Guidance Molecules in Cancer and Tumor Angiogenesis

273 Pages·2012·5.616 MB·1-274\273

by  Ira O. Daar (Eds.)

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Contributors
Numbersinparenthesesindicatethepagesonwhichtheauthors’contributionsbegin.
Mimmi S. Ballard, Department of Molecular, Cell and Developmental
Biology,University ofCalifornia, SantaCruz,California, USA(187)
Jin Chen, VA Medical Center, Tennessee Valley Healthcare System;
Departmentof Medicine; Departmentof Cancer Biology; Departmentof
Cell and Developmental Biology and Vanderbilt-Ingram Cancer Center,
Vanderbilt University Schoolof Medicine,Nashville,Tennessee, USA (1)
Christian Gespach, INSERM U938, UMR_S938, Molecular and Clinical
Oncology, UPMC Universite´ Paris VI, Hoˆpital Saint-Antoine, Paris
Cedex, France (87)
LindsayHinck,DepartmentofMolecular,CellandDevelopmentalBiology,
Universityof California,SantaCruz,California,USA (187)
Giovanni Melillo, Discovery Medicine-Oncology, Bristol-Myers Squibb,
Princeton, New Jersey,USA (237)
Claudia Muratori, University of Torino Medical School, Institute for
Cancer Research (IRCC), Candiolo, Turin, Italy (59)
Annamaria Rapisarda, SAIC-Frederick, Inc., Frederick National
Laboratory forCancerResearch, Frederick, Maryland,USA (237)
Ombretta Salvucci, Laboratory of Cellular Oncology, Center for Cancer
Research,National Cancer Institute,Bethesda, Maryland,USA (21)
LucaTamagnone,UniversityofTorinoMedicalSchool,InstituteforCancer
Research(IRCC),Candiolo, Turin, Italy (59)
Giovanna Tosato, Laboratory of Cellular Oncology, Center for Cancer
Research,National Cancer Institute,Bethesda, Maryland,USA (21)
ix
Preface
Progression of human cancer requires the coalescence of many processes
thatarealsocriticalduringdevelopmentofthenervoussystem,suchascell
proliferation, invasion, avoidance of apoptosis, and angiogenesis. Several
familiesofneuronalguidancemolecules,forexample,semaphorins,ephrins,
netrins,andslit,alongwiththeircognatereceptors(neuropilin/plexin,Eph,
Unc/DCC, and Robo, respectively), have been identified that control guid-
anceandmorphogeneticfunctionsinothertissuesandthevascularsystem.
One additional family, the vascular endothelial growth factors (VEGFs),
mayplayaroleinneurogenesisbutarebestknownascriticalregulatorsof
vasculardevelopment.Severalantiangiogenictherapieshavebeendeveloped
andusedclinicallythattargetVEGForthecognatereceptor,buttheclinical
outcomes have varying success. The role of the nervous system in tumor-
igenesis was originally thought to be that of a path by which the invading
tumor cells travel or to act as a neoangiogenic supply line to tumors.
However,nowithasbeensuggestedthatthenervoussystemisfunctionally
relatedtotumorprogressionbyregulatingacomplexnetworkofmediators
(soluble factors, membrane-bound molecules, and receptors) that influence
tumorprogression.Emergingevidenceindicatesthatthereisalsocross-talk
between tumor cells and the nervous system, particularly between neural
factorsorguidancemoleculesandcancercells.Understandingtherelation-
ship between guidance molecules (as well as neurotrophic factors and neu-
rotransmitters), the tumor, and the tumor microenvironment may prove to
be critical for devising appropriate therapies for intervention in cancer
growth, invasion, and progression. Thisvolumewill focus on severalguid-
ancemoleculesystemsincludingEph/ephrin,semaphorins/plexinsandneu-
ropilins, slit/robo, netrins/unc and DCC, and VEGF/VEGFR and what is
known(orimplicated)abouttheirroleincancer.
