Tissue invasion and metastasis: Molecular, biological and clinical perspectives

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ContentslistsavailableatScienceDirect

Seminars

in

Cancer

Biology

j ou rn a l h om ep a ge :w w w . e l s e v i e r . c o m / l o c a t e / s e m c a n c e r

Review

Tissue

invasion

and

metastasis:

Molecular,

biological

and

clinical

perspectives

W.G.

Jiang

a,∗

_,

_A.J.

_Sanders

a

_,

_M.

_Katoh

b

_,

_H.

_Ungefroren

c

_,

_F.

_Gieseler

c

_,

_M.

_Prince

d

_,

S.K.

Thompson

e

_,

_M.

_Zollo

f,g

_,

_D.

_Spano

g

_,

_P.

_Dhawan

h

_,

_D.

_Sliva

i

_,

_P.R.

_Subbarayan

j

_,

M. Sarkar

j

_,

_K.

_Honoki

k

_,

_H.

_Fujii

k

_,

_A.G.

_Georgakilas

l

_,

_A.

_Amedei

m

_,

_E.

_Niccolai

m

_,

_A.

_Amin

n

_,

S.S.

Ashraf

n

_,

_L.

_Ye

a

_,

_W.G.

_Helferich

o

_,

_X.

_Yang

o

_,

_C.S.

_Boosani

p

_,

_G.

_Guha

q

_,

_M.R.

_Ciriolo

r

_,

K. Aquilano

r

_,

_S.

_Chen

s

_,

_A.S.

_Azmi

t

_,

_W.N.

_Keith

u

_,

_A.

_Bilsland

u

_,

_D.

_Bhakta

q

_,

_D.

_Halicka

v

_,

S. Nowsheen

w

_,

_F.

_Pantano

x

_,

_D.

_Santini

x

a_Cardiff_University,_Cardiff,_United_Kingdom

b_National_Cancer_Center,_Tokyo,_Japan

c_University_Hospital_{Schleswig-Holstein,}_Lübeck,_Germany

d_University_of_Michigan,_Ann_Arbor,_MI,_USA

e_Royal_Adelaide_Hospital,_Adelaide,_Australia

f_Department_of_Molecular_Medicine_and_Medical_{Biotechnology}_(DMMBM),_University_of_Naples_Federico_II,_Naples,_Italy

g_CEINGE_{Biotecnologie}_Avanzate,_Naples,_Italy

h_University_of_Nebraska_Medical_Center,_Omaha,_USA

i_Purdue_Research_Park,_{Indianapolis,}_IN,_USA

j_University_of_Miami,_Miami,_FL,_USA

k_Nara_Medical_University,_Kashihara,_Japan

l_Physics_Department,_School_of_Applied_Mathematical_and_Physical_Sciences,_National_Technical_University_of_Athens_(NTUA),_Athens,_Greece

m_University_of_Florence,_Florence,_Italy

n_United_Arab_Emirates_University,_Al_Ain,_United_Arab_Emirates_and_Faculty_of_Science,_Cairo_University,_Egypt

o_University_of_Illinois_at_{Urbana-Champaign,}_Urbana,_IL,_USA

p_Creighton_University,_Omaha,_NE,_USA

q_SASTRA_University,_Thanjavur,_India

r_University_of_Rome_Tor_Vergata,_Rome,_Italy

s_Ovarian_and_Prostate_Cancer_Research_Trust_Laboratory,_Surrey,_United_Kingdom

t_Wayne_State_University,_Detroit,_MI,_USA

u_University_of_Glasgow,_Glasgow,_United_Kingdom

v_New_York_Medical_College,_Valhalla,_NY,_USA

w_Mayo_Clinic_College_of_Medicine,_Rochester,_MN,_USA

x_University_Campus_Bio-Medico,_Rome,_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Availableonline10April2015

Keywords:

Cancermetastasis

Invasion

Cancertherapy

a

b

s

t

r

a

c

t

Cancerisakeyhealthissueacrosstheworld,causingsubstantialpatientmorbidityandmortality.Patient

prognosisistightlylinkedwithmetastaticdisseminationofthediseasetodistantsites,withmetastatic

diseasesaccountingforavastpercentageofcancerpatientmortality.Whileadvancesinthisareahave

beenmade,theprocessofcancermetastasisandthefactorsgoverningcancerspreadandestablishmentat

secondarylocationsisstillpoorlyunderstood.Thecurrentarticlesummarizesrecentprogressinthisarea

ofresearch,bothintheunderstandingoftheunderlyingbiologicalprocessesandinthetherapeutic

strate-giesforthemanagementofmetastasis.ThisreviewliststhedisruptionofE-cadherinandtightjunctions,

keysignalingpathways,includingurokinasetypeplasminogenactivator(uPA),phosphatidylinositol

3-kinase/v-aktmurinethymomaviraloncogene(PI3K/AKT),focaladhesionkinase(FAK),␤-catenin/zinc

ﬁngerE-boxbindinghomeobox1(ZEB-1)andtransforminggrowthfactorbeta(TGF-␤),togetherwith

inactivationofactivatorprotein-1(AP-1)andsuppressionofmatrixmetalloproteinase-9(MMP-9)

activ-ityaskeytargetsandtheuseofphytochemicals,ornaturalproducts,suchasthosefromAgaricusblazei,

Albatrellusconﬂuens,Cordycepsmilitaris,Ganodermalucidum,PoriacocosandSilybummarianum,together

∗ Correspondingauthorat:Cardiff-PekingCancerInstituteandCardiff-CapitalMedicalUniversityJointCentreforBiomedicalResearch,CardiffUniversitySchoolofMedicine,

CardiffUniversity,HenryWellcomeBuilding,HeathPark,CardiffCF144XN,UnitedKingdom.Tel.:+442920687065.

E-mailaddress:[email protected](W.G.Jiang).

http://dx.doi.org/10.1016/j.semcancer.2015.03.008

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withdietderivedfattyacidsgammalinolenicacid(GLA)andeicosapentanoicacid(EPA)andinhibitory

compoundsasusefulapproachestotargettissueinvasionandmetastasisaswellasotherhallmarkareas

ofcancer.Together,thesestrategiescouldrepresentnew,inexpensive,lowtoxicitystrategiestoaidin

themanagementofcancermetastasisaswellashavingholisticeffectsagainstothercancerhallmarks.

creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

Thechainofeventsleadingtothemalignanttransformationof

cells,whetherthroughgeneticorepigeneticalterations, is

com-plex.Malignantcellspossesskeyhallmarks,namely,uncontrolled

growthpotentialsandtheabilitytoinvadesurroundingtissuesand

metastasize[1].Cancercellslikelypossesstheseinnateabilitiesto

someextent,thoughthedegreeandtimingofinvasionand

metas-tasismayvarydue tothegeneticand epigeneticheterogeneity

withinthetumorandfurthersignalsfromextrinsicfactors,such

asthosewithinthatparticularmicroenvironment[2].

Despitesubstantialeffortdedicatedtotheearlydetectionand

preventionofcancer,mostpatientsarelikelytohavemicro-(not

visibleusingconventionalmethods)ormacro-metastasesbythe

timetheycometomedicalattention[3,4].Cancerpatients,both

earlyandlatestage,dependentonlifespan,arelikelytodevelop

metastasis.Thismetastaticspreadoftheprimarytumoraccounts

for over 90% of patientmortality associated withsolid cancers

[1,4,5].Despitethis,researchintotheﬁeldofmetastasis,in

compar-isontootherkeyeventssuchasproliferation,etc.,islagging.Thisis

partlyduetothecomplexityofthemetastaticprocessbutalsodue

toalackofsufﬁcientfundingandeffortsintothisareaofresearch.

However,signiﬁcantprogressinthisvitalareaofcancerresearch

hasbeenwitnessedoverthepastdecade,thoughmuchremainsto

beelucidatedbeforewefullyunderstandthisperniciouscondition

andanumberofsigniﬁcantgapsremaintobeﬁlledbeforewecan

trulyunderstandthiscomplexprocess.

Diagnosisandtreatmentofmetastaticdiseasearevitalareasin

theconstantbattlemanypatientsfaceagainstcancer,yeteffective

treatmentsarelimitedandsubstantialmorbidityandmortalityare

stillassociatedwithmetastaticdisease[5,6].This,togetherwith

thecomplexitiessurroundingthemetastaticprocess(summarized

inFig.1)andthecomplexnatureandheterogeneityofmetastatic

tumors,fullysupportsandjustiﬁesfurtherresearchdedicatedto

thediscoveryofalesstoxicmeanstotreatthiscondition.Thisisthe

majormissionofgettingtoknowcancer(GTKC).Thisreviewaims

todiscusskeyknowledgegaps,explorepotentialtargetsintackling

metastasisandalsopotentialmethods,includingphytochemicals,

smallmoleculeinhibitorsandnaturalcompoundsindevisingnew

strategiesfortreatingmetastasis.

