Restoration of p53 function leads to tumour regression in vivo. p53 locus. Targeting vector DTA LSL. Targeted allele

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1 Vol Ferury 27 oi:1.138/nture5541 Restortion of funtion les to tumour regression in vivo Anre Ventur 1, Dvi G. Kirsh 1,2, Mrgret E. MLughlin 1, Dvi A. Tuveson 1, Jn Grimm 3, Lur Lintult 1, Jmie Newmn 1, Elizeth E. Rezek 1, Rlph Weissleer 3 & Tyler Jks 1,4 Tumorigenesis is multi-step proess tht requires tivtion of onogenes n intivtion of tumour suppressor genes 1. Mouse moels of humn ners hve reently emonstrte tht ontinuous expression of ominntly ting onogene (for exmple, Hrs, Krs n My) is often require for tumour mintenne 2 5 ; this phenotype is referre to s onogene ition 6. This onept hs reeive linil vlition y the evelopment of tive ntiner rugs tht speifilly inhiit the funtion of onoproteins suh s BCR-ABL, -KIT n EGFR 7 1. Ientifying itionl gene muttions tht re require for tumour mintenne my therefore yiel linilly useful trgets for new ner therpies. Although loss of funtion is ommon feture of humn ners 11, it is not known whether sustine intivtion of this or other tumour suppressor pthwys is require for tumour mintenne. To explore this issue, we evelope Cre-loxP-se strtegy to temporlly ontrol tumour suppressor gene expression in vivo. Here we show tht restoring enogenous expression les to regression of utohthonous lymphoms n sroms in mie without ffeting norml tissues. The mehnism responsile for tumour regression is epenent on the tumour type, with the min onsequene of restortion eing poptosis in lymphoms n suppression of ell growth with fetures of ellulr senesene in sroms. These results support efforts to tret humn ners y wy of phrmologil retivtion of. Biohemil n geneti stuies hve emonstrte tht respons to genotoxi n onogeni stresses y inuing ell yle rrest or poptosis 11. Beuse onogeni stress n persist fter intivtion, loss of funtion my not only ply role in the erly stges of tumour evelopment, ut lso e require for the ontinue prolifertion or survivl of n estlishe tumour To test this hypothesis, we use geneti strtegy to restore enogenous expression in primry, utohthonous tumours. Mie rrying retivtle knokout llele were generte y inserting trnsription trnsltion stop ssette flnke y loxp sites (LSL) in the first intron of the enogenous wil-type lous (Fig. 1, ). When in ple, the STOP ssette effiiently prevents expression of the gene (Fig. 1, ). Cells from homozygous LSL/LSL (referre to herefter s -LSL) mie re funtionlly equivlent to null ( 2/2 ) ells, s emonstrte y the sene of ell-ultureinue senesene (Fig. 1e) n y their mrke geneti instility lox 1 STOP lox STOP lous DTA Trgeting vetor Trgete llele LSL LSL/LSL LSL/ Cre + LSL/LSL / + p21 Atin DAPI Anti- e f A-LZ A-Cre Figure 1 Genertion of the -LSL llele., The -LSL llele ws generte while engineering point-mutnt llele 31 when homologous reomintion ourre etween the LSL ssette n the point muttion (sterisk)., Genotyping of -LSL mie y PCR (polymerse hin retion) mplifition of til DNA., Western lot of pssge 5 MEFs infete with empty or Cre-expressing reominnt enoviruses Popultion oulings LSL/LSL Pssges 1 Center for Cner Reserh, Msshusetts Institute of Tehnology, Cmrige, Msshusetts 2142, USA. 2 Deprtment of Rition Onology, 3 Center for Moleulr Imging Reserh, Msshusetts Generl Hospitl, Boston, Msshusetts 2129, USA, n Hrvr Meil Shool, Boston, Msshusetts 2115, USA. 4 Howr Hughes Meil Institute, Chevy Chse, Mryln 2815, USA. These uthors ontriute eqully to this work. 27 Nture Pulishing Group / P3 P9 2n 4n / LSL/LSL 2n 4n (Aeno-empty n Aeno-Cre)., Anti- immunostining of LSL/LSL (tht is, -LSL) MEFs infete with Aeno-LZ or Aeno-Cre n then trete with oxoruiin (.2 mgml 21 ; 13 h). DAPI, 4,6-imiino-2- phenylinole. e, 3T3 protool on LSL/LSL, 2/2 n wil-type () MEFs. f, DNA ontent of erly (P3) n lte (P9) pssge LSL/LSL or 2/2 MEFs. Diploi (2n) n tetrploi (4n) DNA ontent is inite. 661

