New signals from the invasive front Gerhard Christofori 1

Transcription

1 NATURE Vol My 2006 doi: /nture04872 New signls from the invsive front Gerhrd Christofori 1 Approximtely 90% of ll ner deths rise from the metstti spred of primry tumours. Of ll the proesses involved in rinogenesis, lol invsion nd the formtion of metstses re linilly the most relevnt, ut they re the lest well understood t the moleulr level. Reveling their mehnisms is one of the min hllenges for explortory nd pplied ner reserh. Reent experimentl progress hs identified numer of moleulr pthwys nd ellulr mehnisms tht underlie the multistge proess of metstsis formtion: these inlude tumour invsion, tumour-ell dissemintion through the loodstrem or the lymphti system, oloniztion of distnt orgns nd, finlly, ftl outgrowth of metstses. The pst two dedes hve seen our knowledge of the geneti nd epigeneti events involved in the erly events of nerogenesis inrese onsiderly. By ontrst, despite the ppreition of the linil relevne of tumour metstsis, there is n emrrssing lk of therpies tht n effiiently prevent metstsis. Multiple signl-trnsdution pthwys, hnges in the dhesive nd migrtory pilities of tumour ells, nd the tumour miroenvironment hve ritil roles in mlignnt tumour progression. At this stge of tumour development, tumour ells migrte into nd invde the surrounding tissue either s single ells or in olletive lusters, therey forming n invsive front. Here, I summrize exiting new insights into the moleulr mehnisms tht underpin tumour invsion nd metstsis. The intertions nd reltionships etween these proesses re now eing studied on vrious levels, leding to n inresed understnding of the system involved nd, it is hoped, to new ner therpies. Epithelil mesenhyml trnsition nd metstsis Aout 90% of ners originte from epithelil tissue. The most pprent morphologil hnge tht ours during the trnsition from enign tumour to mlignnt nd metstti one is tht tumour ells hnge from highly differentited, epithelil morphology to migrtory nd invsive phenotype. Metstti tumour ells then permete the sl lmin rrier nd invde the neighouring tissue (Fig. 1). During this proess of epithelil mesenhyml trnsition (EMT), ells progressively redistriute or downregulte their pil nd solterl epithelil-speifi tight nd dherens juntion proteins (inluding E-dherin nd ytokertins) nd re-express mesenhyml moleules (inluding vimentin nd N-dherin) 1 3. These hnges led to the loss of ell ell ontts nd the gin of ell motility hnges tht re neessry for invsion. EMT is indued y severl growth ftors, whih re produed either y tumour ells themselves or y stroml ells, nd inlude trnsforming growth ftor- (TGF-), heptoyte growth ftor (HGF; lso known s stter ftor), epiderml growth ftor (EGF), insulin-like growth ftors (IGFs) nd firolst growth ftors (FGFs), nd lso y the upregulted proteolyti tivity of mtrix metlloproteses (MMPs) (Box 1). The role of EMT in the progression of epithelil ners (rinoms) nd their metstti dissemintion is hotly deted, euse in most ners full EMT the omplete loss of epithelil mrkers nd the gin of mesenhyml mrkers is rrely oserved. In ft, the presene of ertin epithelil mrkers, suh s ytokertins, is routinely used to detet nd hrterize metstti epithelil ners in ptients. New insights into lssil signlling pthwys TGF- hs dul role during tumour progression: it represses tumour growth during the erly phses of tumorigenesis y induing ell-yle rrest nd progrmmed ell deth (poptosis), ut during the lte phses of rinogenesis, it promotes EMT, tumour invsion nd metstti dissemintion of tumour ells 4. Consistent with its tumour-suppressor funtions, severl omponents of the TGF--medited signlling pthwys re impired in vrious humn ner types, suh s TGF- reeptor II (TGF-RII) in hereditry non-polyposis oloretl ner, TGF-RI in ovrin, rest nd pnreti ners, nd the signl trnsduers SMAD4 (lso known s DPC4) nd SMAD2 in pnreti, oloretl nd lung ners. TGF- lso promotes tumour progression y exerting n immunosuppressive funtion. It represses the expression of mjor