API5 Confers Tumoral Immune Escape Through FGF2- Dependent Cell Survival Pathway

Published OnlineFirst April 25, 2014; DOI: 10.1158/0008-5472.CAN-13-3225

Cancer Therapeutics, Targets, and Chemical Biology Research

API5 Confers Tumoral Immune Escape through FGF2- Dependent Cell Survival Pathway

Kyung Hee Noh1, Seok-Ho Kim1,3, Jin Hee Kim1, Kwon-Ho Song1, Young-Ho Lee1, Tae Heung Kang4, Hee Dong Han4,6, Anil K. Sood5,6, Joanne Ng2, Kwanghee Kim2,7,9, Chung Hee Sonn2, Vinay Kumar8, Cassian Yee7,9, Kyung-Mi Lee2, and Tae Woo Kim1,2

Abstract Identifying immune escape mechanisms used by tumors may define strategies to sensitize them to immunotherapies to which they are otherwise resistant. In this study, we show that the antiapoptotic gene API5 acts as an immune escape gene in tumors by rendering them resistant to apoptosis triggered by tumor antigen-specificT cells. Its RNAi-mediated silencing in tumor cells expressing high levels of API5 restored antigen-specific immune sensitivity. Conversely, introducing API5 into API5low cells conferred immune resistance. Mechanistic investiga- tions revealed that API5 mediated resistance by upregulating FGF2 signaling through a FGFR1/PKCd/ERK effector pathway that triggered degradation of the proapoptotic molecule BIM. Blockade of FGF2, PKCd, or ERK phenocopied the effect of API5 silencing in tumor cells expressing high levels of API5 to either murine or human antigen-specific T cells. Our results identify a novel mechanism of immune escape that can be inhibited to potentiate the efficacy of targeted active immunotherapies. Cancer Res; 74(13); 3556–66. 2014 AACR.

Introduction of immunosuppressive cytokines such as TGFb and IL10 and – Despite the presence of a competent immune system, tumor the accumulation of regulatory cells (1 4) can exacerbate the cells may elude detection from host immune surveillance immune inhibitory milieu, whereas intrinsic genetic instability through a process of cancer immune editing. In this process, can generate cells resistant to immune eradication (5). There- elimination of tumor cells sensitive to host immune attack fore, successful anticancer therapies depend on the control of leads to the selection and survival of immune-resistant cancer tumor cell growth and their microenvironment along with cells. For this reason, immune-based strategies can engender strategies to overcome immune tolerance in patients. Howev- an initial response, but recurrences are common as immune- er, the current understanding of molecular mechanisms and resistant tumor cell variants develop under immunoselective signaling pathways underlying tumor immune evasion fi pressure. Extrinsic mechanisms associated with upregulation remains nascent and calls for the identi cation of master factors governing immune escape. In an effort to elucidate potential targetable pathways of Authors' Affiliations: 1Laboratory of Infection and Immunology, Graduate immune resistance and restore immune sensitivity, we dis- 2 School of Medicine, Korea University; Global Research Lab, Division of sected the immune resistance phenotype with the prospect of Brain Korea 21 Program for Biomedical Science and Department of Biochemistry, Korea University College of Medicine, Seoul; 3Immunother- identifying a master gene regulating tumor immune escape. apy Research Center, Korea Research Institute of Bioscience & Biotech- Our studies in the murine model utilized a highly immune- nology, Daejeon, Korea; 4Department of Immunology, College of Medicine, Konkuk University, Chungju, South Korea; 5Department of Gynecologic resistant cervical tumor cell subline, TC-1/P3/A17, generated Oncology and 6Center for RNA Interference and Non-coding RNA; 7Depart- by serial in vivo selection of its immune-susceptible parental ment of Melanoma Medical Oncology and Immunology, U.T. MD Anderson 8 cell line TC-1/P0 expressing the CTL target antigen, HPV16/E7 Cancer Center, Houston Texas; Department of Pathology, University of fi Chicago, Chicago, Illinois; and 9Clinical Research Division, Fred Hutch- (6). This model allowed us to use E7-speci c CTL to assess inson Cancer Research Center, Seattle, Washington immune sensitivity both in vitro and in vivo tumor models. Note: Supplementary data for this article are available at Cancer Research Comparative microarray analysis revealed selective overex- Online (http://cancerres.aacrjournals.org/). pression of an antiapoptotic gene, apoptosis inhibitor 5 (API5), in the immune-resistant phenotype. Through a series of in vitro K.H. Noh and S.-H. Kim contributed equally to this work. and in vivo assays assessing immune sensitivity, we found that Corresponding Authors: Tae Woo Kim, Laboratory of Infection and Immu- nology, Graduate School of Medicine, Korea University, 516 Gojan-1 Dong, API5 plays a critical role as a master regulator of tumor Ansan-Si, Gyeonggi-Do 425-707, Republic of Korea. Phone: 82-31-412- immune escape in mouse. We also validate the role of API5 6713; Fax: 82-31-412-6718; E-mail: [email protected]; Kyung-Mi Lee, as an immune escape factor in human cancer cells by using a E-mail: [email protected]; and Cassian Yee, Department of Melanoma Medical Oncology and Immunology, U.T. MD Anderson Cancer Center, 1515 CTL clone generated from patients with melanoma that Holcombe, Unit 904, Houston, TX 77030. Phone: 713-563-3750; Fax: 713- recognizes an endogenous tumor-associated antigen, MART- 563-3424; E-mail: [email protected] 1. Furthermore, we define a new pathway involved in API5- doi: 10.1158/0008-5472.CAN-13-3225 induced immune resistance that is dependent on the secretion 2014 American Association for Cancer Research. of FGF2 and downstream FGFR1 receptor signaling, which

