Protein tyrosine phosphatase 1B deficiency or inhibition delays ErbB2-induced mammary tumorigenesis and protects from lung metastasis

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1 Protein tyrosine phosphtse 1B defiieny or inhiition delys ErB2-indued mmmry tumorigenesis nd protets from lung metstsis Sofi G Julien 1,5, Ndi Dué 1,6, Mihelle Red 1, Jnie Penney 1, Mrilene Pquet 2, Yongxin Hn 3, Brin P Kennedy 3, Willim J Muller 4,5 & Mihel L Tremly 1,5 We investigted the role of protein tyrosine phosphtse 1B (PTP1B) in mmmry tumorigenesis using oth geneti nd phrmologil pprohes. It hs een previously shown tht trnsgeni mie with deletion muttion in the region of Er2 enoding its extrellulr domin (referred to s NDL2 mie, for Neu deletion in extrellulr domin 2 ) develop mmmry tumors tht progress to lung metstsis. However, deletion of PTP1B tivity in the NDL2 trnsgeni mie either y reeding with Ptpn1-defiient mie or y tretment with speifi PTP1B inhiitor results in signifint mmmry tumor lteny nd resistne to lung metstsis. In ontrst, speifi overexpression of PTP1B in the mmmry glnd leds to spontneous rest ner development. The regultion of ErB2-indued mmmry tumorigenesis y PTB1B ours through the ttenution of oth the MAP kinse (MAPK) nd Akt pthwys. This report provides rtionle for the development of PTP1B s new therpeuti trget in rest ner. Brest ner is the most frequent mlignny mong women, with n estimted 1 million new ses per yer worldwide 1. In individuls with rest rinoms, the proto-onogene ERBB2 (lso known s NEU), whih enodes reeptor tyrosine kinse (RTK) elonging to the epiderml growth ftor reeptor (EGFR) fmily, hs een oserved to e mplified 2. Although the other fmily memers, EGFR (lso known s HER-1 or ErB1), ErB3 (lso known s HER-3) nd ErB4 (lso known s HER-4) re lso expressed in vrious ners, mplifition of ErB2 ours in pproximtely 25% of ll rest ners 3 nd orreltes with negtive prognosis. Onogeni tivtion of ErB2 n e triggered either y point muttions in the region enoding its trnsmemrne domin 4,5 or y deletion or insertion in the extrellulr domin (ECD) 4,6,7. To explore the importne of ECD muttions in humn rest ner, trnsgeni mie (NDL2 mie) hve een generted tht overexpress, in mmmry glnd, n ltered Er2 (lso known s Neu) trnsgene ontining n in-frme deletion in its ECD 6. Trnsgeni NDL2 femles develop multiple mmmry tumors with frequent lung metstsis lesions. Tumor progression in this strin is ssoited with elevted levels of ErB2 nd ErB3 with phosphorylted tyrosine (ref. 7), whih hve lso een shown to e oexpressed in the mjority of humn rest ners 8,9. Another importnt similrity etween this mouse model nd humn rest ner is tht n lterntive splied form of ErB2 tht is nlogous to the Er2 deletion mutnt in NDL2 mie hs een deteted in humn rest ner 6,7,1,11. For exmple, when the tivted Er2 onogene is introdued into n immortlized nontumorigeni humn rest epithelil ell line, it results in mrked nd speifi inrese in PTP1B expression 12. Furthermore, 9% of ll rest tumors overexpress oth ErB2 nd PTP1B 13. The signifine of this link is unknown. Both geneti nd iohemil studies hve implited this enzyme s negtive regultor of RTK signling. For exmple, geneti evidene indites tht PTP1B is involved s negtive modultor in metoli diseses suh s oesity nd dietes Similrly, we reently reported the first in vivo geneti study suggesting tht removing PTP1B in p53-dependent tumorigenesis 17 leds to slight inrese in B ell lymphom, proly euse of n inresed numer of p53-defiient pre B-lymphoytes. Yet, we were surprised to find tht our in vitro experiments in immortlized firolsts demonstrted tht PTP1B exerts positive effets on Rs signling through p62 Dok nd p12rsgap regultion 18. In order to lrify the role of PTP1B in onogenesis, we used oth geneti nd phrmologil pprohes to investigte the funtion of PTP1B in rest ner. We show here tht mie lking Ptpn1 or mie dministered PTP1B inhiitor show ErB2-indued rest tumor lteny. Finlly, mouse mmmry tumor virus (MMTV)-dependent overexpression of PTP1B in mmmry glnd leds to spontneous 1 MGill Cner Centre, MGill University, 3655 Sir Willim Osler Promende, Montrel, Quee H3G 1Y6, Cnd. 2 Veterinry Pthology Servies, MIntyre Medil Sienes Building, MGill University, 3655 Sir Willim Osler Promende, Montrel, Quee H3G 1Y6, Cnd. 3 Merk Frosst Center for Therpeuti Reserh, Pointe-Clire, Quee H9R 4P8, Cnd. 4 Moleulr Onology Group, MGill University Helth Centre, Royl Vitori Hospitl, Montrel, Quee H3A 1A1, Cnd. 5 Deprtment of Biohemistry, MGill University, Montrel, Quee H3G 1Y6, Cnd. 6 Present ddress: Deprtment of Physiologil Chemistry, University Medil Center Utreht, Universiteitsweg 1, 3584 CG Utreht, The Netherlnds. Correspondene should e ddressed to M.L.T. (mihel.tremly@mgill.). Reeived 31 Otoer 26; epted 18 Deemer 26; pulished online 28 Jnury 27; doi:1.138/ng VOLUME 39 [ NUMBER 3 [ MARCH 27 NATURE GENETICS

