Roles of the PI-3K and MEK pathways in Ras-mediated chemoresistance in breast cancer cells

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1 ritish Journl of Cner (23) 89, & 23 Cner Reserh UK All rights reserved 7 92/3 $2. Roles of the PI-3K nd MEK pthwys in Rs-medited hemoresistne in rest ner ells W Jin 1,LWu 1, K Ling 1, Liu 1,YLu 1 nd Z Fn*,1 1 Deprtment of, The University of Texs MD Anderson Cner Center, Houston, TX 773, USA Ativted Rs utilises severl downstrem pthwys, inluding the mitogen-tivted protein kinse (MAPK) kinse (MEK)/MAPK pthwy nd the phosphoinositide 3-kinse (PI-3k)/Akt pthwy, to promote ell prolifertion nd to inhiit poptosis. To investigte whih pthwy plys mjor role in Rs-indued drug resistne to hemotherpeuti gents in rest ner ells, we trnsfeted rest ner ells with onstitutively tive H-RsG12V nd exmined the toxiities of three ommonly used rest ner hemotherpeuti gents, plitxel, doxoruiin, nd -fluorouril in these ells under the onditions tht PI-3K or MEK were seletively inhiited y their respetive speifi inhiitors or dominnt negtive expression vetors. We found tht Rs-medited drug resistne is well orrelted with resistne to poptosis indued y ntiner gents in rest ner ells. Although inhiition of MEK/MAPK or PI-3K/Akt n eh enhne the ytotoxiity of plitxel, doxoruiin, or -fluorouril, inhiition of the PI-3K/Akt pthwy seems to hve greter effet thn inhiition of the MEK/MAPK pthwy in reversing Rs-medited drug resistne. Our results indite tht the PI-3K pthwy my ply more importnt role in reeptor tyrosine kinse-medited resistne to hemotherpy nd suggest tht PI-3K/Akt might e ritil trget moleule for ntiner intervention in rest ner. ritish Journl of Cner (23) 89, doi:1.138/sj.j & 23 Cner Reserh UK Keywords: rest ner; drug resistne; Rs; Akt; MAPK It hs een well-demonstrted tht hemotherpy is effetive in prolonging oth disese-free nd overll survivl of women with rest ner. A further question is how to improve the existing regimens of rest ner hemotherpy for etter therpeuti outomes. Reent studies hve provided onvining evidene tht the therpeuti outome of hemotherpy my e ffeted y the expression nd tivity of reeptor tyrosine kinses suh s epiderml growth ftor (EGF) reeptor, HER2, nd their relted moleules. Pursune of this onept my provide importnt lues for optimising the linil pplitions of rest ner hemotherpy, nd for designing new therpeuti pprohes. Memers of the Rs superfmily of GTPses t s importnt moleulr swithes to oordinte extrellulr stimuli with tivtion of intrellulr signlling pthwys for pproprite iologil responses. Although Rs geneti muttionl tivtion is infrequent in rest ner, Rs is often pthologilly tivted in rest ner due to its link to the EGF reeptor fmily or other tyrosine kinses tht signl through Rs nd tht re ommonly overexpressed in this disese (Kroll et l, 22). Ativted Rs sttus hs een ssoited with redued oestrogen dependene nd inresed invsiveness of rest ner (Ksid et l, 198; Sukumr et l, 1988; Sommers et l, 199). Owing to this importnt role of Rs in tumorigenesis, the Rs-signlling pthwy hs ttrted onsiderle ttention s trget for ntiner therpy. Novel ner therpeuti pprohes sed on the inhiition of *Correspondene: Dr Z Fn, The University of Texs MD Anderson Cner Center, Unit 36, 11 Holome lvd, Houston, TX , USA; E-mil: zfn@mdnderson.org Reeived 18 Novemer 22; revised 1 April 23; epted 17 April 23 Rs-medited signlling, inluding inhiition of Rs proessing, inhiition of Rs protein synthesis, nd lokge of downstrem Rs effetors, re eing evluted (Adjei, 21). Ativted Rs is involved not only in tumour progression ut lso possily in the development of resistne of tumour ells to hemotherpy nd to ionising rdition. While the role of Rs in mediting resistne to