3556 Cancer Res; 74(13) July 1, 2014

API5 Is a Novel Tumor Immune Escape Factor

triggers specific degradation of the proapoptotic molecule, formed with SPSS version 12.0 software (SPSS), depending on BIM, by PKCd-dependent ERK activation. Therefore, our data the data. Comparisons between individual data points were uncover a major axis of tumor immune resistance regulated by analyzed by Student t test. Where P value was less than 0.05, the API5 and underline the necessity for combinatorial strategies result was considered significant. that include targeting API5 to circumvent tumor immune resistance in patients with cancer. Results Api5 expression in murine tumor cells is associated with þ Materials and Methods immune resistance to antigen-specific CD8 T cells Chemical kinase inhibitors To identify the master regulators governing the immune- LY294002 (Calbiochem Corp) for PI3K, API-2 (Calbiochem resistance phenotype of A17 tumors, we analyzed a previous Corp) for AKT, SB203580 (Calbiochem Corp.) for p38, PD98059 comparative microarray data of parental TC-1/P0 and TC-1/P3 (Stressgen) for ERK, and rottlerin for PKCd (Sigma) were used (A17) cells (NCBI accession number GSE2774). Among several to specifically suppress the activity of indicated kinases. candidate genes, API5, an antiapoptotic factor expressed both in humans and mice, was found to be highly overexpressed in Flow-cytometric analysis and CTL assays A17 cells as compared with their parental P0 cells both at the For in vitro CTL assays, 1 105 E7-expressing or MART-1– mRNA and protein levels (Supplementary Fig. S1; P < 0.05). expressing/HLA-A2–restricted M27 peptide pulsed tumor Transfer of siRNA-targeting Api5 (siApi5) abolished protein þ target cells were incubated with murine E7-specificCD8 expression of Api5 in A17 cells and led to a significant increase þ þ TcellsorMART-1–specific human CD8 T cells, respec- in active caspase-3 A17 cell populations exposed to E7- þ tively, at 1:1 ratio for 4 hours. The percentages of active specific CD8 T cells (Fig. 1A). Conversely, ectopic expression þ caspase-3 tumor cells were measured by flow cytometry to of Api5 in TC1/P0 parental cells rendered them resistant to determine the level of apoptotic cell death. All analysis was CTL-mediated apoptosis (Fig. 1B). Api5 was also overexpressed performed using a Becton Dickinson FACScan with CELL- in murine cancer cells of skin and colorectal origin (B16 and Quest software (BD Biosciences). CT26, respectively; Supplementary Fig. S2A). Downregulation of Api5 led to enhanced CTL-mediated killing, whereas forced Inhibition of BIM degradation expression in Api5-negative targets (EL-4) led to immune To measure the stability of BIM, MG132 (Calbiochem) was resistance to antigen-specific lysis (Supplementary Fig. S2B dissolved in DMSO and then added to a final concentration of and S2C). 25 mmol/L for 3 hours to inhibit proteasome activity. To confirm the role of Api5 as an immune escape factor in vivo, C57BL/6 mice were inoculated subcutaneously with A17 Real-time quantitative RT-PCR cells and administered either siApi5-orsiGFP-loaded chitosan The total RNAs of the cells were isolated using TRIzol nanoparticles (CNP) starting 7 days after initial tumor chal- þ reagent (Invitrogen). First-strand synthesis were performed lenge followed by adoptive transfer of E7-specific CD8 T cells by using RT&Go Mastermix (MP Biomedicals) and real-time (Fig. 1C). As expected, mice receiving E7-specific CTL dem- PCR was performed by a Lightcycler Fast start DNA SYBR onstrated poor control of tumor growth; however, combina- Green Master Mix (Roche) using specific primers with the tion with siApi5 restored the immune sensitivity, resulting in following sequences (rates normalized to the expression level significantly lower tumor volumes (P < 0.05). Conversely, of actin): hAPI5, 5;-GGGCAAAAGAGAGCCAGTGA-30 (forward) forced expression of Api5 in parental TC-1/P0 cells led to and 50- AAAGTTGCCCAAATTGCTGCT-30 (reverse); hFGF2,50- immune resistance and unchecked tumor growth in the pres- GGCTATGAAGGAAGATGGAAGATT-30 (forward) and 50- ence of antigen-specific CTL (Fig. 1D). These results demon- TGCCACATACCAACTGGTGTATTT-30 (reverse); b-actin,50- strate that Api5 directly controls immune resistance in tumor CGACAGGATGCAGAAGGAGA-30 (forward) and 50-TAGAAG- cells both in vitro and in vivo. CATTTGCGGTGGAC-30 (reverse). Api5-mediated immune resistance results from p-Erk– FGF2 assessment of medium supernatants dependent degradation of the proapoptotic molecule, The cells were grown in 6-well plates and incubated with Bim 0.1% FBS containing medium for 48 hours at 37C in a 5% CO2 Because the resistance of Api5-expressing TC-1/P0 cells to incubator. Supernatants were collected and centrifuged to CTLs could simply be due to inhibition of apoptosis or remove cell debris. FGF2 levels in the supernatant were deter- increased cell survival, the expression of pro- and antiapoptotic mined by following the eBioscience FlowCytomix detection kit molecules was assessed by Western blot analysis (Fig. 2A). The instructions. For Western blot analysis, supernatants were protein levels of Xiap, Bcl-2, and Bcl-XL antiapoptotic mole- further concentrated in the 10 by Centricon Plus-70 centrif- cules and Bad, Bak, Bax, and Bid proapoptotic molecules were ugal Filter Units-10 kDa (Millipore). comparable between TC-1/P0/Api5 and TC-1/P0/no insert cells. However, the expression of Bim, a proapoptotic protein, Statistical analysis was substantially diminished in TC-1/P0/Api5 cells. Exposure All data are representative of at least three separate experi- to the proteasome inhibitor, MG 132, restored Bim levels in TC- ments. Nonparametric one-way or two-way ANOVA was per- 1/P0/Api5 cells (Fig. 2B), indicating that Bim was undergoing

