelley & Parker 2010). In a studyof immune cell infiltration into tumours, a high level ofthe chemokine CCL22 (secreted by tumour-associatedmacrophages) was detected in tumour ascites, possiblyinducing Treg trafficking into tumours and suppressingthe response of TILs (Curiel et al. 2004). In an apparentcontradiction, however, under certain conditions in thetumour microenvironment, CXCL9, CXCL10 and thecytokine IL17 can also be produced by tumour-associated macrophages and contribute to positiveoutcomes in EOC (Kryczek et al. 2009).

An explanation as to why TILs are effective atimproving EOC patient outcomes can be partiallyattributed to the recent identification of T helper17 (Th17) cells. These cells can contribute to pathogenesisof autoimmune disease and initiate tumour growth incertain models but drive anti-tumour immunity in someepithelial malignancies (Zou & Restifo 2010, Wilke et al.2011). Th17 cells are postulated not to be immunosup-pressive, as they do not express FOX3P (Wilke et al.2011). An important study by Kryczek et al. (2009)showed that in addition to NK cells and Th1 effector

Figure 3 Promotion of a favourable outcome inEOC involving CXC chemokines. (1) Th17 cells(without Treg suppression in this example) secreteIL17 and anti-tumour promoting IFN-g, afterinduction by tumour-associated macrophagessecreting IL1b (Kryczek et al. 2009); (2) IL17secretion and IFN-g induce tumour cells tosecrete CXCL9 and CXCL10; (3) angiostaticchemokines establish a gradient whereby adap-tive and innate immune cells migrate to thetumour site. A high CD8CT-cell to low CD4CT-cell/Treg ratio can lead to positive outcomes inEOC (Sato et al. 2005).

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T-cells, Th17 cells secreting IFN-g and IL17 werecorrelated with the elevation of Th1-type chemokinesCXCL9 and CXCL10 in the ovarian cancer microenvir-onment. CXCL9 and CXCL10 were produced by primaryovarian cancer cells and macrophages, leading to therecruitment of CXCR3-expressing effector CD8CT-cellsand NK cells to the tumour. A significant association wasfound with the presence of IL17 in patient ascites, withhigh IL17 levels (median patient survival 78 months)significantly improving survival time compared with lowIL17 levels (median patient survival 27 months) (Kryczeket al. 2009). The levels of CXCL9 and CXCL10 weredirectly correlated with tumour and tumour-infiltratingNK cells and CD8CT-cells, highlighting the importanceof these ELR-chemokines in limiting the spread of EOC.Figure 3 attempts to summarise the effect of increasedCXCL9 and CXCL10 concentrations in the EOC micro-environment, which are reported to lead to a positivesurvival outcome.

Angiostatic chemokine ligands: CXCL9 and CXCL10expression can inhibit EOC progression

The ELRK chemokines CXCL9, CXCL10 and CXCL11can influence tumour progression through their angio-static effects (Strieter et al. 2006). Primary ovarian cancercells stimulated with IL17 and IFN-g can be induced tosecrete CXCL9 and CXCL10 (Kryczek et al. 2009), whileCXCL11 (and CXCL9) expression has been detected inserous ovarian carcinoma cell lines and tissue byimmunohistochemistry and reverse transcriptase PCR(Furuya et al. 2007). These CXCR3-binding chemokinescan inhibit endothelial cell migration and proliferation,attract cells involved in innate and adaptive immunityand increase MHC class I processing by tumour cells,

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thereby enhancing recognition by the immune system(Groom & Luster 2011, Wilke et al. 2011; Fig. 3).

CXC-mediated regulation of the EOC microenviron-ment appears to be primarily due to CXCL9 and CXCL10;the anti-tumour effect of CXCL11 in EOC is reportedlyless efficient in mounting an anti-tumour response(Lasagni et al. 2003, Furuya et al. 2007). Similar to theexpression of CXCL9 and CXCL10 ligands, increasedexpression of CXCR3 has been reported on the surface ofEOC cells (Furuya et al. 2007) and clear cell ovariancancer cells (Furuya et al. 2011). CXCR3 has been foundto be expressed by endothelial cells while undergoingG2M and late S phases of the cell cycle (Romagnani et al.2001). Although the expression of CXCR3 in multiplecell types and specific cell cycle phases have beenidentified (Romagnani et al. 2001), their biologicalrelevance has yet to be conclusively demonstrated(Lasagni et al. 2003, Giuliani et al. 2006, Gacci et al.2009, Campanella et al. 2010).