rdition hs een reltively well-studied nd is nonontroversil (Gupt et l, 21), the ext role of Rs in mediting resistne to vrious hemotherpeuti gents is reltively less doumented, nd in ft, remins ontroversil or even prdoxil. Constitutive expression of the H-Rs onogene inhiits doxoruiin-indued poptosis in rt rhdomyosrom ell line (Nooter et l, 199). In ontrst, the expression of RsG12V in FR3T3 rt firolsts sensitised the ells to tretment with ispltin (Viktorsson et l, 2). Similrly, nother reent study found tht, in the sene of multidrug resistne, humn tumour ell lines with tivted Rs onogenes were uniformly more sensitive to most topoisomerse II inhiitors thn were ell lines ontining wild-type Rs lleles (Koo et l, 1999). In the setting of humn rest ner, the mehnisms of drug resistne ssoited with dvned or metstti hormoneindependent rest rinom re not yet fully understood. The humn rest rinom ell line represents typil in vitro model of rest ner with positive oestrogen reeptors; the originl ells re hormone-dependent nd noninvsive nd eome hormone-independent nd invsive when these ells re trnsformed with n tivted Rs onoprotein (Ksid et l, 198; Sukumr et l, 1988; Sommers et l, 199). In the present study, we investigted the development of drug resistne in ells ssoited with the expression of n tivted Rs (RsG12V) in these ells. As expeted, the expression of RsG12V in ells improved the survivl of ells in hormone-depleted ulture

2 186 medium. We found tht, ompred with ontrol-vetor-trnsfeted ells, the RsG12V ells exhiited inresed resistne to severl hemotherpeuti gents urrently used for treting rest ner ptients. We further ompred the role of two well-known Rs downstrem pthwys, the PI-3K/Akt pthwy nd the MAPK/ ERK kinse (MEK)/MAPK pthwy, in drug resistne medited y tivted Rs. The results of our urrent study provide further evidene for trgeting growth ftor reeptor nd Rs-medited signl trnsdution s n pproh for enhning the therpeuti outome of urrent rest ner hemotherpy. MATERIALS AND METHODS Antiodies nd regents Antiodies direted ginst totl Akt, ser473-phosphorylted Akt1, nd thr22-phosphorylted MAPK (p42/p44) were otined from Cell Signling Tehnology, In. (everly, MA, USA), nti-rs ntiody ws from D iosiene Trnsdution Lortory (Sn Jose, CA, USA), nti-mapk (Erk2) ntiody ws from Snt Cruz iotehnology In. (Snt Cruz, CA, USA), nti-his G ntiody nd nti-ha ntiody were from Rohe Dignostis Corp (Indinpolis, IN, USA), nd LY2942 nd PD989 were otined from Cliohem Corp. (Sn Diego, CA, USA). The ntineoplsti gents used in this study were plitxel (Txol) (ristol Lortories, Prineton, NJ, USA), doxoruiin (Adrimyin) (Gensi Sior Phrmeutils, Irvine, CA, USA), nd - fluorouril (Adruil) (Phrmi, Klmzoo, MI, USA). All other regents were purhsed from Sigm Chemil Co. (St Louis, MO, USA) unless otherwise speified. Cells, ell ulture, nd trnsfetion humn rest ner ells were otined from Amerin Type Culture Colletion (Mnsss, VA, USA). The ells were grown nd routinely mintined in Duleo s modified Egle s medium/f12 medium supplemented with 1% foetl ovine serum (FS), 2 mm glutmine, 1 U ml 1 peniillin, nd 1 mgml 1 streptomyin. Cells were inuted t 371C with % CO 2 nd 9% ir. The RsG12V DNA (psr-h-rsv12, whih ws kindly provided y Dr Rihrd AJ Jnssen), ws suloned into pdna3.1 expression vetor (Invitrogen, Crlsd, CA, USA). pdna3.1 RsG12V or pdna3.1 kone vetor ws trnsfeted into ells with the Fugene-6 trnsfetion kit (Rohe Dignostis), followed y seletion with medium ontining 1 mgml 1 neomyin (G418), nd mintenne in medium ontining 4 mgml 1 G418. Stle lones were evluted for his-tg expression y Western lot nlysis with nti-his G ntiodies, or nti-rs ntiodies. Cell prolifertion ssy ws performed y seeding ells onto sixwell tissue ulture pltes t density of ells well 1, followed y -dy ulture in medium supplemented with FS (1 or.