2. Cellularpropertiesandmetastasis

2.1. Cell–celladhesion

Incancersderivedfromtheepithelium,inter-cellularstructures

and cell–cell adhesion are key factors in maintaininga

coher-entprimarytumormass[7,8].Abnormalitiesinthesestructures,

throughmutationor dysregulation,canleadtothedissociation

oftheprimarytumorandanenhancedpotentialfor

dissemina-tionandmetastaticspreadofcancercellstosecondarylocations

[7–9]. Keystructures involvedin maintainingthis adhesiveness

betweencellsincludeadherensjunctions(includingdesmosomes),

tightjunctions(TJ)and gapjunctions.While gapjunctions

con-feraweakadhesionstructureandTJs,amodestone,theadherens

junctionsprovidethemostpowerfulsourceofadhesionin

epithe-lialcells.Perhapsoneofthestrongestandmoststudiedregulators

ofadhesionisE-cadherin(cadherin-1orCDH1),amemberofthe

cadherinfamilyofproteins.E-cadherin,togetherwithassociated

catenins,playsakeyroleinmaintainingcell–celladhesionandis

alsoinvolvedintheregulationofthecellcycleregulatorsp27kip1

andp57kip2_,_which_are_involved_in_cell–cell_contact_inhibition_in

normalepithelium,butwhicharelostordisturbedincancercells,

mainly due to the loss of E-cadherin in cancer cells [8,10,11].

Hence,reducedcell–celladhesionnotonlyenhancesthe

poten-tialformetastaticdisseminationofcancercellsbutalso,through

lossofcontactinhibition,promotesuncontrolledcellgrowth[7].

E-cadherin has alsobeen established as a key mediator of the

epithelial–mesenchymaltransition(EMT)process(discussedin

Section2.4).Thus,enhancedexpressionofkeycadherinmolecules

couldofferpotentialasastrategytocontrolmetastatic

dissemina-tion,thoughrealizingthispotentialhasproveddifﬁcult;thusfar

therehavebeenfewreportsidentifyingviabletreatmentoptions

inthisregard.However,thereareanumberofnoteworthyoptions,

namelythepolyunsaturatedfattyacidsgammalinolenicacid(GLA)

anddihomo-␥-linolenicacid(DGLA),bothobtainablethroughthe

diet.ThesehavebeenreportedtobekeyregulatorsofE-cadherin

and desmosomal cadherins in cancer cells and have also been

reportedtohavebeneﬁcialeffectsforpatientswithseveralcancer

typesincludingpancreaticcancerandbreastcancer[12–15].The

desirableeffectsoftheseessentialfattyacids(EFAs)wereblocked

bynon-EFA,aslongchainedoleicderivativesonhumancelllines

[16].

2.1.1. Claudinsincancer

TheTJcomplexistheapicalmostjunctionalcomplexinmost

typesofepithelialandendothelialcells.TJsarethegasket-likeseals

thatencircleeachcolumnarepithelialcellarounditsapicalpole.

Theyservetworoles:(1)theyhelptomaintaincellpolarityby

phys-icallyseparatingtheapicalandthebasolateralmembranedomains

and(2)theypreventfreeinterchangeofsubstancesbydiffusion

alongtheparacellularpathwaybetweentheluminaland

antilu-minaltissue ﬂuidcompartments.TJs andtheirpermeabilityare

importantintheformationofthebloodbrainbarrier,bloodretinal

barrierandbloodtestisbarrier.TheTJproteinscanbesub-divided

intotheintegralmembraneproteinssuchasoccludin,tricellulin,

marvelD3,junctionaladhesionmolecules(JAMs)andtheclaudin

family(currently27members[17])andthecytoplasmicproteins.

ThecytoplasmicadaptorproteinsarethezonulaoccludensorZO

proteins,andaredesignatedZO-1,-2,and-3.Theseproteinslink

themembrane proteinstotheactin cytoskeleton.Traditionally,

researcheffortsfocusedonbarrierandfencefunctions,however,

thereisanewmovementintheﬁeld,whichistounderstandhow

TJ proteinsparticipate in cellproliferation, transformation, and

metastasissuppression.Recentstudieshavedemonstratedtherole

ofTJsduringepithelialtissueremodeling,woundrepair,

inﬂamma-tion,andtransformationintotumors.Epithelialmultilayeringwas

associatedwithincreasedTJpermeability[18],activationofprotein

kinaseC(PKC)-␣[19]andphosphorylationofTJproteins[20].

StudiesfocusingonthemoleculararchitectureoftheTJhave

now conﬁrmed that theclaudin family of proteinsis the

inte-gralcomponentoftheTJ.Lossofcell–celladhesioniscentralto

thecellulartransformationand acquisitionofmetastatic

poten-tial,however,theroletheclaudinfamilyofproteinsmayplayin

aseriesofpathophysiologicalevents,includinghumancarcinoma

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Fig.1. Themetastaticcascadeandpotentialfortherapeuticinterruption.Changesincellularpropertiesarenecessarytoallowthedevelopmentofaninvasivephenotype

andprogressionthroughthemetastaticcascade.Keyeventsofthecascadeareoutlined.Targetingsuchproperties/eventsortheunderlyingsignalingpathwaysusinglow

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claudinmouseknockoutmodelshavedemonstratedtheir

impor-tantroleinthemaintenanceoftissueintegrityinvariousorgans.

Themechanismsofclaudinregulationandtheirexactrolesin

nor-malphysiologyanddiseasearebeingelucidated,butmuchwork

remainstobedone.

Thereare27typesofclaudinsinmammals[17,21]andtheyare

dividedinclassicandnon-classicclaudinsbasedontheirsequence

similarity[21].Classicclaudinsincludeclaudins1–10,14,15,17

and 19and non classicclaudins11–13,16, 18 and 20–24[21].

Claudinsarefoundinepithelial,mesothelial,glialand

endothe-lialcells[22–24]withamolecularweightofaround20kDaand

incellmembranestheyarecomposedoftwoextracellularloops,

EL1andEL2,fourtransmembranedomains,onesmall20amino

acidlongintracellularpartbetweenthetwoextracellularloopsand

theintracellularaminoterminalandcarboxyterminalends[21,25].

The carboxyterminalend hasregions which recognize thePDZ

(postsynapticdensityprotein,Drosophiladisklargetumor

sup-pressor,andzonulaoccludens-1protein)domainsofZO-1,ZO-2

andZO-3[25].ThelargerEL1loopinﬂuencesparacellularcharge

selectivitywhereasthesmallerEL2loopbindstothe

correspond-ingclaudinoftheneighboringcell[25].Claudinexpressionand

functionsareregulatedatmultiplelevelsandbydiverse

mech-anisms [26,27]. Animportant question related to regulation of

claudinexpressionandcanceristherolethatclaudinsmayplay

intheEMTprocess[28,29].Theparacellularbarriermodulatedby

claudinmemberscanbeaffectedbyawiderangeof

physiologi-calfactorsincludingcellsignalingpathways,hormones,cytokines,

anddisruptionofthecell–cellcontacts.Post-translational

modiﬁ-cations,includingphosphorylation,lipidmodiﬁcationandremoval

ofclaudinsbyendocytosis,appeartobepotentialmechanismsfor

theregulationofclaudins.Phosphorylationhasbeenlinkedtoboth

increasesanddecreasesinTJassemblyandfunction.Mostclaudin

proteinshaveputativeserineand/orthreonine phosphorylation

sitesintheircytoplasmiccarboxyterminaldomains.Forinstance,

proteinkinaseA(PKA)–mediatedphosphorylationhasbeenshown

todecreaseassemblyofclaudin-3intoTJs[30],yetisnecessary

forclaudin-16assemblyandfunction[31].Claudin-3and-4can

bephosphorylatedin ovariancancercellsbyPKA, akinase

fre-quentlyactivatedinovariancancer[30].Claudinphosphorylation

associatedwithTJdisassemblyisalsoenhancedbyEPHreceptor

A1(EphA1),whichisrecruitedtobindtoclaudin-4byforminga

complexwithephrin-B1[32].StudieshaveimplicatedPKCinthe

regulationofTJs throughphorbolesterstimulation[30,33].

Fur-thermore,modulationofmitogen-activatedproteinkinase(MAPK)

signaling,speciﬁcallyextracellularsignal-regulatedkinase(ERK)

1/2andp38,aswellasphosphatidylinositol3-kinase(PI3K)have

aprofound effectonTJsealingand claudinexpression[30].TJs

arealsoremodeledatamoremacroscopiclevelthrough strand

breaksandreformation[34].Clathrin-mediatedendocytosisplays

animportantroleinthisprocess[35,36].Claudinsareinternalized

byauniquemechanism,wherethetightlyopposedmembranes

oftheTJareendocytosedtogetherintooneoftheadjoiningcells

[24].HostfactorsandcytokinescanalsoinﬂuenceTJturnoverand

claudinexpression[37],forinstance,interferon(IFN)-␥increases

claudinendocytosisandTJpermeability[38].Otherinﬂammatory

cytokines,suchastumor-necrosisfactor(TNF)-␣andinterleukin

(IL)-13,downregulateclaudinsandinduceamarkedincreasein

paracellularpermeabilitybyepithelialcellsinculture[39,40].