2 NATURE Vol Ferury 27 (Fig. 1f). However, owing to the presene of flnking loxp sites, the STOP ssette n e exise y the Cre reominse 15, thus restoring expression of the enogenous gene (Fig. 1, ). Beuse mie lking re tumour-prone 16,17, this system offers n opportunity to stuy the onsequenes of retivtion in primry tumours in vivo. To temporlly ontrol retivtion in vivo, we hve lso generte mie rrying Cre-reominse-Oestrogen-Reeptor-T2 (Cre-ER T2 ) llele trgete to the uiquitously expresse ROSA26 lous (Supplementry Fig. 1, ). The ER T2 moiety retins the Cre reominse in the ytoplsm until tmoxifen ministrtion releses this inhiition 18, thus permitting the reomintion of genomi loxp sites. Effiient tmoxifen-inue Cre-meite reomintion throughout the oy ws emonstrte y rossing Cre-ER T2 mie to mie rrying Cre-responsive -gltosise reporter llele 19 (Supplementry Fig. 1). Next, Cre-ER T2 n -LSL mie were rosse to generte ohorts of -LSL homozygous mutnt nimls rrying the Cre- ER T2 llele ( LSL/LSL ;Cre-reominse-Oestrogen-Reeptor-T2, herefter referre to s -LSL;Cre-ER T2 ) s well s -LSL homozygotes without the Cre-ER T2 llele (herefter referre to s - LSL). To elerte tumour formtion, some of these mie were irrite shortly fter irth, s rition ereses tumour lteny in null mie 2. These nimls unerwent perioi mgneti resonne imging (MRI) to etet the presene of ner (Fig. 2). After tumour etetion, the mie were trete with tmoxifen n then re-imge t ifferent times therefter. As shown in Fig. 2, in the mjority of -LSL;Cre-ER T2 mie, tmoxifen tretment use regression of utohthonous lymphoms n sroms (Fig. 2,, f; Supplementry Fig. 2, Supplementry Movies). This effet ws ue to retivtion rther thn tmoxifen tretment lone, euse in Cre-ER T2 -negtive -LSL mie, tumours rpily progresse espite tretment (Fig. 2 f). Figure 2f summrizes the responses oserve in the nimls stuie. Out of 1 Cre-ER T2 -positive tumours trete (6 thymi lymphoms, 3 sroms n 1 intr-ominl lymphom), 7 showe regression rnging from 46% to 1%. In two tumours ( thymi lymphom n n osteosrom), tmoxifen prevente tumour progression ut file to erese the size of the neoplsi (Fig. 2f; Supplementry Fig. 3). In one thymi lymphom tumour progression ws oserve espite tmoxifen tretment (Fig. 2f; Supplementry Fig. 3). DNA n immunohistohemil nlysis inite tht in this se resistne to tmoxifen ws ue to the speifi eletion of the Cre-ER T2 llele in the tumour (Supplementry Fig. 3e, f). In ontrst, ll Cre- ER T2 -negtive tumours (two thymi lymphoms, two ominl lymphoms n three sroms) progresse espite tmoxifen ministrtion (Fig. 2 f). These results emonstrte tht sustine intivtion is require for tumour mintenne in utohthonous lymphoms n sroms in the mouse. To stuy the mehnisms responsile for tumour regression, tumours from -LSL;Cre-ER T2 mie were nlyse 24 or 48 h fter tmoxifen tretment. In thymi lymphoms, tumour regression ws lrey pprent t 48 h (pproximtely 75% reution in tumour volume; see Fig. 3). In ition, expression ws etetle s erly s 24 h following tmoxifen tretment n ws ssoite with wiespre poptosis (Fig. 3). We next erive three thymi