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A CTL assay (siRNA knockdown) B CTL assay (overexpression) P P < 0.005 30 < 0.003

15 +

TC-1/P3 (A17) + TC-1/P0 25 siGFP siApi5 w/o CTL No insert Api5 w/o CTL 10 w/ CTL 20 w/ CTL Api5 50 kD Api5 55 kD 1 0.2 15 45 kD 1 10.7 β-Actin 16 kD E7 10 1 1.0 5 1 1.0 45 kD β-Actin 5

1 1.0 casp-3 active of %

apoptotic tumor cells apoptotic tumor 0 % of active casp-3 active of % 0 apoptotic tumor cells apoptotic tumor siGFP siApi5 No insert Api5 TC-1/P0 C D E7-specific CTL

0 7 14 25 (days) 0 7 25 (days)

TC-1/P3 (A17) siRNA CNP (i.v.) TC-1/P0 E7-specific CTL TC-1/P0/Api5 Adoptive transfer 250 1,0001000 P < 0.05 TC-1/no insert – ) TC-1/P3(A17) tumor ) 3 3 TC-1/Api5 –

) 800 TC-1/no insert

200 3 800 + TC-1/Api5 CNP P < 0.04 + 150 600600 Api5 55 kD

100 400400 E7-specific P

45 kD < 0.05 β-Actin CTL 200 50 Tumor volume (mm 200 Tumor volume (mm Tumor volume (mm volume Tumor 0 00 siGFP-CNP ++–– 00 5 1010 15 15 20 20 25 25 siApi5-CNP ––++ Days afterDays after tumor tumor challenge challenge E7-specific CTL –+–+

Figure 1. Identification of API5 as a candidate gene conferring immune resistance in TC-1 tumor cells in vitro and in vivo. A, left, TC-1/P3 (A17) cells were þ treated with siGFP (control) or siApi5 and the levels of Api5 protein analyzed. Right, the percentages of active caspase-3 A17 cells were treated with siGFP or siApi5 generated during CTL assay in response to E7-specific CTL. Data presented are representative of three independent experiments. B, left, þ TC-1/P0/no insert or TC-1/P0/Api5 was analyzed for expression of Api5, E7, or b-actin. Right, the percentages of active caspase-3 TC-1/P0/no insert or TC-1/P0/Api5 cells generated during CTL assay. Data presented are representative of three independent experiments (mean SD). C, schematic representation illustrating in vivo challenge of A17 tumors and subsequent treatment protocol. Left, Western blot analysis of Api5 and b-actin in siGFP-or siApi5-CNP–treated A17 tumor mass. Right, the tumor volumes measured at day 25 in mice treated with either siGFP or siApi5-CNP in the presence or absence of adoptive transfer of E7-specific CTL. Each group contained 5 mice and all data shown are representative of three independent experiments. D, adoptive transfer protocol for E7-specific CTL following in vivo challenge of TC-1/P0/no insert or TC-1/P0/Api5 tumors. Tumor volume was measured over the course of 25 days after tumor challenge. Each group contained 5 mice and all data shown are representative of three independent experiments.

posttranscriptional degradation. Furthermore, silencing Bim challenged with TC-1/P0/Api5 cells and intratumorally using siBim in TC-1/P0/no insert cells conferred resistance to injected with PD98059-loadedchitosan hydrogel along with CTL-mediated lysis (Fig. 2C). Because phosphorylation of Bim E7-specific CTL. Delivery of PD98059 almost completely by MAPKs has been shown to be critical in the degradation of restored immune sensitivity of TC-1/P0/Api5 to E7-specific Bim via proteasomal pathway (7), we next investigated wheth- CTL (Fig. 2G). Notably, PD98059 alone seemed to slightly er Erk and other members of MAPKs were activated in TC-1/ reduce the tumor volume, but was not found to be statistically P0/Api5 cells. Expression of the active form of Erk, Thr202/ significant (P < 0.12). Taken together, these data corroborate Tyr204-pErk, was found to be highly elevated in TC-1/P0/Api5 the essential role of Erk in the degradation of Bim and the cells compared with TC-1/P0/no insert cells (Fig. 2D). Treat- development of immune resistance in vivo. ment with an Erk inhibitor, PD98059 (PD), reduced pErk and restored levels of Bim (Fig. 2E), leading to enhanced killing of API5 regulates immune resistance in human tumor cells TC-1/P0/Api5 cells by E7-specific CTL (Fig. 2F). Inhibition of To explore the physiologic relevance of API5 in the devel- p38, Akt, or PI3K activity using their specific inhibitors did not opment of immune resistance in patients with cancer, the exert significant effect on apoptotic death of TC-1/P0/Api5 expression of human API5 (hAPI5) was investigated in several cells. To further confirm the roles of Erk in vivo, mice were human tumor cell lines arising from various tissue types

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API5 Is a Novel Tumor Immune Escape Factor