2 Perentge of tumor free nimls T5 = (n = 12) T5 = 16 (n = 15) 18 NDL2-Ptpn1 +/ 15 NDL2-Ptpn1 +/ 12 T5 = 25 (n = 18) Age (d) rest ner development fter multiple pregnnies. Our findings thus support inhiition of PTP1B tivity s potentil new therpeuti pproh in humn rest ner. RESULTS Predominnt phenotype of MMTV-NDL2 Ptpn1-defiient mie To explore the role of PTP1B in ErB2-dependent rest tumor development, we rossed PTP1B-defiient mie (Ptpn1 / ) with the NDL2 trnsgeni mouse strin, whih hs een shown to develop mmmry rinoms 7. In order to elerte nd synhronize mmmry tumor onset, we initited multiple pregnnies y mting the femles with FVB mles 7. Tumors rose in synhronous mnner etween d of ge in ll NDL2 Ptpn1 +/+ mie, wheres the NDL2 Ptpn1 / mie did not develop tumors y tht stge (Fig. 1). Compred with the NDL2 Ptpn1 +/+ mie (medin time to tumor onset (T5) ¼ d), the time ourse of mmmry tumor development in the NDL2 Ptpn1 / mie ws signifintly delyed (T5 ¼ 25 d, P o.1). In ft, tumor development ws delyed y pproximtely 85 d in the NDL2 Ptpn1 / strin ompred with the NDL2 Ptpn1 +/+ mie. The first plple tumor in the NDL2 Ptpn1 / strin ourred t 147 d of ge, nd y dy 23, 9% of the nimls hd developed tumors. Notly, NDL2 Ptpn1 +/ mie showed n intermedite profile of tumor ourrene, suggesting tht the sene of one llele of Ptpn1 is suffiient to dely the onset of rest tumor development y 35 d ompred with NDL2 Ptpn1 +/+ mie (Fig. 1). These results indite tht the Ptpn1 gene dosge is limiting for tumorigenesis. Susequently, we killed mie t weeks.3, 4 nd 6 fter tumor ourrene nd onfirmed dignosis of mmmry tumorigenesis nd tumor multipliity y visul exmintion fter neropsy. Up to 4 weeks fter tumor formtion, there ws no signifint differene in tumor urden etween the groups. However, 6 weeks fter tumor ourrene, the NDL2 Ptpn1 +/+ mie showed twie s mny tumors (15 ± 2) thn the NDL2 Ptpn1 / mie (6.7 ± 1.8, P o.1) (Fig. 1). In ddition to monitoring mmmry tumor onset, we lso monitored the ourrene of lung metstti lesions. Four weeks fter rest tumor onset, ll NDL2 Ptpn1 +/+ mie developed lung metstses (Fig. 1), wheres the level of lung metstses ws ttenuted in Tumors per mouse (4) (5) (6) (4) (5) (6) (4) (5) (6) Stge of tumors (weeks) Perentge of nimls with lung metstsis weeks 4 weeks 6 weeks (/4)(5/5)(6/6) (/4)(1/5)(4/6) (/4)(2/5)(2/6) NDL2-Ptpn1 +/ Figure 1 Redued rte of tumor development, numer of tumors nd lung metstses in NDL2 Ptpn1 / mie. () Kpln-Meier kineti nlysis of tumor ourrene in NDL2 Ptpn1 femle trnsgeni mie. In order to detet mmmry tumors, mie were exmined twie per week fter wening of the seond litter. The urves were drwn nd nlyzed using Prism softwre. Log rnk tests of survivl plots of the dt indited sttistilly signifint differene etween eh survivl urve: NDL2 Ptpn1 +/+ versus NDL2 Ptpn1 +/ (P ¼.4); NDL2 Ptpn1 +/+ versus NDL2 Ptpn1 / (P ¼.1); nd NDL2 Ptpn1 +/ versus NDL2 Ptpn1 / (P ¼.1). The numer of nimls nlyzed for eh genotype (n) nd the medin time to tumor onset (T5) re lso shown. () Femle mie desried in were killed t different time points (.3, 4 or 6 weeks) fter tumor initition, nd the numer of tumors ws determined (men ± s.d.). Numers in prentheses represent the numer of mie per group. () Lung metstses were monitored y histopthologil nlysis from hemtoxylin nd eosin stined lung setions from ll nimls presented in. Numers in prentheses represent the frequeny of lung metstses. oth the heterozygous nd homozygous PTP1B-defiient NDL2 strins (Fig. 1). For exmple, 6 weeks fter mmmry tumor formtion, the penetrne of lung metstses ws deresed y 66.6% nd 33.3% (P ¼.1) in the sene of one or two lleles, respetively, of Ptpn1 in the NDL2 strin ompred with the prent strin (Fig. 1). Tken together, these results onstitute the first geneti evidene tht PTP1B ontriutes to ErB2-indued mmmry tumor ourrene nd mlignny. Although their mrosopi ppernes were very similr (Supplementry Fig. 1 nd Supplementry Methods online), mirosopi nlyses showed tht the histopthology of the mmmry glnd in the MMTV- NDL2 Ptpn1 / mie ws mrkedly different from tht of the NDL2 Ptpn1 +/+ mie (Fig. 2). Indeed, t.3 weeks fter plple tumor ourrene, NDL2 Ptpn1 +/+ mie presented mmmry prolifertive dutl lesions (Fig. 2), ompred with the enign dutl hyperplsi present in rest of NDL2 Ptpn1 / femles (Fig. 2). Four weeks lter, the morphology of the mmmry tissue ws dominted y denorinom infiltrting the ft pd in NDL2 Ptpn1 +/+ nimls (Fig. 2d), wheres the NDL2 Ptpn1-null mie showed enign intrdutl ppillom (Fig. 2e). Finlly, 6 weeks fter tumor ourrene, denorinoms were lso present in NDL2 Ptpn1-null femles (Fig. 2g). We oserved lrger tumor ell size, higher nuler-to-ytoplsmi rtio, nuler pleomorphism nd more prominent nuleoli in NDL2 Ptpn1 +/+ tumors (inset in Fig. 2f) s ompred with NDL2 Ptpn1 / tumors (inset in Fig. 2g), whih still presented some heterohromti ells within denorinoms. These results lerly demonstrte tht PTP1B defiieny in the NDL2 strin delys the progression from hyperplsi to lunt rinom, t lest during the first 6 weeks of mmmry glnd tumor development. ErB2 nd ErB3 levels during mmmry tumor progression In order to ssess the reltionship etween the rpid onset of multifol denorinoms nd high expression of ErB2 driven y the MMTV promoter in our niml model, we first exmined the level of tivted ErB2 during mmmry tumor development t.3, 4 nd 6 weeks fter tumor onset (Fig. 3). Tumor smples t 4 nd 6 weeks demonstrted high levels of totl nd tyrosine-phosphorylted ErB2 (referred to s phospho-erb2 ) ompred with levels in norml NATURE GENETICS VOLUME 39 [ NUMBER 3 [ MARCH