histoomptiility omplex (MHC) lss II nd exerts negtive effets on ntigen-presenting ells. However, t the moleulr level, the metstsis-promoting funtion of TGF- is diffiult to explin. Chnges in omponents of the TGF- signl-trnsdution mhinery nd funtionl synergies with other signlling pthwys seem to ontriute to TGF- s tumour-promoting funtion 1 (Box 1). Moreover, it indues ngiogenesis y upregulting the expression of ngiogeni ftors, suh s vsulr endothelil growth ftor-a (VEGF-A) nd ngiopoietin-1. HGF, whih is produed minly y stroml ells, nd the -Met reeptor tyrosine kinse (RTK), its ognte reeptor on tumour ells, re key meditors of invsive growth during emryoni development nd tumour progression 5 7. Germline nd somti muttions, nd mplifitions of the -met gene tht led to inresed -Met tivity, re frequently found in vrious ner types. Reently, -Met tivtion hs een shown to use thromohemorrhgi events tht filitte tumour metstsis ondition lredy desried in the lte nineteenth entury s Trousseu s sign 8. HGF -Met-reeptor signl trnsdution now serves s n exmple of unntiipted sophistition in RTK-medited signlling (Box 1). In its lssil mode, -Met is stimulted y its on fide lignd, HGF, to ssemle signlling omplex tht indues ll the signls required for ell sttering nd invsive growth 7. Besides (or perhps s prt of) its lssil signl-trnsdution role, -Met lso seletively interts with the ell-dhesion moleule α 6 4 integrin, whih is itself phosphorylted y -Met kinse tivity (Box 1). Suh phosphoryltion genertes dditionl doking sites for other dptor moleules nd signlling effetors, whih, in turn, potentite HGF-indued invsion nd metstsis independently of the integrin s dhesion tivity 9. The hyulronn reeptor CD44 lso oopertes with -Met-medited signl trnsdution (Box 1). 1 Deprtment of Clinil Biologil Sienes, Center of Biomediine, University of Bsel, Mttenstrsse 28, CH 4058 Bsel, Switzerlnd. 444

2 NATURE Vol My 2006 INSIGHT REVIEW Adenom Epithelil Crinom Mesenhyml Figure 1 The trnsition from epithelil tumour to invsive ner.,, Histopthology of tumours from trnsgeni mouse model of pnreti -ell rinogenesis (Rip1Tg) 25. Note the differenes etween the epithelil orgniztion of enign denom () nd the ell invsion nd nuler typi of mlignnt rinom (). This trnsition oinides with prtil epithelil mesenhyml trnsition (EMT) tht is, loss of E-dherin ut not ytokertin expression, nd gin of N-dherin ut not vimentin expression (not shown)., d, Cultured norml murine mmmry glnd (NMuMG) epithelil ells express E-dherin nd grow in epithelillike sheets. On stimultion with TGF-, the ells undergo full EMT tht is, they hnge to mesenhyml, migrtory phenotype through the loss of epithelil nd the gin of mesenhyml gene expression, inluding the dherin swith. -Met shres struturl homology with plexins, whih re widely expressed reeptors for semphorins (Sem). The semphorin fmily onsists of sereted nd memrne-ound memers tht t minly s guidne ues for neurons. Plexin-B1 physilly ssoites with -Met 10. Notly, inding of Sem4D to plexin-b1 trnstivtes the tyrosinekinse tivity of -Met independently of the presene of HGF 11. By ontrst, tivted Noth signlling provokes downregultion of -Met expression nd impirment of HGF-medited signlling 12. Conversely, -Met tivtion leds to the expression of Noth lignds nd tivtion of the Noth pthwy, resulting in negtive-feedk loop for finetuning -Met signlling tivities. Hene, TGF-- nd HGF-indued signlling ply n importnt prt in tumour progression, notly y providing funtionl onnetions mong severl moleulr plyers previously implited in the metstti proess. They re ertinly first-line trgets for the development of nti-metstsis therpies. Initil proof-of-onept experiments in niml models, using solule -Met deoy reeptors, hve een highly enourging 13. Moreover, tretment of mie with solule version of TGF-RII prevents the formtion of metstses 14. New kids on the metstsis lok During the pst few yers, mny reserhers hve set out to disover new genes nd ftors with usl involvement in tumour metstsis. Surprisingly, mny of these