lymphom ell lines, two from LSL/1 ;Cre-ER T2 mie n one from -LSL;Cre-ER T2 mouse. An itionl line, to e use s ontrol, ws erive from thymi lymphom rising in 1/2 mouse. In ll ses, expression ws unetetle y western lot (t not shown), suggesting tht in the tumours rising in LSL/1 n 1/2 mie expression of the wil-type llele h een lost. 4-hyroxytmoxifen (4-OHT) tretment use ell eth in the -LSL;Cre-ER T2 -positive ell lines ut not in the ontrol ells (Fig. 3). A time-ourse experiment with the -LSL;Cre-ER T2 thymi lymphom ell line showe tht ell eth egn within 12 h fter 4-OHT ministrtion n ws virtully omplete y 96 h (Fig. 3). Moreover, just s the primry thymi lymphoms showe poptoti Irrite Monitor y MRI No Tumour present? Yes i.p. to retivte Repet MRI Neropsy/erive tumour ell lines m Dy Dy 12 Dy 18 Dy 28 No tumour (m 3 ) Dy Dy Srom (m 3 ) e f m Dy Dy 8 m Dy Dy 7 Figure 2 restortion les to tumour regression in vivo., Flow hrt of the strtegy use to etermine tumour response. i.p., intrperitonel., MRI imges (top) n tumour volumes (ottom) of -LSL;Cre-ER T2 (, ) n -LSL (, e) mie in response to tmoxifen (rrows). The tumours (sterisks) were n ominl lymphom (), two thymi lymphoms (t. lymphom; n, white sterisks) n two sroms ( n 662 Srom Srom Osteosrom Srom Srom Osteosrom 27 Nture Pulishing Group Chnge in tumour volume (%) CRE-ER + CRE-ER e, re sterisks). The volumes were lulte from the ville MRI sequenes (n 5 2 to 6) for eh time point, n re shown s men 1 1 s.. f, Summry of mximl responses to tmoxifen of tumours from Cre-ER T2 - positive (grey rs) n Cre-ER T2 -negtive (lue rs) mie. Asterisks inite tumours from Cre-ER T2 -positive mie with limite or no response (see lso Supplementry Fig. S3).

3 NATURE Vol Ferury 27 ell eth (Fig. 3), immunolot for tivte spse-3 n flowytometry nlysis of nnexin-5 positive ells (Fig. 3e n t not shown) emonstrte tht poptosis ws the primry onsequene of retivtion in this ontext. Interestingly, the ell yle istriution of the vile lymphom ells i not hnge in response to 4-OHT ministrtion (Fig. 3e), espite inution of the p21 ylinepenent kinse inhiitor (t not shown). Compre to lymphoms, srom regression upon tmoxifen tretment ws more elye, with only moest reution t 48 h (Fig. 4). Furthermore, lthough ws lerly expresse in the sroms t 24 n 48 h (Fig. 4 n t not shown) extensive poptosis ws not evient t these time points (Fig. 4). Despite the sene of ler inrese in poptosis, prolonge tmoxifen tretment le to regression of sroms (Fig. 2, f; Supplementry Figs 4 n 5), with the resiul mss lrgely ompose of neroti tissue (Supplementry Fig. 5). In orer to etermine the mehnism unerlying srom regression, we investigte the onsequenes of restortion in two ell lines erive from -LSL sroms. m PI + ells (%) Dy Dy 2 4-OHT A B C D Vile ells (%) Mok 4-OHT Time fter 4-OHT (h) H&E Anti- TUNEL e Apoptoti ells (%) Cell-yle istriution (%) Cre-ER T2 pos Cre-ER T2 neg Time fter 4-OHT (h) CC3 Vinulin G2/M Figure 3 restortion in lymphoms les to poptosis., MRI imges (top) n tumour volumes (ottom) of -LSL;Cre-ER T2 lymphom. Error rs, 1 1 s.., immunohistohemistry n TUNEL stining in thymi lymphoms fter tmoxifen tretment. H&E, hemtoxylin n eosin., Viility of thymi lymphom ell lines erive from Cre-ER T2 - positive mie (lines A C), n 1/2 mouse (line D) trete with vehile (ethnol) or 25 nm 4-OHT for 48 h. De ells were ientifie y their retention of propiium ioie (PI1)., Time ourse of ell viility of line A trete with vehile (mok) or 25 nm 4-OHT. e, Line A ws trete with 4-OHT n nlyse for poptosis (nnexin