Growth factor receptors that are important in the

regula-tionofcellproliferationandsurvivalincludingepidermalgrowth

factor (EGF), hepatocyte growth factor (HGF) and insulin like

growthfactor(IGF)receptorsregulateclaudinexpressionand

cel-lulardistributionthoughonceagainincell/tissuespeciﬁcmanner

[28,29,41].ClaudintranscriptioncanberegulatedbytheSnail/Slug

family[42].ItiswellestablishedthatoverexpressionofSnailin

epithelialcellsinducesEMTandtheacquisitionofmigratoryand

invitroinvasivebehavior.SnailandSlugbindtotheE-boxmotifs

presentinthehumanclaudin-1promoterwhichplayacritical

neg-ativeregulatoryroleinbreastcancercelllinesthatexpressedlow

levelsofclaudin-1[42].Caudaltypehomeobox2(Cdx-2),

hepa-tocyteNuclearFactor1-alpha(HNF-␣),andGATAbindingprotein

4(GATA-4)[43,44]canbindtothepromoterregionsofvarious

claudingenesandaffecttheirexpression.Furthermore,ithasbeen

shownthatcolonicclaudin-1transcriptsareregulatedbySmad-4,

aknowntumorsuppressoraswellashistonedeacetylase(HDAC)

inhibitorsandthussupportacomplexregulationatmultiple

lev-els[45,46].Collectively,thedataprovidesanemergingpictureof

theimportanceofclaudinhomeostasisinnormalandpathological

tissuefunction,butthereremainsmuchtobelearned,especially

regardingwhetheritmaybepossibletoidentifyadistinctclaudin

signatureintheinitiationandprogressionofvarioustumortypes.

Alterationsinclaudinexpressionproﬁlesduring

tumorigene-sisbegsthequestionof howclaudinsareregulatedin different

tissuesinbothnormalandpathologicalsituations.Tanetal.[47]

haveshownthattheexpressionanddistributionofclaudin-1is

associatedwithcelldissociationstatusinpancreaticcancercells

throughMAPK2activation.Bycontrast,claudin-7hasbeenfound

tobedecreasedininvasiveductalcarcinomas[48],headandneck

cancer[49]andmetastaticbreastcancer[37].Ontheotherhand,

claudin-3and-4arefrequentlyelevatedinvariouscancers

includ-ingpancreaticductaladenocarcinoma,prostate,uterine,ovarian

cancer[38]andbreastcancer[50]whilehepatocellularandrenal

carcinomasexpressedlowerlevelsofclaudins-4and-5[22].While

lowerexpressionofclaudin-2wasalsoseeninbreastand

pros-taticcarcinomas,expressionsofclaudin-1andclaudin-7thatwere

undetectableinnormalcervicalsquamous epitheliumincreased

inthecervicalneoplasia[22,51].Intriguingly,recentstudieshave

shownthatexpressionofcertainclaudins,especiallyclaudin-1and

claudin-4, increasesduring metastasisandgeneticinhibition of

theirexpressionhasaprofoundeffectonthemetastaticabilities

ofcancercellsthoughinatissuespeciﬁcfashion[52–54].Thereis

thepossibilitythatmutationsinclaudinsmaybecausaltotumor

formation.However,todatethereisnosystematicsequencedata

onclaudinsinanytumortype.Ontheotherhand,gene

silenc-ing due topromoter hypermethylationis a commonfeatureof

humancancers[55]andithasbeensuggestedtounderliethe

down-regulationofclaudinsincertaintumors.Forexample,aCpGisland

wasidentiﬁedwithinthecodingsequenceoftheclaudin-4gene,

andtreatmentwithamethyl-transferaseinhibitorrestored

expres-sionoftheproteininprimaryculturespreparedfromhigh-grade

humanbladdertumors [56].Furthermore,claudin-4expression

alsocorrelatedwithitsgenemethylationproﬁleinhealthyand

tumoralbladdersfrom20patientsandclaudin-6expressionis

par-tiallysilencedbypromoterCpGislandhypermethylationinMCF-7

breastcarcinomacells,whileasynergisticeffectofademethylator

andhistonedeacetylaseinhibitorsupregulatestheexpressionof

endogenousclaudin-6,andsensitizesthecellsforapoptosis[57].

Intuitively,themechanismbywhichdecreasedclaudinexpression

might lead to the compromised TJ function and thus,

neopla-siaiseasytocomprehend,buthowincreasedclaudinexpression

contributestoneoplasticprogressionislessclear.Oneplausible

mechanismisthatupregulationoraberranttissueexpressionof

certainclaudinsmaycontributetoneoplasiabydirectlyalteringTJ

structureandfunction.Furthermore,itispostulatedthatclaudins

mayalsoaffectcellsignalingpathways.Claudinproteinsarelikely

involvedinsignalingpathwaysviabindingdomainstoZO-1attheir

carboxylterminus[58].

Cell–celladhesionproteinsareknowntoplayanimportantrole

incellulartransformationwhendisplacedfromtheirnormal

mem-brane localizationand could serve asoncogenic molecules, the

beststudiedmoleculebeing␤-catenin[59].Asimilarfunctional

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studiesareneededtosupportsuchanotion.Anincreasein

claudin-1expressionhasbeenreportedinhumanprimarycoloncarcinoma,

inmetastasissamplesandinthecelllinesderivedfromprimary

andmetastatictumorscomparedtotheirnormalcounterparts[54].

Crucially,therewasnuclearlocalizationofclaudin-1ina

signiﬁ-cantsubsetofcoloncancersamples,particularlyamongthesubset

oflivermetastaticlesions.Nuclearlocalizationofseveralcell

junc-tionproteins(␤-catenin,ZO-1,ZO-2)isknowntobecorrelatedwith

oncogenictransformationandcellproliferation[60].Mutantsofthe

TJproteinZO-1thatnolongerlocalizeattheplasmamembrane

inducedramaticEMTinMadin-DarbycaninekidneyIcells[61].

Similarly,geneticmanipulationsofclaudin-1expressionincolon

cancercelllinesinducedchangesincellularphenotype,with

struc-turalandfunctionalchangesinmarkersofEMT,andhadsigniﬁcant

effectsuponthegrowthofxenograftedtumorsandmetastasisin

athymicmice.Notably,regulationofE-cadherinexpressionand

␤-catenin/Tcfsignalingemergedasoneofthepotentialmechanism

underlyingclaudin-1dependentchangesand therebysuggested

complexinterplaybetweendifferentcell–celladhesionmolecules

[54].Expressionofspeciﬁcclaudin familymemberscanbe

reg-ulatedbythewingless-typeMMTVintegrationsitefamily(Wnt)

signalingpathway.Claudin-1andclaudin-2areshowntobetarget

genesregulatedby␤-cateninsignaling[62,63].

Metastasisisacomplexphenomenonthatrequiresanumber

of speciﬁc steps such as decreased adhesion, increased

motil-ity and invasion, proteolysis, and resistance to apoptosis [64].

Claudin-5promotesprocessingofpro-matrixmetalloproteinase-2

(pro-MMP-2)bymembranetype1-MMP(MT1-MMP).Expression

of claudin-5 not only replaced tissue inhibitors of

metallopro-teinases (TIMP)-2 in pro-MMP-2 activation by MT1-MMP but

also promoted activation of pro-MMP-2 mediated by all

MT-MMPsandMT1-MMPmutantslackingthetransmembranedomain

(DeltaMT1-MMP)[65].It appearsthat interactionof MMPwith

claudins might play an important role in tumorigenesis,

inva-sionandmetastasismediatedbyclaudinexpression.Ithasbeen

observedthat overexpression ofclaudin-1in colon cancercells

increasedactivityofbothMMP-2andMMP-9whileinhibitionof

claudin-1resultedinasigniﬁcantdecreaseinMMP-9activity[54].

Similarly,overexpressionofclaudin-3or4inovarianepithelialcells

increasedMMP-2activity[52].AnincreaseinmRNAtranscription

andproteinexpressionofMT1-MMPwasalsoobservedin

claudin-10overexpressingcells,inwhichclaudin-1,-2,and-4werealso

upregulated,suggestingthattheexpressionofclaudin-10in

can-cercellsmaydysregulatetheexpressionofotherclaudinfamily

members[66].

Mostmalignanttumorsarederivedfrom,andmostpathogens

invadethebodyviatheepithelium.Theepitheliumisthereforea

potenttargetforimprovingdrugabsorption,treatingcancer,and

curinginfectiousdiseases.ModulationofTJsealsisapopular

strat-egyforimprovingdrugabsorption.TJscompartmentalizetheapical

andbasalmembranedomainsofepithelialcells,leadingtothe

for-mationofcellularpolarity.Lossofcell–cellinteractionandcellular

polarity,whichoftenoccursin cancercells during

carcinogene-sis,leadstoexposureof TJcomponentsonthecellularsurface.