experiments unovered numer of wellknown genes y novel iologil funtions. For exmple, n elegnt expression-loning pproh reently identified TrkB, neurotrophi RTK for rin-derived neurotrophi ftor (BDNF), s suppressor of ell deth indued y the lk of ell-mtrix dhesion (noikis) 15. TrkB pprently onfers survivl on ells tht would otherwise die when dislodged from their sustrte. Insted, they migrte nd invde new sites, nd form metstses. TrkB nd BDNF re frequently expressed in metstti humn ners. Another exmple is the IGF-1 reeptor (IGF-1R), whih is highly expressed in mny types of humn ner. Besides its importnt funtion in ell growth nd survivl, it promotes tumour invsion nd metstsis in trnsgeni mouse model of pnreti -ell rinogenesis 16. IGF-1R n impir E-dherin funtion, therey induing tumour-ell migrtion nd invsion (Box 1). IGF-1R d seems to offer n exellent multipurpose trget for the development of ner therpies. Indeed, oth NVP-AEW541, smll hemil IGF- 1R inhiitor, nd neutrlizing ntiodies ginst IGF-1R prevent the growth of tumour ells y loking prolifertion nd induing poptosis in ulture nd in niml models 17,18. A muh more dvned tehnique tht is in routine linil use is the speifi repression of ErB2 (lso known s HER2) n EGF reeptor (EGFR) fmily memer tht is frequently upregulted in vrious ner types, prtiulrly rest ner y humnized neutrlizing ntiodies (Hereptin). ErB2 expression orreltes with poor prognosis nd hs reently een onneted to tumour-ell migrtion y the identifition of signlling moleule, known s Memo, tht relys signls to the mirotuule ytoskeleton 19. The hedgehog signlling pthwy, nother well-studied pthwy tht is essentil in developmentl ptterning, hs een implited in the origin of mny types of ner. In the prostte, for exmple, sustined hedgehog signlling n indue tumour formtion nd metstsis, proess tht relies on the presene of the hedgehog signl trnsduer smoothened. Notly, smoothened is not expressed in enign prostte epithelil ells nd my, therefore, serve s moleulr mrker to distinguish etween enign nd mlignnt prostte ner 20. Indution of metstti signlling pthwys y hypoxi Tumour hypoxi not only indues tumour ngiogenesis, ut lso modultes the expression of severl genes tht hve een implited in tumour metstsis. An importnt exmple is the hypoxi indution of -met gene expression, whih mplifies HGF signlling y sensitizing ells to HGF signlling 21. Thus, hypoxi seems to ffet tumour ells in two wys: it indues ngiogenesis (for instne, through hypoxi-induile ftor (HIF)-1α- nd HIF-2α-driven expression of the ngiogeni ftor VEGF-A) nd lolly dpts the tumour environment for optiml tumour growth. Moreover, y stimulting -Met signlling nd fvouring tumour-ell migrtion nd invsion, it promotes metstti dissemintion, for exmple, through the hypoxi- nd -Met-dependent upregultion of hemokine reeptor CXCR4, whih promotes rest ner invsiveness nd orgn-speifi metstsis 22,23 (see elow). Chnges in ell dhesion The dherin swith Among the mny hnges in gene expression nd protein funtion tht our during tumour progression, ltertions in ell ell nd ell mtrix dhesion seem to hve entrl role in filitting tumourell migrtion, invsion nd metstti dissemintion. E-dherin, the proto type memer of the dherin fmily of lium-dependent ell ell dhesion moleules, is lost onomitntly with tumour progression in most epithelil ners 24, nd fored downregultion of E-dherin funtion in mouse model of rinogenesis promotes tumour invsion nd metstsis 25. The E-dherin promoter is frequently repressed y speifi trnsriptionl repressors, inluding Snil, Slug, SIP1, δef1, Twist nd E12/E47, nd y susequent promoter hypermethyltion Some of these repressors re speifilly expressed t the invsive front of humn ners, nd their expression seems to e highly regulted y pthwys known to promote tumour progression, inluding Wnt, TGF-, FGF, EGF, signl trnsduer nd tivtor of trnsription 3 (STAT3) nd nuler ftor-κb (NF-κB) signlling 27,30,31 (Box 1). Of the trnsriptionl repressors, the est-studied is Snil, highly unstle protein. It is rpidly phosphorylted