V stining, top pnel), expression of, leve spse 3 (CCR) n vinulin (western lots, mile pnel) n ell yle istriution (PI stining, ottom pnel). S G1 m3 Srom line A C Srom line B h fa A-Cre Bright fiel SAβGl h Cre-ER Ki67 ph3 p16 p15 27 Nture Pulishing Group Dy Dy 2 CC3 Vinulin Cell numer X h 48 h Control Anti- H&E TUNEL A-empty A-Cre 2 4 6ys g Cre-ER + Cre-ER 4-OHT + + BrU positive ells (%) p15 p16 DR2 De1 Tuulin e i LSL/LSL p-h3-positive ells per fiel Cre-ER LSL/LSL Cre-ER / Cre-ER + Figure 4 restortion in sroms les to growth suppression with fetures of ellulr senesene., MRI imges (top) n tumour volumes (ottom) of -LSL;Cre-ER T2 soft tissue srom (STS). Error rs, 1 1 s.., immunohistohemistry n TUNEL stining of -LSL;Cre- ER T2 STSs fter tmoxifen tretment. The ontrol is Cre-ER T2 -negtive srom., -LSL srom ells were infete with Aeno-Cre n hrveste for immunolot. Lne C, positive ontrol for leve spse 3., Prolifertion ssy. Averge of two inepenent ell lines 6 1 s.. is shown. e, BrU (5-romoeoxyuriine ) inorportion in srom ells infete with Aeno-GFP (lk rs) or Aeno-Cre-GFP (grey rs). Error rs, 1 1 s.. f, Senesene-ssoite -gltosise (SAGl) stining of srom ell line 5 ys fter infetion with Aeno-empty or Aeno-Cre. g, Immunolot nlysis of senesene mrkers in -LSL;Cre-ER T2 n -LSL srom ells 11 ys fter 4-OHT tmoxifen tretment. h, Immunohistohemistry nlysis of four sroms 5 6 ys fter in vivo tmoxifen ministrtion. i, Quntifition of phospho-histone H3 positive ells in sroms from h. Averge of seven rnom 43 fiels 1 1 s.. 663

4 NATURE Vol Ferury 27 Consistent with the results in vivo, expression of Cre reominse in these ell lines file to inue signifint poptosis (Fig. 4). Inste, restortion of expression suppresse prolifertion (Fig. 4) n inue ell yle rrest (Fig. 4e). In ition, these ells lost their spinle morphology, ppere flt n enlrge, n mny of them expresse senesene-ssoite -gltosise (Fig. 4f). Reent work hs le to the ientifition of numer of proteins whose expression is inrese in senesent pre-neoplsti lesions 21. These mrkers inlue the k-inhiitors p15-ink4 n p16-ink4, s well s DR2 n De1. We exmine their expression in srom ell lines erive from -LSL;Cre-ER T2 n -LSL mie. Only the Cre-ER T2 -positive srom ells inue p15-ink4, p16-ink4, DR2 n (to lesser extent) De-1 in response to tmoxifen tretment (Fig. 4g). We next exmine whether ell yle rrest n senesene mrkers re similrly inue y restortion in sroms in vivo. Senesene-ssoite -gltosise positive ells were oserve in one out of three Cre-ER T2 -positive sroms trete with tmoxifen for 5 6 ys, ut in none of the Cre-ER T2 -negtive ontrols (Supplementry Fig. S6, n t not shown). Consistent with the t otine in srom ell lines, p15-ink4 n p16-ink4 expression ws higher in the tmoxifen-trete Cre-ER T2 -positive sroms, ompre to the Cre-ER T2 -negtive ones (Fig. 4h). Although Ki67 stining ws not signifintly ifferent fter restortion in these tumours, n nlysis of phospho-histone H3 revele mrkely lower frtion of prolifertion in the tmoxifen-trete Cre-ER T2 -positive sroms (Fig. 4h n i). Tken together, these results inite tht the min onsequene of restortion in sroms is ell yle rrest with some fetures of senesene. We i not oserve upregultion of ll senesene mrkers in every srom stuie in vivo, whih my reflet the geneti n histologil heterogeneity etween the spontneous mesenhyml tumours nlyse here n epithelil pre-neoplsti lesions reporte y others 21. Furthermore, owing to the reltively low iniene of sroms in null nimls, our nlysis of senesene mrkers in vivo hs