Activation of Akt as a Mechanism for Tumor Immune Evasion

Transcription

1 The Amerin Soiety of Gene Therpy originl rtile Ativtion of Akt s Mehnism for Tumor Immune Evsion Kyung Hee Noh 1, Te Heung Kng 1, Jin Hee Kim 1, Sr I Pi 2, Ken Y Lin 3, Chien-Fu Hung 4, T-C Wu 4 7 nd Te Woo Kim 1 1 Divison of Infetion nd Immunology, Grdute Shool of Mediine, Kore University, Seoul, South Kore; 2 Otolryngology/Hed nd Nek Surgery, The Johns Hopkins Medil Institutions, Bltimore, Mrylnd, USA; 3 Gyneology & Reprodutive Sienes, Deprtment of Ostetris, Yle University Shool of Mediine, New Hven, Connetiut, USA; 4 Deprtment of Pthology, The Johns Hopkins Medil Institutions, Bltimore, Mrylnd, USA; 5 Deprtment of Ostetris nd Gyneology, The Johns Hopkins Medil Institutions, Bltimore, Mrylnd, USA; 6 Deprtment of Onology, The Johns Hopkins Medil Institutions, Bltimore, Mrylnd, USA; 7 Deprtment of Moleulr Miroiology nd Immunology, The Johns Hopkins Medil Institutions, Bltimore, Mrylnd, USA Immune evsion is n importnt reson why the immune system nnot ontrol tumor growth. To eluidte the mehnism for tumor immune evsion, we generted n immune-resistnt humn ppillomvirus type 16 (HPV-16) E7-expressing tumor ell line y sujeting suseptile tumor ell line to multiple rounds of in vivo immune seletion with n E7-speifi vine. Comprison of prentl nd immune-resistnt tumors reveled tht Akt is highly tivted in the immune-resistnt tumors. Retrovirl trnsfer of onstitutively tive form of Akt into the prentl tumor signifintly inresed its resistne ginst E7-speifi CD8 T-ell medited poptosis. The oserved resistne ginst poptosis ws found to e ssoited with the upregultion of ntipoptoti moleules. We lso oserved tht intrtumorl injetion of n Akt inhiitor enhned the therpeuti effiy of E7-speifi vine or E7-speifi CD8 T-ell doptive trnsfer ginst the immune-resistnt tumors. Thus, our dt indite tht the tivtion of PI3K/ Akt pthwy represents new mehnism of immune espe nd hs importnt implitions for the development of novel strtegy in ner immunotherpy ginst immune-resistnt tumor ells. Reeived 6 August 28; epted 17 Otoer 28; pulished online 23 Deemer 28. doi:1.138/mt Introdution Cner immunotherpy hs een resonly suessful in generting tumor-speifi immune responses, leding to signifint ntitumor effets. However, in some ses, it is oserved tht the genertion of tumor-speifi immune responses does not trnslte into tumor regression in ner ptients. 1 One potentil explntion is tht tumors, with their norml ptterns of gene expression nd prolonged, insidious growth, n possily influene nd impir the immune system in mny wys. Thus, it is importnt to fous on the moleulr normlities of ertin tumors tht my filitte their ility to espe ttk from the immune system. To generte suitle prelinil model to study the mehnisms of tumor immune evsion, we employed n in vivo seletion strtegy using previously developed humn ppillomvirus type 16 (HPV-16) E7-expressing ner ell line, lled /P, whih hs served s prelinil tumor model for testing vrious E7-speifi ner immunotherpies. 2,3 We hve previously generted n HPV-16 E7-expressing vini virus vine termed V- Sig/E7/LAMP-1, whih enodes fusion protein onsisting of n endoplsmi retiulum signl sequene, HPV-16 E7 gene, nd the trnsmemrne nd ytoplsmi domins of lysosome-ssoited memrne protein-1 (LAMP-1). 4 Vintion with V-Sig/E7/ LAMP-1 led to sustntil inrese in oth E7-speifi CD8 nd CD4 T-ell immune responses s ompred to wild-type E7 vini virus, preventing the growth of /P in 6 8% of immunized mie. 2 Using the /P ell line nd the Sig/E7/LAMP-1 vini vine, we generted n immunoresistnt tumor ell line, P3, from the /P prentl tumors. 5 Briefly, we immunized mie with Sig/E7/LAMP-1 vini nd hllenged them with / P tumors. We then explnted n outgrowth tumor from the immunized mouse nd expnded it in vitro. This espe vrint ell line ws designted P1 nd ws injeted into new group of mie immunized with Sig/E7/LAMP-1 vini. A tumor from immunized mie ws explnted nd expnded in vitro into nother ell line (P2). These repeted hllenges with vine resistnt tumor ells llowed us to perform n in vivo immune seletion nd resulted in inresing resistne to immuniztion. After three rounds of immune seletion, we otined the P3 ell line, whih ws ompletely resistnt to the vine-indued immune response. Both the P nd P3 ell lines grew with similr growth kinetis. However, when these ell lines were injeted into mie immunized with Sig/E7/LAMP-1 vini, P3 ws developed plple tumors in ll hllenged mie within 7 dys, while only 2 out of 5 mie hllenged with the P tumor ell developed tumors fter severl weeks. Thus, we were suessfully The first two uthors ontriuted eqully to this work. Correspondene: T-C Wu, Deprtment of Pthology, The Johns Hopkins University Shool of Mediine, CRB II Room 39, 155 Orlens Street, Bltimore, Mrylnd 21231, USA. E-mil: wut@jhmi.edu or Te W Kim, Lortory of Infetion nd Immunology, Grdute Shool of Mediine, Kore University, 516 Gojn-1 Dong, Ansn-Si, Gyeonggi-Do , South Kore. E-mil: twkim421@kore.om Moleulr Therpy vol. 17 no. 3, mr