Theclaudinfamily ofproteinsis anattractivetargetfor

antitu-mortherapy consideringtheepithelium-speciﬁcexpressionand

thehighspeciﬁcityofclaudinexpressionpatternsincancer.Itis

worthmentioningthatclaudinfamilymembersareexpressedin

aprecisetissue-speciﬁc mannerand thuscouldserve astumor

speciﬁcbiomarkers.Inthisregard,asetoffourmarkers,

includ-ingclaudin-3, wasfoundto besufﬁcient to accuratelyidentify

all158ovariancancerstested,includingeightearly-stageserous

cancers[67].Furthermore,claudin expressionmaybeusedasa

prognosticindicatorbecausehighclaudin-1expressionhasbeen

showntobeassociatedwithtumorprogressionandmetastasisin

coloncancer[68].Atthesametime,in breastcancer,claudin-1

expressionisdifferentialbetweenthesubtypesandlowversushigh

claudin-1expressionhelpsidentifyhighlyaggressivetriple

nega-tivebreastcancer[69].Similarly,claudin-10expressionhasbeen

showntobeanindependentprognosticfactorforhepatocellular

carcinomarecurrenceaftercurativehepatectomy[70].Regarding

theidentiﬁcationoftheclaudinfamilyofproteinsastoolsto

iden-tifyand/orclassifytumortypes,serialanalysisofgeneexpression

(SAGE)studiesof thebreast [71]andovarian [72]cancershave

allowedforthefirsttimetheidentificationofspecificclaudin

fam-ilymembersaspotentialbiomarkersforthesecancers.Although

largescaleanalysisinaclinicalsettingwillberequiredto

estab-lishsuchpotentialofclaudins,basicresearchonclaudinsislikely

toremainvaluableforprovidingimportantinsightsintonormal

andneoplasticcellularphysiology.Preclinicalstudieshaveshown

thattumorcellsoverexpressingclaudinscanbesuccessfully

tar-geted viaseveral approaches, includingthe useof anti-claudin

antibodiesaswellasthecytolytic enterotoxin fromClostridium

perfringens.However,mostofthestudieshaveconcentrated

pri-marilyuponclaudin-3andclaudin-4[73].Bothoftheseproteins

havebeenidentiﬁedastargetsofC.perfringensenterotoxin(CPE)

andhave beenreportedtobeoverexpressedinmultipletumor

typesincludingovarianandprostatecancer. Yetanother

poten-tialapproachthathasbeensuggestedistheuseofclaudinsasdrug

deliverysystemusingPseudomonasaeruginosaexotoxinA(PE).PE

iswidelyusedincancer-targetingstudiesasitbindstothecell

surfaceandisinternalizedviaendocytosis.Followingthis,aPE

frag-ment,proteinsynthesisinhibitoryfactor(PSIF),escapesfromthe

endosometothecytosol[74],whereitinhibitsproteinsynthesis

byinhibitingelongationfactor2.PSIFlacksthereceptor-binding

domainofPE,andfusionofatumorantigensuchasclaudinswith

PSIFisapromisingstrategyforcancer-targetingtherapy.Therapies

speciﬁctocertainclaudinfamilymemberscouldalsoserveas

adju-vanttherapies.Highlyincreasedandcytosolic/nuclearclaudin-1

expressionincoloncancerhasbeenreported[54,75]andclaudin-1

dysregulationmodulatesthebalancebetweentheNotch-and

Wnt-signalingtodysregulatecolonocytedifferentiationandpromotes

tumorgrowthandprogression.SinceNotchandorWnt-signaling

inhibitioncarriesinherenthightoxicity,theuseofclaudin-1based

therapymayprovideanalternative.

2.2. Cell–matrixadhesion

Interactionand adhesionbetweencells andthe surrounding

extracellularmatrix(ECM)classicallyinvolvescellsurfaceintegrins

whichinteractandbindECMproteincomponents[76].Functional

integrinsconsistofaheterodimerstructuremadeupofdifferent

␣-and␤-subunitsanddifferentintegrinstructurespossess

dif-feringafﬁnitiesfordifferentmatrixproteins[76].Theinteraction

betweenintegrinsandtheECMtriggersa seriesofintercellular

eventsthatnotonlyresultsintheadhesionofthecelltotheECM

butalsoformsamechanismforcommunicationbetween

intracel-lulareventsandthesurroundingECM.Thisprocessofcell–matrix

adhesionisessentialfortheattachmentofcancercellstothe

sur-roundingmatrixandsubsequentlythedegradationofthematrix

barrier[9].Anumberofintegrinshavebeenlinkedtometastatic

likelihoodandcancerand/orstromalcellsmaydepositECM

pro-teinsthatagaincanenhancemetastaticprogression.Blockingthe

extracellularpartofthecell–matrixadhesionbymeansof

antibod-ies,smallpeptides,andothernatural-andphytochemicalshasbeen

demonstratedandhasbeencoveredbyanotherarticleinthisissue.

However,blockingtheintracellularsignalingeventhasalsoproved

tobeausefulapproachininhibitingthisimportanteventduring

cancermetastasis.Keyeventsfollowingthematrix–integrin

inter-actionincludeactivationofthefocaladhesionkinase(FAK),paxillin

anddownstreamchainsignalingevents[77].Thus,inhibitingFAK

(6)

CD44representsanotherkeycell adhesionmoleculethat holds

potentialasanantimetastatictargetboththroughitsrolein

inter-actingwithothercelltypesandtheECM.TheCD44gene,located

athumanchromosome11p13,encodestheCD44sandCD44v

iso-forms,whicharisethroughalternativesplicing.CD44sandCD44v

isoforms share the extracellular globular region that includes

binding sites for hyaluronan,collagen, laminin and ﬁbronectin

aswellasthecytoplasmictailregionthatincludesbindingsites

for ERM domainproteins (Ezrin, Radixin and Moesin),Ankyrin

andS6kinaserelatedkinase(SRK).CD44functionsasa

hyaluro-nanreceptor,co-receptorforgrowthfactorsandasanadhesion

molecule[78–82].CD44isinvolvedinthemalignantphenotypes

ofcancerstem cells,includingEMT,invasion,metastasis,

recur-rence, resistance to chemotherapy and resistance to radiation

therapy[82–85],whichclearlyindicatesthatCD44isapotential

targetofcancertherapy.Humanizedanti-CD44v6monoclonal

anti-body BIWA-4 (bivatuzumab), paclitaxel-conjugated hyaluronan

prodrugHYTAD1-p20(ONCOFID-P),SN-38-conjugatedhyaluronan

prodrug ONCOFID-S, hyaluronan–irinotecan complex and other

hyaluronan-conjugateddrugsorsiRNAshavebeendevelopedas

cancer therapeutics [86]. Therapeutics targeted to cell–matrix

adhesionmayrepresentausefulstrategytoblockcancercellsfrom

settlingonandsubsequentlypenetratingvascularorcavitylining

andhencenegativelyimpactingtheirabilitytoestablishsecondary

tumorsinthenewsite.

2.3. Cellularmigration

Whileessentialtonormaldevelopmentandhomeostasis,the

processofcellularmigrationisalsoatraitessentialfor

metasta-sis.Enhancedmigrationiskeyacrossthemetastaticcascadeand

isinvolvedintheinitialscatteringofcellsandmigrationfromthe

primarytumor,thepenetrationofthebasementmembraneand

ECMandintravasationandextravasationofvessels.Themigration

ofcellsrequiresanumberofintra-andextra-cellulareventssuch

asthedetectionofextracellularsignalsbythecells,synthesisofcell

surfaceproteinsandthecoordinationofintracellularsignalingand

cytoskeletonproteins.Throughouttheliterature,cellmigrationhas

beentightlylinkedtocancerprogressionandmetastasis.

Numer-ousproteinsandpathwayshavebeenimplicatedinalteringthe

migratorypotentialsofcancercellsandthereforetheiraggressive

nature[87,88].Hence,givenitsessentialroleincancerprogression,

treatmentsthatinhibitcellmigrationorsuchproteins/pathways

involved in enhancing cellular motility represent an attractive

strategy forcontrollingmetastatic dissemination.While in

nor-malphysiologycellularmigrationislessactive,thereareprocesses

whereitisessential,suchaswoundhealing,andhencemustbe

takenintoconsideration.Currentlytherearemanycompoundsthat

inhibitcellularmigration,althoughveryfewhavebeentestedina

clinicalsetting.

2.4. EMT

The process through which epithelial cells undergo a series

of morphological and biochemical changes to take on a more

mesenchymal phenotype is known as epithelial–mesenchymal

transition.EMT is widespreadthroughout normal development

buthasalsobeenlinkedtotheestablishmentofamoreinvasive,

motilecancercellphenotypefacilitatingdetachmentand

dissem-inationawayfromtheprimarytumor[89–92].EMTinvolvesthe

lossofcell–celladhesionandthepolarizedepithelialmorphology

through thecharacteristic loss of epithelialcell junctional

pro-teins suchas E-cadherin, claudinsand ZO-1, and a subsequent

increaseinmesenchymalmarkerssuchasN-cadherin,vimentin

andﬁbronectinandcytoskeletalreorganization[91,93].Indeed,the

lossofE-cadherinandsubsequentreplacementwithN-cadherin

(‘cadherinswitching’)isacharacteristicofEMT,seeninmany

can-certypesandisthoughttoaccountsomewhatfortheenhanced

invasiveandmotilepropertiesofcancercells[8].Unsurprisingly,

alterationsincelladhesionmolecules(CAM)suchasE-cadherin,

impacttheprocessesofcell–celladhesionandcell–matrix

adhe-sionandsubsequentlytheirmetastaticpotential.E-cadherinplays

anessentialroleintheadhesionofcellsandtissuesandtogether

withothermembersoftheadhesivecomplex,suchas␤-catenin,

regulatescelladhesion,signalingandtranscriptionincancersand

controlmetastaticprogression[94].Indeed,studieshave

demon-stratedanassociationbetweenlossofE-cadherinand␣-catenin

expression with enhanced tumor cell invasiveness [95]. Other

workhasdemonstratedaninversecorrelationbetweenE-cadherin

expressionandtumorcellinvasionandmotilityandsimilarlywith

metastaticdiseaseincancerpatients[96].Thetranslocationof

␤-cateninfromtheadhesivestructuretothenucleus,aneventleading

totranscriptionalactivationofanumberoftargetgeneshasalso

beendemonstratedtocorrelatewithdevelopmentofa

mesenchy-malphenotype[97,98].