een reltively limite to te. We ntiipte tht with further stuies of restortion in sroms, we will e le to lrify the rrest/senesent phenotype of ifferent srom sutypes more fully. Although tmoxifen tretment of -LSL;Cre-ER T2 mie les to exision of the STOP ssette in oth neoplsti n norml tissues, expression of the protein ws etetle y immunohistohemistry only in tumour ells (Figs 3 n 4, n t not shown). Furthermore, histopthologil nlysis revele no signs of - meite toxiity in norml orgns fter ute retivtion (t not shown). These results inite tht in null mie, trnsforme ells re uniquely prime to stilize n tivte. In estlishe tumours, one potentil stimulus for protein stiliztion n tivtion is persistent onogeni stress. Beuse the tumour suppressor gene p19 Arf is inue in response to onogeni stimuli n is well known tivtor of (ref. 22), we nlyse its expression levels in tumours from -LSL nimls. Strikingly, high p19 Arf expression ws frequently oserve in oth lymphoms n sroms ut not in the norml tissues nlyse (Supplementry Fig. 7 n t not shown). These results suggest tht in lymphoms n sroms, p19 Arf might prime tumour ells to respon to retivtion. In this stuy, we hve use novel geneti strtegy for restoring enogenous tivity in ells n in mie. By omining retivtle loss-of-funtion llele together with temporlly regulte Cre reominse, we emonstrte tht sustine intivtion of is require for the mintenne of primry, utohthonous lymphoms n sroms. This pproh my e of generl pplition for the stuy of other tumour suppressor gene pthwys. Interestingly, the mehnism of tumour regression fter restortion ppers to e tumour type speifi. In lymphoms, restortion of use wiespre poptosis, wheres the mjor onsequene of retivtion in sroms ws ell yle rrest with Nture Pulishing Group fetures of ellulr senesene. Beuse sroms lso regresse following retivtion in vivo, it is possile tht senesent ells re rpily lere from the tumour mss. This hypothesis is onsistent with results otine in mouse moel of liver ner in n ompnying pper 23. Alterntively, srom regression my e onsequene of -epenent effets on tumour vsulture 24,25 or on other stroml omponents. The effet is lso tumour speifi in tht norml ells ppere not to respon to retivtion. Thus, this represents n extremely promising ntiner strtegy with ro therpeuti winow. These results re lso onsistent with work in other moel systems. For exmple, mouse emryo firolsts (MEFs) trnsforme y etopilly expressing n onogeni K-Rs llele in the setting of oxyyline-regulte knok-own enter senesene upon restortion of expression 26. Tumours otine y injeting these ells into nue mie lso regresse in response to oxyyline tretment 26. Likewise, immortlize MEFs from oestrogen-reeptor(tmoxifen) fusion knok-in mouse strin unergo senesene fter tretment with tmoxifen 27, n temporrily tivting in these mie elys tumour evelopment 28. Owing to the prevlene of pthwy intivtion in humn ners, severl phrmologil strtegies ime t restoring funtion hve een propose. These inlue smll moleules tht restore point-mutnt proteins to trnsriptionlly ompetent onformtion 13, s well s ompouns tht interfere with the Mm2 intertion 29 n gene-therpy-se pprohes ime t introuing wil-type opy of the gene into tumour ells 3. Provie tht humn ners, like the mouse ners stuie here, remin epenent on sustine intivtion for tumour mintenne, our results len strong support to suh therpeuti efforts. METHODS A etile esription of mterils n methos is given in Supplementry Informtion. Genertion of -LSL mie. The -LSL llele ws otine s y-prout of