3 d f NB-FVB BT.3 BT.3 djent mmmry epithelium (Fig. 3), feture ssoited with rest tumor progression 7.InNDL2Ptpn1 +/+ tumor smples, phospho-erb2 levels were detetle t the time of tumor onset (Fig. 3) nd were higher thn in NDL2 Ptpn1 / tumor smples (Fig. 3, P o.1). As heterodimeriztion of ErB reeptors is ruil for ellulr signling, nd the ErB2-ErB3 reeptor pir forms the most potent signling module of the ErB-reeptor fmily in terms of ell growth nd trnsformtion 8, we exmined the kinetis of ErB3 expression during rest tumor progression from.3 to 6 weeks. Wheres the ErB3 nd ErB2 expression ptterns were quite similr (Fig. 3) nd the ErB3 reeptor ws overexpressed in tumor smples 7, we oserved muh higher expression of ErB3 in tumors expressing PTP1B ompred with the PTP1B-null smples t eh time point exmined (Fig. 3). Numerous tumors nd ell lines overexpressing ErB2 show high levels of tivted Sr 19, nd PTP1B hs een proposed to tivte Sr through dephosphoryltion of the inhiitory tyrosine Tyr529, therey providing mehnism y whih PTP1B overexpression might promote tumorigenesis 2. Some reserhers hve found tht PTP1B expression is higher in humn rest ner ell lines thn in norml rest ell line 2. In ontrst, others hve rgued for negtive role for PTP1B in signling downstrem of HER2, using v-sr in firolst models 21,22. These onfliting in vitro oservtions leve the reltionship etween PTP1B nd HER2 miguous. We exmined the reltive levels of Sr tivity nd expression in rest tumor smples of our NDL2 Ptpn1 +/+ nd NDL2 Ptpn1 / nimls (Supplementry Fig. 2 e g Figure 2 Distint mmmry glnd histopthology in NDL2 Ptpn1 +/+ mie ompred with NDL2 Ptpn1 / mie. ( g) Hemtoxylin nd eosin stined tissue setions of dominl mmmry glnd numer four from 3-monthold nontrnsgeni ontrol FVB mouse () nd from rest tumors (BT) from NDL2 Ptpn1 +/+ mie (,d,f) ndndl2ptpn1 / mie (,e,g) t.3,4 nd 6 weeks (BT.3, nd, respetively) fter tumor ourrene. The representtive setions were otined from the mie desried in Supplementry Figure 1. Arrows in f nd g indite mitoti figures. Sle rs: 5 mm (); 2 mm ( e); 1 mm (f,g) nd 2 mm (insets in f nd g). nd Supplementry Note online). Quntifition of the lots did not unover ny differene in Tyr529 phosphoryltion levels etween NDL2 Ptpn1 +/+ nd NDL2 Ptpn1 / rest tumor tissue. This ontrdits the urrent in vitro prdigm tht tivtion of Sr kinses is essentil for Er2-indued onogenesis. However, our in vivo results re in ordne with reent study tht ws the first to distinguish etween the kinse-dependent nd kinseindependent tions of Sr nd showed tht its kinse-dependent properties re not required for Er2-indued tumorigenesis 23. PTP1B defiieny downregultes the Rs-MAPK signling pthwy We next investigted the role of PTP1B in the ErB2 downstrem signling pthwys tht my ontriute to mmmry tumor development. The ErB2-ErB3 heterodimer potently triggers prolifertive signls through the Rs-MAPK pthwy 8. Moreover, we reently demonstrted tht PTP1B positively regultes Rs tivity y ting on p62 Dok nd p12rsgap 18. Bsed on these findings, we exmined whether the loss of PTP1B influenes Rs signling downstrem of ErB2-ErB3 in mmmry tumors (Fig. 4). As expeted, p62 Dok ws phosphorylted in smples t 6 weeks fter tumor onset, ut phosphop62 Dok ws signifintly enhned (y 48%) in PTP1B-defiient tumor smples (Fig. 4, P o.5). Previous studies hve shown tht p62 Dok inds to p12rsgap, resulting in inhiition of ERK tivtion 24,25. To determine whether tivted p62 Dok my hve diret impt on the Rs-MAPK pthwy through RsGAP, we immunopreipitted p12rsgap from rest tumor lystes t different time points fter tumor initition (.3, 4 or 6 weeks) nd performed protein lot nlysis using ntiodies to phosphotyrosine. Brest tumor smples 4 nd 6 weeks fter onset showed RsGAP phosphoryltion (Fig. 4) tht inversely orrelted with p62 Dok phosphoryltion (Fig. 4). RsGAP phosphoryltion ws ttenuted in the sene of PTP1B ompred with the wild-type smples (Fig. 4, P o.5), nd onsistent with our previous dt 18 there ppered to e deresed levels of RsGAP protein in the PTP1B-expressing smples ompred with the PTP1B-defiient smples. Furthermore, when we exmined p42/p44 MAPK phosphoryltion in these sme smples, we found tht MAPK tivtion ws totlly inhiited in NDL2 Ptpn1 / rest tumor smples (Fig. 4, P o.5). These results suggest tht the sene of PTP1B ttenuted the Rs-MAPK indued ErB2 tivtion in rest tumor progression. Indeed, PTP1B my t s positive regultor downstrem of ErB2 t lest through the downregultion of p62 Dok tivity, leding to inresed Rs nd p42/p44 MAPK tivtion. PTP1B defiieny downregultes the PI3K-Akt pthwy As previously mentioned, the ErB2-ErB3 heterodimer hs the pity to signl very potently through Rs-MAPK for prolifertion ut lso through the phosphoinositide-3 kinse (PI3K)/Akt pthwy for survivl 8. Akt is one of the most tivted serine/threonine kinse in humn ner 26, nd its tivtion ours during ErB2 34 VOLUME 39 [ NUMBER 3 [ MARCH 27 NATURE GENETICS