Initiationsignals,suchasHGF,EGFandtransforminggrowth

factor ␤(TGF-␤) arebelieved toonsettheEMTprocess,

result-inginupregulationofEMT-inducingtranscriptionalfactorssuch

asSnail,SlugandTwist[99–102].Slug,SnailandTwisthavebeen

implicatedininﬂuencingtheexpressionofEMTproteinsandare

hencelinkedtometastasis[103–105].ForexampleSlugandSnail

areinvolvedinthedown-regulationofE-cadherin[99,106]andthe

expressionbetweenSnailandE-cadherin isinverselycorrelated

ina numberof cancersincludingbreastcancer[107].Similarly,

asdiscussedinSection2.1.1,Snailexertsregulatoryeffectsover

membersoftheTJsuchastheclaudins.Theseinitiationfactorsalso

actonothereffectormoleculestobringaboutEMT, suchasthe

MMPfamily.Membersofthisfamilyofproteinasesplaykeyroles

inmatrix-degradation,invasion,motilityandadhesionandare

fre-quentlydysregulatedincancerprogression.SlugandSnailhave

bothbeenimplicatedintheupregulationofMMP-2andMMP-9

andsubsequentEMTinitiation[108].

The process of EMT and subsequent acquisition of an

inva-sive,motilephenotypewithenhancedlikelihoodofinvasionand

disseminationrepresentsakeyinterestincancerresearch.

Thera-peuticstrategiesthatcanspeciﬁcallytargetthisprocessincancer

cellsarelikelytobeeffectiveinreducingthemetastaticpotential

oftumorcells.

2.5. Molecularnetworksinthetumormicroenvironment

It is now well established that solid tumors are not

sim-plyaggregates of replicatingneoplasticcells but arealsoliving

entities, composed of numerous cell types, whose complexity

approaches,andmayevenexceed,thatofnormalhealthytissues.

Manynon-malignantcelltypes,referredtoasthestroma,populate,

atmajority,thesolidtumors.Thesenon-malignantcellsinclude

ﬁbroblasts,residentepithelialcells,pericytes,myoﬁbroblasts,

vas-cularandlymphovascularendothelialcellsandinﬁltratingcellsof

theimmunesystem.Duringmalignantprogression,neoplasticcells

acquiretheabilitytorecruit,incorporateandreprogramthe

biol-ogyandthefunctionofthesehealthyhostcells, thusproviding

themwithsupport,essentialnutrientsand weaponstohamper

antitumorimmuneactivity.Inturn,therecruitednon-malignant

cellsrespondbyenhancingtheneoplasticphenotypeofthenearby

cancercells,whichagainfeedsignalingbacktothestromato

con-tinueitsreprogramming.Thus,thepreviousideathatthemalignant

phenotype of tumor cells was exclusively determined by

cell-autonomousgeneticandepigeneticalterationsisnowreplacedby

thehypothesisthatthemalignantprogressionofcancernotonly

dependsontumorcells’geneticaberrationsbutalsoonthe

(7)

Fig.2.Cellularinteractionswithinthetumormicroenvironment.Numerousinteractionsbetweencelltypesareinvolvedthroughouttumorprogressionandmetastasis.

Communicationbetweenmaincomponentsofthesurroundingmicroenvironmentplayvitalrolesinenhancingmetastaticpotential,epithelialtomesenchymaltransition

(8)

thecellsofthestromaandcancercellswithinthetumor

microen-vironment[109,110](Fig.2).

Among the non-malignant cells that inhabit the tumor

microenvironment,cancer-associatedﬁbroblasts(CAFs)andtumor

inﬁltrating-immuneinﬂammatory cells arenoteworthybecause

oftherolestheyplayintumordevelopmentandmalignant

pro-gression. CAFs secrete factors that act on tumor cells in both

paracrineandautocrinefashions,thusresultinginamore

aggres-sivecancerphenotype.Acrossmostcancers,activatedCAFssecrete

a wide variety of growth factors, chemokines, collagens, and

ECM-modifyingenzymes,whichcollectivelysupplya

communica-tionnetworkandanalteredthree-dimensionalECMscaffoldthat

togethergovernproliferationofcancercellsandtumorinvasion

andmetastasisacrosstissuetypes.Theyalsocontributetotumor

progressionbyrecruitingtumor-promotingimmunecellsand

sup-portingangiogenesis.Thetumorinﬁltrating-immunecellsinclude

thetumor-associatedmacrophages(TAMs),myeloid-derived

sup-pressorcells (MDSCs),dendritic cells(DCs), tumorinﬁltratingT

cells,regulatoryTcells(Tregs)andmastcells[109].Tumorcells

secretechemokinesandcytokinesthatareabletorecruitmastcells,

DCs,TAMsandMDSCs.Tumorcellsalsoactivatemastcells,promote

theexpansionoftheMDSCsandthepolarizationofTAMs.

Further-moretumorsbothinhibitDCmaturationthroughIL-10secretion,

thusleadingtoantigen-speciﬁcanergy,andreprogramtheDCs,

inducingthemtoexertimmunosuppressive orangiogenic

func-tions,thusresultinginanimmunosuppressiveandinﬂammatory

tumormicroenvironment.Oncerecruitedtothetumor

microen-vironment,theseimmune cellscancontributetothemalignant

progression of the cancer-cell phenotypeby supporting tumor

proliferation,survival,invasion,metastasis,angiogenesisandECM

remodeling.

Incancercells,theconstitutiveactivationofvarioussignaling

pathways (including MAPK, signal transducer and activator of

transcription 3 (STAT3)and ␤-catenin pathways) resultsinthe

secretion of cytokines which modulate the recruitment and

function of the stromal cells. In particular, the tumor-derived

regulated on activation, normal T cell expressed and secreted

(RANTES)/Chemokine(C–Cmotif)ligand5(CCL5)cytokine

stimu-latesCAFs toexternalizethe S100A4 protein,which stimulates

tumor-cell survival and migration, up-regulation of theMMPs,

down-regulationofTIMPs,activationofthenuclearfactorofkappa

lightpolypeptidegeneenhancerinBcells(NF-␬B)andMAPK

path-ways,inﬁltrationofTcellsandﬁnally,up-regulationofRANTES,

thusgeneratinga signalampliﬁcationloop.RANTESalsoinduce

angiogenesisand actaschemoattractantsforadditionaleffector

immunecells.Tumor-derivedstemcellfactor(SCF)promotesthe

recruitmentand activation of mastcells and theMDSC

expan-sion.Tumorsalsosecretethethymicstromallymphopoietin(TSLP)

and bone marrow stromal cell antigen 2 (BST2). TSLP induces

DCstoexpressOX40ligand, whichdirectsCD4+ _T_cells_to

gen-erateTH2cellssecretingIL-4andIL-13.Thesecytokinesprevent

tumorcellapoptosisandindirectlypromotetheproliferationof

tumorcellsbystimulatingTAMstosecreteEGF.BST2isaligand

ofimmunoglobulin-liketranscript7(ILT7),whichisexpressedon

DCssurface.TheinteractionofILT7onDCswithBST2ontumor

cellsresultsininhibitionofIFN-␣andpro-inﬂammatorycytokines

productionbyDCswithimmunosuppressiveeffects.

Oncogeneactivationandsubsequentsignalactivationin

can-cercellstriggermultiplecascadesthusresultinginthesecretion

ofseveralimmunosuppressivemolecules,includingTGF-␤,IL-10,

IL-6, vascularendothelial growthfactor(VEGF), CCL2/monocyte

chemoattractantprotein1(MCP1),cyclooxygenase-2(COX2),that

induce the immunosuppressive immune cells. Production and

secretionofthese factorsbyboth cancerand surroundingcells

enhancetumorcellproliferation,migrationandinvasion.

Further-moreitenhancestheproductionofimmunosuppressivecytokines

and chemokines, including TGF-␤ itself, IL-10 and CCL2/MCP1.

TGF-␤and thepotentialfortargetingthis signalingpathwayin

canceris discussed in Section4.2. A plethora of recent reports

haspaintedaconsistentpictureofhowstromalcells(CAFsand

inﬂammatorycells)canpromotemalignantprogression.Indeed,

within theprimary tumormicroenvironment, thestromal cells

providepotentoncogenicsignals,suchasTGF-␤,HGF,EGF,Wnt,

andbasicﬁbroblastgrowthfactor(bFGF),whichstimulate

cancer-cellproliferation,survivalandinvasion,thusfacilitatingmetastasis.