%) or hrol-stripped (CS) FS (1 or.%). Cells were ounted with Coulter ounter (Coulter Corp., Mimi, FL, USA). Western lot nlysis Cells were lysed in lysis uffer ontining mm Tris, ph 7.4, 1 mm NCl,.% NP-4, mm NF, 1 mm N 3 VO 4, 1mM phenylmethylsulphonyl fluoride, 2 mgml 1 leupeptin, nd 2 mgml 1 protinin. The lystes were lered y entrifugtion, nd the superntnts were olleted. Equl mounts of lyste protein were used for the Western lot nlyses with the indited ntiodies (Fn et l, 199). Speifi signls were visulised using the enhned hemoluminesene (ECL) detetion kit (Amershm, Arlington Heights, IL, USA). Chemotherpy ytotoxiity ssy Cells were seeded onto 24-well ulture pltes. After 4-h pulse exposure of the ells to vrious doses of hemotherpeuti gents, the ells were ultured for n dditionl 7 dys with drug-free medium ontining 1% CS-PS. Cell viility ws then ssessed y dding ml of1mgml 1 MTT (3-[4,-dimethylthizol-2-yl]- 2,-diphenyltetrzolium romide) (Sigm) to. ml of ulture medium nd inuting the ells for 3 h t 371C in CO 2 inutor, followed y ell lysis with ml of lysis uffer ontining 2% sodium dodeyl sulphte (SDS) in dimethyl formmide/h 2 O(1:1,vv 1 ), ph 4.7, t 371C for more thn 6 h. Cell viility ws then determined y mesuring the optil sorne of ell lystes t wvelength of 9 nm nd normlising the vlue with the vlue of the orresponding ontrol (untreted ells). Quntifition of poptosis y enzyme-linked immunosorent ssy (ELISA) An poptosis ELISA kit (Rohe Dignostis) ws used to quntittively mesure ytoplsmi histone-ssoited DNA frgments (mononuleosomes nd oligonuleosomes) fter indued ell deth. This photomeri enzyme immunossy ws performed extly ording to the mnufturer s instrutions. Terminl deoxynuleotidyl trnsferse (TdT)-medited dutp-x nik end lelling (TUNEL) ssy The TUNEL ssy ws performed s desried previously (Liu et l, 2). riefly, following fixtion with 1% prformldehyde on ie for 1 h, the ells were wshed one with PS nd then postfixed in ie-old 7% ethnol overnight. The next dy, the ells were wshed one with PS efore inution with ml of TdT retion solution ontining U of TdT,. nmol of iotin-dutp, nd 2. mm CoCl 2 in TdT uffer (Rohe Dignostis) t 371C for 1 h. After the retion, the ells were stined with uffer ontining 2. mgml 1 FITC-vidin,.1% Triton X-1, % dried low ft milk in 4 SSC (.6 M sodium hloride-6 mm sodium itrte, ph 7) in the drk t room temperture for 1 h. efore flow ytometri nlysis (FCM) with FACSn flow ytometer (eton Dikinson, Sn Jose, CA, USA), the ells were ounterstined with solution ontining mgml 1 propidium iodide nd 1 mgml 1 RNse A in PS. The FITC signl ws nlysed using Epis Elite softwre (Coulter Corp.). RESULTS Chrteristion of rest ner ells expressing n tivted Rs We trnsfeted ells with onstitutively tive Rs (RsG12V) expression vetor nd seleted the trnfetnts with 4 mgml 1 G418 for positive lones. Over 1 lones expressing vrious levels of RsG12V were otined. Four representtive lones with the highest levels of RsG12V expression re shown in Figure 1A (gels nd ). Compred with prentl or ontrolvetor-trnsfeted ells, the RsG12V ells exhiited mrked inreses in the levels of phosphorylted Akt nd MAPK (Figure 1A gels nd e), inditing tht the trnsfeted RsG12V ws funtionlly onstitutively tive. The inreses in the tivtion of Akt nd MAPK were not ompnied y hnges in their individul protein levels (Figure 1A gels d nd f). While there ws no signifint differene in the prolifertion rte etween the RsG12V-trnsfeted ells nd ontrol-vetor-trnsfeted ells growing in regulr ulture medium (Figure 1), expression of the RsG12V enhned the survivl of the ells ultured in low- ritish Journl of Cner (23) 89(1), & 23 Cner Reserh UK