y glyogen synthse kinse-3 (GSK-3) nd susequently degrded y the uiquitin protesome pthwy 32. Conversely, inhiition of GSK-3 funtion results in upregultion of Snil y n NF-κB-dependent pthwy, loss of E-dherin expression nd EMT 33. Additionl protein modifition y lysyl oxidses 2 nd 3 further stilizes Snil protein nd promotes EMT nd tumour invsion 34. E-dherin n lso e downregulted t the protein level. RTKs, suh s EGFR, -Met, IGF-1R, FGF reeptors (FGFRs) nd the non- RTK -Sr n indue phosphoryltion of E-dherin nd tenins, resulting in their uiquityltion y the E3 ligse Hki, nd susequent endoytosis nd degrdtion 35 (Box 1). Finlly, sereted proteses, suh 445

3 NATURE Vol My 2006 Box 1 Signlling pthwys eliiting epithelil mesenhyml trnsition, tumour-ell invsion nd metstsis Tumour-ell migrtion, invsion nd metstti dissemintion ll depend on hnges in ell ell nd ell mtrix dhesion. E-dherin is n importnt ell-dhesion moleule, nd in mny epithelil ners is lost onomitntly with tumour progression. The reeptor omplexes shown here n ll ontriute to loss of E-dherin funtion y mens of downstrem effetor signlling pthwys tht indue the expression of trnsriptionl repressors of E-dherin expression, suh s Snil, SIP1 nd Twist 26,27. During tumour progression, TGF- stimultes SMAD-medited signlling y inding to nd tivting its reeptors TGF-RI nd TGF-RII (). Suh TGF-- nd Rs-indued ltestge tumour progression seems to depend ritilly on NF-κB signlling 72. Moreover, Pr6, regultor of epithelil-ell polrity nd ell-juntion ssemly, interts with TGF- reeptors nd is itself sustrte for TGF-RII kinse. Phosphoryltion of Pr6 is required for TGF--indued epithelil mesenhyml trnsition, whih leds to uiquityltion of the smll GTPse RhoA y the E3 ligse SMURF1, RhoA degrdtion y the protesome, nd loss of tight juntions nd stress fires 73. Severl metstsis genes re regulted y TGF-, inluding those enoding CTGF, IL-11, nd prthyroid-hormone-relted peptide (PTHrP) ll ftors tht support osteolst differentition, osteolysis nd, therefore, one metstsis 64. Binding of HGF to the -Met reeptor results in the ssemly nd tivtion of signlling omplex () onsisting of the non-rtk -Sr, PI(3)K, the signlling dptor moleules GRB2, SHC nd GAB1, phospholipse Cγ (PLC-γ), the tyrosine phosphtse SHP2 nd the trnsription ftor STAT3. Binding of integrin α 6 4 to the -Met reeptor results in the integrin s phosphoryltion, therey generting dditionl doking sites for other dptor moleules nd signlling effetors, whih potentite HGF-indued invsion nd metstsis independent of the integrin s dhesion tivity 9. -Met lso oopertes with the hyulronn reeptor CD44. Its lterntively splied isoform CD44v6, whih hs previously een implited in tumour metstsis, seems to TGF-RII TGF- TGF-RI R-Smd Co-Smd CD44 HGF -Met Nuleus e required for the effiient stimultion of -Met y HGF 74. Interestingly, the ytoplsmi til of CD44, known to ind the tin-ytoskeletonmodulting ERM (ezrin/rdixin/moesin) proteins, is essentil for this funtion. ERM proteins link dhesion moleules to filmentous tin nd prtiipte in memrne nd ytoskeletl remodelling during ell migrtion. CD44v3, heprin-inding isoform of CD44, promotes HGF signlling y sequestering the HGF lignd nd presenting it to -Met reeptors, Snil, SIP1, Twist ERM ERM GRB2 SHP2 Rs GAB1 SHC PLC-γ STAT3 Twist Snil Twist SIP1 E-dherin α 6 4 integrin PI(3)K Cytoplsm d E-dherin IGFs α α IRS1 PI(3)K Rs PLC-γ Protesome IGF-1R leding to inresed -Met kinse tivity 75. IGF-1R-medited signl trnsdution is known to involve PI(3)K, PLC-γ, the Rs Rf MAPK pthwy () nd mmmlin trget of rpmyin (mtor), therey modulting protein synthesis, ell prolifertion nd survivl (see the min text for detils). IGF-1R lso interts with nd phosphoryltes E-dherin nd tenins, leding to their susequent internliztion nd protesoml degrdtion (d). α, α-tenin;, -tenin. s MMPs, indued y TGF- nd HGF/SF, n leve E-dherin nd disrupt dherin-medited ell ell ontts. Wht re the tumour-invsion-promoting signls eliited y the loss of E-dherin funtion? First, E-dherin loss disrupts dhesion juntions