IraO.Daar
xi
Regulation of Tumor Initiation and
Metastatic Progression by Eph
Receptor Tyrosine Kinases
JinChen
VAMedicalCenter,TennesseeValleyHealthcareSystem,
Nashville,Tennessee,USA
DepartmentofMedicine,VanderbiltUniversitySchoolofMedicine,Nashville,
Tennessee,USA
DepartmentofCancerBiology,VanderbiltUniversitySchoolofMedicine,
Nashville,Tennessee,USA
DepartmentofCellandDevelopmentalBiology,VanderbiltUniversitySchoolof
Medicine,Nashville,Tennessee,USA
Vanderbilt-IngramCancerCenter,VanderbiltUniversitySchoolofMedicine,
Nashville,Tennessee,USA
I. Introduction
II. DysregulationofEphReceptorsinCancer
III. RoleofEphReceptorsinTumorCellProliferation
IV. RoleofEphReceptorsinInvasionandMetastasis
V. RegulationofTumorAngiogenesisbyEphReceptors
A. Ephrin-A1andEphA2inTumor–EndotheliumInteraction
B. Ephrin-B2andEphB4inTumorAngiogenesis
C. Ephrin/EphinEndothelial–MuralCellInteraction
VI. ConcludingRemarks
Acknowledgments
References
Inrecentyears,agrowingbodyofevidencehasindicatedthatsignalingmolecules
previouslyimplicatedinaxonguidanceareimportantregulatorsofmultisteptumor-
igenesis and progression. Eph receptors and ephrins belong to this special class of
molecules that playimportant roles inboth axon guidance and cancer. Tremendous
progresshasbeenmadeinthepastfewyearsinbothunderstandingtheroleofEph
receptors and ephrins in cancer and designing therapeutic strategies for cancer
therapy. This review will focus on new advances in elucidating the contribution of
Eph/ephrinmoleculestokeyprocessesintumorinitiationandmetastaticprogression,
includingcancercellproliferation,invasionandmetastasis,andtumorangiogenesis.
#2012ElsevierInc.
AdvancesinCANCERRESEARCH,Volume114 0065-230X/12$35.00
Copyright2012,ElsevierInc.Allrightsreserved. DOI:10.1016/B978-0-12-386503-8.00001-6
1
2 Jin Chen
I. INTRODUCTION
Cancer initiation and malignant progression are multistep processes that
involvelossofgrowthcontrol,evasionofapoptosis,sustainedangiogenesis,
tissue invasion, and metastasis (Hanahan and Weinberg, 2000, 2011).
Emerging evidence has indicated that signaling molecules previously
implicated in axon guidance are important regulators of multistep tumor
initiation and progression (Adams and Eichmann, 2010; Eichmann et al.,
2005).TheseincludeEph/ephrin,semaphorins/plexins,VEGF/VEGFR,che-
mokines/receptors, netrins/DCC UN5, Slit/Robo, and Notch/Delta. This
review will focus on recent advances on dissecting the role of Eph/ephrin
molecules in cancer and tumor angiogenesis. Due to space and scope con-
straints,wehavelimitedourdiscussioninthetexttotheEphreceptorsand
ephrins for which the most data are available. Tumor and vascular pheno-
types of Eph/ephrin knockout and transgenic animals are summarized in
Tables I and II. More comprehensive reviews on Eph–ephrin signaling in
physiologyanddiseasecanbefoundelsewhere(KullanderandKlein,2002;
Pasquale,2005,2008,2010).