2 Akt-medited Tumor Immune Evsion The Amerin Soiety of Gene Therpy le to generte n immune-resistnt tumor model (P3), therey developing system tht would llow us to identify genes tht my ontriute to tumor espe from vine-medited immune responses. 5 On the sis of this model, we hve employed novel strtegy to identify moleules involved in tumor immune evsion. Sine downregultion of MHC lss I moleules is ommon mehnism of tumor immune evsion, we generted sulones of the immunoresistnt P3 tumors nd hrterized the MHC lss I expression profile of these lones ompred to P nd P3 tumors. While the mjority of sulones from P3 tumors demonstrted deresed MHC lss I expression ompred to the prentl /P tumors, one prtiulr sulone,, demonstrted omprle level of MHC lss I expression ompred to / P tumors, suggesting other mehnisms tht my ontriute to immune espe. In this study, we oserved tht the immune-resistnt sulone, ws resistnt to poptoti ell deth indued y E7-speifi CD8 T ells in vitro nd in vivo. The tumor ells demonstrted upregultion of severl ntipoptoti proteins, whih ws medited y Akt tivtion, thus resulting in the immune resistne phenotype of tumors. In ddition, Akt inhiition ws ple of reversing the immune-resistnt phenotype of tumors, rendering them suseptile to effetive immunotherpy. Thus, we hve suessfully identified Akt tivtion s n innovtive mehnism for tumor immune evsion. The linil implitions of the urrent study re disussed. Results Immunoresistnt tumor ells demonstrte omprle levels of E7 expression nd the ility to tivte E7-speifi CD8 T ells ompred to tumors Using the immunoresistnt P3 tumor model, we generted vrious P3 sulones; A1 to A2. We determined the surfe MHC lss I expression of P nd P3 tumors. We found tht the mjority of sulones of P3 tumors hve low levels of MHC lss I expression ompred to P/ tumors. However, three sulones inluding A3, A9 nd demonstrted omprle level of MHC lss I expression ompred to /P tumors (Supplementry Figure S1). Among the three sulones, showed quite similr MHC lss I expression levels s /P tumors. Therefore, we further evluted the phenotypi nd funtionl hrteristis of the tumor ells. As shown in Figure 1, we oserved tht tumor ells demonstrted omprle expression levels of MHC lss I ompred to the /P ells. The nd / P tumor ells lso showed omprle expression levels of E7 (Figure 1). We lso sequened the HPV-16 E7 gene within the tumor ells nd found no muttions (dt not shown), thus preluding the ltertion of the immunodominnt epitope of E7. Furthermore, to determine the ility of to proess nd present the E7 peptide through the MHC lss I pthwy, we hrterized the ility of tumor ells to tivte E7-speifi CD8 T ells ompred to /P tumors. As shown in Figure 1, we oserved tht tumor ells demonstrted omprle ility IFN-γ (intrellulr) Counts MHC lss I expression Numer of tumor ells % % % % 1.36% % CD8 (surfe) d E7 V Dys fter tumor hllenge P >.9 Figure 1 Phenotypil nd funtionl hrteriztion of tumor ells. () Flow ytometry nlysis to hrterize MHC lss I expressions on nd tumor ells. PE-onjugted ntimouse H-2D monolonl ntiody ws used to detet MHC lss I expression. The isotype ntiody ws used s the negtive ontrol (gry profile). () Western lot nlysis to hrterize the expression of E7 in the nd tumor ells. Equl mounts of ell lystes (5 μg) from nd tumor ells were loded nd seprted y sodium dodeyl sulphte polyrylmide gel eletrophoresis using 15% polyrylmide gel. After eletrolotting, the memrnes were proed with E7-speifi ntiody, nd then inuted with got ntimouse IgG onjugted to horserdish peroxidse for visuliztion of E7 protein using Hyperfilm-enhned hemiluminesene. () Intrellulr ytokine stining nd flow ytometry nlysis to determine the numer of IFN-γ sereting E7-speifi CD8 T ells indued y nd tumor ells. nd tumor ells were inuted with E7-speifi CD8 T ells t.1:1,.1:1, nd 1:1 rtio of tumor ells:t ells for 16 hours. After inution, ells were stined for CD8 nd IFN-γ, nd were sujeted to flow ytometry nlysis to detet tivted E7-speifi CD8 T ells. Dt shown re representtive of three independent experiments. (d) C57BL/6 mie (five per group) were inoulted suutneously with /mouse of or tumor ells. Five dys fter tumor hllenge, mie were treted with V-Sig/E7/LAMP-1. Tumor volumes (mm 3 ) from nd groups were reorded twie per week for 18 dys following immuniztion. Tumor tretment experiments were performed three times to generte reproduile dt vol. 17 no. 3 mr. 29

3 The Amerin Soiety of Gene Therpy Akt-medited Tumor Immune Evsion to tivte E7-speifi CD8 T ells in vitro ompred to / P tumors. This result suggests tht the ntigen proessing nd presenttion of E7 through the MHC lss I ws not impired in tumors reltive to /P tumors. We further determined whether the tumor ells were immune resistnt similr to the prentl P3 tumors. C57BL/6 mie (five per group) were inoulted suutneously with /mouse of /P or tumor ells. Five dys fter tumor hllenge, mie were treted with V-Sig/E7/LAMP-1. Tumor volumes were reorded twie per week for 18 dys following immuniztion. As shown in Figure 1d, tumor-hllenged mie showed signifintly higher tumor volumes over time ompred to tumor-hllenged mie, thus demonstrting immune resistne. This indites tht the tumor ell line is Numer of live tumor ells ( 1 4 ) Counts % % % % % % Ative spse-3 d E:T.1:1.5:1 1: E7 CTL.1:1 E7-speifi CD8 T ell.5:1 1: Dys fter tumor hllenge Figure 2 Chrteriztion of poptoti ell deth of the nd tumors indued y E7-speifi CD8 T ells in vitro nd in vivo. () Flow ytometry nlysis to hrterize tive spse-3 expression in nd tumor ells indued y E7-speifi CD8 T ells. nd tumor ells were inuted with E7-speifi CD8 T ells t different E:T rtios (1:1,.5:1 or.1:1) for 4 hours. Detetion of poptoti ells in the nd tumor ells ws performed using PE-onjugted rit nti tive spse-3 ntiody. The perent of poptoti ells ws nlyzed using flow ytometry for tive spse-3 expression. Dt shown re representtive of three independent experiments. () Grphil representtion of the perentge of poptoti nd tumor ells t different E:T rtios. () Br grph depiting numer of vile nd tumor ells. nd tumor ells were inuted with n E7-speifi CD8 T-ell line t 1:1 rtio of tumor:t ells for 16 hours. The numer of vile tumor ells ws determined using Trypn lue stining (men ± SD). (d) Grphil representtion of the tumor volume in mie hllenged with or tumor ells with or without doptive trnsfer of E7-speifi CD8 T ells. C57BL/6 mie (five per group) were hllenged suutneously with /mouse of nd tumor ells in the left leg. Seven dys fter tumor hllenge, mie reeived / mouse of E7-speifi CD8 T ells intrvenously vi til vein. Mie were