etween neighouring ells nd therey supports dethment of mlignnt ells from the epithelil-ell lyer. Seond, loss of E-dherin hs diret effets on signlling pthwys involved in tumour-ell migrtion nd tumour growth, inluding the nonil Wnt signlling pthwy nd Rho fmily GTPse-medited modultion of the tin ytoskeleton (Fig. 2). However, s prt of EMT, the loss of E-dherin is frequently ontrsted y the gin of expression of mesenhyml dherins, suh s N-dherin, whih enhne tumour-ell motility nd migrtion 24. Hene, in ddition to the loss of E-dherin, the gin of N-dherin (in other words, the dherin swith) my mke ritil ontriution to tumour invsion nd metstti dissemintion, not only y hnging the dhesive repertoire of tumour ell, ut lso y modulting vrious signlling pthwys nd trnsriptionl responses (Fig. 3). Immunogloulin-domin ell-dhesion moleules (IgCAMs) Memers of the immunogloulin superfmily hve ritil roles in tumour progression. For exmple, the ell-dhesion moleule (CAM) L1 is diret trget of Wnt/-tenin signlling in oloretl ner ells 36. It is highly expressed t the invsive front of oloretl ners, nd promotes motility, trnsformtion nd tumorigeniity in experimentl ell systems. L1 is frequently o-expressed with ADAM10, metlloprotese tht leves L1 nd uses shedding of its extrellulr domin. In similr mnner, neuronl CAM (NrCAM) is on fide trget of Wnt signlling nd onfers tumorigeniity nd motility on vrious tumour-ell types. It is highly upregulted in oloretl ners nd in melnoms 37. By ontrst, neurl CAM (NCAM) is downregulted in severl types of ner, nd its loss leds to the formtion of lymph node metstses in the Rip1Tg2 trnsgeni mouse model of pnreti -ell rinogenesis 38. Notly, nd similrly to N-dherin (Fig. 3), NCAM nd L1 ind to nd tivte FGFRs in neurons nd tumour ells, 446

4 NATURE Vol My 2006 INSIGHT REVIEW Filopodi Lmellipodi Wnt Dsh Frz Stress fires Cd42 α α R1 α Rho APC Axin GSK-3 PP P Axin TrCP APC Cytoplsm E-dherin E-dherin α α TCF Nuleus Protesome PP P Uiquitin Figure 2 Potentil signlling pthwys downstrem of the loss of E-dherin funtion., Following the loss of E-dherin funtion, -tenin () is sequestered y the denomtous polyposis oli (APC) xin GSK-3 omplex nd phosphorylted y GSK-3. This phosphorylted -tenin is speifiilly ound nd uiquitylted y TrCP, suunit of the E3 uiquitin ligse omplex. Uiquityltion ermrks -tenin for protesoml degrdtion., When the Wnt signlling pthwy is tivted, GSK-3 is repressed nd insted of eing phosphorylted, -tenin trnslotes to the nuleus. Together with TCF/Lef-1 trnsription ftors, it modultes the expression of severl trget genes involved in ell prolifertion nd tumour progression., On disssemly of the E-dherin dhesion omplex, displed tn represses the smll G protein RhoA nd tivtes R1 nd Cd42, whih together modulte the tin ytoskeleton nd the migrtory ehviour of tumour ells. (Filopodi re indued y Cd42, lmellipodi re indued y R1, nd stress fires re indued y RhoA.) α, α-tenin; Dsh, dishevelled; Frz, frizzled. therey modulting 1 integrin-medited ell mtrix dhesion, neurite outgrowth nd ell migrtion 39. Role of integrins Integrins re heterodimeri ell-surfe reeptors tht onsist of two trnsmemrne suunits, α nd, whih form distint integrin sutypes linking extrellulr mtrix (ECM) lignds, suh s fironetin, vitronetin, lminin nd ollgen, to the intrellulr tin ytoskeleton 40,41. Importntly, inding to these ECM omponents tivtes integrins, whih, in turn, indue intrellulr signlling sdes tht modulte ell prolifertion, survivl, polrity, motility nd differentition 42. Beuse there re mny integrin fmily memers with different funtions, the role of integrins in tumour progression remins elusive. To deth nd migrte, tumour ells depend on hnges not only in ell ell, ut lso in ell mtrix, intertion. Therefore, it is titly ssumed tht strong ell mtrix dhesions need to e resolved, wheres trnsient nd wek dhesions re prerequisite for migrtion. This omplex sitution hs mde it diffiult to determine the funtionl ontriution of ell mtrix dhesion to mlignnt tumour progression experimentlly 42. One