A B Proteasomal D TC-1/P0 degradation of BIM Signal pathways No insert Api5 TC-1/P0 TC-1/P0 Xiap 55 kD No insert Api5 No insert Api5 64 kD 1 0.8 MG132 – + – + pAkt (T308) Bcl2 22 kD 22 kD 1 0.9 Bim 64 kD 1 0.7 1 3.6 0.2 3.5 pAkt(S473) 45 kD 1 1.1 BclXL 22 kD β-Actin 64 kD 1 1.1 Akt 1 1.0 1.0 1.0 1 1.1 pBad 22 kD 45 kD 1 0.8 pp38 1 0.9 45 kD Bad 22 kD C TC-1/P0 p38 1 1.0 + P 30 kD < 0.001 1 0.9 45 kD Bak 30 pErk 1 0.9 22 kD 25 1 5.6 45 kD Bax 20 w/o CTL Erk 1 0.9 15 w/ CTL 1 1.0 β 45 kD 22 kD 10 -Actin Bid 1 1.0 1 0.8 5 Bim 22 kD 0 % of active casp-3 active of %

1 0.3 cells apoptotic tumor siGFP siBim 45 kD In vivo β-Actin G intra-tumoral 1 1.0 ERK inhibition E F Kinase inhibition P < 0.12 In vitro ERK inhibition 120 P < 0.05 )

P < 0.05 3 100 P TC-1/P0/Api5 80 < 0.05 w/o CTL DMSO PD + 80 45 kD 3 w/ CTL pErk 60 60 1 0.1 TC-1/P0/Api5 45 kD 40 Erk 40 1 1.1 20 Bim 22 kD

20 volume(mm Tumor 0 1 4.3 45 kD β-Actin CH-DMSO + + –– % of active casp- active of % 1 1.1 cells apoptotic tumor 0 CH-PD98059 ––++ DMSO SB API-2 LY PD E7-specific CTL –+–+ Kinase inhibitors

Figure 2. Identiﬁcation of BIM as a proapoptotic molecule downregulated by API5. A, Western blot analysis characterizing the expression of pro- and antiapoptotic molecules in TC-1/P0/no insert and TC-1/P0/Api5 cells. B, proteasomal degradation of Bim in TC-1/P0/no insert or TC-1/P0/Api5 cells was assessed by Western blot analysis in cells treated with or without MG132. C, TC-1/P0 cells were transfected with siGFP or siBim and exposed to E7-speciﬁc þ CTL. Fractions of apoptotic tumor cells induced by CTL killing are represented by the percentage of activated caspase-3 cells. D, putative MEK kinase intermediates upstream of Bim were analyzed by Western blot analysis. Expression of pAkt, Akt pp38 MAP kinase, p38 MAP kinase, pErk, Erk, and b-actin in the TC-1/P0/no insert and TC-1/P0/Api5 tumor cells are shown. E, TC-1/P0/Api5 tumor cells were incubated with either DMSO or PD98059 for 12 hours and the level of pErk, total Erk, Bim, and b-actin was analyzed by Western blot analyses. F, TC-1/P0/Api5 tumor cells were incubated with DMSO, SB203580, API-2, LY294002, or PD98059 for 18 hours and the percentage of apoptotic TC-1/P0/Api5 tumor cells was measured following exposure to E7-CTL. G, C57BL/6 mice were inoculated subcutaneously with 1 105 TC-1/P0/Api5 cells/mouse and chitosan hydrogel loaded with either PD98059 or DMSO was intratumorally administered at day 7. One day later, mice were adoptively transferred with E7-CTL. Bar graphs represent tumor volumes at day 18 from TC-1/P0/Api5-challenged mice treated with or without PD98059-loaded chitosan hydrogel in the presence or absence of E7 CTL. The data are representative of three separate experiments and bar graphs represent the tumor volume of 5 mice in each group (mean SD).

(Fig. 3A). Among those tested, API5 was found to be markedly were engineered to express single-chain trimer (SCT) of MHC elevated in HeLa, PC-3, MCF-7, HCT116, and 526mel, while the class I (H-2Db) linked to an HPV-16 E7 immunodominant CTL expression of API5 in other tumor lines was comparable with epitope (aa 49–57; HeLa/SCT-E7) for recognition by murineE7- that of nontumorigenic HEK293 cells (Fig. 3A).When the specific CTL (Fig. 3C; ref. 8). The human melanoma cell line expression of hAPI5 was silenced in HeLa, PC3, HCT116, and 526mel was chosen for high endogenous expression of the 526mel cells using siAPI5-loaded CNP (Fig. 3B), pERK level was tumor-associated antigen, MART-1, and presentation of its significantly reduced with concomitant elevation of cellular HLA-A2–restricted epitope for recognition by MART-1–spe- BIM, confirming the role of API5 in regulating the activity of cific CTL (clone KKM; Supplementary Fig. S3). When HeLa/ ERK and BIM degradation in human cancer cells. To further SCT-E7 and 526mel cells were transfected with siAPI5, examine the immune sensitivity of API5-silenced human can- enhanced killing by E7-specificandMART-1–specificCTL cer cells, we chose two API5-expressing tumor lines: HeLa cells was observed as compared with siGFP controls (Fig. 3C). and 526mel. HeLa cells, expressing the highest levels of API5, Similar enhancement was noted when the cellular activity of

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A Kidney Cervix Lung Liver Kidney Prostate Kidney Breast Colon Skin

API5 50 kD 1 6.8 0.9 0.2 0.5 0.3 1 1.0 3.5 1.5 1 3.7 0.7 0.9 0.7 6.2 0.7 0.5 4.2 β-Actin 45 kD 1 1.0 0.9 1.0 1.0 1.0 1 1.0 1.0 0.8 1 1.6 1.2 1.0 1.2 1.2 1.1 1.3 1.0

