ARTICLES. mtor inhibition reverses Akt-dependent prostate intraepithelial neoplasia through regulation of apoptotic and HIF-1-dependent pathways

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1 mtor inhiition rvrss Akt-pnnt prostt intrpithlil noplsi through rgultion of poptoti n HIF-1-pnnt pthwys Prip K Mjumr 1,2,Phillip G Fo 1,2,Rhl Bikoff 1,2,Rnn Brgr 1,2, Qi Xu 1,2,Louis M MMhon 3, Juith Mnol 1,Jms Brugrols 1,2,Timothy J MDonnll 4,To R Golu 1,2,5,Mssimo Lo 1,2,Hii A Ln 6 & Willim R Sllrs 1,2,5 Loss of PTEN funtion ls to tivtion of phosphoinositi 3-kins (PI3K) signling n Akt. Clinil trils r now tsting whthr mmmlin trgt of rpmyin (mtor) inhiition is usful in trting PTEN-null nrs. Hr, w rport tht mtor inhiition inu poptosis of pithlil lls n th omplt rvrsl of noplsti phnotyp in th prostt of mi xprssing humn AKT1 in th vntrl prostt. Inution of ll th rquir th mitohonril pthwy, s prostt-spifi oxprssion of BCL2 lok poptosis. Thus, thr is n mtor-pnnt survivl signl rquir ownstrm of Akt. Bl2 xprssion, howvr, only prtilly rstor intrluminl ll growth in th stting of mtor inhiition. Exprssion profiling show tht Hif-1α trgts, inluing gns noing most glyolyti nzyms, onstitut th ominnt trnsriptionl rspons to AKT tivtion n mtor inhiition. Ths t suggst tht th xpnsion of AKT-rivn prostt pithlil lls rquirs mtor-pnnt survivl signling n tivtion of HIF-1α, n tht linil rsistn to mtor inhiitors my mrg through BCL2 xprssion n/or uprgultion of HIF-1α tivity. Intivtion of th tumor-supprssor gn PTEN ours in gliolstom multiform, nomtril nr n prostt nr, mong othrs. Th tumor-supprssor funtion of PTEN is link to its lipi phosphts tivity; loss of this tivity ls to umultion of its sustrt, phosphtiylinositol 3,4,5-trisphospht, n tivtion of th PI3K signling pthwy 1. On onsqun of PTEN loss is hyprtivtion of th onogni srin/thronin kins AKT n phosphoryltion of ownstrm AKT sustrts, inluing BAD 2,FOXO protins 3,4 n GSK3 (rf. 5). Phosphoryltion n intivtion of ths protins n l to nhn ll survivl, inrs ll prolifrtion n ltr llulr mtolism. PI3K signling hs n implit in th rgultion of mtor (in mous, no y th gn Frp1) n S6K. In Drosophil mlnogstr,loss of Tor is pistti to loss of Ptn, n in mous n ll-s mols loss of PTEN snsitizs lls to mtor inhiition 6 10.This pthwy hs n furthr luit through stuis in D. mlnogstr n mmmlin lls showing tht turin, th protin prout of Ts2,rgults Tor n is n AKT sustrt. Thus, AKT-pnnt phosphoryltion inhiits turin, ling to tivtion of mtor n S6K On th sis of ths umultiv osrvtions, rivtivs of th mtor inhiitor rpmyin r ing tst in linil trils in ptints with nr 16. W prviously show tht prosin promotr-myr-ha-akt1 trnsgn irts proution of tivt AKT1 sptilly rstrit to th luminl pithlil lls of th mous vntrl prostrt n, s rsult, ths mi vlop highly pntrnt prostti intrpithlil noplsi (PIN) phnotyp 17.Th phnotyp rs mny of th hllmrks of mtor tivtion inluing inrs ll numr, inrs ll siz, n tivtion of th ownstrm kins S6K, togthr suggsting tht tivtion of mtor ownstrm of AKT my link to th vlopmnt of PIN in ths mi. Hr, w show tht th Akt-inu PIN phnotyp is ompltly pnnt on mtor. Spifilly, trtmnt with th mtor inhiitor RAD001 l to rpi loss of intrluminl pithlil lls mrk y th inution of poptosis, n rvrs th PIN phnotyp within 14. Mi rrying oth AKT1 n BCL2 trnsgns wr rsistnt to RAD001- inu poptosis n h PIN phnotyp prtilly rsistnt to RAD001. Furthr nlysis of this prtil rsistn show tht Hif- 1α trgt gns, inluing thos noing nzyms ssntil for glyolysis, wr th prinipl onstitunts of th trnsriptionl rspons to lvt Akt tivity n of th rspons to mtor inhiition. Ths t suggst tht th rspons to mtor inhiition is mit through inpnnt poptoti n Hif-1α rgultory pthwys. 