monitored twie week for tumor growth. Tumor tretment experiments were performed three times to generte reproduile dt. % Of poptoti tumor ells 8 immune resistnt. Tken together, our dt suggest tht lthough tumor ells express high levels of MHC lss I, demonstrte omprle levels of E7 expression nd n ility to tivte E7-speifi CD8 T ells, they mintin the immune resistne of the prentl P3 tumor ell line. tumors re resistnt to poptoti ell deth indued y E7-speifi CD8 T ells in vitro nd in vivo To determine whether the immune resistne of tumor ells is relted to the poptoti ell deth indued y ytotoxi T-lymphoyte (CTL) killing, /P nd tumor ells were inuted with E7-speifi CD8 T ells t different effetor:trget (E:T) rtios (1:1,.5:1, or.1:1) for 4 hours nd the perentge of poptoti ells ws nlyzed using flow ytometry for tive spse-3 expression. As shown in Figure 2, there ws signifintly lower perentge of poptoti tumor ells ompred to poptoti /P tumor ells t different E:T rtios ompred to / P tumors. A grphil representtion of the perentge of poptoti tumor ells t different E:T rtios is depited in Figure 2. We further hrterized the numer of vile /P nd tumor ells whih were inuted with E7-speifi CD8 T ells t 1:1 rtio of tumor:t ells for 16 hours y stining with Trypn lue. As shown in Figure 2, the numer of vile tumor ells ws signifintly higher in the tumors ompred to the tumors. This oservtion ws lso onfirmed y diret oservtion of the ell morphology of nd tumor ells using light mirosopy fter inution with E7-speifi CD8 T ells overnight. To ssess the immune resistne of the tumor ell lines to the poptoti ell deth indued y doptively trnsferred E7-speifi CD8 T ells in vivo, C57BL/6 mie (five per group) were hllenged suutneously with or tumor ells. Seven dys fter tumor hllenge, mie were doptively trnsferred E7-speifi CD8 T ells intrvenously through the til vein. Mie were monitored twie week for tumor growth. As shown in Figure 2d, tumor-hllenged mie treted with E7-speifi CD8 T ells demonstrted signifintly lower tumor volume over time ompred to tumor-hllenged mie treted with E7-speifi CD8 T ells. In ontrst, there ws no signifint differene in the tumor volumes of untreted or tumor-hllenged mie. Thus, our dt indite tht tumor ells re highly resistnt to poptoti ell deth indued y E7-speifi CD8 T ells in vitro s well s in vivo. Akt tivtion plys role in the resistne of poptoti tumor ell deth indued y E7-speifi CD8 T ells To hrterize the expression of the vrious key nti- nd propoptoti proteins expressed y the nd /P tumor ells, we performed western lot nlysis using nd /P tumor ell lystes. As shown in Figure 3, the expression of the ntipoptoti proteins inluding Bl-2, Bl-xL, phosporylted Bd (p-bd), Bl-w, IAP-2, nd survivin ws signifintly inresed in the tumor ells ompred to /P tumor ells. In omprison, the expression of key propoptoti proteins inluding Bk, Bx, Bd, Bim, nd Bid ws not signifintly different etween ells nd /P ells. Thus, our results suggest the glol upregultion of ntipoptoti proteins. Moleulr Therpy vol. 17 no. 3 mr

4 Akt-medited Tumor Immune Evsion The Amerin Soiety of Gene Therpy We then performed western lot nlysis to determine the expression of the vrious signling moleules tht my ply role in the glol ontrol of poptosis. We nlyzed the expression of totl Akt, Ser 473 phosphorylted pakt, totl Erk, Thr 22/Tyr 24 phosphorylted perk, totl p38 MAP kinse nd Thr 18/ Tyr 182 phosphorylted pp38 MAP kinse. We oserved tht the expression of pakt ws signifintly inresed in the tumor ells ompred to /P s ompred to ll of the poptoti proteins hrterized (Figure 3). Interestingly, no differene ws oserved in the expression of Akt in the /P nd tumors. Thus, our dt indite tht Akt tivtion plys role in the immune resistne of tumor ells ginst poptoti tumor ell deth indued y E7-speifi CD8 T ells. Ativtion of Akt in /P ells indues n immune resistne phenotype in vitro nd in vivo To determine whether tivtion of Akt in /P ells results in n immune resistne phenotype, we trnsfeted /P ells with DNA onstrut enoding HA-tgged myristylted Akt (myrakt) (/CA-Akt). Beuse myrakt hs myristoyltion signl t its N terminus, the protein is not only onstitutively lolized to the plsm memrne ut lso mintins onstitutively tive sttus in the ell. 6,7 /P ells trnsdued with no insert (pmscv) were used s ontrol (/ no insert). We performed western lot with these onstruts to hrterize the expression of Akt, pakt nd HA. As shown in Figure 4, the /CA-Akt tumor ells expressed pakt nd HA ompred to the /no insert. We further hrterized the expression of the vrious key nti- nd propoptoti proteins in these two tumor ell lines. We found tht the expression of ntipoptoti proteins (Bl-2, Bl-xL, p-bd, Bl-w, IAP-2, survivin) ws signifintly inresed in the /CA-Akt tumor Antipoptoti proteins Bl-2 Bl-xL p-bd Bl-w CIAP-1 CIAP-2 Propoptoti proteins Bk Bx Bd Bim Bid (ontrol) pakt Akt perk Erk pp38 p38 Figure 3 Akt tivtion ontriutes to the resistne of poptoti tumor ell deth indued y E7-speifi CD8 T ells. Equl mounts of ell lystes (5 µg) from nd tumor ells were loded nd seprted y sodium dodeyl sulphte polyrylmide gel eletrophoresis using 1% polyrylmide gel. ws used s loding ontrol. () Western lot nlysis to hrterize the expression of vrious key ntipoptoti proteins inluding Bl-2, Bl-xL, p-bd, Bl-w, IAP-2, survivin nd key pro poptoti proteins inluding Bk, Bx, Bd, Bim, Bid in the nd tumor ells. () Western lot nlysis to hrterize the expression of totl Akt, Ser 473 pakt, totl Erk, Thr 22/Tyr 24 perk, totl p38 MAP kinse nd Thr 18/Tyr 182 pp38 MAP kinse in the nd tumor ells. ells ompred to the /no insert ells. In omprison, the expression of key propoptoti proteins (Bk, Bx, Bd, Bim, Bid) ws deresed in /CA-Akt ells ompred to /no insert ells (Figure 4). We then performed flow ytometry nlysis to ompre the resistne