4 P-ErB2 ErB2 FVB mmmry tissue Totl lyste IP supernttnt BT.3 BT IP: ErB2 IB: 4G1 IP: ErB2 IB: ErB2 Phospho-ErB2/ErB2 rtio (ritrry units) overexpression. Thus, we exmined the effets of PTP1B defiieny on Akt tivtion in this ErB2-indued mmmry tumor model. In NDL2 Ptpn1 +/+ rest tumor smples s erly s tumor initition (.3 weeks) nd up to 6 weeks fter tumor onset, there ws sustined Ser473 Akt phosphoryltion in ll tumor smples (Fig. 5, P o.5). This ws in ontrst to the NDL2 Ptpn1 / smples, whih showed n ttenuted Akt phosphoryltion level ompred with NDL2 Ptpn1 +/+ smples nd deresed further during rest tumor progression (Fig. 5, P o.5) Stge of tumor (weeks) PTP1B ErB2 ErB3 Gr2 Both MAPK nd PI3K/Akt signling pthwys regulte ell yle progression y modulting the levels of effetors suh s ylin D1 nd the CDK inhiitor p27 kip (refs. 27,28). Moreover, ylin D1 is required for ErB2-indued tumorigenesis 29. Bsed on this dt, we explored the expression levels of these effetors in NDL2 Ptpn1 +/+ nd NDL2 Ptpn1 / rest tumor smples (Fig. 5,). Both groups showed BT.3 BT Figure 3 Redued expression levels of ErB2 nd ErB3 in rest tumors of NDL2 Ptpn1 / trnsgeni mie. Brest tumor tissue (BT) ws isolted from NDL2 Ptpn1 +/+ nd NDL2 Ptpn1 / trnsgeni mie t weeks.3 (BT.3), 4 () nd 6 () fter tumor ourrene. Norml mmmry epithelium djent to the tumors t week 6 fter tumor onset () ws used s ontrol. () ErB2 ws immunopreipitted from rest tumor tissue lystes, nd immunopreipittes were sujeted to SDS-PAGE. Immunolot (IB) nlysis ws performed using ntiodies to phosphotyrosine (4G1), followed y reproing with ntiodies to ErB2. Phosphorylted ErB2 (P-ErB2) nd ErB2 nds re indited y the rrows t moleulr weight 185 kd. Mmmry tissue from nontrnsgeni FVB mouse (lne 1) ws inluded s ontrol for endogenous ErB2 expression. The experiment ws performed three times, using three mie per time point in totl. Grph t right illustrtes the densitometry nlysis of the phosphorylted ErB2/ErB2 rtio otined from ll the experiments (men ± s.d.). P o.1 for NDL2 Ptpn1 +/+ versus NDL2 Ptpn1 /.() Brest tumor tissue (BT) lystes from NDL2 Ptpn1 +/+ nd NDL2 Ptpn1 / trnsgeni mie were seprted y 4% 12% grdient SDS-PAGE nd were trnsferred to PVDF memrne. The memrne ws lotted with ntiodies to ErB2 nd reproed with ntiodies to ErB3. The nti-gr2 immunolot is used s loding ontrol. The experiment ws repeted three times, using three mie per time point. Figure 4 Deresed Rs-MAPK signling in rest tumors of NDL2 Ptpn1 / mie. Brest tumor tissue (BT) ws isolted from NDL2 Ptpn1 +/+ nd NDL2 Ptpn1 / trnsgeni mie t weeks.3 (BT.3), 4 () nd 6 () fter tumor ourrene. Norml mmmry epithelium djent to the tumors t week 6 fter tumor onset (norml rest, ) ws used s ontrol. () Brest tumor tissue lystes (2 mg) were nlyzed y protein lotting with ntiodies to phospho-tyr449-p62dok nd were reproed with ntiodies to Dok1 to ensure equl loding. () p12rsgap phosphoryltion ws nlyzed y immunopreipitting p12rsgap, proing with ntiodies to phosphotyrosine (4G1) nd then reproing with ntiodies to p12rsgap. () Phosphoryltion of p42/p44 MAPK in rest tumor smples desried in. The smples were nlyzed for p42/p44 MAPK protein nd phosphoryltion levels using phosphospeifi ntiodies (ntiodies to phospho-thr22/ Tyr24-MAPK). Grphs t right illustrte the densitometry nlysis of the lots. Results represent the men ± s.d. of three independent experiments with totl of n ¼ 3 mie per group.p o.5 for NDL2 Ptpn1 +/+ versus NDL2 Ptpn1 /. P-p62Dok Y449 P-RsGAP RsGAP p62dok P-p44MAPK P-p42MAPK p44mapk p42mapk BT.3 BT.3 BT.3 BT BT.3 BT.3 IP: RsGAP IB: 4G1 IP: RsGAP IB: RsGAP Phospho-p62Dok/p62Dok rtio (ritrry units) Phospho-RsGAP/RsGAP rtio (ritrry units) P-p42p44/p42p44 rtio (ritrry units) BT.3 BT.3 BT.3 NATURE GENETICS VOLUME 39 [ NUMBER 3 [ MARCH