1 Dprtmnts of Mil Onology, Pitri Onology n Biosttistil Sins, Dn-Frr Cnr Institut, 44 Binny Strt, Boston, Msshustts 02115, USA. 2 Dprtmnts of Miin n Pthology, Brighm n Womn s Hospitl, Hrvr Mil Shool, 75 Frnis Strt, Boston, Msshustts 02115, USA. 3 Novrtis Phrmutil Corportion, On Hlth Plz, Est Hnovr, Nw Jrsy 07936, USA. 4 Dprtmnt of Molulr Pthology, Th Univrsity of Txs M.D. Anrson Cnr Cntr, Houston, Txs 77030, USA. 5 Th Bro Institut t Hrvr n MIT, 320 Chrls Strt, Cmrig, Msshustts 02142, USA. 6 Novrtis Institut for Biomil Rsrh, Onology, CH-4002 Bsl, Switzrln. Corrsponn shoul rss to W.R.S. (willim_sllrs@fi.hrvr.u). Pulish onlin 23 My 2004; oi: /nm VOLUME 10 NUMBER 6 JUNE 2004 NATURE MEDICINE

2 Figur 1 An mtor inhiitor (RAD001) rvrss th PIN phnotyp of AKT1-Tg mi. ( h) Wil-typ (WT) (,,,f) n AKT1-Tg mi (,,g,h) wr trt for 14 with ithr plo (,,,g) or 10 mg/kg/ RAD001 (,,f,h). Shown r H&E-stin tissu stions from vntrl prostt (VP) hrvst from oth untrt (,) n trt mi (,). Dt r rprsnttiv of 12 mi in h trtmnt group. Tissu stions from oth ontrol n RAD001-trt mi wr stin with ntioy to Zo1 (α-zo1) n img y onfol mirosopy ( h). Sl r, 25 µm. RESULTS Rvrsl of AKT-pnnt PIN y mtor inhiition To trmin whthr n Akt1-inu PIN phnotyp rquirs mtor tivity, 8- to 12-wk-ol trnsgni mi xprssing humn AKT1 in th vntrl prostt (AKT1-Tg) n wil-typ mi wr trt with plo or th mtor inhiitor RAD001 (vrolimus), n orlly tiv rpmyin rivtiv 18.At this g th PIN phnotyp is fully vlop in ll AKT1-Tg mi 17.Pk n trough onntrtions for loo n for vntrl prostt x th onntrtion prouing 50% growth inhiition (IC 50 ) of humn tumor ll lins m snsitiv to RAD001 (Supplmntry Fig. 1 onlin; H.A.L. n T. O Rilly, unpulish t), n t 10 mg/kg/, RAD001 ws wll tolrt with no hng in oy wight (Supplmntry Fig. 1 onlin). Aftr 14 th vntrl prostt histology of plo or RAD001- trt wil-typ mi ws norml, whrs PIN prsist in AKT1- Tg mi trt with plo. Howvr, trtmnt of AKT1-Tg mi with RAD001 rstor ll siz, pithlil ll polriztion (msur y ZO-1 stining) n th luminl rhittur to norml (Fig. 1 h n Supplmntry Figs. 2 n 3 onlin). Most notly, th utl lumns wr now voi of th xssiv lls sn in plo-trt AKT1-Tg mi. Ths t suggst tht th gnsis n/or survivl of th intrluminl lls rivn y Akt xprssion is mtor pnnt. Phnotyp rgrssion spit ontinu AKT tivity To trmin whthr RAD001 sltivly inhiit mtor without ltring othr lmnts of Akt signling, tissu stions n protin xtrts wr pro with ntisr rognizing phosphorylt Sr235/236 on S6RP n phosphorylt Sr1108 on IF4G. Th lttr is oth srum pnnt n inhiit y rpmyin 19. Phosphoryltion of S6RP n IF4G ws lvt in AKT1-Tg mi s ompr with ontrol mi, n ws ru y RAD001 (Fig. 2,i). Nxt, w xmin th phosphoryltion of Akt n GSK3, wllfin Akt sustrt. In AKT1-Tg mi, RAD001 h no fft on th lvt lvls of ps473-akt or ps9/21-gsk3 ompr with plo-trt ontrols (Fig. 2 i), suggsting tht in ths mi RAD001 ts sltivly ownstrm of AKT to inhiit mtor tivity. Thus, th AKT-inu PIN phnotyp is mtor pnnt. An mtor-pnnt survivl pthwy To trmin th tim ours of th rspons to