B siRNA knockdown CDCTL sensitization ERK inhibition HeLa PC-3 HCT116 526mel HeLa/SCT-E7 526mel HeLa/SCT-E7 526mel + P P P 40 < 0.05 P < 0.03 80 < 0.05 60 < 0.04 60 API5 50 kD + w/o CTL w/o CTL 30 50 1 0.1 1 0.1 1 0.2 1 0.1 50 w/ CTL 45 kD w/ CTL 40 60 20 pERK 40 30 1 0.1 1 0.6 1 0.5 1 0.1 45 kD 10 20 ERK 40 30 10

1 1.2 1 1.2 1 0.9 1 1.0 20 apoptotic cells 0 0 DMSO PD DMSO PD BIM 22 kD 20 casp-3 active of % 45 kD 10 pERK 1 8.2 10.1 1 4.5 1 2.2 1 1.9 10.2 45 kD 45 kD casp-3 active of % β-Actin cells apoptotic tumor 0 0 ERK 11.0 11.0 1 1.0 1 1.2 1 0.9 1 1.2 BIM 22 kD 13.0 14.0

EFOverexpression CTL resistance GERK inhibition

HEK 293Db A375 b A375 HEK 293 Db/API5 A375/API5

HEK 293D + P < 0.03 No insert API5 No insert API5 80 P < 0.05 40 80 50 P + P API5 < 0.05 < 0.03 50 kD 60 w/o CTL 30 14.2 13.5 40 w/ CTL 45 kD 60 w/o CTL pERK w/ CTL 40 20 1 4.1 12.5 30 45 kD 20 10 ERK 40 20

11.2 1 1.0 Apoptotic cells 0 0 DMSO PD DMSO PD

20 casp-3 active of % 45 kD BIM 22 kD 10 pERK 1 0.2 10.2 10.1 10.345 kD % of active casp-3 active of % β-Actin 45 kD cells apoptotic tumor 0 0 ERK 11.1 11.0 11.1 10.9 API5 API5 BIM 22 kD No insert No insert 13.5 12.7

Figure 3. API5 controls immune resistance in human cancer cells. A, API5 expression was determined in HeLa, CaSki, MCF-7, MDA-MB-231, DU145, PC-3, SNU-C4, SNU-368, HCT116, HepG2, A549, A375, and 526mel. B, pERK, ERK, BIM, and b-actin expression was assessed by Western blot analysis in HeLa, PC-3, HCT116, and 526mel cells silenced with either siGFP or siAPI5. C, HeLa cells stably transfected with SCT-E7 or 526mel tumor cells transfected þ with siGFP or siAPI5 were subject to CTL assays using murine E7-specific or human MART-1–specific CD8 T cells, respectively. D, HeLa/SCT-E7 or 526mel þ tumor cells treated with DMSO or PD98059 were subject to CTL assay using murine E7-specific or human MART-1–specific CD8 T cells. E, expression of API5, pERK, ERK, BIM, and b-actin in HEK293Db and A375 cells. F, left, HEK293Db/no insert and HEK293Db/API5 cells were subjected to CTL assays using E7 CTL. Right, A375 tumor cells transfected with empty vector or API5 were subject to CTL assays. G, HEK293Db/API5 or A375 tumor cells treated with DMSO or PD98059 were subject to CTL assays. The data are representative of three separate experiments (mean SD).

ERK was suppressed by treatment with PD98059 (Fig. 3D). in antigen processing through the MHC class I pathway and Thus, inhibition of ERK activity led to reduced BIM degra- activation of T cells nor triggering of T-cell death (Supplemen- dation and restored immune sensitivity to antigen-specific tary Fig. S4). Taken together, our data indicate that API5 CTL in human. represents a shared immune escape factor in human cancer To further demonstrate the role of hAPI5 in immune resis- cells and endogenous overexpression of API5 confers immune tance, API5-negative HEK293Db cells and A375 melanoma cells resistance through an ERK-dependent mechanism while gene were retrovirally transduced with hAPI5 and their immune silencing of API5 restores immune sensitivity to antigen-spe- sensitivity monitored. As expected, overexpression of API5 in cific CTL. both cell types caused elevation of pERK (>4 fold), and downregulation of BIM (80% reduction) compared with no-insert API5 activates ERK via the FGF2/FGFR1 pathway controls (Fig. 3E). Furthermore, expression of API5 in As a transcription factor, the ability of API5 to deliver an HEK293Db cells pulsed with E7-peptides or A375 melanoma activation signal to ERK is not well defined. Because multiple cells pulsed with MART-1-peptides mounted resistance to receptor tyrosine kinases (RTK) can mediate ERK signaling apoptotic killing by their cognate antigen-specific CTL (Fig. in tumor cells, an antiphosphotyrosine receptor antibody 3F), whereas inhibition of ERK by PD98059 restored immune array was performed to identify an upstream RTK, which þ sensitivity against API5 target cells (Fig. 3G). The immune might have been involved in API5-mediated ERK activation resistance by API5 overexpression was neither due to the defect (Supplementary Fig. S5A). These arrays detected increased