TableI TumorPhenotypesofEph/ephrin-MutantAnimals
Mousestrain Tumormodel Phenotype Reference
(cid:1)/(cid:1)
EphA2 knock- MMTV-Neu Decreasedtumorincidence, Brantley-Sieders
outmice breastcancer tumorburden,and etal.(2008)
metastasis
i/i
EphA2 genetrap DMBA/TPAskin Increasedtumornumber, Guoetal.(2006)
mice cancer tumorburden,and
invasiveness
i/i Min/þ
EphA2 genetrap APC Decreasedtumornumberand Boganetal.(2009)
mice coloncancer sizeinsmallandlarge
intestine
Min/þ
Villin-ephrinA1 APC Increasedtumornumbersand Shietal.(2008)
transgenicmice coloncancer invasiveness
MMTV-EphB4 MMTV-Neu Acceleratedtumoronsetand Munarinietal.
transgenicmice breastcancer increasedmetastasis (2002)
DC Min/þ
Villin-EphB2 APC Increasedtumornumberand Batlleetal.(2005),
(cid:1)/(cid:1)
EphB3 coloncancer tumorinvasion Cortinaetal.
Vil-Cre/ephrin- (2007)
D/D
B1
TableII VascularPhenotypesofEph/ephrin-MutantAnimals
Geneknockout/knockin Phenotype Reference
(cid:1)/(cid:1)
ephrinA1 Defectsinheartvalves;impaired Friedenetal.(2010)
cardiacfunction
(cid:1)/(cid:1)
EphA3 Perinatallethality;hypoplasticheartvalve Stephenetal.(2006)
(cid:1)/(cid:1)
EphA2 Defectsintumorangiogenesis Brantley-Siedersetal.
(2005,2008)
EphB2(cid:1)/(cid:1)EphB3(cid:1)/(cid:1) Embryoniclethal,dieatE10.5((cid:3)30%);defectivevesselremodeling, Adamsetal.(1999)
(cid:1)/(cid:1)
similartothoseobservedinephrinB2
(cid:1)/(cid:1)
EphB4 Embryoniclethal,dieatE10.5;defectivevesselremodeling,similarto Geretyetal.(1999)
(cid:1)/(cid:1)
thoseobservedinephrinB2
(cid:1)/(cid:1)
ephrinB2 Embryoniclethal,dieatE10.5; Wangetal.(1998)
defectivevesselremodelingandsprouting
DC/DC
ephrinB2 (deletionof Defectsinangiogenicremodelingsimilartothoseobservedin Adamsetal.(2001)
cytoplasmicdomain) ephrinB2(cid:1)/(cid:1)
DV/DV
ephrinB2 (deletionofVal.residuein Defectsinangiogenicremodelingsimilartothoseobservedin Ma¨kinenetal.(2005),
PDZ-bindingmotif) ephrinB2(cid:1)/(cid:1);defectsintumorangiogenesis Sawamiphaketal.(2010)
ephrinB25Y/5Y(knockin Mildlymphaticphenotype Ma¨kinenetal.(2005),
mutationinfivetyrosineresidues) Sawamiphaketal.(2010)
iDEC
ephrinB2 (VE-cadherinpromoter-driven Defectsinangiogenicsproutinginbothbloodvesselsandlymphatic Wangetal.(2010)
CreERT2,EC-specificknockout) vessels
iGOF
ephrinB2 (Tie2-rtTAdriven Increasedangiogenicsprouting Wangetal.(2010)
tetO-ephrinB2transgenic)
ephrinB2DPC/vSMC(PDGFRbpromoter- Perinatallethality;vasculardefectsinmultipleorgans;abnormalmi- Fooetal.(2006)
drivenCre,muralcell-specificknockout) grationofsmoothmusclecellstolymphaticcapillaries
4 Jin Chen
II. DYSREGULATION OF EPH RECEPTORS IN CANCER
TheEphfamilyofreceptortyrosinekinases(RTKs)isthelargestidentified
inthevertebrategenomeandissubdividedintoclassAandclassBbasedon
sequencehomologyandbindingaffinityfortwodistincttypesofmembrane-
anchoredephrinligands.Ingeneral,classAreceptorsinteractwithglycosyl-
phosphatidylinositol-linked classAephrins, whileclassBreceptorsbindto
classBephrinsthatareattachedtothecellmembranebyatransmembrane-
spanning domain, although interclass binding does occur among certain
family members (reviewed in Pasquale, 2005, 2008, 2010). Binding of
ephrin to Eph receptor induces receptor clustering and activation. Recent
structural studies of the Eph/ephrin interactions have not only identified
ligand–receptorinteractionsitesbutalsodiscoveredreceptor–receptorinter-
facesthatfunctioninassemblyofhigher-ordersignalingclusters(Himanen
et al., 2010; Seiradake et al., 2010). Because both ligand and receptor are
membrane bound, engagement of ephrin with Eph receptor between adja-
centcellsinducesbidirectionalsignalingthroughbothligand-andreceptor-
expressing cells (reviewed in Kullander and Klein, 2002; Pasquale, 2008).