to CTL-indued poptosis in vitro etween the /No insert nd /CA-Akt tumor ells. For this, /No insert nd /CA-Akt tumor ells were inuted with E7-speifi CD8 T ells t different E:T rtios (1:1,.5:1 or.1:1) for 4 hours. The perentge of poptoti ells ws nlyzed using flow ytometry for tive spse-3 expression. As shown in Figure 4, there ws signifintly lower perentge of poptoti ells in the /CA-Akt tumor ells ompred to the /no insert ells. pakt Akt HA % Of poptoti tumor ells No insert CA Akt /No insert /CA Akt.1:1.5:1 1:1 Antipoptoti proteins Bl-2 Bl-xL p-bd Bl-w CIAP-1 CIAP-2 d No insert CA Akt Propoptoti proteins /No insert /No insert /CA Akt /CA Akt Bk Bx Bd Bim Bid (ontrol) V No insert CA Akt V- Sig/E7/LAMP Dys fter tumor hllenge Figure 4 Chrteriztion of poptoti ell deth of /no insert nd /CA Akt ells indued y E7-speifi CD8 T ells in vitro nd in vivo. (,) Equl mounts of ell lystes (5 µg) from / No insert nd /CA Akt tumor ells were loded nd seprted y sodium dodeyl sulphte polyrylmide gel eletrophoresis using 1% polyrylmide gel. ws used s loding ontrol. () Western lot nlysis to hrterize the expression of totl Akt, Ser 473 pakt, nd hemgglutinin (HA) in the /No insert nd /CA Akt tumor ells. () Western lot nlysis to hrterize the expression of key ntipoptoti proteins inluding Bl-2, Bl-xL, p-bd, Bl-w, IAP-2, survivin nd key pro poptoti proteins inluding Bk, Bx, Bd, Bim, Bid in the /No insert nd /CA Akt tumor ells. () Grphil representtion of the perentge of poptoti /no insert or /CA Akt tumor ells t different E:T rtios (1:1,.5:1 or.1:1) for 4 hours. Detetion of poptoti ells in the /No insert nd /CA Akt tumor ells ws performed using PE-onjugted rit nti tive spse-3 ntiody. The perentge of poptoti ells ws nlyzed using flow ytometry for tive spse-3 expression. Dt shown re representtive of three independent experiments. (d) Grphil representtion of the tumor volume in mie hllenged with /No insert nd /CA Akt tumor ells with or without vintion with V-Sig/E7/LAMP-1. C57BL/6 mie (five per group) were inoulted suutneously with /mouse of /No insert nd /CA Akt ells. Five dys fter tumor hllenge, mie were treted with V-Sig/E7/LAMP-1. Tumor volumes (mm 3 ) from /No insert nd /CA Akt groups were reorded twie per week for 7 dys following immuniztion. Tumor tretment experiments were performed three times to generte reproduile dt vol. 17 no. 3 mr. 29

5 The Amerin Soiety of Gene Therpy Akt-medited Tumor Immune Evsion We further ssessed resistne of the /CA-Akt tumor ells to poptoti tumor ell deth indued y E7-speifi CD8 T ells in vivo. C57BL/6 mie (five per group) were hllenged suutneously with /no insert or /CA-Akt tumor ells. Seven dys fter tumor hllenge, mie reeived either V-Sig/E7/LAMP-1 vintion or E7-speifi CD8 T ells intrvenously through the til vein. Mie were monitored twie week for tumor growth. / no insert tumor-hllenged mie treted with either V-Sig/E7/ LAMP-1 (Figure 4d) or E7-speifi CD8 T ells (Supplementry Figure S2) demonstrted signifintly lower tumor volume over time ompred to /CA Akt tumor-hllenged mie treted with either V-Sig/E7/LAMP-1 or E7-speifi CD8 T ells. In ontrst, there ws no signifint differene in the tumor volumes of untreted /no insert or /CA-Akt tumor-hllenged mie. Thus, tken together, our dt indite tht /CA-Akt tumor ells re resistnt to poptoti tumor ell deth indued y E7-speifi CD8 T ells in vitro s well s in vivo. Tretment of tumor ells with the Akt inhiitor,, redues the expression of ntipoptoti proteins resulting in n inrese in the poptosis of tumor ells To onfirm the role of Akt in the resistne of poptoti tumor ell deth indued y E7-speifi CD8 T ells, we employed phrmologil inhiitor of Akt, Akt/protein kinse B signling inhiitor-2 (). 8 hs een shown to suppress the kinse tivity nd phosphoryltion level of Akt leding to inhiition of ell growth nd indution of poptosis. 8 As shown in Figure 5, Bl-xL p-bd Bl-w IAP2 pakt Akt pakt inhiitor DMSO Without T ell With T ell Figure 5 Chrteriztion of the expression of ntipoptoti proteins nd perentge of poptosis fter tretment with the Akt inhiitor, API-1. () Western lot nlysis to hrterize the level of totl Akt nd Ser 473 pakt in the tumor ells in the presene or sene of API-1. tumor ells were inuted with the phrmologil pakt inhiitor for 24 hours prior to lyste preprtion. Blots re representtive of three seprte experiments. () Western lot nlysis to hrterize the expression of the key ntipoptoti proteins in the presene nd sene of inhiitor. tumor ell ws inuted with (1 nmol/l) for 24 hours prior to lyste preprtions. ws used s loding ontrol. () Grphil representtion of the perentge of poptoti tumor ells in the presene or sene of E7-speifi CD8 T ells. tumor ells were pretreted with dimethyl sulfoxide (DMSO) or nd inuted with n E7-speifi CD8 T ell t E:T rtios.5:1 for 4 hours. After inution, ells were stined using PE-onjugted rit nti tive spse-3 ntiody. % Of poptoti ells fter CTL ssy we demonstrted suessful inhiition of pakt using the inhiitor. We then hrterized the influene of the inhiitor in the expression of key ntipoptoti proteins in tumor ells. As shown in Figure 5, the expression of the ntipoptoti proteins (Bl-xL, p-bd, Bl-w, IAP-2) in the tumor ells ws signifintly redued in the presene of the pakt inhiitor,. Furthermore, the tumor ells treted with the inhiitor demonstrted inresed suseptiility to poptoti ell deth indued y E7-speifi CD8 T ells (Figure 5). Thus, our dt indite tht tretment with the Akt inhiitor,, redues the expression of ntipoptoti proteins nd results in inresed poptosis of tumor ells, onfirming the role of Akt in the resistne of poptoti tumor ell deth indued y E7-speifi CD8 T ells. Tretment with the Akt inhiitor reverses the immune-resistnt phenotype of tumors nd renders them suseptile to effetive immunotherpy To determine whether the omintion of immunotherpy