of these pthwys involving integrin funtions is disussed in the ontext of -Met signlling 9 (Box 1). Another exmple of integrin-medited pro-metstti signlling ws provided y the disovery of periostin, protein tht is highly expressed in metstti oloretl ner 43. Periostin seems to prevent poptosis in oth ner ells nd endothelil ells y tivting the protein kinse B/Akt-medited survivl pthwy through integrin α v 3. Contriution of the tumour miroenvironment During the pst few yers, results from the ellulr nd moleulr dissetion of tumour progression hve led to the ide tht, esides the ellulr proesses nd moleulr pthwys tht exist in tumour ells themselves, n eqully importnt ontriution to mlignnt tumour progression omes from ells nd omponents of the tumour miroenvironment. These inlude endothelil ells nd murl ells of lood or lymphti vessels, tumour firolsts, infiltrting ells of the immune system nd the tumour s ECM 44. Crinom-ssoited firolsts A key experiment for estlishing the ontriution of stroml firolsts to tumour progression ws the demonstrtion tht firolsts of mlignnt ners re not identil to firolsts of the orresponding norml orgn. Non-tumorigeni prostte epithelil ells o-implnted with rinom-ssoited firolsts (CAFs) into immunoompromised mie form tumours, wheres norml firolsts n impir the growth of mlignnt tumour ells 45. Similrly, CAFs isolted from rest rinoms promote the growth of rest ner ells 46. CAFs resemle myofirolsts nd ffet ner ells, t lest in prt, through the prodution of stroml-ell-derived ftor 1 (SDF1), whih inds its ognte reeptor CXCR4 on tumour ells. CAFs re lso le to stimulte tumour ngiogenesis y ttrting endothelil preursor ells (EPCs). Finlly, expression of dominnt-negtive form of TGF-RII in stroml firolsts results in tumours of the prostte nd forestomh, proly through the upregulted expression of HGF 47. Cells of the immune system The immune system hs dul funtion in modulting tumour progression: repression of tumour growth y the dptive immune system (immunosurveillne) nd support of tumour progression y the innte immune system (inflmmtory response). But this view hs een hllenged. For exmple, the presene of T nd B ells is usully tken s 447

5 NATURE Vol My 2006 sign of tive immunosurveillne of the tumour nd, therefore, good prognosis. However, reent report using trnsgeni mouse model of skin rinogenesis demonstrtes tht T nd B ells re required for tumour mlignny 48. The innte immune system lso hs dul role in tumour progression. The presene of ells of the myeloid linege, suh s mrophges, grnuloytes, neutrophils nd mst ells, is inditive of good prognosis in some ners, ut seems to signify d news in mny other ner types. In prtiulr, monoytes nd their differentited/tivted derivtives, infiltrting tumour-ssoited mrophges (TAMs), re found in high numers in tumours 49,50. On the one hnd, they serete ytokines tht support the immune response, nd they promote ntigen proessing nd presenttion, resulting in n nti-tumour immune response. On the other hnd, there hve een mny reports tht the lne of sereted ftors is tilted towrds ytokine/hemokine milieu tht promotes tumour progression for exmple, y ngiogeni ftors, inflmmtory ytokines nd MMPs 51. Bone-mrrow-derived preursor ells, s well s monoytes nd TAMs, express numer of reeptors for lignds tht re sereted y tumour ells, inluding VEGF reeptor 1 (VEGFR1) for inding of VEGF-A nd plentl growth ftor (PlGF), whih, in turn, results in the reruitment of monoytes nd TAMs to the tumour environment. The funtionl ontriution of TAMs to tumour progression hs een demonstrted for instne, in mouse model of skin rinogenesis, in whih tumour ngiogenesis is indued y the expression of MMP9 y TAMs 52. Conversely, depletion of mrophges represses lte-stge tumour progression nd metstsis, ut not the development of primry tumours 53,54. Identifition of mny TAM sutypes, with potentilly different funtions, hs just egun. For exmple, in experimentl models, infiltrting Tie2-reeptor-expressing monoytes (TEMs) promote ngiogenesis nd ontriute to