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phosphorylation of FGFR1 and EGFR in HEK293/hAPI5 cells target effects of rottlerin, we performed the same experi- compared with HEK293/no insert cells. Because both API5 ment using siRNA targeting PKCd and observed similar and FGF2 have been found to be upregulated in some malig- results (left panels, Fig. 5C and D). Consistent with this nancies (Supplementary Fig. S5B; ref. 9), we postulated that result, siPKCd-treated HeLa/SCT/E7 cells and 526mel cells the FGF2/FGFR1 signaling pathway may be involved in deli- were more susceptible to CTL-mediated killing (right vering API5 signals downstream to ERK and initiating the panels,Fig.5CandD).Likewise,thein vivo tumor growth subsequent antiapoptotic cascade by transcriptionally reg- of 526mel-bearing NOD/SCID mice, in vivo siPKCd treatment ulating FGF2 production. As expected, HEK293/hAPI5 cells using CNP before the adoptive transfer of MART-1–specific þ expressed higher levels of phosphorylated FGFR1 (4.2-fold) CD8 T cells (Fig. 5E), demonstrated significantly lower and accompanying downstream signals (p-PKCd,p-ERK) tumor volume compared with those receiving control siGFP compared with HEK293/no insert cells (Fig. 4A; refs. 10, 11). treatment (Fig. 5F). Together, these data reveal that the We also found that both mRNA and protein levels of FGF2 activation of PKCd by FGF2/FGFR1 pathways in API5-over- were elevated among API5-overexpressing cells (Fig. 4B–D). expressing human tumor cells can lead to ERK activation Knockdown or neutralization of secretable 18 kDa FGF2 and BIM degradation, hence controlling API5-mediated by its specific siRNA (Fig. 4E) or mAbs (10 mg/mL of anti- immune resistance both in vitro and in vivo. FGF2; Fig. 4F), respectively, led to a significant decrease in phosphorylated FGFR1 and downstream phosphorylation Silencing of hAPI5 renders the tumor susceptible to intermediates (p-PKCd, p-ERK) with concomitant elevation immune-mediated control in a preclinical human of cellular BIM, suggesting a direct role for FGF2 in the API5- melanoma model induced antiapoptotic axis (9, 12). Functionally, anti-FGF2mAb To demonstrate the potential translational relevance of treatment of HKE293/hAPI5 cells led to increased sensitization API5-targeting and its downstream FGF2/FGFR1 signaling to immune-mediated apoptosis when exposed to antigen- pathways in human tumor immunity, the efficacy of anti- specificCTL(Fig.4G). gen-specific CTL recognizing MART-1 was tested in NOD/ þ To further validate the significance of FGF2 in API5- SCID mice bearing established API5 human tumors. Mice mediated immune resistance, we assessed for a correlation receiving MART-1–specific CTL together with siAPI5, accord- in protein expression levels between FGF2 and API5 in ing to the schedule described in Fig. 6A, experienced signifi- various tumor cell lines (Fig. 4H). A highly significant near cantly lower tumor burden at 49 days after tumor challenge 1:1 correlation was observed between API5 and FGF2 level, compared with siAPI5 alone or CTL with control siRNA (Fig. suggesting close relationship between these two proteins in 6B). Tumors excised on day 49 were also substantially smaller allcelllinestested(Fig.4H). Consistent with these findings, by weight and size among mice receiving both MART-1– we observed increased mRNA and protein levels of FGF2 specific CTL and siAPI5 compared with either treatment alone among the A375 cells engineered to express hAPI5, and (Fig. 6C). Western blot analysis of ex vivo isolated tumors at day decreased levels of FGF2 when API5-expressing tumor cells 49 after challenge demonstrated decreased FGF2, pFGFR1, (HeLa and 526mel) were treated with siAPI5 (Fig. 4I–K). pPKCd, and pERK and a concurrent increase in BIM proteins Furthermore, antibody blockade of FGF2 led to a decrease in among those mice receiving siAPI5 treatment (Fig. 6D), dem- p-FGFR1, p-PKCd, and pERK, and reciprocal increase in BIM onstrating that in vivo delivery of siAPI5 to the tumor and expression in API5-positive A375/hAPI5, HeLa, and 526mel modulation of the tumor immune resistance pathway was cells (Fig. 4L). These intracellular signaling events, occurring successfully achieved. Treatment with siAPI5 resulted in an in the presence of neutralizing anti-FGF2 Abs, were accom- þ increased percentage of apoptotic tumor cells (Fig. 6F), not as a panied by increased sensitization of API5 tumor targets to result of increased CTL infiltration at the tumor site (Fig. 6E), antigen-specific CTL-mediated lysis (Fig. 4L). Taken togeth- but rather due to enhanced lytic capacity of infiltrating CTL. er, these results provide strong evidence for FGF2 in medi- Taken together, our data demonstrate that targeting and ating API5-induced immune resistance and degradation of silencing of API5 or any of its downstream elements represents BIM via the FGFR1/PKCd/ERK axis. an attractive strategy for restoring immune sensitivity to resistant tumor cells. Silencing of PKCd leads to reconstitution of immune sensitivity in API5-expressing tumor cells We next determined whether silencing of PKCd,adown- Discussion stream molecule of FGFR1 signaling proximal to the ERK/ In this study, we identify API5 as a novel shared immune MAPK (13), was sufficienttoinhibitAPI5-inducedtumor escape gene that plays a significant role in controlling immune immune resistance. Exposure of HeLa/SCT-E7 and526mel to resistance to antigen-specific T cells both in mouse and human rottlerin, an inhibitor of PKCd, resulted in a dose-dependent cancer cells. Using murine TC-1/P3 (A17) lung cancer and decrease in pERK levels and accompanying increase in BIM human 526mel tumor cells that endogenously overexpressed expression (Fig. 5A). Furthermore, rottlerin treatment API5, the role of API5 in controlling immune resistance was restored immune sensitivity of HeLa/SCT-E7 or 526mel cells validated both in vitro and in vivo tumor models receiving their to antigen-specificCTLsin vitro (Fig. 5B). No effect of antigen-specific T-cell adoptive transfer. Specific knockdown rottlerin was observed on API5low HeLa/SCT-E7 or 526mel of API5 in API5-positive tumor cells restored antigen-specific cells treated with siAPI5 (Fig.5B).Toexcludepotentialoff- immune sensitivity, whereas the introduction of API5 into