Angiogenic chemokine ligands can promote tumourdevelopment

Pathological angiogenesis involves multiple cell typesand growth factors and is observed in wound healing,arthritis and diabetic retinopathy. It is closely related tochronic inflammation and is an important event in theprogression and spread of cancer (Keeley et al. 2008).ELRC chemokines including CXCL8 (IL8), CXCL5(ENA-78) and CXCL1 (GRO-a) are known to induceendothelial migration and proliferation through bindingto CXCR2, leading to metastatic spread (Mukaida &Baba 2012). They can also act indirectly via theexpression of CXCR2 on leukocytes, leading to thesecretion of pro-angiogenic factors such as VEGF(Kiefer & Siekmann 2011). All three CXC chemokines

Figure 4 Modification of CXCL5 increaseschemotactic potency and neutrophil recruit-ment. (1) As in normal wound healing, neutro-phils containing CXCR2 continue to migrate tothe wound site towards CXCL1 and -8;(2) neutrophils secrete cathepsin G and cleaveCXCL5, increasing chemotactic potency.(3) Enhanced CD11b/CD18 expression on theneutrophil surfaces increases adhesion toendothelial tumour cells and initiatesremodelling of the endothelium, making it easierfor tumour cells to metastasise.

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CXC chemokines in epithelial ovarian cancer 311

have the ability to attract polymorphonuclear leukocytesincluding neutrophils (Mukaida & Baba 2012).

The significance of inflammation in the potentialinitiation and progression of EOC makes CXCL5 apotentially important (and largely unreported) factor inthis disease. CXCL5 is expressed in both lung andpancreatic cancer and is up-regulated in endometriosisand ovarian carcinoma where it can attract neutrophils(Wislez et al. 2004, Furuya et al. 2007, Frick et al. 2008,Li et al. 2011). Once present, neutrophils secrete a broadspectrum of proteases including cathepsin G that cancleave CXCL5; N-terminal truncation of CXCL5 signi-ficantly enhances the chemotactic potency of CXCL5both in vitro and in vivo (Wuyts et al. 1999, Mortier et al.2010). Neutrophils isolated from the peripheral blood ofovarian cancer patients also produce increased reactiveoxygen species and display increased adhesion toovarian cancer cells through enhanced CD11b/CD18 expression on their cell surfaces (Klink et al.2008). The adhesion of neutrophils to endothelial cellsinitiates remodelling of the endothelium, making iteasier for tumour cells to metastasise (Wu et al. 2001,De Larco et al. 2004, Klink et al. 2008). Figure 4 attemptsto summarise the influence of neutrophils and modifiedCXCL5 in the metastasis of ovarian tumour cells.

Both CXCL8 (Schutyser et al. 2002) and CXCL1(Bolitho et al. 2010) are up-regulated in ascites fluid,while CXCL8 protein and anti-CXCL8 antibodies are alsopresent in serum from advanced EOC patients (Lokshinet al. 2006). Stimulation by CXCL8 of various ovariantumour cell lines leads to their proliferation in vitro(Wang et al. 2005) and is correlated with increasedmetastatic potential in SKOV3 cells (Yin et al. 2011).CXCL8 also has a direct role in angiogenesis, stimulatingcapillary tube formation via CXCR1 and CXCR2 (Li et al.2003). In the context of advanced EOC, development

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of ascites and possible metastasis is promoted byangiogenesis through the key regulator VEGF (MasoumiMoghaddam et al. 2011). In an early study to determinethe genes expressed in angiogenesis, five differenthuman ovarian carcinomas were implanted into theperitoneal cavities of nude mice (Yoneda et al. 1998).The formation of ascites was associated with theexpression of VEGF and CXCL8, and this was inverselyassociated with survival.

Similar to CXCL5, CXCL1 has chemotactic activity forneutrophils and its N-terminal truncation leads toenhanced chemotaxis in vitro (Wuyts et al. 1999).Over-expression of CXCL1 in vitro also results inincreased cell proliferation (Bolitho et al. 2010).CXCL1 has been shown to promote malignant transfor-mation of epithelial cells through a complex RASpathway-mediated transformation, which inducesnormal stroma to ‘re-program’ and become tumorigenic(Yang et al. 2006). Silencing of CXCL1 using siRNAeliminated RAS-induced transformation (Yang et al.2006). In the same study, CXCL1 expression waselevated in both serum and tumour tissue from ovariancancer patients (Yang et al. 2006). CXCL1 (in com-bination with CCL18) has also been suggested as a serumbiomarker of ovarian cancer (Wang et al. 2011).Multivariate ELISA gave a sensitivity of 92% andspecificity of 97% for detecting ovarian cancers, whichwas significantly higher than values obtained using CA125 alone (Wang et al. 2011).