gliomgenesis 55. Infiltrting ells of the immune system, together with CAFs nd tumour ells themselves, seem to provide mny pro-invsive ftors, suh s proteses, survivl ftors nd ngiogeni ftors. Surprising insights into the potent role of proteses in tumour progression hve een otined through the trnsgeni expression of MMPs in rest epithelil ells, whih, unexpetedly, resulted in full-lown ner. For exmple, MMP3 expression results in trnsriptionl upregultion of the smll GTPse R1 nd upregulted levels of retive oxygen speies (ROS), whih, in turn, indue expression of Snil, loss of E-dherin expression, genomi instility nd tumour progression 56. MMPs n lso promote tumour-ell invsion nd metstti dissemintion y tivting the protese-tivted reeptor 1 (PAR1), G-protein-oupled reeptor implited in metstsis of vrious ner types. Although thromin is usully the min lignd for PARs, in xenogrft mouse model of rest ner it is predominntly MMP1, sereted y firolsts, tht inds nd tivtes PAR1 in ner ells, resulting in tumour-ell migrtion nd invsion 57. Metstti dissemintion Lymphogeni versus hemtogeni metstsis The formtion of new lood vessels during tumour progression is prerequisite for tumour outgrowth nd n e viewed s ontriuting to mlignnt tumour progression on the sis of two min oservtions 58,59. First, ngiogenesis is frequently indued y trnsforming signls tht promote tumour progression nd diretly upregulte the expression of ngiogeni ftors. For exmple, VEGF-A expression is indued y the Rs Rf MAPK (mitogen-tivted protein kinse) pthwy, or y hypoxi, whih lso indues the expression of other proto-onogenes, suh s -Met (see ove). Seond, ongoing ngiogenesis nd the susequent inrese in mirovessel density, together with the presene of inflmmtory sites, filitte invsive tumour ells to intrvste nd disseminte through the loodstrem. Conversely, reent orreltion studies in ner ptients s well s funtionl studies in mouse models hve indited tht lymphngiogenesis, the outgrowth of new lymphti vessels, n diretly promote the formtion of lymph node metstses, minly t the drining regionl lymph nodes of tumour 60,61. Lymphngiogenesis is indued y the lymphngiogeni memers of the VEGF fmily: y inding to VEGFR3 on the surfe of lymphti endothelil ells, VEGF-C nd VEGF-D stimulte the formtion of new lymphti vessels. Inflmmtory responses for exmple, those triggered y TNF-α, interleukin-1 (IL-1) nd NF-κB signlling seem to e involved in the regultion of VEGF-C expression. VEGF-D is the produt of n immedite erly, Fos-regulted gene nd its expression my, therefore, e regulted y vrious onogeni signlling pthwys 60. In onlusion, oth ngiogenesis nd lymphngiogenesis ontriute not only to primry tumour growth ut lso to the metstti dissemintion of tumour ells nd, together, offer ttrtive trgets for the development of nti-metstti therpies. Although the first ngiogenesis inhiitors re in linil use, we need to wit results on the omintoril inhiition of ngiogenesis nd lymphngiogenesis, nd its speifi effets on tumour metstsis. Cytoplsm Extrellulr spe FGFR Sr N-dherin PLC-γ FRS2 PKC PI(3)K MAPK d Sr PLC-γ PI(3)K FRS2 MMP γ-seretse CBP Solule N-dherin Nuleus Cytoplsm C-terminl domin of N-dherin Figure 3 The pleiotropi funtions of N-dherin. In response to loss of E-dherin, N-dherin is frequently upregulted during EMT ( proess known s the dherin swith). N-dherin hs severl funtions, ll of whih my ontriute to tumour invsion nd metstsis., Cell ell dhesion to N-dherin-expressing ells of the strom., Binding nd tivtion of FGFRs, whih results in the ssemly of lssil FGFR signlling omplex nd tivtion of downstrem phospholipse Cγ (PLC-γ), PI(3)K nd MAPK signlling pthwys, therey promoting ell survivl, migrtion nd invsion 39,70., Clevge nd shedding of the extrellulr domin of N-dherin y MMPs. Shedded N-dherin my neutrlize N-dherin-medited ell ell dhesion nd/or stimulte FGFR signlling on neighouring ells. d, Clevge of N-dherin y γ-seretse-like protese results in trnslotion of the roxy-terminl frgment of N-dherin to the nuleus, where it represses CREB-inding protein (CBP)-medited gene expression