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ABFGFR signaling FGF2 mRNA CFGF2 expression DFGF2 secretion HEK293Db HEK293Db P < 0.02 2.0 No insert 98 kD HEK293Db FGF2 No insert pFGFR1 16 kD Y653/654 (Secreted) 1 4.2 98 kD 1.5 1 4.3 FGFR1 No insert 500 P < 0.05 δ 1 1.0 1.0 pPKC 70 kD API5 50 kD 400 Y311 1 3.5 1 5.2 δ 0.5 300 PKC of Relative 70 kD FGF2 mRNA FGF2 16 kD 1 1.0 45 kD (internal) 1 7.5 200 0.0 45 kD pERK β-Actin 100 1 2.3 45 kD ERK 1 1.0 FGF2 (pg/mL) 0 1 0.9 45 kD No insert β-Actin HEK293Db 1 1.0 No insertHEK293Db

EFFGF2 knockdown FGF2 neutralization GCTL assay H FGF2 expression

HEK293Db/hAPI5 HEK293Db/hAPI5

P < 0.05 8 98 kD 30 R FGF2 16 kD pFGFR1 + = 0.859 1 0.3 Y653/654 6 P 98 kD 1 0.3 98 kD 25 < 0.05 pFGFR1 FGFR1 w/o CTL Y653/654 1 0.3 20 w/ CTL 98 kD δ 1 1.0 4 FGFR1 pPKC 70 kD 1 1.1 Y311 1 0.3 15 δ 2 pPKC 70 kD PKCδ 70 kD Y311 1 0.5 10 1 1.0 45 kD PKCδ 70 kD pERK 5 0 1 1.0 apoptotic cells 45 kD 1 0.5 45 kD

pERK % of active casp-3 0

ERK FGF2 1 0.1 45 kD 1 1.0 02468 ERK BIM 22 kD 1 1.0 1 3.0 API5 BIM 22 kD 45 kD β-Actin b 1 5.6 45 kD 1 1.0 HEK293D /hAPI5 β-Actin 1 1.0

I FGF2 expression (mRNA,protein) L CTL sensitization

P < 0.005 P < 0.002 P < 0.001 1.2 1.2 2.0 1.0 1.0 A375 1.5 0.8 HeLa 0.8 526mel A375/hAPI5 HeLa 526mel 1.0 0.6 0.6 0.4 0.4 P < 0.04 P < 0.03 P < 0.04 0.5 0.2 0.2 + 40 40

Relative of Relative 20 FGF2 mRNA 0.0 0.0 0.0 w/o CTL w/ CTL 30 30 No insert 15 J A375 HeLa 526mel 10 20 20 5 10 10 apoptotic cells API5 50 kD 0 0 0 1 6.5 1 0.3 1 0.2 % of active casp-3 α α FGF2 α-IgG α-FGF2 α-IgG α-FGF2 -IgG -FGF2 16 kD 98 kD (internal) pFGFR1 1 3.1 1 0.1 1 0.1 45 kD Y653/654 1 0.6 1 0.5 1 0.5 β-Actin 98 kD 1 1.1 1 1.0 1 1.1 FGFR1 1 1.0 1 1.0 1 1.0 pPKCδ 70 kD K FGF2 secretion Y311 1 0.4 1 0.1 1 0.3 PKCδ 70 kD P P P 1 1.0 1 1.0 2,500 < 0.003 4,000 < 0.01 250 < 0.02 1 1.0 45 kD 2,000 200 pERK 3,000 1 0.3 1 0.4 1 0.3 45 kD 1,500 150 2,000 ERK 1,000 100 1 1.0 1 1.0 1 1.0 1,000 22 kD 500 50 BIM 1 3.2 1 4.0 1 2.5 FGF2 (pg/mL) 45 kD 0 0 0 β-Actin 1 1.0 1 1.0 1 1.0 No insert A375 HeLa 526mel

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API5 Is a Novel Tumor Immune Escape Factor

In vitro PKCδ inhibition In vitro PKCδ knockdown (KD) ACDConc. of rottlerin (nmol/L) 0 10 100 1,000 HeLa/SCT-E7 P < 0.03 526mel pPKCδ 30 P < 0.05 70 kD siGFP siPKCd siGFP siPKCd 60 1 0.8 0.4 0.2 HeLa/SCT δ + 25 PKC 70 kD pPKCδ pPKCδ 70 kD + 50

70 kD 3

1 1.0 1.1 1.0 - 45 kD 1 0.2 20 1 0.3 pERK 45 kD 45 kD 40 w/o CTL 1 0.9 0.2 0.1 pERK w/o CTL pERK 45 kD 15 w/ CTL ERK 1 0.3 w/ CTL 1 0.2 30