In normal ovarian function, both CXCL8 and CXCL1are produced by the ovulating follicle to attract CXCR1C

and CXCR2C leukocytes to assist with ovulation(Karstrom-Encrantz et al. 1998). Using mouse modelsof peritoneal ovarian cancer, it has been shown thatmatrix metalloprotease-1 (MMP1) activation of theG protein-coupled receptor, protease-activated

Figure 5 Promotion of an unfavourable outcome inEOC involving CXC chemokines. (1) Tumour-associated macrophages secrete CCL22 to attractTreg cells. This results in suppression of Th17 cells,thereby reducing IL17 and IFN-g and impairingproduction of CXCL9 and -10 (Kryczek et al.2009). CXCL1 and -8 continue to be produced,enhancing angiogenesis (Romagnani et al. 2004);(2) CXCL12 production by tumour cells andenhanced CXCR4 expression by epithelial cancercells facilitate metastasis; (3) the lack of angio-static chemokines and presence of Treg cellsprevent immune cell migration to the immuno-suppressive tumour site (Sato et al. 2005).

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receptor-1 (PAR1), is also an important stimulator ofangiogenesis and metastasis in EOC (Agarwal et al.2008). The paracrine control of chemokine productionby CXCR1 and CXCR2 receptors on ovarian carcinomaendothelial cells is stimulated by the MMP1-PAR1signalling system (Agarwal et al. 2010). After MMP1-PAR1 stimulation, it was shown that ovarian carcinomacells both in vitro and in mice secreted CXCL8 andCXCL1, with the introduction of X1/2pal-i3 pepducincompletely inhibiting angiogenesis (Agarwal et al.2010). Inhibition of this pathway blocks tumour growthand metastasis in breast cancer models (Yang et al.2009); the MMP1-PAR1-CXCR1/2 pathway may there-fore be a new target for preventing EOC-relatedangiogenesis.

Direct involvement of the ELRK chemokine CXCL12in metastatic spread

In early studies to determine the influence of chemokinegradients on the metastatic spread of ovarian tumours, itwas shown that the receptor CXCR4 induced calciumflux and could direct migration of tumour cells (Scottonet al. 2001, 2002). The expression of CXCR4 mRNAwas detected in six ovarian cancer cell lines, ascites (19 of20 samples), and primary ovarian tumours (eight of tensamples). The study showed that the cell lines displayedchemotaxis towards CXCL12, which was also present athigh levels in ovarian cancer ascites fluid from 63 patients(Scotton et al. 2001). However, not all tumour cells in theprimary ovarian tumours expressed CXCR4 mRNA,suggesting that other factors present in the tumourmicroenvironment contributed to receptor expression.After in vitro stimulation of the ovarian cancer cell lineIGROV with CXCL12, cells could invade through Matrigeltowards a CXCL12 gradient (Scotton et al. 2002). This alsopromoted the activation of Akt/protein kinase B, biphasicphosphorylation of p44/42 MAP kinase and induction ofthe cytokine TNF-a (Scotton et al. 2002).

The metastatic spread of EOC tumour cells to theperitoneal mesothelium also involves the CXCL12/CXCR4axis (Kajiyama et al. 2008, Miyanishi et al. 2010).Furthermore, a significant correlation has been shownbetween CXCR4 expression in both primary and meta-static ovarian tumours, as well as lymph node metastases(Jiang et al. 2006). In a mouse model of ovarian cancer,siRNA-mediated silencing of CXCL12 reduced tumourgrowth in vivo, and a CXCR4 antagonist (AMD3100)could increase T-cell-mediated anti-tumour responses(Righi et al. 2011). Human primary ovarian tumour cellsalso express CXCL12 and VEGF following exposure to thehypoxic tumour microenvironment (Kryczek et al. 2005).In this in vivo model, CXCL12 and VEGF promotedangiogenesis and the migration of human vascularendothelial cell expressing CXCR4 (Kryczek et al. 2005).Importantly, CXCL12 and CXCR4 are not detected in

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normal ovarian tissues (Jiang et al. 2006, Kajiyama et al.2008), even in women with a family history of ovariancancer (Scotton et al. 2002).