using vintion or doptive T-ell trnsfer with Akt inhiitor tretment n enhne the ntitumor effets ginst tumor ells, C57BL/6 mie (three per group) were inoulted suutneously with tumor ells per mouse. Seven dys fter tumor hllenge, mie were immunized with either V-Sig/E7/LAMP-1 or V-WT. Another group of tumor-hllenged mie were treted either with doptively trnsferred E7-speifi CD8 T ells or norml sline. Three dys lter, ll the mie were intrtumorlly injeted with. Tumor volumes were reorded twie per week for 1 dys following immuniztion. As shown in Figure 6, tumor-hllenged mie treted with V-Sig/E7/LAMP-1 omined with tretment demonstrted signifintly lower tumor volume over time ompred to tumor-hllenged mie treted with V-WT omined with. A grphil representtion of the tumor weights nd representtive imges of the tumor re depited in Figure 6. Furthermore, tumor-hllenged mie treted with doptively trnsferred E7-speifi CD8 T ells omined with tretment demonstrted signifintly lower tumor volume over time ompred to tumor-hllenged mie treted with nd norml sline (Figure 6). A grphil representtion of the tumor weights nd representtive imges of the tumors re depited in Figure 6d. We lso oserved tht inhiition of Akt rendered humn ner ells sensitive to CTLindued poptosis in vitro nd in vivo (Supplementry Figure S3). Thus, tken together, our dt suggest tht Akt inhiition is ple of reversing the immune-resistnt phenotype of different tumors, rendering them suseptile to effetive immunotherpy. Disussion In this study, we used novel pproh to identify the pthwys involved in tumor immune evsion. We identified prtiulr sulone of immune-resistnt P3 tumors () tht expressed omprle levels of MHC lss I ompred to /P tumors yet were resistnt to poptoti ell deth indued y E7-speifi CD8 T ells in vitro nd in vivo. The tumor ells demonstrted upregultion of severl ntipoptoti proteins, whih ws medited y Akt tivtion, thus resulting in the immune resistne phenotype of tumors. In ddition, Akt inhiition ws Moleulr Therpy vol. 17 no. 3 mr

6 Akt-medited Tumor Immune Evsion The Amerin Soiety of Gene Therpy (5 µg) CH V-Sig/E7/LAMP-1 V-W.T V P <.1 V- Sig/E7/LAMP-1 V-W.T V-SEL V-SEL V-W.T V-W.T Dys fter tumor hllenge (5 µg) CH E7-speifi CD8 T ell Sline E7 CTL P <.2 d E7-speifi CD8 T ell Sline Tumor weight (mg) E7 CTL DMSO E7 CTL Sline DMSO Sline Dys fter tumor hllenge Tumor weight (mg) 4 Figure 6 Chrteriztion of ntitumor effets generted y V-Sig/E7/LAMP-1 vintion or E7-speifi CD8 T-ell doptive trnsfer omined with Akt inhiitor () tretment. C57BL/6 mie (three per group) were inoulted suutneously with /mouse of tumor ells. Seven dys fter tumor hllenge, (,) one set of mie were immunized with pfu/mouse of V-Sig/E7/LAMP-1 while (,d) nother set of mie were treted with /mouse of doptively trnsferred E7-speifi CD8 T ells. Three dys fter immuniztion, mie were injeted intrtumorlly with hitosn hydrogel ontining 5 µg. (,) Tumor volumes from tumor ws reorded twie per week for 1 dys following immuniztion. Line grph representing the tumor volume of tumor-hllenged mie treted with nd () with or without the DNA vintion or () in the presene or sene of E7-speifi CD8 T ells. (,d) Br grph nd representtive imges representing tumor weights nd morphology from hllenged mie treted with nd () with or without the DNA vintion or (d) in the presene or sene of E7-speifi CD8 T ells on 17 dys fter tumor hllenge. ple of reversing the immune-resistnt phenotype of tumors, rendering them suseptile to effetive immunotherpy. Thus, our study identifies tivtion of the Akt pthwy s mehnism for tumor immune espe. Expression of the tive form of Akt hs een shown to ontriute to the resistne of tumor ells to hemotherpy 8,9 nd/ or rdition therpy. 1 In this study, we lso oserved tht tumors demonstrte resistne to hemotherpy nd rdition ompred to P tumors (dt not shown). Furthermore, inhiition of Akt ws le to render the tumor ells more suseptile to hemotherpy nd rdition (see Supplementry Figure S4). Thus, our study is onsistent with previous results, showing tht expression of tive Akt in tumor ells enhnes tumor resistne to hemotherpy nd rdition therpy. Furthermore, Akt signling hs een shown to medite resistne ginst tumor suppression y ntigen-speifi T ells in vitro nd doptively trnsferred ellulr immune effetors in vivo. 11 Tken together, these dt suggest tht resistne to vrious ner therpeuti gents inluding tumor-speifi CD8 T-ell immune response my e relted to the Akt signling pthwy. Thus, the evsion of poptoti ell deth indued y T ells or onventionl ner killing gents my e onsidered hllmrk of multi-resistne to ner therpy. The identifition of expression of the tive form of Akt s mehnism for tumor immune evsion hs signifint linil implitions. Severl linil trils hve demonstrted tht the suessful genertion of tumor-speifi immune responses in vinted ptients did not neessrily orrelte with tumor regression in ner ptients (for review, see ref. 1). We hve lso oserved similr findings in ptients with HPV-16 ssoited highgrde ervil intrepithelil neoplsi lesions who were vinted with n HPV-16 E7 therpeuti DNA vine. We oserved tht in some vinted ptients ppreile levels of HPV-16 E7-speifi CD8 T-ell immune responses were generted whih orrelted with lesion regression; however, in some ptients filed to demonstrte lesion regression in spite of generting ppreile levels of E7-spefi immune responses (T.-C. Wu, unpulished results). Thus, it would e of interest to hrterize the expression levels of the tive form of Akt in these linil speimens. The demonstrtion of upregultion of Akt in the linil speimens from ptients who filed to show lesion regression would onfirm tht the tive form of Akt plys n importnt role in immune evsion. We lso oserved tht tretment reverses the immuneresistnt phenotype of tumors nd renders them suseptile to effetive immunotherpy. This finding hs immense linil relevne sine the Akt inhiitor;, hs een widely used in severl linil trils. is syntheti smll moleule ompound, whih ws previously identified s triiriine or triyli nuleoside. A numer of phse I nd II linil trils using hve een onduted in ptients with dvned tumors, inluding rinoms of the pnres, lung, rest, olon, ldder, nd ovry (for review see ref. 12). Thus, it is oneivle tht n e used in onjuntion with immunotherpy to result in etter therpeuti effets in the linil ren vol. 17 no. 3 mr. 29