5 P-Akt S473 p27 Gr2 Akt Cylin D1 Gr2 7 8 BT.3 BT.3 BT Phospho-Akt/Akt rtio (ritrry units) Cylin D1/gr2 rtio (ritrry units) mrked derese in p27 kip expression during tumor progression (Fig. 5). In ontrst, nd in ordne with previous reports, ylin D1 ws overexpressed t the tumor initition stge nd up to 6 weeks fter tumor ourrene ompred with djent tissue in ontrol smples (Fig. 5) 3,31. Moreover, the ylin D1 protein levels showed reiprol response to p27 kip, with elevted expression oserved in sene of p27 kip (Fig. 5 versus Fig. 5). Consistent with the tumor progression dt desried ove, ylin D1 expression ws signifintly higher when PTP1B ws present (Fig. 5). These results strongly suggest tht the requirement for ylin D1 in ErB2-indued mmmry glnd tumorigenesis is dependent on PTP1B. PTP1B defiieny triggers erlier poptosis As hypertivtion of the PI3K/Akt pthwy in tumors overexpressing ErB2 hs een reported to prevent tumor ell poptosis in rest ner 32, we monitored the poptoti sttus in tumor smples y levge nlysis of poly(adp-riose) polymerse (PARP). Full-length PARP (115 kd) is known to e leved y tivted spse-3, whih yields n 85-kD levge produt. In NDL2 Ptpn1 +/+ nimls, PARP levge ws visile only 6 weeks fter tumor onset, wheres we redily deteted this levge event 2 weeks erlier in NDL2 Ptpn1 / mie (Fig. 6). To further onfirm these results, we lso determined spse-3 tivtion in these smples. As expeted, the 17-kD tivted spse-3 levge produt ws present in higher mounts in NDL2 Ptpn1 / rest tumor smples 6 weeks fter tumorigenesis onset thn in NDL2 Ptpn1 +/+ smples (Fig. 6), suggesting higher numer of poptoti events in rest tumor in the sene of PTP1B. As determined y immunohistohemistry of rest tumor smples t 6 weeks fter tumor ourrene, the perentge of proliferting ell nuler ntigen (PCNA)-positive nulei ws signifintly higher in NDL2 Ptpn1 +/+ tumors (85 ± 11%) thn in NDL2 Ptpn1 / tumors (36 ± 15%; P o.5; Fig. 6). In ontrst, the perentge of leved BT.3 BT.3 BT.3 p27/gr2 rtio (ritrry units) BT.3 BT.3 BT.3 Figure 5 Downregultion of Akt tivtion in NDL2 Ptpn1 / trnsgeni mie. Brest tumor tissue (BT) ws isolted from NDL2 Ptpn1 +/+ nd NDL2 Ptpn1 / trnsgeni mie t weeks.3 (BT.3), 4 () nd 6 () fter tumor ourrene. Norml mmmry epithelium djent to the tumors t week 6 fter tumor onset (norml rest, ) ws used s ontrol. () Wenlyzed2mg ofthesmples for Akt protein nd phosphoryltion levels using phosphospeifi ntiodies (ntiodies to phospho-ser473-akt). (,) p27 nd ylin D1 expression levels were determined in the rest tumor smples desried in. Gr2 expression ws used s loding ontrol. Grphs t right illustrte the densitometry nlysis of the lots. Results represent the men ± s.d. of three independent experiments; totl of n ¼ 3 mie per group.p o.5 for NDL2 Ptpn1 +/+ versus NDL2 Ptpn1 /. spse-3 positive ells ws lmost undetetle in NDL2 Ptpn1 +/+ tumors (1.2 ±.7%) ompred with NDL2 Ptpn1 / tumors (2.9 ± 1.1%; P o.5; Fig. 6). This suggests tht protetion from invsiveness in NDL2 Ptpn1- null mie my e due to oth deresed prolifertion nd inresed ell loss. PTP1B inhiitor dministrtion delys rest tumor development The geneti results presented here provide strong evidene tht PTP1B hs ritil nd positive role in humn ErB2-indued rest ner, nd they suggest tht inhiition of this enzyme my e enefiil in the tretment of this disese. To determine if PTP1B inhiition would provide the sme dely in tumorigenesis s ws oserved for the PTP1B geneti defiieny, we used speifi orlly ville PTP1B inhiitor 33 to tret NDL2 Ptpn1 trnsgeni mie. We monitored the effet of PTP1B inhiition in these nimls y mesuring gluose levels during the 21 d of tretment. PTP1B defiieny is known to enhne insulin sensitivity nd derese lood gluose levels. Tretment of the NDL2 trnsgeni mie with the PTP1B inhiitor resulted in lowering of lood gluose over the time ourse of tretment to the levels omprle to those oserved in the NDL2 Ptpn1 / mie. Tretment of NDL2 Ptpn1 / mie with the ompound hd no effet on lood gluose levels, suggesting speifi trget enggement (Supplementry Fig. 3 nd Supplementry Methods online). We determined the time ourse of tumor development (Fig. 7) nd found signifint dely in the onset of mmmry tumor development in the NDL2 Ptpn1 +/+ mie treted with PTP1B inhiitor (T5 ¼ 28 d) ompred with NDL2 Ptpn1 +/+ group tht ws treted with vehile (T5 ¼ 75 d, P o.1). Tumors rose in synhronous mnner etween 2 to 5 d fter the end of tretment in ll NDL2 Ptpn1 +/+ mie tht were treted with vehile. The first plple tumor in the NDL2 Ptpn1 +/+ group treted with PTP1B inhiitor ourred 48 d fter the end of tretment, nd 2 d lter, 9% of the nimls hd developed tumors, with signifint dely of pproximtely 29 d ompred with the NDL2 Ptpn1 +/+ mie treted with vehile. Two mie still remined tumor-free fter more thn 75 d fter the end of tretment. These results strongly suggest tht PTP1B my represent vlule therpeuti trget for ntiner pplitions. 342 VOLUME 39 [ NUMBER 3 [ MARCH 27 NATURE GENETICS