3 A C 1 1% FS 1.% FS d e f neo HisG Rs G12V Rs p Akt 473 Akt p MAPK (p44/42) D MAPK (p42) Cell numer ( 1 4 ) Cell numer ( 1 4 ) neo neo 1% CS-FS E 1.% CS-FS neo Cell numer ( 1 4 ) Cell numer ( 1 4 ) 1 1 neo 187 Figure 1 Estlishment nd hrteristion of humn rest ner ells expressing onstitutively tive RsG12V. (A) Prentl ells, ontrol-vetor-trnsfeted ells, nd four seleted ell lones expressing RsG12V were ultured overnight in medium ontining.% hrol-stripped fetl ovine serum (CS-FS). The ells were then hrvested for Western lot nlyses with vrious indited ntiodies. ( E) The ells desried in (A) were ultured for dys in medium ontining 1 or.% fetl ovine serum (FS) (, C) or 1 or.% CS FS. Cells were ounted with Coulter ounter. A Perentge of vile ells (OD t 9 nm) Indution of poptosis (OD t 4 nm) neo neo (μm) RsG12V neo neo (μm) RsG12V neo neo (μm) RsG12V Figure 2 Inresed resistne of RsG12V rest ner ells to tretment with doxoruiin, plitxel, nd -fluorouril. (A) The ells desried in Figure 1 were pulse-exposed to doxoruiin, plitxel, or -fluorouril for 4 h t the indited onentrtions. The ells were then ultured for n dditionl 7 dys with drug-free medium supplemented with 1% CS-FS medium. Cell viilities fter hemotherpy were determined y n MTT ssy s desried in Mterils nd Methods nd were expressed s the perentge of the vlue of untreted ells. () One representtive ell lone (lone C) ws treted with doxoruiin, plitxel, or -fluorouril for 8 h, followed y dditionl ulture for 16 h with 1% CS-FS medium. Cells were hrvested nd sujeted to poptosis ELISA. & 23 Cner Reserh UK ritish Journl of Cner (23) 89(1),

4 188 serum medium (Figure 1C) or in medium with CS serum (hormone-depleted) (Figure 1D nd E). Resistne of RsG12V ells to doxoruiin, plitxel, nd -fluorouril (-FU) Compred with ontrol-vetor-trnsfeted ells, RsG12V lones showed inresed resistne to the tretment with doxoruiin, plitxel, or -fluorouril (Figure 2A). The IC of doxoruiin nd plitxel inresed pproximtely 1-fold, with lesser inrese in the IC of -fluorouril. The inresed resistne to eh of the individul drugs ws ssoited with redued indution of poptosis y the ntineoplsti gents, ssyed y n poptosis ELISA (Figure 2). Involvement of PI-3K nd MEK/MAPK in Rs-medited drug resistne We then ritrrily seleted the RsG12V lone (lone C) nd investigted the potentil roles of the two mjor Rs downstrem pthwys, PI3K/Akt nd MEK/MAPK, in Rsmedited resistne of RsG12V ells to tretment with plitxel, doxoruiin, nd -fluorouril. We first exmined whether inhiition of PI-3K with its speifi inhiitor, LY2942, enhned the poptosis of the RsG12V ells following A d e d e neo LY2942 (μm) PD989 (μm) neo Rs G12V Rs p - Akt 473 Akt p - MAPK (p44/42) MAPK (p42) Rs G12V Rs p - Akt 473 Akt p - MAPK (p44/42) MAPK (p42) Figure 3 Nonrossing inhiition of RsG12V-indued tivtion of PI-3K nd MEK y their respetive speifi inhiitors (PD989 nd LY2942). RsG12V lone C ells were exposed to inresing onentrtions of PD989 s indited (A) or inresing onentrtions of LY2942 s indited () in medium ontining.% CS FS for 16 h, ompnied y prllel ultures of prentl ells nd ontrol-vetor- trnsfeted ells in.% CS FS medium without PD989 or LY2942. Cells were olleted for Western lot nlysis for the levels of Rs (), phosphorylted Akt (), totl Akt (), phosphorylted MAPK (d), nd MAPK (p42) (e). tretment with the hemotherpeuti gents. Within the dose rnge from to 4 mm LY2942, there ws n LY2942-dosedependent inhiition of PI-3K tivity, s demonstrted y redued phosphoryltion of Akt y Western lot nlysis with Akt serine-473 phosphoryltion-speifi ntiodies. At the dose of 2 or 4 mm, LY2942 nerly ompletely inhiited RsG12Vindued PI-3K/Akt tivtion (Figure 3A, gels, lnes 7 or 8 vs lnes 1 or 2). In