Recently, sequential genetic changes were found in theTP53 allele as well as the CXCR4 gene locus (chromo-some 2q21.3) of human transformed ovarian surfaceepithelial cell lines (Archibald et al. 2012). These wereinvestigated to determine early events in the develop-ment of serous EOC. Following the screening of 50 serousEOC patient biopsies, the mutated TP53 expression genesignature (with decreased TP53 target gene expression)was associated with activation of epidermal growthfactor receptor pathways, as well as high expression ofCXCR4 genes (with corresponding mRNA and protein).This suggests that these events may occur early in thedevelopment of EOC (Archibald et al. 2012). Amongstother recently discovered genetic changes to potentiallyup-regulate CXCR4 expression in EOC is the abrogationof breast cancer metastasis suppressor 1 (BRMS1; Shenget al. 2012). Suppression of BRMS1 by short-hairpin RNAtransfection of OVCAR3 cell lines significantly enhancedCXCR4 expression at the mRNA and protein levels, withenhanced expression mediated by the NF-kB signallingpathway (Sheng et al. 2012).

Figure 5 offers a simplistic summary of the immuno-suppressive tumour microenvironment in EOC. The lackof angiogenic ELR-chemokines results in uncheckedangiogenesis due to the presence of Treg cells, while CXCchemokines and their receptors, in particular theCXCL12/CXCR4 axis, can promote tumour metastasis.

CXC chemokines as future therapeutic targetsfor EOC

Due to the importance of CXC chemokine signalling,there are active development strategies to exploit thesepathways to reduce the morbidity and mortalityassociated with EOC. One strategy involves the stimu-lation of Th17 cells through the use of vaccines orimmunotherapy to stimulate IFN-g and IL17, resulting inelevated CXCL9 and CXCL10 levels (Munn 2009,Cannon et al. 2011, Goyne et al. 2011). However,animal or clinical trials are yet to be reported. The use ofCXC chemokine inhibitors and antagonists for a varietyof different cancers, including EOC (Zlotnik et al. 2011,Balkwill 2012), is emerging as a promising strategy fornew treatments.

Of the CXC chemokine ligands and receptors involvedin EOC, inhibition of CXCR4 has received a great deal ofattention with several molecules found that are able toantagonise activity in response to CXCL12 (Barbieri et al.2010). In two separate studies involving mouse modelsof EOC, AMD3100 (a clinically approved CXCR4inhibitor) significantly reduced ovarian cancer cellgrowth (Ray et al. 2011, Righi et al. 2011). In mice,administration of AMD3100 significantly reduced

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CXC chemokines in epithelial ovarian cancer 313

intraperitoneal dissemination and angiogenesis(measured by vessel density with the tumour), increasedT-cell-mediated anti-tumour immune responses andapoptosis and reduced FoxP3C Treg cell numbers withinthe tumour (Righi et al. 2011). Subsequently, it wasshown that AMD3100 blocked CXCL12/CXCR4 binding,resulting in prolonged survival and reduced tumourgrowth in mice with disseminated ovarian cancer (Rayet al. 2011). The use of this important chemokineinhibitor in human trials is yet to be reported.

Conclusion

A significant body of evidence indicates the involvementof chemokines in EOC and suggests that initiation,progression and metastasis are strongly influenced by theCXC chemokines. In particular, their roles in inflam-mation, angiogenesis and immune cell recruitment haveall been shown to exert profound influence in the tumourmicroenvironment; moreover, there is a complex inter-play of these factors that influences clinical outcomes.Research to exploit these chemokine-signallingpathways, and translate the findings to a clinicalenvironment, remains at a very early stage. Relevantstudies in murine models using the inhibitor AMD3100targeting the CXCR4/CXCL12 axis have been reported,but there are no human trials yet reported. Similarly, theenhancement of CXCL9 and CXCL10 expression throughstimulation of Th17 cells, or CXCL5 inhibitor develop-ment to prevent neutrophil adhesion and potentialmetastasis, is also at an early stage.

The groundwork for understanding the combinedaction and mechanisms of CXC chemokine activity inEOC is underway. Exploitation of CXC chemokine-signalling pathways to reduce EOC morbidity andmortality remains as a future avenue to potentiallyimprove outcomes for patients with this disease.

Declaration of interest

The authors declare that there is no conflict of interest thatcould be perceived as prejudicing the impartiality of theresearch reported.

Funding

This work was supported by the Ovarian Cancer ResearchFoundation and the Victorian Government’s OperationalInfrastructure Support Program.

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Received 24 April 2012

First decision 19 June 2012

Accepted 5 July 2012

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