6 BT.3 BT.3 A PCNA B Cleved spse 3 PARP 116 kd 85 kd Cspse-3 Cleved spse BT.3 Tissue-speifi overexpression of PTP1B in rest In order to evlute the potentil of overexpressed PTP1B to promote mmmry glnd tumor initition nd progression, we generted trnsgeni mie in whih wild-type PTP1B, fused to enhned green fluoresent protein (EGFP), ws expressed speifilly in the mmmry glnd vi MMTV (Fig. 8). Trnsgeni protein expression diretly orrelted with inresing numers of litters (Fig. 8), with onomitnt mrked inrese in endogenous PTP1B protein levels (Fig. 8) nd mmmry glnd trnsformtion (Fig. 8). Indeed, mirosopi nlyses demonstrted tht the histopthology of the mmmry glnd in MMTV-EGFP-PTP1B trnsgeni mie differed mrkedly depending on the numer of litters delivered y the mothers. After only two pregnnies, mie presented usul mmmry BT.3 35 kd 17 kd 85%/11% 1.2%/.7% C 36%/15% dutl hyperplsi (Fig. 8) wheres fter seven litters, mie showed indued denorinom infiltrting the ft pd (Fig. 8). Supplementry Tle 1 online summrizes the phenotypi normlities derived from four different mouse lines. Tken together, our findings onfirm tht PTP1B lone is ple of driving the oserved rinom nd tht PTP1B possesses onogeni properties or t lest ontriutes positively to the trnsformtion proess on its own. DISCUSSION Previous studies of PTP1B tivity nd expression in vrious ner ells nd tumors hve unovered vrile expression levels nd onfliting effets of this phosphtse in onogenesis 16,34. To dte, the ext role of PTP1B in tumorigenesis remins unler. To nswer this question, we used geneti pproh y reeding Ptpn1-null mie 35 with rest ner trnsgeni strin expressing the Er2 mutnt (NDL2) driven y the MMTV-LTR promoter 6,7. The NDL2 mouse is well-estlished rest ner model 6 nd replites mny of the events tht our in humn rest tumor progression, inluding the ErB2 in-frme deletion muttion 7,1. In the present study, we report tht the geneti deletion or phrmologil inhiition of PTP1B results in delyed ErB2-indued rest tumorigenesis. In ddition, NDL2 Ptpn1 / mie re resistnt to lung metstsis, nd the effets my our through the regultion of ylin D1 expression s well s through poptosis triggered y the downregultion of MAPK nd AKT signling pthwys. To our knowledge, our results onstitute the first geneti nd phrmologil evidene tht PTP1B is ritil in rest tumor development. We found our results surprising, s PTP1B ts s negtive regultor of multiple RTKs, nd inhiition of this enzyme leds to inresed tivtion of these reeptors nd should promote onogeni signling. We hve reently reported tht this is indeed the se in p53- defiient kground, where geneti ltion of PTP1B elertes the rte of tumor development 17. However, in this se, the totl removl of PTP1B seems to led to n inrese in the pre B ell popultion, whih in the sene of p53 is seemingly more prone to develop D 2.9%/1.1% Figure 6 Inresed poptosis in rest tumor smples of NDL2 Ptpn1 / mie. () Protein lot nlysis of PARP expression nd levge from rest tumor lystes from NDL2 Ptpn1 +/+ nd NDL2 Ptpn1 / trnsgeni mie t.3 (BT.3), 4 () nd 6 () weeks fter tumor ourrene. The full-length (113-kD) nd the leved (85-kD) frgments re indited y the rrows. () Immunolot nlysis ws performed with ntiodies to spse-3 on rest tumor totl lystes s desried in. Full-length (35-kD) nd leved (17-kD) spse-3 frgments re indited y the rrows. The experiments desried in nd were repeted three times using three different mie for eh time point, nd similr results were otined. () Immunohistohemil detetion of PCNA-positive nd leved spse-3 positive ells (lk nd white rrows, respetively) in rest tumor smple 6 weeks fter tumor ourrene. Smples shown re representtive of three independent experiments with totl of n ¼ 3 mie per genotype. Results represent men ± s.d. The verge perentges of PCNA-positive nd leved spse-3 positive ells re indited in the lower left orner of eh pnel. Mgnifition: 63. Perentge of tumor free nimls T5 = 28 (n = 6) T5 = 75 (n = 6) Dys fter end of tretment - vehile - PTP1B inhiitor - vehile - PTP1B inhiitor Figure 7 PTP1B inhiitor dministrtion delys the onset of mmmry tumor development in NDL2 Ptpn1 +/+ mie. Kpln-Meier kineti nlysis of tumor ourrene in NDL2 Ptpn1 femle trnsgeni mie tht reeived the PTP1B inhiitor or vehile. Mie were exmined twie per week fter the end of tretment in order to detet mmmry tumors. The urves were drwn nd nlyzed using Prism softwre. Log rnk tests of survivl plots of the dt indite sttistilly signifint differene etween NDL2 Ptpn1 +/+ reeiving the inhiitor nd NDL2 Ptpn1 +/+ mie reeiving the vehile (P o.1). The numer of nimls nlyzed for eh genotype (n) nd the medin time to tumor onset (T5) re indited on the grph. NATURE GENETICS VOLUME 39 [ NUMBER 3 [ MARCH