Moreover, thesecells produce severalangiogenesis-modulating

enzymes,suchasVEGF,thymidinephosphorylase,MMP-2,

MMP-7,MMP-9,MMP-12,COX2,urokinaseplasminogenactivator(uPA)

andcathepsinsBandD,whichtogetherdegradetheECM,again

promotingmetastasis.TAMspromotecarcinoma-cellmotilityand

invasion throughaparacrine signalingloopbetweenthetumor

cells and the TAMs. Withinthis loop the macrophages express

EGF,whichpromotesformationofelongatedprotrusionsandcell

invasionbycarcinomacells.Inaddition,EGFpromotesthe

expres-sionofcolonystimulatingfactor1(CSF-1)bythecarcinomacells,

which further promote the expression of EGF by macrophages

generating a positive-feedback loop. The secretion of

stromal-cell-derived factor 1 (SDF1), alsoknown as chemokine (C–X–C

motif) ligand12 (CXCL12), by TAMs and CAFsat a tumor site

canenhancetheinvasion,intravasation,metastasisformationand

recruitmentofMDSCs,TAMsandendothelialcellstotheprimary

tumor.Thisenhancementofinvasionandintravasationdepends

uponchemokine(C–X–Cmotif)receptor4(CXCR4)signaling,andit

ismostlikelytooccurthroughactivationofCXCR4onmacrophages,

whichresultsinincreasedparacrineinteractionswithtumorcells

inthetumormicroenvironment.IncreasedCXCL12/SDF1secretion

alsogivesrisetoanincreasedmicrovesseldensity,whichmight

alsobemediatedbyTAMsandmightcontributetoanalteredtumor

architecture,thusresultinginincreasedintravasationthroughthe

presenceofahigherdensityofentrancesitesintotheblood,with

acorrespondingincreaseintheformationofmetastases.The

sig-niﬁcanceofCXCL12/CXCR4signalinginbreastcancerinvasionand

metastasisiswidelyappreciated.CXCR4expressioninbreast

can-cercellshasbeenshowntoincreasemetastasisthroughthehoming

oftumorcellstositesofincreasedCXCL12expression,suchasthe

lymphnodes.Similarly,theinteractionofCXCL12/SDF1andCXCR4

expressedonmammaryadenocarcinomaMTLn3cellsincreasesthe

chemotacticandinvasivebehaviorofthesecellstoCXCL12/SDF1,

aswellastheirmotilebehaviorwithintheprimarytumorandtheir

abilitytointravasate.TAM-derivedCCL17andCCL22chemokines

preferentiallyattract Tcellsubsets that aredevoidof cytotoxic

functions,suchasTregsandTh2lymphocytes.TAM-derivedCCL18

recruitsnaïveTcells,whichinduceTcellanergy.Withinthetumor

microenvironmentIL-10,secretednotonlybyimmunecells,but

alsoby CAFsand tumorcells, is themain cytokineresponsible

fortheestablishmentoftheimmunosuppressivemilieu.

Further-more,IL-10,togetherwithIL-4,IL-6andIL-13,inducesmonocyte

differentiationtowardamatureM2-polarizedphenotypethatis

characteristic of TAMs. At the tumor site, the IL-1␤ and IL-6

cytokines,S100A8andS100A9pro-inﬂammatoryproteinsandthe

chemoattractantmoleculesCCL2/MCP1,CXCL12/SDF1andCXCL5

arethemainfactorsthatareresponsiblefortherecruitmentand

theinductionofMDSCs.VEGFisoneofthemainfactors

responsi-blefortheexpansionofMDSCs,whileIL-4,IL-13,IFN-␥,IL-1␤and

TGF-␤turnontheirsuppressivefunctions.MDSCsproducehigh

levelsofIL-17,whichfurtherexacerbatestheinﬂammatorytumor

microenvironment.

Thegrowingbodyof evidenceregardingtherolesplayedby

non-malignantcells ofthetumormicroenvironmentin

promot-ingtumor progressionindicatethat it isconceivable that these

cellscanserveasnoveltherapeutictargetsinthecancertreatment.

(9)

Table1

Effectsofapprovedandexperimentaltargetedagentsontumorcellsandtumormicroenvironmentstromalcells.

Drug Drugclass Target Effectontumor Effectontheimmunesystem References

STI571(Gleevecor imatinibmesylate) Smallmolecule inhibitor PDGFRand c-Kit

Reducesmicrovesseldensity Preventsmastcellproliferation andsurvival

[283,284]

Bevacizumab Monoclonal

antibody

VEGF Blocksangiogenesis IncreasesDCmaturation,shiftsDC

differentiationtowardmatureDCs

insteadofMDSCsandincreasesDC

primingofTcells

[285,286]

IM-2C6 Antibody VEGFR Blocksangiogenesis NA [287]

SU5416 Smallmolecule

inhibitor

VEGFR Reducesvasculardensity NA [288]

MMI-166 Smallmolecule

inhibitor

MMP-2and

MMP-9

SuppressesMMP-2andMMP-9

activities;inhibitsangiogenesis

andtumorgrowth

NA [289] S-3304 Smallmolecule inhibitor MMP-2and MMP-9 InhibitsMMP-2andMMP-9 NA [290]

Dasatinib Smallmolecule

inhibitor

c-Kit,ABL,SRC,

PDGFR

Inducesapoptosisinleukemiccell Inducesapoptosisinmastcell [117]

Dipyridamole Smallmolecule Wnt,MAPK

andNF-␬B

pathways

Decreasestumorgrowthand

metastasis

DecreasesTAMandMDSC

inﬁltration

[118]

Bindarit Smallmolecule CCL2/MCP1 Decreasestumorgrowthand

metastasis

DecreasesTAMandMDSC

inﬁltration

[119]

Upanap-126 RNAaptamer uPA Delaystheproteoliticconversion

ofpro-uPAtoactiveuPA;inhibits

tumorcellinvasion;reducesthe

tumorcellintravasationand

dissemination NA [111] ATN-658 Monoclonal antibody uPAreceptor (uPAR)

Decreasestumorcellinvasionand

migrationandtumorvolume

NA [112]

L2G7/Rilotumumab Monoclonal

antibody

HGF Inhibitsthetumorgrowth NA [113,291,292]

Trastuzumab Monoclonal

antibody

HER2 Blocksgrowthsignals PrimesantitumorCTLs,boostsNK

secretionofIFN-␥andmediates

potentantibody-dependent cellularcytotoxicity [293] Cetuximab Monoclonal antibody EGFreceptor (EGFR)

Blocksgrowthsignals Immuneactivating:increasesMHC

classIandMHCclassIIexpression;

augmentsDCprimingof tumor-speciﬁcCTLs. [294] MGA271 Monoclonal antibody B7-H3 NA Mediatespotent antibody-dependentcellular cytotoxicity [295]

AMD3100 Smallmolecule CXCR4/CXCL12

(SDF1) signaling

Sensitizescancercellsto

chemotherapy:inhibitstumor

growth

Reducestherecruitmentof

bone-marrowderivedcells

[114–116]

Celecoxib Smallmolecule

inhibitor

COX2 NA DecreasesbothMDSCnumbers

andfunction

[296]

5-Fluorouracil Smallmolecule Thymidylate

synthase

Promotesthecytotoxicityoftumor

cells

InducesMDSCapoptoticcelldeath [297]

All-trans-retinoicacid

(ATRA)

VitaminA

derivative

NA NA ReducesMDSCs [298]

Sclareol Phytochemical NA Decreasesthetumorsize DecreasesthenumberofTregs [120]

Temozolomide Smallmolecule DNA Promotesthecytotoxicityoftumor

cells

ReducesthenumberofTregs [121]

moleculeinhibitors,antibodiesorphytochemicalsthatspeciﬁcally

targetmoleculesandsignalingpathwaysinvolvedinthe

recruit-ment,activationandfunctionoftumorinﬁltratingnon-malignant

cellshavebeentestedinbothanimalmodelsandhuman.Table1

summarizesthemost up-to-datedrugsavailablewithpotential

useincancertherapy,knowneffectsontumorcellsandactivity

againsttumor-stromalmicroenvironmentcommunications.

Sev-eral strategies to inhibit either CAF activation or CAF-derived

factors(e.g.HGF,uPA,CXCL12/SDF1)have beenappliedin

pre-clinicalstudiesof cancertherapies and theresultshave shown

efﬁcacyintheinhibitionoftumorgrowthandinvasion[111–116].

Similarly,severalimmunotherapeuticapproacheshavebeen

devel-oped to target immune cells that inﬁltrate the tumor. Some

anti-angiogenicagentsimpairproliferationandsurvival ofmast

cellsandinduceDCmaturationandtheirantitumoractivity(e.g.