7 The Amerin Soiety of Gene Therpy Akt-medited Tumor Immune Evsion We lso oserved tht tretment with lone n itself generte ppreile ntitumor effets ginst tumors (see Figure 6). Sine the Akt pthwy plys pivotl role in mlignnt trnsformtion y induing ell survivl, growth, migrtion, nd ngiogenesis, the inhiition of Akt using my potentilly ply role in suppression of ell growth nd indution of poptosis in humn ner ells tht express Akt. Indeed, studies hve shown tht potently inhiits tumor growth in humn ner ells in whih Akt is errntly expressed/tivted. 8 Thus, the employment of my potentilly ontriute to the ntitumor effet independent of therpeuti vines. In our study we hve monitored the tumor size in tumor ering mie treted with V-Sig/E7/LAMP-1 or doptive T-ell trnsfer omined with or without tretment twie per week for 1 dys following immuniztion. However, the durtion for follow-up of the tumor growth is limited y the relese of from the hitosn hydrogel (CH). We oserved more ovious differene in the ntitumor effet strting from dy 14 nd peks t dy 16 (see Figure 6). However, the differene in the ntitumor effets mong different groups grdully disppers y dy 23 (dt not shown). In our previous study, we hve demonstrted tht CH system used in this study ws ompletely degrded nd disppered 7 dys fter injetion under our experimentl onditions. 13 Thus, the follow-up of tumor growth fter tumor hllenge nd tretment is quite limited in this se. Nevertheless, our dt suggest tht Akt inhiitor tretment n signifintly enhne the therpeuti effiy of tumor ntigen-speifi T-ell immune responses ginst the immune-resistnt tumors. In summry, we hve identified tht upregultion of the tive form of Akt is n innovtive mehnism of tumor immune evsion. In ddition, we hve demonstrted tht inhiition of Akt using n render humn ner ells tht upregulte pakt suseptile to effetive immunotherpy. Thus, our findings hve immense potentil for future linil trnsltion using Akt inhiitors in omintion with immunotherpy for the ontrol of ner. Mterils nd Methods Mie. Six- to 8-week-old femle C57BL/6 mie nd the nude mie were purhsed from Dehn Biolink (Chunguk, Kore). All niml proedures were done in ordne with reommendtions for the proper use nd re of lortory nimls. DNA onstruts. For the genertion of the pmscv/ca Akt onstrut, the DNA frgment enoding HA-tgged CA-Akt Δ4-129, whih ontins sr myristoyltion signl sequene ws mplified from pece-myrakt, ( kind gift from Dr Jongkyeong Chung, KAIST; Dejeon, Kore) 14 using set of primers: 5 -GGAGATCTACCATGGGGAGTAGCAAGAGCAAG-3 nd 5 -GGCTCGAGTCACAGTCCAGGTCCCAGAC-3. The mplified DNA ws susequently loned into the Bgl II/Xho I sites of pmscv retrovirl vetor (Clonteh, Mountin View, CA). For the genertion of pmscv/sct E7 onstrut, DNA frgment enoding the immunodominnt E peptide nd flnking AgeI/NheI restrition enzyme sites ws mde y nneling two singlestrnded oligo-nuleotides 5 -CCGG AGA GCC CAT TAC AAT ATT GTA ACC TTT-3 nd 5 -CTAG TCT CGG GTA ATG TTA TAA CAT TGG AAA-3. It ws then loned into pires-e6-k (ref. 15) using AgeI/NheI sites to reple the E6 epitope, generting pires-e7-2m- K. E7-2m ws then mplified with PCR using pires-e7-2m-k s the templte nd set of primers, 5 -AGA TCT AGA GCC CAT TAC AAT ATT GTA ACC TTT-3 nd 5 -CTC GAG GGT GGT GGA GGT AGT GGC GGG GCG ATG GCT CCG CGC ACG CTG C-3. The mplified produt ws then loned into the BglII/XhoI sites of pmscv/d vetor, whih ws inserted with PCR-mplified D gene using DNA lirry from dendriti ell line, DC2.4, s the templte nd set of primers 5 -CTC GAG ATC CGG TGG TGG AGG TAG TGG CCC ACA CTC GAT GCG GTA TT-3 nd 5 -GAA TTC AAC AAT TGT CAC GCT TTA CAA TCT CGG AGA G-3 into XhoI/EoRI sites of pmscv (Clonteh, CA). Plsmid onstruts were onfirmed y DNA sequening. Regents nd ell lines. The drug, (Cliohem, Sn Diego, CA) ws used for inhiition of the Akt pthwy. Four HPV-16 E7-expressing ell lines,, P3 (), /No insert, nd /CA Akt, were used s murine tumor models. The prodution nd mintenne of ells hs een desried previously. 2 ws generted from P3 lones, outgrowth tumors from vinted mie in vivo, whih hs een previously desried. 5 Among the P3 lones, representtive lone with norml level of MHC lss I expression ws isolted, expnded, nd designted s the P3 () tumor ell line. For the genertion of /CA Akt ell line, the onstruted pmscv/ CA Akt or pmscv/no insert DNAs were trnsfeted into the Phoenix pkging ell line, nd the virus-ontining superntnt ws olleted 48 hours fter trnsfetion. The superntnt ws immeditely onentrted using Centrifugl filter devies (Millipore, Bedford, MA) nd used to infet trget ells () in the presene of 8 µg/ml polyethylenimine (Sigm, St Louis, MO). One dy fter retrovirl trnsdution, the virus superntnt ws repled with norml ulture medium, nd when the ells rehed 7% onflueny, puro myin (5 µg/ml) ws used to selet for ells with integrted pmscv/ca Akt. Four ell lines (,, /No insert, nd /CA Akt) were ultured in RPMI 164 medium ontining 2 mmol/l l-glutmine, 1 mmol/l sodium pyruvte, nonessentil mino id, 1 IU peniillin/streptomyin nd 1% fetl ovine serum in 37 C inutor with 5% CO 2. The CSki/SCT E7 is CSki ell line expressing single-hin trimer of MHC lss I (H-2D ) linked to n HPV-16 E7 immunodominnt CTL epitope ( 49 57), whih n e reognized nd killed y n HPV-16 E7- speifi CD8 T-ell line. 16 For the genertion of the CSki/SCT E7 ell line, the onstruted pmscv/sct E7 ws trnsfeted into the Phoenix pkging ell line for olleting the virus-ontining superntnt. One dy fter retrovirl trnsdution of CSki with the virus superntnt, the trnsdued CSki/SCT E7 ells were seleted using puromyin (5 µg/ ml) s desried ove. The CCD-112Sk (ATCC numer: CRL-251), firolst ell line estlished from humn skin tken from norml tissue, ws used s norml epithelil ell for omprison of Akt tivtion. Genertion of E7-speifi T-ell line. The genertion of the E7-speifi T-ell line hs een desried previously. 