Originally characterized as axon guidance regulators, ephrins and Eph
RTKs are subsequently recognized to regulate physiologic and pathologic
processesduringembryonicdevelopment,innormaltissuehomeostasis,and
indisease(reviewedinPasquale,2008,2010).
Recent technological advances in analyzing the human cancer genome
permitthestudyofgenecopynumber,expressionlevel,andmutationstatus
in tumor tissue. These studies demonstrated that Eph receptors are often
dysregulatedincancer(reviewedinBrantley-Sieders,2011;Brantley-Sieders
et al., 2011b; Pasquale, 2010). Gene expression studies by microarray
analysis and immunohistochemistry on tumor tissue microarrays have
correlated expression of some Eph receptors in tumor epithelium and/or
vasculature with disease stage, metastasis, recurrence, and survival. For
example, EphA2 expression is elevated in many types of cancer (Landen
et al., 2005b; Wykosky and Debinski, 2008) and high levels of EphA2
correlatewithtumormalignancyandpoorpatientsurvivalinbreastcancer
(Brantley-Sieders,2011;Fournieretal.,2006;Martinetal.,2008;Zhuang
etal.,2010)andlungcancer(Brannanetal.,2009;Faoroetal.,2010;Kinch
etal.,2003).Likewise,levelsofseveralEphBreceptorsarealsoelevatedin
different stages of colon cancer, lung cancer, or breast cancer (Batlle et al.,
2002;Brantley-Sieders,2011;Jietal.,2011;Kumaretal.,2009;Norenand
Pasquale, 2007). However, decreased Eph receptor levels have also been
reported in certain types of human cancer (Batlle et al., 2005; Kumar
et al., 2009). Dysregulation of Eph receptor expression in tumor has been
attributed to multiple mechanisms, including chromosomal abnormality,
EphReceptorTyrosineKinaseinCancer 5
epigeneticregulation,mRNAstability,andtranscriptionalcontrol(reviewed
inPasquale,2010).Asidefromchanges inreceptor level, somatic mutations
havebeenfoundinnearlyallEphreceptors.Notably,11somaticmutationsin
EphA3 receptor were identified in 5–10% of lung cancers, placing EphA3
among 27 most frequently mutated genes in human lung adenocarcinoma
(Davies et al., 2005; Ding et al., 2008; Greenman et al., 2007; Wood et al.,
2006).However,thesemutationsarescatteredthroughoutthereceptor,andit
isunclearwhetherthesenonrecurrentmutationsare“driver”orbiologically
neutral“passenger”geneticalterations.Elucidatingtheeffectsofthesemuta-
tionswillgreatlyimproveourunderstandingofhowEphreceptorsfunctionin
cancer.
III. ROLE OF EPH RECEPTORS IN TUMOR CELL
PROLIFERATION
Sustainedproliferativesignalsandevasionofgrowthsuppressorsaretwo
hallmarksofcancer.However,Ephreceptorswereinitiallythoughtnottobe
involved in regulation of cell growth (Brambilla et al., 1995; Bruce et al.,
1999; Lhotak and Pawson, 1993). More recent studies found that Eph
receptors maintain tissue homeostasis by controlling the proliferation of
stem and progenitor cells in adult. For example, in the intestinal stem cell
niche,EphBsignalingpromotescell-cyclereentryofprogenitorcellsthrough
Abl and Cyclin D1 and accounts for approximately 50% of the mitogenic
activityintheadultmousesmallintestineandcolon(Genanderetal.,2009).