STI571andbevacizumab).Theimpairmentofthestemcellfactor

(SCF)/c-Kit signalingpathwaybydasatinibinduces apoptosisof

bothtumorcellsandmastcells[117].Severalimmuno-therapeutic

strategiesthattargetMDSCsandthatcanneutralizetheir

immuno-suppressiveeffectshavebeenreportedinbothanimalmodelsand

human.Thesestrategiesincludeapproachesthatareaimedatthe

inductionofdifferentiationoftheseimmaturecells[e.g.

all-trans-retinoicacid(ATRA)],orofadecreaseintheirnumberandtumor

inﬁltration(e.g.dipyridamoleandbindarit),oratinterferingwith

theirimmunosuppressivefunctions(celocoxib),orkillingMDSCs(5

ﬂuorouracil-or5FU).Interestingly,dipyridamole[118]andbindarit

[119]decreasetheinﬁltrationnotonlyofMDSCsbutalsoofTAMsin

breastandprostatecancerproofofconceptanimalmodelstudies.

Finallysclareolandtemozolomidereducetumorgrowthandthe

numberoftumorinﬁltratingTregs[120,121].Therefore,although

furtherstudieswillbeneededtodeterminewhichcell(s)is/arethe

(10)

andselective,thereisnodoubtthatthetherapeutictargetingof

tumormicroenvironmentcells representsa valuablestrategyto

complementconventionalanticancerstrategies.

2.6. Cancerstemcells(CSC)

Cancerstemcells(CSC)presentanexcitingyetsomewhat

con-troversialﬁeldincancerresearch.Inthecancerstemcellmodelof

carcinogenesisthereisahierarchicalorganizationofcancercells.

TheCSCrepresentahighlytumorigenicsub-populationofcancer

cellsthatcanbeisolatedfromothercancercellsinthesametumor.

Thesehighlytumorigeniccellshavebeenproposedascrucialto

thegrowthanddevelopmentofprimarytumorsandarebelieved

toberesistanttoconventionaltherapyandthereforelikelytobe

responsiblefordiseaserecurrenceandtreatmentfailures.CSCwere

ﬁrstisolatedinacutemyelogenousleukemia(AML)andsubsequent

investigationsofsolidtumorshaverevealedthepresenceofhighly

tumorigeniccancercells(CSC)inessentiallyeverysolidcancertype

includingbreast,lung,colon,pancreas,headandneck[122–127].

ThecriticalcharacteristicsofaCSCrequirethesecancercells

to be: tumorigenic, able to reproduce the original tumor

het-erogeneityincludingboth thetumorigenicandnon-tumorigenic

subpopulationsofcancercells,self-renewing,andseparablefrom

theothercancercells. CSCtypicallyrepresentonlyasmall

sub-population(<10%)oftheentirecancercellpopulation.Avariety

ofcellssurfacemarkersandbiologicalmarkershavebeenusedto

isolatetheCSCpopulationfromothercancercells,includingCD24,

CD44,CD26,CD133,epithelialspeciﬁcantigen(ESA),andaldehyde

dehydrogenaseactivity(ALDH)[128].Sofar,nosinglemarkeror

combinationofmarkershasprovenusefulforisolatingCSCfrom

everytumorsite.TheexpressionofCSCmarkersinprimarytumors

hasbeenfoundinsomestudiestobeassociatedwithtumorstage,

prognosisandresponsetotherapy.Itisalogicalextensionofthe

CSCtheorytohypothesizethat,asCSCaretheonlycancercells

thatcanproduceaprimarytumor,CSCmustalsobeessentialfor

thedevelopmentofmetastaticdisease.

Inbreastcancer,cellscharacterizedbyhighCD44expression

andlowlevelsofCD24expression(CD44+CD24−/low)havebeen

showntoencompasstheCSCsubpopulationofcancercells[125].

AnothermarkerfortheidentiﬁcationofCSCinbreastcancerishigh

levelsALDHexpression,alsoamarkerformanynormalstemcell

populations[123].Bycomparinggeneexpressionproﬁlesbetween

ALDH+andALDH−breastcancercells,a413-genebreastCSC

sig-naturewasdetermined.Amongthedifferentiallyexpressedgenes,

thegeneencodingfortheIL-8receptorCXCR1/IL-8RA,previously

describedtobeinvolvedintheregulationofcancergrowthand

metastasis,wasfound.TheALDH+CSCderivedfrombreast

can-cercelllineswereshowntobesigniﬁcantlymoremetastaticthan

ALDH−cellsbyintracardiacinjectioninNonobesediabetic/severe

combinedimmunodeﬁciency(NOD-SCID)miceindicatinga

pos-sibleroleforCXCR1/IL8-RAinthemetastaticpotentialofbreast

CSC.AdditionallythesameCSC-enrichedpopulationsgaveriseto

extra-pulmonarymetastasesinthepancreas,liver,spleenand

kid-ney[129].Similarresultshavebeenshowninheadandneckcancer

wheretheCSCcollectedbasedonCD44expressionwereshown

tobeessentialformetastaticformationinatailveinmodeland

inanorthotopicheadandneckcancermodel[130,131].Thisdata

supportstheconceptthatCSCarecriticaltothedevelopmentof

metastasis.

There is accumulating evidence of cellular heterogeneity

within the CSC compartment with some CSC exhibiting an

enhancedpotentialforthedevelopmentofmetastasis.Evidence

formetastaticandnon-metastaticCSCwasﬁrstraisedincancerof

thepancreas.ThepancreaticCSCpopulationisdeﬁnedbyCD133

expression.InvivostudiesoftheCD133+CSCrevealeda

subpopu-lationofmigratingCSCdeﬁnedbysurfaceexpressionofCXCR4.

CXCR4 isa proteinthat haspreviouslybeenimplicated in

can-cercellmetastaticpotential[132].Usinganorthotopicmodelof

pancreaticcanceronlythemigratingCSCpopulation,expressing

CD133+CXCR4+,wereabletoestablishlivermetastasis.Inhibition

ofCXCR4signiﬁcantlyreducedtheCSCmetastaticpotential[133].

Theseresultsaresigniﬁcantastheﬁrstobservationofthe

impor-tanceoftheCSCphenotypetotheirmetastaticpotentialaswellas

theroleofCXCR4inregulatingthisbehaviorinCSC[134].

AdditionalevidenceoftheexistenceofdifferentCSCsubtypes

responsible for speciﬁc behaviorsexists in colon cancer. It has

beenreportedthatcolonCSChavethreedistinctphenotypes;

self-renewing long-term (LT-TICs), tumortransient amplifying cells

(T-TAC),anddelayedcontributing(DC-TICs).Interestinglythe

self-renewing LT-TICs were the only subpopulation of CSCable to

contributetometastasisformation[135].Morerecently,CD26was

conﬁrmedasamarkerformetastaticCSCincoloncancer[136].

NoneofthepatientswithoutCD26-expressingcancercellsintheir

primarytumorsdevelopedmetastases,whilethemajorityofthose

whosetumorscontainedCD26+cellsdid.Inanimalmodels,both

CD26+and CD26− cells werecapableof giving risetoprimary

tumors,however, onlyCD26+cells hadthecapacity toproduce

metastasis[137].These reportsconﬁrm theimportanceof CSC,

andmorespeciﬁcallythemigratorysubpopulationsofCSC,tothe

developmentofmetastasisincoloncancer.

EMThasbeenimplicatedasanimportantmechanismbywhich

cancercellsgainmetastaticpotential.Manycancertypeshavebeen

showntoexhibitEMT.EMTisbelievedtorepresentacrucialstep

towardcancercellsacquiringinvasivenessandthepotentialto

pro-duce metastasis[138].Thereis accumulatingevidencethatCSC

undergoEMTandthisabilityhasimportantregulatoryfunctions

relatedtoCSCbehavior.StudieshaverevealedthatEMTcaninduce

apparentlydifferentiatedcancercellstogainaCSC-likephenotype

increasingtheirtumorgenicityandtheirabilitytomigratetoand

invadetissuesdistantfromtheprimarytumor.Additionally,

can-cercellsundergoingEMThavebeenshowntobeenrichedforCSC

[139].CSCexpressmanyEMTregulatingfactorsincludingTWIST,

SnailandSlugsuggestingthesegenesplayanimportantregulatory

roleinCSCbehavior[140].

ThepreferentiallocationofEMTcellsalongtheinvasivefrontof

tumorsandtheassociationofEMTwithhighWntsignalinglevels

havebeendemonstrated.Thisincludesthenuclearaccumulationof

␤-catenin,evidenceofWntactivation,observedincellsundergoing

EMTattheinvasivefront[134].Signalsfromthetumor

microenvi-ronmenthavebeendemonstratedtoelicitWntsignalingincolon

cancercells,inducingEMTandallowingfortheirdetachmentand

spreadfromtheprimarytumorsite.ObservationsregardingEMT

andWntexpressioninlocationswithinprimarytumorswhereCSC

typicallyresideledtotheconceptofmigratingCSCasproposedby

Brabletzetal.in2005[134].