RAD001, w trt 228 AKT1-Tg n wil-typ mi for 6 h, 12 h, or with ily osing for 1, 2, 3, 9 n 14 with ithr RAD001 or plo (Fig. 3). No histologi hngs wr not through 3 of RAD001 trtmnt, ut on y 9 luminl lls ppr vuolt n wr ru in numr. At this tim thr ws lso n inrs in intrluminl llulr ris follow y omplt lring of th lumns y y 14. In th vntrl prostt of AKT1-Tg mi, th rt of llulr prolifrtion (msur y 5-romooxyuriin (BrU) inorportion) ws only mostly lvt ompr with wil-typ ontrols g f h n, s ntiipt, is ru y RAD001 (Supplmntry Fig. 4 onlin). Th lk of lrg numrs of prolifrting lls, th ytopthi hngs sn t y 9 n th rpiity of th phnotypi rvrsion suggst tht loss of intrluminl lls ftr RAD001 xposur might u to progrmm ll th. In support of this i, DNA frgmnttion, msur y fluorsnt TT-mit UTP nik n lling (TUNEL) t 6 h, 12 h, 24 h n 48 h ftr RAD001 initition, show sustntilly highr prntg of poptoti lls ftr 48 h thn i plo ontrols (Fig. 4 h,m). Aitionl xprimnts ovr longr trtmnt prios show ontinu inrs in th numr of poptoti lls whil th xtnt of prolifrtion inhiition rmin onstnt (Fig. 4n). In prlll, sps-3 tivtion, msur y immunostining, ws tt within 24 h of RAD001 trtmnt (Fig. 4i l). Ths t suggst tht th intrluminl pithlil lls ontriuting to th PIN phnotyp pn on n mtor-pnnt, ntipoptoti signling pthwy for thir ontinu survivl, n tht th onsquns of mtor inhiition in rvrsing th AKT phnotyp ris, t lst in prt, from propoptoti ffts. Apoptosis inu y mtor inhiition is lok y BCL2 To trmin whthr poptosis inu y mtor inhiition rquir mitohonril pthwy tivtion, AKT1-Tg mi wr intrross with trnsgni mi xprssing BCL2 spifilly in th vntrl prostt (BCL2-Tg) 20.W trt tn mi of h gnotyp from this intrross with RAD001 or plo for 14. Consistnt with th rsults otin in AKT1-Tg mi, RAD001 fully rvrs th PIN in F1 mi ring only th AKT1 trnsgn. In ontrst, in mi rrying oth AKT1-Tg n BCL2-Tg, RAD001 ministrtion NATURE MEDICINE VOLUME 10 NUMBER 6 JUNE

3 Figur 2 Sltiv in vivo intivtion of mtor n S6K tivity y RAD001. ( h) AKT1-Tg mi wr trt with plo (,,,g) or RAD001 (,,f,h) for 14, n vntrl prostt stions wr pro with ntiois to ps6rp (,), pif4g (,), pakt (,f) or pgsk3 (g,h). Th t r rprsnttiv of rsults otin in 12 mi pr trtmnt group. Sl r,,g,h, 50 µm;,f, 25 µm. (i) Protin lysts from th vntrl prostt of AKT1-Tg n wil-typ mi trt with RAD001 (+) or plo ( ) for 72 h wr sujt to immunolot nlysis with ntiois to ps6rp, pif4g, pakt, pgsk3 or tuulin s init. ompltly inhiit mtor signling, ut prtil rthr thn omplt rgrssion of th phnotyp ourr (Fig. 5-h n Supplmntry Fig. 5 onlin). Inhiition of mtor supprss prolifrtion in oth wil-typ AKT1 n AKT1-Tg/BCL2-Tg mi (Supplmntry Fig. 5 onlin), whrs TUNEL stining ftr 48 h show tht RAD001 inu n inrs in poptoti lls in wil-typ Akt1 mi, ut no hng in poptosis in AKT1-Tg/BCL2-Tg mi (Supplmntry Fig. 5 onlin). Thus, BCL2 xprssion lok th inution of poptosis y mtor inhiition n l to prtil phnotyp rsistn to RAD001. α-ps6 α-plf4g α-pakt α-pgsk3 mtor-pnnt rgultion of Hif-1α trgt gns Although xprssion of BCL2 lok RAD001-inu poptosis, th PIN phnotyp ws only prtilly rstor. Spifilly, in th ompoun trnsgni mi trt with RAD001, PIN onsisting of two to thr pithlil ll lyrs ws sn, ut mor xtnsiv luminl filling, s sn in untrt mi, ws not osrv. Thus, to furthr unrstn th pthwys miting th xpnsion of th intrluminl lls n th rspons to RAD001, w rri out xprssion