7 Figure 8 Overexpression of PTP1B in rest indues mmmry glnd tumorigenesis. () Shemti digrm of the humn Ptpn1 trnsgene. The unshded region t left represents sequenes within the pbskk vetor kone. Arrow indites the trnsriptionl strt site within the MMTV- LTR, nd the shded gry ox to the right of the rrow indites the 5 nonoding sequenes derived from the MMTV-v-H-Rs vetor pa9. Relevnt restrition endonulese sites re lso identified in the figure. () We seprted 2 mg of totl rest or tumor tissue lystes from MMTV- EGFP-PTP1B trnsgeni mie y SDS-PAGE, trnsferred it to PVDF memrne nd immunolotted it with ntiodies to PTP1B. Brest tissue lyste from nontrnsgeni mouse (FVBN) ws used s negtive ontrol (lne 3). Equl loding ws verified y reproing the memrne with ntiodies to Gr2. () Hemtoxylin nd eosin stined tissue setions of dominl mmmry glnd numer four from MMTV-EGFP-PTP1B trnsgeni mie tht hd no litters (upper right), two litters (lower left) or seven litters (lower right). Tissue setion from nontrnsgeni FVBN mouse tht hd two litters is presented s ontrol (upper left). The representtive setions were otined from the mie desried in. Br¼ 2 mm. B lymphoms. Yet, in ordne with our present geneti dt, we hve previously demonstrted tht immortlized PTP1B-defiient firolsts show deresed Rs signling despite enhned signling of other pthwys 18. The question whether PTP1B ts s n onogene or tumor suppressor, s with other geneti modultors, depends lrgely on the preexisting geneti kground, lol environment nd ell type 36,37. For exmple, PTP1B-null mie do not develop tumors even t dvned ges. Alterntively, tissue-speifi overexpression of PTP1B in mmmry glnd leds to rest tumorigenesis, suggesting true onogeni property for this phosphtse. Notly, we oserve mrked inrese of endogenous PTP1B in mmmry glnd tissue with inresing numers of litters, onomitnt with rest trnsformtion. We hve previously oserved similrly inresed levels of endogenous PTP1B in immortlized ell lines ompred with levels in primry emryoni firolsts 38. Altogether, these findings lerly support positive role for PTP1B during immortliztion nd trnsformtion, proly in ell-utonomous mnner. Heterodimeriztion of the ErB2 nd ErB3 reeptors results in the tivtion of lrge network of signling pthwys tht medite enhned prolifertion nd/or survivl, leding to rest onogenesis nd metstsis 8,39. Previous studies hve reported tht ErB2 reversily redues p27 kip nd inreses ylin D1 levels through oth the MAPK nd Akt pthwys 31,4,41, leding to inresed survivl. Our results demonstrte tht loss of PTP1B dereses ErB2 tivity s well s ErB2 nd ErB3 expression levels during mmmry tumor progression. Consequently, derese in tivtion of downstrem effetors suh s MAPK nd Akt is oserved. This is in greement with reent studies reporting Akt tivtion in rest ner owing to preferentil oupling of ErB3 with the PI3K-Akt pthwys 32,36,42. Finlly, we propose tht PTP1B my diretly link ErB2/ErB3 signling to ylin D1 regultion nd p27 stiliztion vi its sustrte p62 Dok, thus susequently ting in synergisti fshion on oth the MAPK nd Akt pthwys. The indution of the ell-deth pthwy in NDL2 Ptpn1-null mie is n importnt feture, s it ould e omined with hemotherpy in the tretment of rest ner. For instne, resistne to hemotherpy-indued poptosis ould e overome y loking the nti ell deth pthwy with PTP1B inhiitor. Notly, ltion of PTP1B hs no onsequenes on dult mouse physiology, suggesting tht nti-ptp1b therpy might e highly seletive in preventing rest tumorigenesis. Moreover, s NDL2 Ptpn1 / nimls re resistnt to lung metstsis, shorter tretments with PTP1B inhiitor should e investigted in linil studies in individuls with rest ner. EGFP-PTP1B PTP1B Gr2 pbssk Mmm. Gl-2 litters Mmm. Gl-No litter FVBN Mmm. Gl Mmm. Gl-7 litters MMTV-LTR Tumor mmm. G-7 litters 77 kd 5 kd v-h-rs EGFP PTP1B WT SV4PolyA NheI BglII Trnsription termintor EoRI FVBN No litter 2 litters 7 litters Indeed, we hve otined promising results with PTP1B inhiitor in our in vivo ssy (Fig. 7). Finlly, ellulr signling driven y ErB2 must not e viewed s liner pthwy ut rther s omplex network with rosstlk nd redundny ourring etween multiple effetors 39. Indeed, reent reports hve demonstrted tht the simultneous lokge of different trgets produes more signifint inhiition of growth ompred with inhiition of only single omponent of speifi network 43,44. Numerous linil trils re urrently underwy in individuls suffering from rest rinom to improve strtegies using omintions of tretments ple of loking multiple effetors 44. Our findings hve therpeuti implitions, nd we propose tht individuls with ErB2-positive rest ner might enefit from phrmologil inhiition of PTP1B tivity in omintion with nti-erb2 therpies. METHODS Antiodies. For immunolotting nd immunopreipittion, we used ntiodies ginst the following proteins: phospho-thr22/tyr24-p42/p44 MAPK, p42/ p44 MAPK, spse-3, leved spse-3, phospho-ser473-akt, AKT nd p27 (Cell Signling Tehnology); phosphotyrosine 4G1, ErB3 nd PTP1B (Upstte Biotehnology); Gr2, ylind1, PARP nd Dok1 (Snt Cruz Biotehnology); p12rsgap (Trnsdution Lortories); phospho-tyr529-sr nd Sr (Biosoure Interntionl); nd ErB2 nd PCNA (EMD Biosienes). Antiody to phospho-tyr449p62dok ws gift of Amerin Proteomix. Mouse experimentl proedure. Ptpn1 +/ nd Ptpn1 / mie were generted s previously desried 35 nd were krossed with FVB/J wild-type mie for seven genertions to introdue the trgeted Ptpn1 lleles onto n FVB kground. Er2 trnsgeni mie