6 NATURE Vol My 2006 INSIGHT REVIEW Orgn-speifi metstsis One unexplined phenomenon of tumour metstsis is the speifiity with whih ertin ner types metstsize to speifi orgns. Although the ntomy of the loodstrem nd lymphti dringe n explin some ptterns of metstsis, other guiding ues seem to e involved. For exmple, the hemokine reeptors CXCR4 nd CCR7 re frequently expressed on metstti rest ner ells, nd their lignds, SDF1/ CXCL12 nd CCL21, respetively, re expressed y lung nd regionl lymph nodes frequent sites of rest ner metstsis. Blokde of these lignd reeptor intertions hs resulted in redution of metstsis in experimentl mouse models 62. In similr mnner, metstsis to the liver nd lungs of oloretl ner ell line is impired when CXCR4 funtion is loked on tumour ells 63. Reent gene-expression profiling experiments with rest ner ell lines metstsizing to speifi trget orgns hve reveled list of interesting genes nd ftors. Signifint genes tht re expressed in rest ner ell lines olonizing one inlude IL-11, onnetive-tissue growth ftor (CTGF), CXCR4 nd osteopontin. Comintoril expression of these genes indues one metstsis in ells tht would not otherwise olonize one 64. Notly, the expression of CTGF nd IL-11 is under ontrol of the TGF- SMAD signlling pthwy desried ove. Genes tht diret rest ner ells to the lung inlude SPARC (sereted protein, idi, ysteine-rih), Id1 (inhiitor of DNA inding 1), MMP1, MMP2, VCAM1 (vsulr ell-dhesion moleule 1), IL-13Rα (interleukin 13 reeptor-α), COX2 (ylooxygense 2) nd CXCL1 (ref. 65). Their omintoril expression onfers the ility to metstsize to the lungs, wheres omintoril ltion of their expression impirs lung-speifi metstsis. Pthophysiologil hnges in the trget orgns lso ffet orgnspeifi metstsis. In rt metstti prostte ner model, MMP7 nd other proteses re upregulted t the one tumour interfe nd proteolytilly tivte the reeptor tivtor for NF-κB lignd (RANKL), whih susequently inds to its reeptor RANK on osteolsts. In turn, osteolsts re tivted to resor one nd indue osteolysis, therey relesing more MMP7 nd RANKL. Consistent with this notion, MMP7- defiient mie show redued osteolysis nd impired one metstsis 66. The intertion etween speifi reeptors on the surfe of disseminting tumour ells nd the trget orgn endothelium ould lso explin orgnspeifi metstsis. For exmple, phge-disply experiments designed to identify rest ner surfe proteins tht intert with the vsulture of the lung hve reveled metdherin, ell-surfe protein highly expressed in rest ner, s funtionl plyer in lung-speifi metstsis of rest ner ells 67. A reent thought-provoking report demonstrtes tht one-mrrow-derived VEGFR1-positive ells ply n intriguing prt in direting metstti tumour ells to speifi trget orgn: metstti tumour ells serete thus fr unknown ftors tht indue expression of fironetin speifilly in the metstti trget orgns. In turn, VEGFR1 ells re reruited to these sites, prepring the metstti nihe for the rrivl of tumour ells 68. Finlly, the outgrowth of seondry tumours my e nother level for the design of nti-metstti therpies, euse fter dissemintion to speifi orgns, tumour ells hve to dpt to nd ommunite with the new environment nd indue ngiogenesis 69. Outlook The mny stges of development of ftl metstsis re sed on multitude of ellulr nd moleulr mehnisms. There is good reson to elieve tht mny of the moleulr pthwys nd plyers tht re uslly involved my offer suitle trgets for the development of effiient nti-metstti therpies. 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Experimentl work in the uthor s lortory is supported y NCCR Onology, Swiss Ntionl Siene Foundtion, EU-FP6 frmework progrmme LYMPHANGIOGENOMICS LSHG-CT , EU-FP6 frmework progrmme BRECOSM LSHC-CT , Swiss Bridge Awrd, Kreslig Beider Bsel, nd Rohe Reserh Foundtion. Author Informtion Reprints nd permissions informtion is ville t npg. nture.om/reprintsndpermissions. The uthor delres no ompeting finnil interests. Correspondene should e ddressed to G.C. (gerhrd.hristofori@unis.h). 450