profiling follow y gn-st nrihmnt nlysis (GSEA) 21. First, totl RNA ws prpr ftr 12 or 48 h of RAD001 or plo trtmnt in wil-typ n AKT1-Tg mi, n mssg unn ws trmin for 22,626 gns using mirorrys. To intify gns ltr y AKT xprssion n y susqunt mtor g Plo f h RAD001 inhiition, rtifiil vlus for gn xprssion rprsnting n iliz rspons to AKT xprssion n mtor inhiition wr st, n th orrltion twn ths vlus n th tul xprssion t ws trmin using th Prson offiint. Thr wr 654 fturs (rprsnting 571 uniqu gns or xprss squn tgs) with signifint orrltion to this vtor (Supplmntry Tl 1 onlin; P 0.001). To trmin whthr th iffrntil xprssion of this st of gns rsult from ltrtions in th tivity of spifi molulr pthwys, w ppli GSEA to this t st 21.W intifi fin gn sts hving sttistilly signifint inrs or rs oorint gn xprssion twn two xprimntl onitions. W tst 192 prviously spifi Biort gn sts in GSEA y ompring AKT1-Tg smpls trt with RAD001 (n = 9) or plo (n = 10). Gn sts tht hiv nrihmnt grtr thn xpt i RAD001 trtmnt (ys) Figur 3 Tim ours of th phnotypi rspons to mtor inhiition. ( ) AKT1-Tg mi wr trt with 10 mg/kg/ of RAD001 for 1, 2, 3, 9 n 14. Tissu stions from th vntrl prostt wr stin with H&E. Shown r stions rprsnttiv of th rsults otin in t lst 12 mi vlut ftr h spifi trtmnt prio. Sl r, 50 µm. 596 VOLUME 10 NUMBER 6 JUNE 2004 NATURE MEDICINE

4 RAD001 Plo RAD001 Plo m Numr of poptoti lls/100 uts y hn lon wr intifi y prmuting th plo n RAD001 smpl lls. In this nlysis, svn-mmr, hypoxiinuil ftor 1 gn st tht inlu Vg f, En1 (nothlin-1), Lh1 (ltt hyrogns-1) n Hif1 (Hif-1α) ws th only st to hv vlition mo signifin lss thn 0.05 (Fig. 6). To furthr xplor this fining, GSEA ws rri out using list inpnntly urt from th litrtur of puttiv Hif-1α trnsriptionl trgt gns 22.This nlysis show mrk nrihmnt for th xprssion of Hif-1α trgt gns in AKT1-Tg s ompr to wil-typ mi n sttistilly signifint olltiv loss of xprssion of Hif-1α trnsriptionl trgts in RAD001- vrsus plo-trt mi (Fig. 6 n Supplmntry Fig. 6 onlin). This st of Hif-1α trgt gns inlus Sl21 (Glut1), Vg f, Hmox1 (hm oxygns-1) n Ap2p (NIP3); howvr, most prominntly nrih in this st wr gns noing nzyms omprising th glyolysis pthwy (Fig. 6,). In, 9 of 10 gns f g h H&E TUNEL A-Csps-3 n i j k l Figur 4 Inution of ll th y mtor inhiition in AKT1-Tg mi. ( h) H&E ( ) or TUNEL stining ( h) of wil-typ (,,,f) n AKT1-Tg mi (,,g,h) trt with plo (,,,g) or RAD001 (,,f,h) for 48 h. (i l) Csps-3 tivtion ws ssss in wil-typ (i,j) n AKT1-Tg mi (k,l) trt with plo (i,k) or RAD001 (j,l) for 24 h. All rsults r rprsnttiv of thos otin in six mi. (m) TUNEL-positiv lls (mn ± s..m. pr 100 uts ftr RAD001 (+) or plo ( ). (n) Mn fol hng (RAD001 vrsus plo) in th poptoti or prolifrtion inx for t lst thr AKT1-Tg mi t h tim point. Sl r, h, 50 µm; i l, 25 µm. noing nzyms involv in th onvrsion of gluos to pyruvt wr mong th top gns in this st n wr strongly uprgult in th Akt1-Tg mous n ownrgult y RAD001 within 12 h n 48 h (Fig. 6). In ition, ltt hyrogns, whih unr nroi onitions onvrts pyruvi i to lti i, ws rgult in similr mnnr (Fig. 6). Morovr, th glyolyti nzyms s n inpnnt gn st wr fourth mong pthwys rnk y GSEA (Fig. 6) n olustr whn ths t wr nlyz y unsuprvis hirrhil lustring (t not shown). Ths t suggst tht Hif-1α tivity is uprgult in th AKT trnsgni nimls n ownrgult ftr RAD001 ministrtion. To inpnntly vlit ths rsults, Glut1 