Likewise,Ephreceptorsignalinginneuralprogenitorcellsinhippocampus
also promotesproliferation (Chumleyetal.,2007). Incontrast, Eph recep-
tors can negatively regulate proliferation of hair follicle and epidermal
progenitor cells in the adult mice (Genander et al., 2010), as well as in
neuralstem/progenitorcellsinthelateralventricle(Conoveretal.,2000).
Incancercells,theroleofEphreceptorsinregulationoftumorcellgrowth
issimilarlycomplex,andtheeffectsofthesereceptorsoncellproliferationare
oftendependentonligandstimulation,signalingcrosstalk,orothercontex-
tualfactors.ThiscomplexityisbestillustratedinthecaseofEphA2receptor.
EphA2 receptor is highly expressed in a number of tumor types, including
breastcancer,prostatecancer,ovariancancer,lungcancer,andglioblastoma
multiforme (Brantley-Sieders, 2011; Landen et al., 2005b; Wykosky and
Debinski, 2008). High levels of EphA2 are correlated with poor patient
survival (Brannan et al., 2009; Brantley-Sieders, 2011; Faoro et al., 2010;
Fournieretal.,2006;Kinchetal.,2003;Martin etal.,2008;Zhuangetal.,
2010). Silencing of EphA2 in cancer cells by siRNA-mediated knockdown,
6 Jin Chen
antisense oligonucleotides, or targeted deletion of EphA2 in knockout mice
inhibitedtumorinitiationandmetastaticprogression(Brantley-Siedersetal.,
2008; Duxbury et al., 2004; Landen et al., 2005a). Interestingly, the tumor
promotioneffectsofEphA2appeartobeligandindependentandresultfrom
crosstalkwithHER2RTK(Brantley-Siedersetal.,2008).Insupportofthis
notion,overexpressionofEphA2innontransformedMCF-10Abreastepithe-
lial cells induces anchorage-independent colony growth in vitro and tumor
formation in vivo (Zelinski et al., 2001). Further investigation revealed that
high levels of EphA2 in these tumor cells are minimally phosphorylated,
suggesting poor ligand-induced receptor forward signaling. Indeed, exoge-
nousligandstimulationofEphA2suppressedcolonyformationon2Dculture
and spheroid growth in 3D Matrigel (Miao et al., 2001; Yang et al., 2011).
Treatment of mammary tumors with a ligand-mimetic activating antibody
against EphA2 suppressed tumor growth in vivo (Brantley-Sieders et al.,
2008; Coffman et al., 2003; Landen et al., 2006), corroborating the notion
that EphA2’s effects in tumor promotion is ligand independent. Similarly,
EphB4isoftenoverexpressedinhumanbreastcancer(Wuetal.,2004),and
EphB4 knockdown inhibited tumor cell survival, migration, and invasion
in vitro, and tumor growth in vivo (Kumar et al., 2006). However, ephrin-
B2-inducedEphB4forwardsignalinginhibitsbreastcancercellviabilityand
proliferation, as well as tumor volume in xenografts (Noren et al., 2006).
Taken together, these data suggest a model in which ephrin-induced Eph
receptor forward signaling inhibits tumor cell proliferation, whereas in the
absenceoftheligand,crosstalkbetweenEphreceptorsandotheroncogenic
proteins leads to enhanced cell proliferation and tumorigenesis, presumably
independentofephrinstimulation.
How does Eph receptor forward signaling inhibit tumor cell growth?