16 Briefly, C57BL/6 (H-2) mie were immunized using intrperitonel injetion of 1 7 pfu. of Sig/E7/ LAMP-1 vini virus. Splenoytes were hrvested t dy 8 fter immuniztion. Autologous, irrdited splenoytes pulsed with 1 µg/ml of E7 peptide (mino id 49 57) were used s stimultors. Reominnt humn IL-2 ws dded to the ulture t finl onentrtion of 3 U/ml. The speifiity of the E7-speifi T-ell line ws onfirmed y IFN-g ELISPOT ssy following stimultion with E7-speifi peptide ( 49 57). 16 Flow ytometry nlysis. For in vitro E7-speifi CD8 T-ell tivtion, nd tumor ells were inuted with n E7-speifi CD8 T-ell line t.1:1,.1:1, nd 1:1 rtio of tumor:t ells for 16 hours. Cell surfe mrker stining of CD8 nd intrellulr ytokine stining for IFN-γ s well s FACSn nlysis ws performed using onditions desried previously To detet MHC lss I expression, PE-leled ntimouse H-2K or H-2D (BD Biosienes, Sn Diego, CA) monolonl ntiody ws used. After inution with primry ntiody, ells were wshed with Moleulr Therpy vol. 17 no. 3 mr

8 Akt-medited Tumor Immune Evsion The Amerin Soiety of Gene Therpy phosphte-uffered sline. Anlysis ws done on Beton Dikinson FACSn with CELLQuest softwre (BD Biosienes). CTL-medited ytotoxiity. For the in vitro CTL ssy,,, / No insert nd /CA-Akt tumor ells were hrvested y trypsiniztion, nd wshed one with RPMI (Hylone, Logn) ontining.1% fetl ovine serum nd resuspended in 1 ml of RPMI ontining.1% fetl ovine serum. Cells were inuted for 1 minute with 1 μmol/ml of roxyfluoresein suinimidyl ester (CFSE). CFSE-leled,, /No insert nd /CA Akt were inuted for 4 hours with E7-speifi CD8 T-ell line t vrious E:T rtios (1:1,.5:1, or.1:1) t 37 C with 5% CO 2. To detet poptoti ells, ells were then leled with PE-onjugted ntitive spse-3 ntiody (BD Biosiene, Sn Diego, CA) ording to mnufturer s instrutions. The perentge of poptoti tumor ells ws nlyzed using flow ytometry nlysis y gting CFSE nd tive spse-3 ells. The numer of live tumor ells were lso determined fter overnight inution with E7-speifi CD8 T ells t the vrious E:T rtios using.4% Trypn lue. The ells were ounted using hemtoytometer nd light mirosopy. For the in vitro CTL ssy, CFSE-leled CSki/SCT E7 tumor ells were pretreted with dimethyl sulfoxide or for 24 hours, nd then inuted with n E7-speifi CD8 T-ell line 16 t different E:T rtios (1:1, 5:1 or 1:1) for 4 hours. The perentge of poptoti ells in CFSE CSki/SCT E7 tumor ells were mesured y flow ytometry using PEonjugted rit ntitive spse-3 ntiody. In vivo tumor tretment experiments. For the in vivo tumor tretment experiments, on the dy of tumor hllenge, tumor ells were hrvested y trypsiniztion, wshed one with Opti-MEM I (Gio) nd resuspended in 1 ml of Opti-MEM I to the designted onentrtion for suutneous injetion. Mie were suutneously hllenged with 1 1 5, P3 (), /No insert or /CA-Akt tumor ells per mouse in the left leg. Seven dys fter tumor hllenge, one set of mie were immunized with pfu/mouse of V-Sig/E7/LAMP-1 while nother set of mie were treted with /mouse of doptively trnsferred E7-speifi CD8 T ells intrvenously through the til vein. 16 For the Sig/E7/LAMP-1 vini virus vintion, the virus ws thwed, trypsinized with trypsin/ EDTA in 37 C wter th for 3 min, nd diluted with Opti-MEM I to the finl onentrtion of PFUs/ml. Seven dys fter tumor hllenge, PFUs of Sig/E7/LAMP-1 vini virus were injeted intrperitonelly into C57BL/6 mie. Three dys fter immuniztion, mie were injeted intrtumorlly with CH ontining 5 µg. Tumor volumes from tumor ws reorded twie per week for 1 dys following immuniztion. For the in vivo tumor tretment experiments using doptive T-ell trnsfer in nude mie hllenged with CSki/SCT E7 ells, nude mie (3 per group) were inoulted suutneously with CSki/SCT E7 tumor ells per mouse. Seven dys fter tumor hllenge, CH ontining 5 µg or dimethyl sulfoxide ws intrtumorlly injeted. One dy fter hydrogel tretment, mie were either doptively trnsferred E7-speifi CD8 T ells/mouse or intrvenously injeted with norml sline, s ontrol. 16 Tumor volumes from CSki/SCT E7 tumor were reorded regulrly for 17 dys following immuniztion. In ddition, tumor weight ws mesured 17 dys fter tumor hllenge. Western lot. For eh experiment, totl of ells were rinsed twie with ie-old phosphte-uffered sline nd dded.2 ml of the protein extrtion solution RIPA (Elpis Bioteh, Dejeon, Kore) (5 mmol/l Tris Cl, ph 8., 15 mmol/l NCl, 1 mmol/l phenylmethylsulphonyl fluoride,.1% sodium dodeyl sulphte, 1% Nonidet P-4 (NP-4),.5 mmol/l EDTA). After inution for 3 minutes on ie, ells were srped nd entrifuged. Protein onentrtions were determined y the Coomssie Plus protein ssy (Piere, Rokford, IL). Equl mounts of protein were soluilized in Lemmli uffer (62.5 mmol/l Tris/HCL ph 6.8, 1% glyerol, 2% sodium dodeyl sulphte, 5% merptoethnol nd.625% romophenol lue), oiled for 5 min, nd then seprted y sodium dodeyl sulphte polyrylmide gel eletrophoresis nd trnsferred to nitroellulose memrnes (Amershm Biosiene, Uppsl, Sweden). The memrnes were proed with primry ntiodies diluted 1:1 for phospho Akt (Ser473), Akt, phospho Erk(T22/Y24), Erk, p38 MAPK, Bl-w, Bid, Bim, Bd, phospho Bd (Ser 136) (1:1, Cell Signling, Bevery, MA), Dul phosphor p38 MAPK (1:1, Stressgen, Vitori, Cnd), Bl-2, IAP-2, Bl-xL, Bx, HA-Proe, survivin (1:1, Snt Cruz Biotehnology, Snt Cruz, CA), Bk (1:1, BD Biosienes) nd E7 (provided y Dr Ju-hong Jun, Seoul Ntionl University, Kore) in Tris-uffered sline-t ontining 5% ovine serum lumin (Snt Cruz Biotehnology) t 4 C overnight nd followed y 3 wshes in Tris-uffered sline-t. The memrnes were inuted with the pproprite seondry ntiodies for 1 hour t room temperture nd wshed. Immunoretive nds were visulized y enhned hemiluminesene (Elpis Bioteh, Dejeon, Kore) retion. loded CH. As depot system of (n Akt inhiitor), CH ws prepred s desried previously. 