our efforts to generte R27H point mutnt llele 31. All exons n intron exon ounries were sequene to ensure the sene of muttions. Oligonuleotie sequenes for genotyping re ville on request. Genertion of R26-Cre-ER T2 mie. A plsmi ontining the Cre-ER T2 omplementry DNA (pcre-er T2 ) ws otine from the lortory of P. Chmon 19. This DNA ws trgete to the ROSA26 lous s esrie in etil in the Supplementry Informtion. tretment. All niml stuies n proeures were pprove y the MIT Institutionl Animl Cre n Use Committee, n y the Suommittee on Reserh Animl Cre t Msshusetts Generl Hospitl. Mie were of mixe 129Sv/Je n C57/B6 kgroun. After eteting tumour y MRI, mie were trete with tmoxifen (Sigm) y intrperitonel injetion. 1 ml of tmoxifen (1 mg ml 21 in orn oil) ws injete every two to three ys. Followup MRI ws generlly otine 7 1 ys fter the first tmoxifen tretment. MRI n tumour volume nlysis. All nimls were sequentilly imge using 4.7 T Bruker Phrmsn (Bruker BioSpin) to sreen for tumour growth n tretment response to tmoxifen. Tumour volume mesurements were performe using T1- n T2-weighte oronl n xil imge stks. The tumours were mnully segmente using Amir softwre (TGS) to otin the tumour volume in m 3. For eh time point, ll mesurements from the ville sequenes were use to lulte men volume 6 stnr evition in orer to limit errors ue to the mnul segmenttion proess. Reeive 26 Septemer; epte 13 Deemer 26. Pulishe online 24 Jnury Hnhn, D. & Weinerg, R. A. The hllmrks of ner. Cell 1, 57 7 (2). 2. Chin, L. et l. Essentil role for onogeni Rs in tumour mintenne. Nture 4, (1999). 3. Jin, M. et l. Sustine loss of neoplsti phenotype y rief intivtion of MYC. Siene 297, (22). 4. Fisher, G. H. et l. Inution n poptoti regression of lung enorinoms y regultion of K-Rs trnsgene in the presene n sene of tumor suppressor genes. Genes Dev. 15, (21). 5. Pelengris, S., Khn, M. & Evn, G. I. Suppression of My-inue poptosis in ells exposes multiple onogeni properties of My n triggers rinogeni progression. Cell 19, (22).

5 NATURE Vol Ferury Weinstein, I. B. Cner. Aition to onogenes the Ahilles heel of ner. Siene 297, (22). 7. Druker, B. J. et l. Effiy n sfety of speifi inhiitor of the BCR-ABL tyrosine kinse in hroni myeloi leukemi. N. Engl. J. Me. 344, (21). 8. Demetri, G. D. et l. Effiy n sfety of imtini mesylte in vne gstrointestinl stroml tumors. N. Engl. J. Me. 347, (22). 9. Lynh, T. J. et l. Ativting muttions in the epierml growth ftor reeptor unerlying responsiveness of non-smll-ell lung ner to gefitini. N. Engl. J. Me. 35, (24). 1. Pez, J. G. et l. EGFR muttions in lung ner: orreltion with linil response to gefitini therpy. Siene 34, (24). 11. Sherr, C. J. Priniples of tumor suppression. Cell 116, (24). 12. Olivier, M. et l. The IARC TP53 tse: new online muttion nlysis n reommentions to users. Hum. Mutt. 19, (22). 13. Bykov, V. J. et l. Restortion of the tumor suppressor funtion to mutnt y low-moleulr-weight ompoun. Nture Me. 8, (22). 14. Snyer, E. L., Mee, B. R., Senz, C. C. & Dowy, S. F. Tretment of terminl peritonel rinomtosis y trnsuile -tivting peptie. PLoS Biol. 2, E36 (24). 15. Brn, C. S. & Dymeki, S. M. Tlking out revolution: The impt of site-speifi reominses on geneti nlyses in mie. Dev. Cell 6, 7 28 (24). 16. Donehower, L. A. et l. Mie efiient for re evelopmentlly norml ut suseptile to spontneous tumours. Nture 356, (1992). 17. Jks, T. et l. Tumor spetrum nlysis in -mutnt mie. Curr. Biol. 4, 1 7 (1994). 