ws tt in tissu stions y immunohistohmistry n ws signifintly ownrgult y RAD001 (14 ) in oth AKT1-Tg n AKT1-Tg/BCL2-Tg prostts (Fig. 6). In kping with th inirt msurs of Hif-1α tivity, immunolot nlysis show mrk uprgultion of th Hif-1α protin in xtrts prpr from th vntrl prostts of AKT1-Tg/BCL2-Tg mi, whih ws ru to sl lvls ftr thr oss of RAD001. Ths ltrtions in Hif-1α protin wr prlll y hngs in Hif-1α mrna (Fig. 6). Thus, it sms likly tht mtor tivity ls to hngs in oth mrna n protin for Hif-1α. Ths t suggst tht, in this mol of AKT tivtion, mtor inhiition ls to inpnnt tivtion of poptoti pthwys n intivtion of Hif-1α trgt gns. DISCUSSION Mny gnti ltrtions, inluing loss of PTEN 23,muttion of th gn noing phosphoinositi 3-kins p110α (PIK3CA 24 ), mplifition of PIK3CA 25 n mplifition of AKT1 n AKT2, n l to tivtion of AKT kins tivity 23.Hr, w show tht n AKTinu PIN phnotyp is ompltly mtor pnnt. Of not, AKT inus this pnny in norml prostt pithlil lls tht vintly hv no pnn on mtor. Th phnotyp rvrsion NATURE MEDICINE VOLUME 10 NUMBER 6 JUNE

5 sn with mtor inhiition is in prt u to th inution of poptosis within th intrluminl lls. Thus, ths intrluminl lls, n not th lls loliz long th smnt mmrn (whih ontinu to hror tivt AKT), om pnnt on ontinuous mtor tivity. This spifi snsitivity of th luminl lls rlls th sltiv rgultion of luminl poptosis sn in thr-imnsionl mols of immortliz mmmry pithlil lls 26, in whih Aktrivn prolifrtion is lso pnnt on mtor 27, n suggsts tht loss of luminl ll intrtions with th mtrix or with sl pithlil lls or th loss of nognous survivl ftors hs rol in rnring lls mtor pnnt for survivl. Ptn +/ mi, humn xnogrfts n trnsform hikn firolsts r snsitiv to mtor inhiition 8 10.Whrs in suh sttings mtor inhiition is primrily ytostti, in th AKT1-Tg mol thr is lr phnotyp rvrsion. Svrl possiilitis might xplin ths iffrns. First, sltiv tivtion of AKT rthr thn loss of PTEN my rnr lls pnnt on mtor rthr thn on mtor togthr with itionl pthwys. Son, th PIN phnotyp is not invsiv. As th ffts of mtor inhiition wr most profoun within th intrluminl lls, it is possil tht, s lls inv through th smnt mmrn, thy gin nw survivl signls (or rgin th sl survivl signls) rnring thm mtor inpnnt for survivl. In, t pulish whil this work ws in progrss monstrt tht Akt-rivn lymphoms trt with rpmyin, lthough prtilly rsponsiv, rrly hiv omplt rsponss, lthough omintion with oxoruiin l to fr grtr ntilymphom tivity g f h thn tht for ithr gnt lon 28.Thir, ll lins n xnogrfts rflt mor omplx gnti kgroun thn th AKT1-Tg mol. Thus, it is possil tht othr prosurvivl n ptiv vnts, inluing inrs BCL2 xprssion or tivtion of HIF-1, hv lry tkn pl. If so, nr lls hroring suh lsions might rmin snsitiv to only th ntiprolifrtiv ffts of mtor inhiition. Th mtor-rgult vnts tht llow lls to surviv in th intrluminl sp r lrly sprl from thos tht rgult ll prolifrtion. Spifilly, mi ring trnsgns noing oth AKT n th prosurvivl protin BCL2 rmin snsitiv to RAD001- inu inhiition of prolifrtion ut r rsistnt to poptosis. Thus, in ths lls th AKT- n mtor-inu survivl tivity os not rsult simply from first quiring n thn losing n xssiv prolifrtion signl. Th spifi mhnism of poptosis is not fully trmin hrin, ut this pthwy involvs sps-3 tivtion n rquirs n intt mitohonril pthwy. Ths lttr t r in kping with th osrvtion tht rpmyin rss th mitohonril mmrn potntil of