EMT has been shown to be a reversible process in that

mesenchymal-to-epithelialtransition(MET)cantransform

mes-enchymalcellsbacktotheirepithelialstate.Similarlymetastatic

CSCmayrespond tolocalcuesto revertfromtheir

mesenchy-malstatebacktotheiroriginalepithelialstate.OncetheCSChave

returnedtotheiroriginalepithelialconditiontheycanform

grow-ing metastatic deposits.It is highly likely that CSClead to the

developmentofmetastasesbyacquiringmesenchymalproperties

thatenhancetheirabilitytomigrateandinvadeandthentransition

backtotheirepithelialphenotypetoformametastasis.The

fac-torsthatregulateEMTincancercellsarebeingstudiedinmultiple

cancertypesbutarenotyetfullyunderstood.Thetumor

microen-vironmenthasbeenproposedasanimportantregulatorofEMTin

CSCincludinglocalfactorssuchashypoxia,cytokinesincludingIL-6

andcancerassociatedcellsincludingtumorassociatedﬁbroblasts,

mesenchymalstemcells,andlymphocytesabletosecretediffusible

(11)

Althoughattractiveandinagreementwithmanygenetic

anal-ysesandwiththeevaluationofCSCinprimarytumorsandanimal

models,todatethecausativeroleofCSCinmetastasisformationhas

notbeenformallyproven.Metastasesrepresentoneofthekey

fac-torsintreatmentfailuresinpatientswithcancer.Recognitionthat

CSCplayacriticalroleinthedevelopmentofmetastasisisan

impor-tantsteptowardincreasingourunderstandingofhowmetastasis

develop.ThefactorsthatmodulatetheCSCmetastaticphenotype

havenotyetbeenfullyelucidatedandrequiremoreintensive

inves-tigation.Thisworkwillleadtomoreeffectiveanticancertherapy

andimprovedoutcomeforpatients.

3. Cancercelldisseminationandthemetastaticcascade

3.1. Organspeciﬁcmetastasis

The predisposition of certain body sites or organs to house

metastaticcellsandestablishsecondarytumorshasbeen

appar-entforcenturiesandhasbeenfamouslyexplainedinPaget’s‘seed

andsoil’theoryofmetastasis[143].Thistheorydictatesthata

spe-ciﬁctumorcell(theseed)willonlyestablishinaparticularsuitable

organorlocation(thesoil).Indeed,manycancershaveanincreased

propensitytoestablishsecondarymetastasisatcertainsites.For

example,breastandprostatecancersappeartobepredisposedto

metastasizingtotheboneenvironmentwhereasgastrointestinal

cancersfrequentlymetastasizetothelungandliver[2,144].Indeed

afeworgansrepresentthemainsecondarydestinationformost

cancers,namely,theliver,lung,boneandbrain,whileorganssuch

asthespleenandheartrarelyhostmetastasis.Thefactors

underly-ingthisorganspeciﬁcpredispositionformetastaticdissemination

bymanycancertypesarelargelyunknownandarewidelybeing

studiedwithinthescientiﬁccommunity.Establishmentofsuch

fac-torsmayagainyieldintuitivestrategiestolimitmetastaticdisease.

3.1.1. Targetingbonemetastasis

Bonemetastasesareacommoncomplicationofseveraltypes

ofcancers,includingbreast,prostateandlungcancer.The

occur-renceofthesebonemetastasesdeeplyimpacttheprognosisand

thequalityof lifeofpatientsandareresponsibleforsigniﬁcant

morbidity.Bonemetastases are oftenosteolytic(due to

signiﬁ-cantbonedestruction),sometimessclerotic(duetoanexcessof

boneformation)ormixed.Numerousmechanismsandfactorsare

involvedintheinvasion,colonizationandestablishmentoftumor

cells in the bone microenvironment. The complex sequence of

eventsthatleadtotheonsetofbonemetastasesnotonlyinvolves

processescommontoanyothermetastasisbutalsoprocessesthat

aremorespeciﬁctothebone tissue(tumor cellinvasioninthe

boneenvironment,implantationoftumorcellsinbonemarrow,

osteomimicry,deregulationofosteoblast\osteoclastactivity)[145].

Thecurrentsectionillustratesprogressmadetowardthe

under-standing,treatmentsandmanagementofthisparticularformof

metastaticdisease.

Thespreadofmetastaticcellsfromthebloodstreamtothebone

marrow involves factors that are produced by osteoblasts and

stromalcellsinthebonemarrow.Amongthesefactors,akeyrole

is played bychemokines (CXCL12, CXCL13, chemokine (C-X3-C

motif)ligand1(CX3CL1),CCL22)thatstimulatecancercell

migra-tiontothebonemarrow,sincetheyexpressmembranereceptors

correspondingtothesechemokines[145].For example,CXCL12

anditsreceptor CXCR4play animportantrole inbone tropism

of cancer cells and treatment with inhibitors of CXCR4 (AMD

3100,T140)orCXCL12(OTP-9908)hasdemonstratedefﬁcacyin

decreasingtheformationofbonemetastasesinexperimental

mod-elsofbreastcancerorprostatecancer[145,146].Inaddition,some

proteins (bone sialoprotein, osteonectin, osteopontin, collagen)

canstimulatebonematrixinvasionbybindingthesurfaceoftumor

cellsthroughspeciﬁcmembranereceptorssuchasintegrins␣V␤3

and␣2␤1[145]andithasbeendemonstratedthatbreastcancer

cells expressing␣V␤3 integrin and prostate cancer expressing

the␣V␤2,havehigherincidenceofbonemetastases[147].There

is preclinical evidence that ␣V␤3 integrin inhibition is ableto

prevent bone colonization by ␣V␤3 expressing human breast

cancercells[148].Severalongoingclinicaltrialsareevaluatingthe

anticancereffectofintegrinantagonistsinadvancedrefractoryand

metastaticcancers,butonlyonephaseIclinicaltrialisevaluating

integrin antagonists (GLPG0187) in cancer patients with bone

metastasis(NCT01313598)[149,150].

It has been demonstrated that the tyrosine kinase c-MET

promotes stemness phenotype, tumor growth, invasion, and

metastasisin severalmalignancies. Inparticular,c-METis

over-expressedin prostatecancercells andisassociated withtumor

progression and metastatic invasion to bone [151].

Cabozan-tinib (XL184) is an oral small molecule inhibitor of multiple

kinasesignalingpathwaysincludingc-METandvascular

endothe-lial growth factor receptor 2 (VEGFR2). A recent phase II

“randomizeddiscontinuation”trialinpatientswithmetastatic

cas-trationresistantprostatecancer(mCRPC)included171menwith

castration-resistantprostatecancer(CRPCfromalargerphaseII

randomized discontinuation trial that included multiple tumor

typestreatedwithcabozantinib).Therandomizationwasstopped

after122patientsbecauseofimprovementsinbonescansanda

decreaseinpain.Atthetimethestudywashalted,agroupof31

patientshadbeenrandomlyassigned.Inthisgroup,therewasa

markedimprovementintheprimaryendpointofprogression-free

survival(PFS)inthepatientsreceivingcabozantinibcomparedwith

placebo(p<0.001)[152].PhaseIIItrialsarecurrentlyonongoing.

Cathepsins are a class of globular lysosomal proteases that

belongtothepapain-likecysteineprotease familyexpressedin

awidevarietyoftissuesincludingthebone,wheretheyappears

tobeakeyenzymeinbone matrixdegradation[153].Different

cancersexpresscathepsinK,includingprostateandbreastcancers

[154].Untilrecently,aroleforcathepsinKinbonemetastasishad

beenmainlyattributedtoitsabilitytodegradenativecollagenI,a

processnecessaryfortheexpansionofthetumorwithinthebone.

Forexample,thehumanbreast,boneseeking,cancercellline

MDA-MB-231/B02secretedcathepsinKandtreatmentofthesecellswith

acathepsinKantagonistcaninhibittumorinvasion[155].Duetoits

selectivity,odanacatibistheonlycathepsinKinhibitorinclinical

development.AphaseIIcontrolledstudyonwomenwithbreast

cancerand established bone metastasis,randomized toreceive

dailyadministrationofodanacatiborasingledoseofzoledronic

acid,showedreducedboneremodelingmarkers(urinaryNTx)after

4weekstreatment,demonstratingthatodanacatibisaseffectiveas

zoledronatetoreduceboneresorptionmarkers[156].

ReceptorActivator ofNuclearFactor-␬B Ligand(RANKL),the

Receptor Activator of Nuclear Factor-␬B (RANK) and thedecoy

receptor osteoprotegerin (OPG) are members of the TNF and

TNF-receptorsuperfamily,whichareabletoinduceproliferation,

differentiation,activationandapoptosisofosteoclasticcells.Bone

remodelingis mediated bythe interactionof RANKL expressed

ontheosteoblasts,RANKexpressedontheosteoclastsurfaceand

OPGwhichpreventsosteoclast activation[157].Murinemodels

haveshownthatRANKLisabletoactaschemoattractantandasa

pro-migratoryfactorinRANK-expressingbreastandprostate

can-cercelllines and thatRANKL inhibition is abletoreduce bone

lesionsandtumorburdeninamelanomamodelofbone

metasta-sis[158].IthasalsobeendemonstratedthatRANKprimarytumor

expressionlevelscorrelatewiththeoccurrenceofbonemetastases

andthatRANK-expressingcancercouldbefoundinupto80%of

bonemetastasesoriginatedfromsolidtumor[159,160],suggesting