ontrst, within the given dose rnge of LY2942, there ws no effet of LY2942 on the tivity of the MEK/MAPK pthwy; the Rs-indued inrese in MEK/MAPK tivity ws unhnged (Figure 3A, gels d, lne 7 or 8 vs lnes 1 or 2), inditing the reltive speifiity of LY2942 in our experimentl system in inhiiting the PI-3K/Akt pthwy. We found tht the tretment of RsG12V ells with LY2942 mrkedly sensitised the ells to doxoruiin-indued poptosis (Figure 1A). Similr enhnement of sensitivity y LY2942 ws found in RsG12V ells treted with plitxel or -fluorouril (Figure 4 nd C). We simultneously exmined whether inhiition of MEK with its speifi inhiitor, PD989, would enhne poptosis of the RsG12V ells following tretment with the hemotherpeuti gents. Similr to the speifiity of inhiition of LY2942 (within the given dose rnge) on PI-3K, there ws PD989-dosedependent inhiition of MEK tivity, s demonstrted y redued phosphoryltion of MAPK y Western lot nlysis with MAPK threonine-22 phosphoryltion-speifi ntiodies. At doses etween 4 nd 8 mm, PD989 nerly ompletely inhiited RsG12V-indued MEK/MAPK tivtion (Figure 3, gel d, lne 7or8vs lnes 1 or 2), ut hd no effet on the tivity of the PI- 3K/Akt pthwy. The Rs-indued inrese in PI-3K/Akt tivity remined, inditing the reltive speifiity of PD989 in our experimentl system in inhiiting MEK/MAPK pthwys. Similr to the results with LY2942 tretment, inhiition of the MEK/ MAPK pthwy with PD989 sensitised the RsG12V ells to tretments with doxoruiin, plitxel, nd -fluorouril, ut it ppers tht, in ll three ses, inhiition of PI-3K with LY2942 is sttistilly more effetive thn inhiition of MEK in enhning drug-indued poptosis (Figure 4). Although oth LY2942 nd PD989 showed reltive speifiity in inhiiting PI-3K nd MEK, respetively, there were still possiilities tht the two ompounds my inhiit dditionl kinses other thn PI-3K or MEK. To further onfirm our results otined with LY2942 nd PD989, we investigted whether expression of dominnt-negtive (dn) PI-3K (delt p8) or dn MEK (M97K) will hve similr effets on sensitising the RsG12V ells to the drug-indued poptosis. Figure A shows the trnsient expressions of dn PI-3K nd dn MEK in RsG12V ells, respetively. Compred with ontrol-vetortrnsfeted ells, expression of dn PI-3K sensitized the RsG12V ells to doxoruiin-, plitxel-, nd -fluorouril-indued poptosis, ssyed y the poptosis ELISA (Figure ). In ontrst, expression of dn MEK showed different effets: nerly no effet on doxoruiin-indued poptosis, slight effet on plitxel-indued poptosis, nd more pprent effet on -fluorouril-indued poptosis. To provide n dditionl ssy to mesure the sensitistion of dn PI-3K nd dn MEK on drug-indued poptosis, we performed TUNEL ssy (Figure C). Trnsient expression of either dn PI-3K or dn MEK showed similr sensitistion of plitxel- nd -fluorouril-indued poptosis in RsG12V ells. We were unle to detet doxoruiinindued poptosis y TUNEL, possily due to the interferene of n inherent fluoresene of doxoruiin. DISCUSSION The mehnism of drug resistne involves not only the delivery of drugs into trgeted ner ells ut lso the ility of ner ells ritish Journl of Cner (23) 89(1), & 23 Cner Reserh UK

5 A Apoptosis (OD t 4 nm) Apoptosis (OD t 4 nm) C Apoptosis (OD t 4 nm) DMSO DMSO DMSO LY2942 LY2942 PD989 LY2942 PD989 PD989 +LY2942 +PD989 to survive under drug hllenge, whih is olletively ontriuted y multiple intrinsi nd extrinsi ftors. Reent interest nd effort in reduing the resistne of ner ells to hemotherpy hve een foused on modultion t vrious levels of signlling pthwys in ner ells. In our urrent study, we demonstrted n unequl ontriution of the two importnt Rs downstrem pthwys, PI-3K nd MEK/MAPK, in mediting inresed drug resistne in humn rest ner ells following the P<.1 P<.1 +LY2942 +PD989 P<. +LY2942 +PD989 Figure 4 Enhnement of drug-indued poptosis y the PI-3K-speifi inhiitor LY2942 nd the MEK-speifi inhiitor PD989 on RsG12V ells. RsG12V ells were ultured for 16-h predrug period with or without 2 mm LY2942 or 4 mm PD989, 8-h pulse exposure of the ells to 2. mm doxoruiin (A), mm plitxel (), or 4 mm -fluorouril (C), nd 16-h postdrug period in 1% CS-FS. Cells were then hrvested for poptosis ELISA. The sttistil differenes etween LY2942 nd PD989 in enhning the ytotoxiity of drugs (doxoruiin, plitxel, or -fluorouril) were lulted y Student s t- test. A Apoptosis (OD t 4 nm) C TUNEL posiltive rte (%) (FITC) P<.1 expression of onstitutively tive Rs (H-RsG12V). This experimentl setting ws undertken to reflet not only the onogeni Rs muttion per se, ut in order sense, the Rs protein tivted y reeptor tyrosine kinse-medited signl trnsdution, whih is ommonly seen in rest ner. We found tht the resistne of ells expressing H-RsG12V to the ommonly used rest ner hemotherpeuti gents (doxoruiin, plitxel, nd -fluorouril) ws well orrelted with poptosis indued y the ntineoplsti gents. P<.1 P< % 8.26% 17.4% 1.4% 23.4% 3.9% 3.39% 13.% 27.8% DNA ontent (PI) NS* β-atin vetor vetor Figure Effets of dn PI-3K (Dp8) nd dn MEK (K97 M) on Rsindued drug resistne. RsG12V ells were trnsfeted with plsmid ontining HA-tgged MEK (K97 M) or HA-tgged PI-3K (Dp8) for 16 h for trnsient expression, followed y 8-h exposure to doxoruiin, plitxel, or -fluoruril, nd 16-h postdrug period in medium ontining 1% CS FS. The ells were then hrvested for detetion of the expressions of the dn forms of PI-3K (A, gel ) or MEK (A, gel ) y Western lot nlyses with n nti-ha ntiody, nd for determintion of poptosis y ELISA () nd y TUNEL (C). The sttistil differenes etween the ells expressing dn PI-3K (Dp8) nd dn MEK (K97 M) in enhning the ytotoxiity of drugs (doxoruiin, plitxel, or -fluorouril) were lulted y Student s t-test. dn: dominnt negtive; NS* ¼ nonspeifi signls. 189 & 23 Cner Reserh UK ritish Journl of Cner (23) 89(1),

6 19 Cells often reeive multiple stimuli. Given the omplexity of Rsmedited signl trnsdution, none of its downstrem pthwys funtion lone. In order to develop moleulr-trgeted pprohes for sensitising ner ells to hemotherpy, it is importnt to eluidte the downstrem pthwys tht ply mjor roles in Rsmedited drug resistne of ner ells to hemotherpy. The MEK/MAPK nd PI-3K pthwys re t the ifurtion point of Rs-medited downstrem pthwys. Our experimentl gol ws to determine whether oth MEK/MAPK nd PI-3K pthwys ontriute to Rs-medited drug resistne, nd if so, whih of the two pthwys plys mjor role in mediting Rs tivtionindued drug resistne. y using oth speifi inhiitors nd dominnt negtive expression vetors of MEK or PI-3K, we found tht PI-3K seems to ply mjor role in drug resistne in our experimentl setting, euse inhiition of PI-3K y tretment of the ells with the PI-3K speifi inhiitor LY2942 or y expression of dominnt negtive PI-3K expression vetor (Dp8) showed sttistilly signifint etter sensitistion thn inhiition of MEK with similr pprohes in RsG12V ells following tretment with doxoruiin, plitxel, or -fluorouril. An importnt vet is tht, in our experimentl setting, we used the dose of LY2942 (2 mm) tht ompletely inhiited Rs-medited PI-3K tivtion (demonstrted y redued phosphoryltion of Akt to the level omprle to tht in ontrolvetor-trnsfeted ells, shown in Figure 3A, gel ), nd tht did not ffet the tivity of MEK (demonstrted y unhnged phosphoryltion of MAPK, shown in Figure 3A, gels d). Similrly, we used the dose of PD989 (4 mm) tht ompletely inhiited the tivity of MEK without ffeting the tivity of PI-3K, s demonstrted y redued phosphoryltion of MAPK to the level omprle to tht for