13 Briefly, hitosn solution (MW 161 kd, visosity 2, ps nd degree of deetyltion 8%) ws otined y dissolving 4 mg of hitosn in 1.8 ml of.1 mol/l HCl solution. Two grm of Glyerol 2-phosphte disodium slt hydrte (β-gp), 5 μg of or dimethyl sulfoxide ws dissolved in.1 ml of distilled wter. The hitosn solution ws ooled to 4 C nd ontinuously stirred while dding.1 ml of mixed solution nd the finl volume ws rought to 2 ml with the ddition of distilled wter. The finl onentrtion of hitosn in the mixture ws 2% wt/vol nd ws suutneously injeted into the mie. The mie did not demonstrte severe side effets nd mintined helthy pperne fter implnttion of the hydrogel. Sttistil nlysis. All dt re representtive of t lest two seprte experiments. Results for intrellulr ytokine stining with flow ytometry nlysis, nd tumor tretment experiments were evluted y nlysis of vrine. Comprisons etween individul dt points were mde using Student s t-test. All P vlues <.5 were onsidered sttistilly signifint. SUPPLEMENTARY MATERIAL Figure S1. Chrteriztion of MHC lss I expression of the vrious sulones derived from P3 tumors. Figure S2. Chrteriztion of poptoti ell deth of /no insert nd /CA Akt ells indued y doptively trnsferred E7-speifi CD8 T ells in vivo. Figure S3. Intivtion of Akt renders humn ner ell sensitive to CTL-indued poptosis in vitro nd in vivo. Figure S4. tumor ells re resistnt to hemotherpy nd irrdition through the Akt pthwy. ACKNOWLEDGMENTS This work ws supported y the Ntionl Cner Institute SPORE in Cervil Cner P5 CA98252, 1 RO1 CA in the United Sttes s well s grnt R from the Reserh Center for Women s Diseses of KOSEF, grnt from the Ntionl R&D Progrm for Cner Control, Ministry of Helth & Welfre (7355) nd grnt from the Innovtive Reserh Institute for Cell Therpy, Repuli of Kore (A6226). REFERENCES 1. Rosenerg, SA, Yng, JC nd Restifo, NP (24). Cner immunotherpy: moving eyond urrent vines. Nt Med 1: Lin, KY, Gurnieri, FG, Stveley-O Crroll, KF, Levitsky, HI, August, JT, Prdoll, DM et l. (1996). Tretment of estlished tumors with novel vine tht enhnes mjor histoomptiility lss II presenttion of tumor ntigen. Cner Res 56: Wu, TC (27). The role of vsulr ell dhesion moleule-1 in tumor immune evsion. Cner Res 67: Wu, TC, Gurnieri, FG, Stveley-O Crroll, KF, Visidi, RP, Levitsky, HI, Hedrik, L et l. (1995). Engineering n intrellulr pthwy for mjor histoomptiility omplex lss II presenttion of ntigens. Pro Ntl Ad Si USA 92: Lin, KY, Lu, D, Hung, CF, Peng, S, Hung, L, Jie, C et l. (27). Etopi expression of vsulr ell dhesion moleule-1 s new mehnism for tumor immune evsion. Cner Res 67: vol. 17 no. 3 mr. 29

9 The Amerin Soiety of Gene Therpy Akt-medited Tumor Immune Evsion 6. Kim, S, Jee, K, Kim, D, Koh, H nd Chung, J (21). Cyli AMP inhiits Akt tivity y loking the memrne loliztion of PDK1. J Biol Chem 276: Kohn, AD, Summers, SA, Birnum, MJ nd Roth, RA (1996). Expression of onstitutively tive Akt Ser/Thr kinse in 3T3-L1 dipoytes stimultes gluose uptke nd gluose trnsporter 4 trnslotion. J Biol Chem 271: Yng, L, Dn, HC, Sun, M, Liu, Q, Sun, XM, Feldmn, RI et l. (24). Akt/protein kinse B signling inhiitor-2, seletive smll moleule inhiitor of Akt signling with ntitumor tivity in ner ells overexpressing Akt. Cner Res 64: West, KA, Cstillo, SS nd Dennis, PA (22). Ativtion of the PI3K/Akt pthwy nd hemotherpeuti resistne. Drug Resist Updt 5: Kim, TJ, Lee, JW, Song, SY, Choi, JJ, Choi, CH, Kim, BG et l. (26). Inresed expression of pakt is ssoited with rdition resistne in ervil ner. Br J Cner 94: Hhnel, PS, Thler, S, Antunes, E, Huer, C, Theold, M nd Shuler, M (28). Trgeting AKT signling sensitizes ner to ellulr immunotherpy. Cner Res 68: Cheng, JQ, Lindsley, CW, Cheng, GZ, Yng, H nd Niosi, SV (25). The Akt/PKB pthwy: moleulr trget for ner drug disovery. Onogene 24: Hn, HD, Song, CK, Prk, YS, Noh, KH, Kim, JH, Hwng, T et l. (28). A hitosn hydrogel-sed ner drug delivery system exhiits synergisti ntitumor effets y omining with vini virl vine. Int J Phrm 35: Kim, D, Kim, S, Koh, H, Yoon, SO, Chung, AS, Cho, KS et l. (21). Akt/ PKB promotes ner ell invsion vi inresed motility nd metlloproteinse prodution. Fse J 15: Hung, CH, Peng, S, He, L, Tsi, YC, Boyd, DA, Hnsen, TH et l. (25). Cner immunotherpy using DNA vine enoding single-hin trimer of MHC lss I linked to n HPV-16 E6 immunodominnt CTL epitope. Gene Ther 12: Wng, TL, Ling, M, Shih, IM, Phm, T, Pi, SI, Lu, Z et l. (2). Intrmusulr dministrtion of E7-trnsfeted dendriti ells genertes the most potent E7-speifi nti-tumor immunity. Gene Ther 7: Hung, CF, Cheng, WF, He, L, Ling, M, Jung, J, Lin, CT et l. (23). Enhning mjor histoomptiility omplex lss I ntigen presenttion y trgeting ntigen to entrosomes. Cner Res 63: Cheng, WF, Hung, CF, Chi, CY, Hsu, KF, He, L, Ling, M et l. (21). Tumor-speifi immunity nd ntingiogenesis generted y DNA vine enoding lretiulin linked to tumor ntigen. J Clin Invest 18: Chen, CH, Wng, TL, Hung, CF, Yng, Y, Young, RA, Prdoll, DM et l. (2). Enhnement of DNA vine poteny y linkge of ntigen gene to n HSP7 gene. Cner Res 6: Hung, CF, Cheng, WF, Hsu, KF, Chi, CY, He, L, Ling, M et l. (21). Cner immunotherpy using DNA vine enoding the trnslotion domin of teril toxin linked to tumor ntigen. Cner Res 61: Moleulr Therpy vol. 17 no. 3 mr