18. Inr, A. K. et l. Temporlly-ontrolle site-speifi mutgenesis in the sl lyer of the epiermis: omprison of the reominse tivity of the tmoxifeninuile Cre-ER T n Cre-ER T2 reominses. Nulei Ais Res. 27, (1999). 19. Sorino, P. Generlize lz expression with the ROSA26 Cre reporter strin. Nture Genet. 21, 7 71 (1999). 2. Kemp, C. J., Whelon, T. & Blmin, A. -efiient mie re extremely suseptile to rition-inue tumorigenesis. Nture Genet. 8, (1994). 21. Collo, M. et l. Tumour iology: senesene in premlignnt tumours. Nture 436, 642 (25). 22. Lowe, S. W. & Sherr, C. J. Tumor suppression y Ink4-Arf: progress n puzzles. Curr. Opin. Genet. Dev. 13, (23). 23. Xue, W. et l. Senesene n tumour lerne is triggere y restortion in murine liver rinoms. Nture vne online pulition, oi:1.138/ nture5529 (24 Jnury 27). 24. Zhng, L. et l. Wil-type suppresses ngiogenesis in humn leiomyosrom n synovil srom y trnsriptionl suppression of vsulr enothelil growth ftor expression. Cner Res. 6, (2). 25. Teooro, J. G., Prker, A. E., Zhu, X. & Green, M. R. -meite inhiition of ngiogenesis through up-regultion of ollgen prolyl hyroxylse. Siene 313, (26). 26. Dikins, R. A. et l. Proing tumor phenotypes using stle n regulte syntheti mirorna preursors. Nture Genet. 37, (25). 27. Christophorou, M. A. et l. Temporl issetion of funtion in vitro n in vivo. Nture Genet. 37, (25). 28. Christophorou, M. A., Ringshusen, I., Finh, A. J., Swigrt, L. B. & Evn, G. I. The pthologil response to DNA mge oes not ontriute to -meite tumour suppression. Nture 443, (26). 29. Vssilev, L. T. et l. In vivo tivtion of the pthwy y smll-moleule ntgonists of MDM2. Siene 33, (24). 3. Hupt, S. & Hupt, Y. Mnipultion of the tumor suppressor for potentiting ner therpy. Semin. Cner Biol. 14, (24). 31. Olive, K. P. et l. Mutnt gin of funtion in two mouse moels of Li-Frumeni synrome. Cell 119, (24). Supplementry Informtion is linke to the online version of the pper t Aknowlegements We thnk N. Willis for helping to generte the LSL mie, H. Zheng for imging mie, G. Wojtkiewiz for generting the movies with three-imensionl reonstrution, D. Crowley for help with histology, R. Bronson for reviewing the pthology, n M. Hemnn for suggestions. A.V. is grteful to D. Ventur n G. Terrnov for ontinuous support n enourgement. This work ws supporte y the Howr Hughes Meil Institute (T.J.), NCI (T.J., R.W., D.G.K.), n prtilly y Cner Center Support grnt from the NCI (M.I.T.), the Amerin Itlin Cner Reserh Fountion (A.V.), n the Lef fun (D.G.K.). T.J. is the Dvi H. Koh Professor of Biology n Dniel K. Luwig Sholr. D.A.T. is Rit Allen Fountion Sholr. Author Contriutions A.V., D.G.K. n T.J. esigne the experiments n wrote the pper. D.T. generte the -LSL mie n M.E.M. generte n hrterize the Cre-ER T2 mie, etermine the optiml osge, ssiste with histopthologil nlysis n ommente on the mnusript. A.V., D.G.K. n L.L. erive n hrterize the tumour ell lines. A.V. performe the immunostinings, the TUNEL ssys the SA--Gl stinings n the western lottings. A.V., D.K. n L.L. performe the tmoxifen intrperitonel injetions. E.E.R. erive the MEFs. L.L. n J.N. mintine the mouse olony n genotype the nimls. J.G. n D.G.K. evlute the mgneti resonne imges, J.G. supervise the mgneti resonne imging, generte the three-imensionl reonstrutions n etermine tumour volumes. R.W. optimize in vivo imging protools, reviewe imging t, isusse the results, n ommente on the mnusript. Author Informtion Reprints n permissions informtion is ville t The uthors elre no ompeting finnil interests. Corresponene n requests for mterils shoul e resse to T.J. (tjks@mit.eu). 27 Nture Pulishing Group 665