lls xprssing tivt AKT 29.Ths t suggst tht th thrputi ffiy of mtor inhiition my rquir th mitohonril poptoti pthwy. BCL2 is ovrxprss in humn prostt nr n PIN 33,34, suggsting tht BCL2 xprssion is ssoit with initition n progrssion of prostt nr. Both loss of PTEN n ovrxprssion of BCL2 hv n link to inrs prostt nr gr n th vlopmnt of nrogn-inpnnt mtstti prostt nr 30,35,36.In, in primry tumors thr sms to som orrltion twn PTEN loss n BCL2 ovrxprssion 37.Clinil trils of rpmyin rivtivs RAD001 or CCI-779 r ongoing in ptints with vn prostt nr 16,38,39, in whom PTEN muttion is frqunt 36.Ifour t r pritiv of humn prostt nr snsitivity, mtor inhiition my lss fftiv in vn prostt nrs hrtriz y BCL2 ovrxprssion. Phs 2 linil trils of gnts tht irtly or inirtly moult BCL2 funtions r lso unrwy Our rsults provi rtionl for omintion thrpy with mtor inhiitors n BCL2 inhiitors. Finlly, our t suggst tht mjor omponnt of th in vivo trnsriptionl rspons to tivtion of AKT is mtor-pnnt rgultion of Hif-1α. It is notl tht ltrtions in Hif-1 tivity pr ny hng in th phnotyp y svrl ys, lning rn to th i tht ths r primry, not sonry, vnts. Thrfor, it is possil tht th filur to onfr full rsistn to RAD001 y BCL2 oxprssion is u to ontinu n for lvt Hif-1α tivity n lvt glyolyti tivity. If so, prition is tht onstitutiv Hif-1α tivity might l to rsistn to mtor inhiition. Downstrm of PI3K signling, oth Akt-pnnt, mtorinpnnt n Akt pnnt, mtor-pnnt rgultion of Hif-1α hv n sri 44,45. In our mol, Akt-pnnt inution of Hif-1α tivity is ntirly mtor pnnt. Ths in vivo rsults support t suggsting tht hypoxi-inu tivtion of Hif-1α rquirs mtor tivity 46,47, tht insulin tivts Hif-1α through n Akt- n mtor-pnnt pthwy 48 n tht, in th stting of loss of Ts2,Hif-1α protin n mrna lvls r lvt, Figur 5 Complt phnotyp rgrssion ftr mtor inhiition rquirs th mitohonril poptosis pthwy. ( h) Wil-typ (WT, WT) (,), wil-typ AKT1-Tg n trnsgni BCL2 (WT, BCL2-Tg) (,), AKT1-Tg, BCL2 wiltyp (AKT1-Tg, BCL2-WT) (,f) n AKT1-Tg,BCL2-Tg (AKT1-Tg, BCL2-Tg) mi (g,h) wr trt for 14 with ithr plo (,,,g) or 10 mg/kg/ RAD001 (,,f,h) ministr orlly. Shown r rprsnttiv H&Estin tissu stions from th vntrl prostt. Sl r, 50 µm. 598 VOLUME 10 NUMBER 6 JUNE 2004 NATURE MEDICINE

6 WT AKT1-TG Low Normliz xprssion High Lvl of Hifα mrna (ritrry vlu) Figur 6 Exprssion profils of mtor inhiition r nrih for Hif-1α trgts n glyolysis gns. () Th top 21 gn sts nrih y GSEA; ( ) n (+) init RAD001 own- or uprgultion. () GSEA for 44 Hif-1α trgts rnk y high xprssion in plo s ompr to RAD001 trtmnt. Exprssion vlus r rprsnt y stnr vitions ov (r) or low (lu) th mn. () Th mn fol xprssion (lk lin) for h glyolyti nzym (r) is shown (sustrts r in lk). Exprssion vlus r lso shown s in. () Glut-1 immunostining in RAD001- or plo-trt vntrl prostt (VP). Sl r, 25 µm. () Hif-1α immunolotting in RAD001 (+) or plo ( ) trt vntrl prostt. Hif-1α mrna xprssion ws trmin in th init mi ftr 48 h of trtmnt (from mirorry t). ling to uprgult xprssion of Hif-1α trgts 49. Elvt Hif- 1α tivity is, in this stting, rvrs y mtor inhiition 49. Although th mhnism ling to mtor-pnnt lvt Hif-1α tivity rmins unlr, rpmyin sms to intrfr with Hif-1α tivtion in hypoxi lls y inrsing th rt of Hif-1α grtion through th oxygn-pnnt omin 46. In th AKT1-Tg vntrl prostt, inrs mtor tivity is ssoit with 3.2-fol inrs in th Hif-1α mrna itslf tht is normliz