Ephrin-B2-induced EphB4 receptor forward signaling activates anantionco-
genic pathway, leading to Abl kinase activation and inactivation of Crk
adaptor function through phosphorylation by Abl (Noren and Pasquale,
2007;Norenetal.,2006).InthecaseofEphA2,severalstudiesshowedthat
stimulation of tumor cells with ephrin-A1 ligand inhibited the activation of
bothRas–MAPKandAkt–mTORpathways(Macraeetal.,2005;Miaoetal.,
2001;Yangetal.,2011).However,inPC3prostatecancercells,MEKinhibitor
doesnotappeartoaffectcellproliferation,whereasPI3KinhibitorandRapa-
mycin efficiently inhibited cell growth, suggesting that Akt–mTOR is the
major pathway inhibited by EphA2 forward signaling (Yang et al., 2011).
It is interesting to note that EphA2 forward signaling does not change the
activityofAktupstreamregulatorssuchasRasfamilyGTPases,PI3kinase,
integrin,ortheSHIP2lipidphosphatase.Rather,EphA2inactivatestheAkt–
mTORC1oncogenicpathwaythroughAktdephosphorylationmediatedbya
PP1-likeserine/threoninephosphatase(Yangetal.,2011).Althoughinhibition
of Akt–mTOR pathway by EphA2forward signaling appearsto be a major
EphReceptorTyrosineKinaseinCancer 7
mechanism in regulation of cell growth in multiple tumor cell lines, the
importance of EphA2-mediated suppression of Akt–mTOR pathway in
tumorgrowthneedstobefurthervalidatedinanimalmodelsinvivo.
Decades of research have demonstrated that “contact inhibition” is an
important mechanism used by normal cells to suppress cell proliferation.
The mechanism of contact inhibition appears to involve strengthening of
cell–cell adhesion and maintaining cell polarity and tissue integrity. In
MDCK cells, ligand-activated EphA2 signaling suppresses Arf6 GTPase
activity, leading to cell compaction and polarization (Miura et al., 2009),
suggestingaroleforEphA2forwardsignalinginE-cadherin-basedcell–cell
adhesion and the apical–basal polarization of epithelial cells. However,
overexpression of EphA2 receptor in MCF10A mammary epithelial cells
destabilizes adherens junctions via a RhoA-dependent mechanism (Fang
et al., 2008b). The seemingly conflicting results in these two studies may
beduetodifferencesinsignalingstrengthandcompositionofthesignaling
complexintherespectivecelltypes,asillustratedinephrin-B1regulationof
tightjunctions(LeeandDaar,2009;Leeetal.,2008).Eitheroverexpression
orlossofephrin-B1candisruptcell–cellcontactand tightjunctions.Over-
expressed or unphosphorylated ephrin-B1 competes with active Cdc42
GTPaseforbindingtoPar-6andinhibitsaPKCactivationintheParpolarity
complex, leading to tight junction disruption. Tyrosine phosphorylation of
ephrin-B1inducesdissociationofephrin-B1andPar-6,whichisnowavail-
abletointeractwithCdc42andestablishtightjunction.However,ephrin-B1
is also associated with Par-6 at adherence junction. Loss of ephrin-B1
entirely may allow Par-6 at adherence junction and compete with Par-6 at
tightjunctionforCdc-42,resultingindisruptionoftightjunction.Interest-
ingly,phosphorylationofephrin-B1canbeEphBreceptorindependentand
can be induced by association with tight junction protein claudin (Tanaka
etal.,2005).Together,thesestudiessuggestthatderegulationofephrin/Eph
signaling can affect cell–cell adhesion, but it remains to be determined
whether dysregulation of cell–cell adhesion directly leads to changes in
cellular proliferation. However, breaking down cell–cell adhesion is often
a first step that leads to epithelial-to-mesenchymal transformation, a pro-
gramthatbroadlyregulatesinvasionandmetastasis.
IV. ROLE OF EPH RECEPTORS IN INVASION AND
METASTASIS
HighendogenouslevelsofEphreceptorsincancercells,oroverexpression
ofEphmolecules,havebeenimplicatedinpromotingtumorcellinvasiveness
andmotilityinvitroanddistantmetastasisinvivo.TargetedEphA2receptor