ontrol- vetor-trnsfeted ells nd y n unhnged level of Akt phosphoryltion (shown in Figure 3 gels nd d). This experimentl ondition llowed us to rule out ny potentil vrition tht my result from insuffiient or ross inhiition of PI-3K or MEK y LY2942 or PD989. Furthermore, euse LY2942 or PD989 my inhiit other kinses in ddition to PI-3K or MEK, we used the dominnt negtive expression vetors of PI-3K nd MEK, respetively, in our experiments nd otined omprle results. Akt is one of the mjor effetor moleules following PI-3K tivtion. Akt is tivted y PDKs following its reruitment to the ell memrne y phosphtidylinositol 3,4,-triphosphte (PtdIns-(3,4,)-P3) nd phosphtidylinositol 3,4-iphosphte (PtdIns-(3,4)-P2), whih re generted y PI-3K. Reent evidene indites tht Akt is frequently tivted in humn rest ner. The tivted sttus of Akt my led to rest ner ptients eing more prone to relpse with distnt metstsis (Perez- Tenorio nd Stl, 22). In rest ner, the inrese in Akt kinse tivity my minly result from the tivities of Akt upstrem regultory signls produed y overexpression or tivtion of the EGF reeptor fmily. Less ommonly, it ould lso result from the muttionl tivtion of Rs onogene tht tivtes Akt through the PI-3K pthwy (Dtt et l, 1996; Liu et l, 1998) or from the muttionl intivtion of the PTEN tumour-suppressor gene tht normlly inhiits Akt tivity y dephosphorylting the PI-3,4,-P 3 nd PI-3,4-P2 produed y PI- 3K (Stmoli et l, 1998; Rmswmy et l, 1999). Although the mehnisms hve not yet een fully hrterised, tivted Akt signlling is elieved to promote ell survivl y regulting severl key moleules tht re involved in poptosis, suh s d (Dtt et l, 1997; del Peso et l, 1997), spse-9 (Crdone et l, 1998), FKHRL1 (runet et l, 1999), CRE (Du nd Montminy, 1998), nd IKK (Ozes et l, 1999). Erlier oservtions reported tht IGF-I n resue ells from doxoruiin-indued poptosis in PI-3K-dependent, ut not MAPK-dependent mnner (Gooh et l, 1999). In ontrst, IGF-I resued ells from plitxel-indued poptosis, whih required oth PI-3K nd MAPK, suggesting tht the drug mehnism-speifi tion in IGF-I ttenuted the response of rest ner ells to doxoruiin nd plitxel (Gooh et l, 1999). In our experimentl setting with the ells expressing onstitutively tive Rs, we found tht the poptosis indued y plitxel or doxoruiin n e enhned y inhiition of either PI-3K or MAPK, ut inhiition of the PI-3K ppers to e more effetive, suggesting tht oth pthwys re uneqully involved in resuing the ells from drug-indued poptosis. In summry, our results indite tht the PI-3K pthwy my ply more importnt role in mediting reeptor tyrosine kinsemedited resistne to hemotherpy nd suggest tht PI-3K/Akt might e ritil trget moleule for ntiner intervention in rest ner. ACKNOWLEDGEMENTS We thnk Dr Rihrd AJ Jnssen (Center for iologis Evlution nd Reserh, Food nd Drug Administrtion, ethesd, MD, USA) for the psr-h-rsv12 plsmid, Dr Gordon Mills (MD Anderson Cner Center, Houston, TX, USA) for pmm9-ha-mek (K97 M) nd psr-dp8-ha plsmids, nd Mr Mihel Worley (MD Anderson Cner Center, Houston, TX, USA) for editoril ssistne with the mnusript. This work ws supported y US Deprtment of Defense Medil Reserh Grnt DAMD , y grnt from The rest Cner Reserh Foundtion (New York), nd y NCI Cner Center Core Grnt CA REFERENCES Adjei AA (21) loking onogeni Rs signlling for ner therpy. J Ntl Cner Inst 93: runet A, onni A, Zigmond MJ, Lin MZ, Juo P, Hu LS, Anderson MJ, Arden KC, lenis J, Greenerg ME (1999) Akt promotes ell survivl y phosphorylting nd inhiiting Forkhed trnsription ftor. Cell 96: Crdone MH, Roy N, Stennike HR, Slvesen GS, Frnke TF, Stnridge E, Frish S, Reed JC (1998) Regultion of ell deth protese spse-9 y phosphoryltion. 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