with mtor inhiition (Fig. 6). Thus, it is likly tht mtor hs oth trnsriptionl n post-trnsltionl ffts on Hif-1α. Th link twn mtor n GLUT1 inution my hv immit linil rlvn, s gluos uptk n img in humns y [ 18 F]fluorooxygluos positron mission tomogrphy (FDG- PET). Thus, in ptints trt with mtor inhiitors, rpi own-rgultion of FDG-PET intnsity might rfltiv of mtor inhiition (tht is, phrmoynmi rspons) rthr thn of gnrl ntitumor tivity, n might thus llow for th intifition of ptints with mtor-pnnt tumors. Finlly, lthough th urrnt linil pplition to mtor inhiition is trgt t ltr-stg nrs, th rspons of this rly noplsti iss mol to RAD001 riss th possiility tht inhiitors of mtor my fin pplition in trting rly nr lsions in humns. METHODS Mous strins, gnotyping n tissu prprtion. Animl xprimnts wr omplint with th guilins of Dn-Frr Cnr Institut. Th lin FVB-Tg(Psn-Akt1)9Wrs (AKT1-Tg, lso known s MPAKT) hs n sri 17.Gnotyping, prostt isstions, tissu fixtion n H&E stining wr rri out s sri 17 (s sllrsl/tsts/inx.html for protools). NATURE MEDICINE VOLUME 10 NUMBER 6 JUNE

7 Th BCL2-Tg mi 20, mintin on mix C57BL6/FVB kgroun, wr intrross with AKT1-Tg htrozygous mi to gnrt F 1 offspring. Compoun gnotyps wr otin in th xpt proportion. Aministrtion n msurmnt of RAD001.W ministr 10 mg/kg/ of RAD001 (40-O-(2-hyroxythyl)-rpmyin) s miromulsion 50 (2% w/w) ilut in istill, ioniz wtr y orl gvg. Bloo n isst vntrl prostts ollt ftr 12 h, 24 h n 14 of trtmnt wr snp frozn n RAD001 onntrtions trmin y liqui hromtogrphy. Antiois, mirosopy, immunohistohmistry n immunolot nlysis. Tissu stions wr hyrt, inut for 30 min with 3% H 2 O 2 in mthnol t room tmprtur, wsh with istill, ioniz wtr n PBS, n ht in mirowv ovn to 199 F (93 C) in 1 mm EDTA (ph 8.0) for 25 min (ntiois to prps6, pif4g, pakt n tivt sps- 3) or in 10 mm soium itrt uffr (ph 6.0) for 30 min (ntioy to Zo1), or ht in prssur ookr in 10 mm itrt uffr (ph 6.0) for 30 min (ntioy to pgsk3). Stions wr lok in 10% got srum (30 min), inut with ntiois to Akt (ps473; 1:400), pgsk3 (ps21/9; 1:25), prps6 (ps235/236), pif4g (ps1108; 1:400; Cll Signling), Glut1 (1:400; Alph Dignosti) n BrU (1:100; BD Phrmingn) in 1% BSA (12 h t 4 C), wsh with PBS, inut with sonry ntioy (1:200; 30 min) n tt with th ABC kit (Vtor). Protin xtrts n immunolots wr prpr s sri 17. Antiois to phospho-akt-s473, phospho-gsk3-s21/9, phospho-if4g- S1108, phospho-s6 S235/236 (Cll Signling), humn BCL2 (6C8; BD PhrMingn), Hif-1α n α-tuulin (B-5-1-2; Sigm) wr us t 1:1,000. For onfol mirosopy, stions wr lok in immunofluorsn uffr (IFB) (130 mm NCl, 7 mm N2HPO4, 3.5 mm NH2PO4, 7.7 mm NN3, 0.1% BSA, 0.2% Triton X-100, 0.05% Twn 20) with 10% got srum n 20 mg/ml got nti-mous F( )2) for 90 min. Antiois to pakt (ps473; 1:200), tivt sps-3 (Asp175; 1:200) n Zo1 (1:400) wr inut ovrnight t room tmprtur in IFB. Aftr wshing in IFB, slis wr inut with Alx-Fluor onjugt nti-rit ntioy (Molulr Pros) in IFB ontining 10% got srum for 60 min, wsh thr tims with IFB, inut for 15 min with DAPI (0.5 ng/ml in PBS; Roh) n mount with Prolong (Molulr Pros). Confol nlysis (Pinh 1.75) ws rri out using n Invrt Confol Lsr Snning Mirosop (Crl Ziss). TUNEL ssy. Prformlhy-fix tissu stions wr prffiniz in xyln, rhyrt in thnol n inut with protins K (0.02 mg/ml) for 20 min t room tmprtur (21 23 ), n TUNEL stining ws rri out using th Fluorsin-FrgEL