were generted in n FVB strin y the expression of Er2 deletion mutnt (NDL2) DNA under the trnsriptionl ontrol of the MMTV-LTR 6,7. MMTV/NDL2 nd PTP1B mie were interred to generte femles of the required genotypes: NDL2 Ptpn1 +/+ (Er2 trnsgeni with wild-type PTP1B), NDL2 Ptpn1 +/ (Er2 trnsgeni with heterozygous PTP1B) nd NDL2 Ptpn1 / (Er2 trnsgeni, PTP1B-null). Genotypes for PTP1B nd Er2 were determined y DNA lot nd PCR nlysis, respetively 7,35. Mie were kept on 12h light/12h drk yle nd were llowed free ess to food nd wter. Animls were monitored dily for physil welleing nd were exmined twie week for tumor ourrene. We generted MMTV-EGFP-PTP1B trnsgeni mie y miroinjeting the linerized expression onstrut into the pronuleus of FVBN mouse emryos t dy.5 post oitum. Injeted emryos were implnted into pseudopregnnt CD-1 femle mie. Progeny were sreened y PCR of til iopsies. Mie positive for the trnsgene were mted to wild-type FVBN mie. All proedures were rried out ording to the Cndin Counil on Animl Cre ethil 344 VOLUME 39 [ NUMBER 3 [ MARCH 27 NATURE GENETICS

8 regultions nd were pproved y the MGill University Reserh nd Ethis Animl ommittee. PTP1B inhiitor tretment. PTP1B inhiitor ws synthesized t Merk Frosst Cnd, nd methods of synthesis hve een desried previously 33. PTP1B ompound ws dministered (3 mg/kg ody weight) dily y orl gvge with n injetion volume of 1 ml/kg ody weight for 21 d. Vehile solution onsisted of.5% methylellulose (Sigm Aldrih). Solutions were freshly prepred efore eh dministrtion. Mie were killed 1 d fter tumor ourrene. Mmmry tissue hrvesting. At hrvest, strom onsisting of dense interloulr onnetive tissue ws refully disseted from the tumor to exlude stroml ells in whole-tumor ell lyste used for the moleulr nlysis y immunolot. Tissue smples were ground into powder under liquid nitrogen nd lysed for 3 min on ie in modified TLCg lysis uffer (5 mm HEPES, ph 7.4; 1% glyerol; 1% Triton X-1, 15 mm NCl; 1 mm EGTA, ph 8.; 1 mm sodium orthovndte; 2 mm NF; omplete EDTA-free protese oktil inhiitors). The ell lystes were lered y entrifugtion t 12,g for 15 min t 4 1C. Immunopreipittion. Totl mmmry glnd lystes (2 mg) were inuted with 2 ml of protein A-Agrose nd with 2 mg of nti-erb2 ntiody for 3 h, rotting t 4 1C in4ml of modified TLCg lysis uffer. After five wshes with the lysis uffer, preipitted proteins were relesed y oiling in smple uffer nd were then sujeted to protein lot nlysis. Immunolotting. We seprted 2 mg of totl mmmry glnd protein y SDS-PAGE using 4% 12% grdient gels or 1% gels nd then trnsferred the protein to PVDF memrnes, whih we then loked with 6% (wt/vol) milk/ 3% BSA in TBS ontining.1% Tween-2. Memrnes were proed overnight with primry ntiodies ( 1:1, dilution ws used for ll ntiodies). After inution with speifi HRP-onjugted ntiodies, protein nds were deteted using the ECL detetion system. Histologil nlysis. Complete neropsies were performed nd tissues used for histology nd immunohistology were fixed in 1% neutrl uffered formlin nd emedded in prffin, setioned t 4 mm, stined with hemtoxylin nd eosin nd exmined for pthologil findings. PCNA-positive nulei nd leved spse-3 positive ells were visulized in prffin setions. Horserdish peroxidse linked seondry ntiodies were used (Cell Signling Tehnology). The olor retion ws developed using 3-3 diminoenzidine tetrhydrohloride (DAB) s hromogen (SigmFst DAB tlets; Sigm). Aession odes. GenBnk: Mus musulus Ptpn1, BC1191. Note: Supplementry informtion is ville on the Nture Genetis wesite. ACKNOWLEDGMENTS We thnk N. Sonenerg nd S. Hrdy for disussions during the ourse of this work. We re grteful to N. Uetni nd M. Nrlis from the MGill Cner Centre Developmentl Histology Fility for histologil nd immunohistohemil protools nd helpful dvie. We knowledge the Merk Frosst Cnd mediinl hemists for the gift of the inhiitor ompound used in this study. N.D. is reipient of Cndin Institutes of Helth Reserh dotorl wrd nd n Alexnder MFee Memoril Fellowship. W.J.M. holds Cnd Reserh Chir in Moleulr Onology. M.L.T. is Cherheur Ntionl of the Fonds de Reherhe en Snté du Quée. This work ws supported y Cndin Institutes of Helth Reserh operting grnt (MOP-62887) nd the Jenne nd Jen-Louis Chir in Cner Reserh (M.L.T.). AUTHOR CONTRIBUTIONS S.G.J. performed the reserh, nlyzed the dt nd wrote the mnusript. Bkrossing ws performed y M.R. nd J.P. Histopthology nlysis ws done y M.P. PTP1B inhiitor ws synthesized y Y.H. nd B.P.K. W.J.W. generted the ErB2 trnsgeni mie. This study ws designed nd oordinted y S.G.J. nd M.L.T. N.D. nd B.P.K. ontriuted ritil omments on the mnusript. COMPETING INTERESTS STATEMENT The uthors delre tht they hve no ompeting finnil interests. Pulished online t Reprints nd permissions informtion is ville online t reprintsndpermissions 1. Bselg, J. & Norton, L. Fous on rest ner. Cner Cell 1, (22). 2. Slmon, D.J. et l. 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