kit (Onogn Rsrh Prouts) pr th mnufturr s instrutions. TUNEL-positiv luminl pithlil lls wr ount in ll uts, n th numr of poptoti pithlil lls pr 100 uts ws lult. Exprssion nlysis. Biotin-ll RNA prpr from 15 µg totl RNA ws frgmnt n hyriiz to oligonuloti mirorrys (430A, Affymtrix) 17. CEL fils, gnrt y GnChip, wr sl to th min intnsity rry s on th mn vlu of ll gns. Bfor nlysis, gns with miniml vrition (lss thn fivfol iffrn or n solut iffrn of <50 twn ny two smpls) or thos outsi th thrshols of 16,000 n 10 wr xlu. Cnit gns rgult y mtor wr intifi using n iliz gn profil (plo wil typ 12 h n 48 h = 0, RAD001 wil typ 12 h = 5, RAD001 wil typ 48 h = 10, plo AKT1-Tg 12 h n 48 h = 24, RAD001 AKT1-Tg 12 h = 12, n RAD001 AKT1-Tg 48 h = 0), n orrlting gn xprssion with this il profil ws trmin using th Prson offiint. Prmuttion of th il profil lls ws us to trmin if th orrltion ws grtr thn xpt y hn lon; 571 uniqu gns or xprss squn tgs pss prmuttion tsting. GSEA ompring RAD001- n plo-trt smpls (n = 9 n 10) ws on using sri mthos n 192 prviously fin gn-pthwy sts 21 or urt list of Hif-1 trgts 22.Pthwys wr rnk oring to th signifin of nrihmnt, n th vlition mo msur of signifin ws us to intify pthwys of grtst nrihmnt. For urt Hif- 1 trgts, nrihmnt ws tst for RAD001 vrsus plo trtmnt, for AKT1-Tg vrsus wil-typ plo-trt prostts, n for ll plo vrsus ll RAD001-trt smpls. Signifin ws tst y ompring th osrv nrihmnt with th nrihmnt sn in t sts in whih smpl lls wr rnomly prmut (n = 1000). GEO ssion numrs. Mirorry t r vill t th NCBI s GEO ( with th ssion numrs GSE1413 n GSM23276 GSM Sttistil nlysis. Anlysis of vrin ws us to tst for iffrns in th prnt poptoti lls or BrU inorportion twn RAD001- n plo-trt mi. Th Wiloxon rnk-sum sttisti ws us to tst for iffrns in th numr of poptoti lls y trtmnt n gnotyp. Stunt s t-tst ws us to tst for iffrns in ll siz y trtmnt. ACKNOWLEDGMENTS W thnk M.A. Brown n W.G. Klin for ritil ommnts; J. Shim, N. Bhtthry, A. Thornr n S. Luo for thnil ssistn; n J. Pouyssgur for th Hif-1 ntioy. This work ws support th Lin n Arthur Gl Cntr for Trnsltionl Rsrh, y th Ntionl Cnr Institut (PO1CA89021), y CPCURE, y th Dmon-Runyon Cnr Rsrh Fountion (W.R.S.) n y Crr Dvlopmnt Awr from th DF/HCC SPORE in Prostt Cnr (P.K.M.). Not: Supplmntry informtion is vill on th Ntur Miin wsit. COMPETING INTEREST STATEMENT Th uthors lr ompting finnil intrsts (s th Ntur Miin wsit for tils). Riv 19 Jnury; pt 28 April 2004 Pulish onlin t 1. Mhm, T. & Dixon, J.E. Th tumor supprssor, PTEN/MMAC1, phosphorylts th lipi son mssngr, phosphtiylinositol 3,4,5-trisphospht. J. Biol. Chm. 273, (1998). 2. l Pso, L., Gonzlz-Gri, M., Pg, C., Hrrr, R. & Nunz, G. Intrlukin- 3-inu phosphoryltion of BAD through th protin kins Akt. Sin 278, (1997). 3. Kops, G.J. t l. Dirt ontrol of th Forkh trnsription ftor AFX y protin kins B. Ntur 398, (1999). 4. Nkmur, N. t l. Forkh trnsription ftors r ritil fftors of ll th n ll yl rrst ownstrm of PTEN. Mol. Cll. Biol. 20, (2000). 5. Cross, D.A., Alssi, D.R., Cohn, P., Anjlkovih, M. & Hmmings, B.A. Inhiition of glyogn synths kins-3 y insulin mit y protin kins B. Ntur 378, (1995). 6. Gorhn, D.C., Priio, N., Goomn, E.C., Mlozik, M. & Wilson, C. Drosophil tumor supprssor PTEN ontrols ll siz n numr y ntgonizing th Chio/PI3-kins signling pthwy. 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