-E7 45 kD 45 kD 1 1.0 1.0 1.0 ERK 10 ERK BIM 22 kD 20 1 1.0 1 1.0 1 1.1 3.2 3.5 5 β 45 kD 22 kD 22 kD 10 -Actin BIM casp-3 active of % BIM apoptotic tumor cells % of active casp active of % δ 1 3.9 0 1 2.5 apoptotic tumor cells 0 pPKC 70 kD 45 kD siGFP siPKCd β 45 kD siPKCd 1 1.1 0.8 0.5 β-Actin -Actin siGFP δ 1 1.0 PKC 70 kD 1 1.1 HeLa/SCT-E7 526mel 1 1.1 1.0 1.1 526mel pERK 45 kD 1 1.1 0.2 0.1 45 kD ERK 1 1.0 1.0 1.0 BIM 22 kD 1 1.1 2.7 2.9 β-Actin 45 kD

BEFCTL sensitization In vivo PKCδ KD 80 HeLa/SCT-E7 526mel 60 tumor P 40 < 0.03 MART-specific CTL P 600 < 0.1 20 siAPI5 HeLa/SCT-E7 P < 0.02 apoptotic cells siGFP HeLa/SCT-E7 500 + 0 0 10 17 24 31 (days) P < 0.05 400 526mel 60 300 526mel 40 siRNA CNP 200

100 20 siAPI5 526mel Tumor weight (mg) Tumor siGFP 526mel 0 0 CTL – + – +

% of active casp-3 active of % 0 10 100 1,000 siGFP siPKCd Conc. of rottlerin (nmol/L)

Figure 5. Identification of PKCd as an immediate target gene for API5 controlling CTL resistance. A, the protein expression in HeLa/SCT-E7 or 526mel tumor þ cells was analyzed in the treatment of 0, 10, 100, or 1,000 nmol/L of rottlerin. B, the percentage of caspase-3 apoptotic cells in siGFP-orsiAPI5-transfected HeLa/SCT-E7 (top) and 526mel cells (bottom) following exposure to antigen-specific CTL was shown in the presence of increasing doses of rottlerin. C, left, þ protein expression was assessed in HeLa/SCT-E7 cells silenced with siGFP or siPKCd. Right, levels of caspase-3 cells in HeLa/SCT-E7 cells silenced with siGFP or siPKCd following exposure to E7-CTL. D, left, protein expression was assessed in 526mel cells silenced with siGFP or siPKCd. Right, levels þ þ of caspase-3 cells in 526mel cells silenced with siGFP or siPKCd following exposure to MART-1–specific CD8 T cells. E, schematic representation illustrating in vivo xenogeneic challenge of 526mel tumors and subsequent treatment protocol. F, tumor volume was analyzed at day 31. The data are representative of three separate experiments.

API5-null tumor cells rendered tumors resistant to immune- API5 has previously been shown to be expressed in mediated cytotoxicity. At the molecular level, we report, for the multiple cancers and contribute to tumor invasion and ﬁrst time, that the tumor immune resistance conferred by API5 metastases (14, 15); however, its precise molecular mecha- is attributable to upregulation of FGF2 and activation of a nism of action remained unclear. Recent studies have shown downstream pathway involving, FGFR1/PKCd/ERK, ultimately that API5 caused suppression of apoptosis by inhibiting leading to the ubiquitin-dependent degradation of the proa- caspase-3–mediated DNA fragmentation (16) and in an poptotic molecule BIM (Fig. 7). E2F-dependent manner (17). Depletion of API5 was shown

Figure 4. API5 activates ERK through the FGF2/FGFR1 pathway. A, FGFR1 signaling following API5 expression is evaluated by Western blot analysis of pFGFR1, FGFR1, pPKCd, PKCd, pERK, and ERK expression in HEK293Db/no insert and HEK293Db/API5 cells. B, mRNA expression analysis of FGF2. C, the protein expression of API5, internal FGF2, and secreted FGF2 in the HEK293Db/API5 cells versus HEK293Db/no insert. D, the amount of FGF2 secreted into the media was measured by ﬂow cytomix. E, Western blot results were shown in siGFP-orsiFGF2-transfected HEK293Db/API5 cells. F, Western blot analysis of expression in IgG isotype controls or FGF2 antibody-treated HEK293Db/API5 cells. G, IgG antibody- or FGF2 antibody-treated HEK293Db/ API5 cells were subject to CTL assays with E7-CTL. H, scatter plot graph shows the linear relationship between expressing API5 (x-axis) and FGF2 (y-axis) in all tumor cell lines tested in Fig. 3A. I–K, mRNA expression of FGF2 (I), protein expression of API5 and internal FGF2 (as surrogates for all FGF2; J), and secretion of FGF2 were monitored in A375 cells transfected with no insert or API5 as well as HeLa and 526mel tumor cells silenced with either siGFP-orsiAPI5 (K). L, top, the percent killing of A375/API5, HeLa, or 526mel cells, treated with either IgG antibody or FGF2 antibody, was measured in CTL assays. Bottom, Western blot results in IgG or FGF2 antibody-treated A375/API5, HeLa, and 526mel cells are shown.

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Noh et al.

ABC 526 mel CTL (Day 49) 400 siGFP – Adoptive transfer of siGFP + ) MART-specific CTL 3 siAPI5 – 400 i.v. 300 siAPI5 + via route P < 0.03 P 300 < 0.02

0 7 14 21 28 35 42 49 200 P 200 < 0.04 P < 0.04 100 526mel 100 Chitosan nanoparticle(CNP) (mm Tumor volume

injection via i.v. Tumor weight(mg) injection via route 0 s.c. route 0 01020304050 CTL – + – + siGFP siAPI5 Days after tumor challenge DEF API5 signaling CTL infiltration Cell death siGFP siAPI5 CTL – + – + siGFP siAPI5 API5 50 kD 1 1.0 0.5 0.4 2.7% 2.4% 10 FGF2 16 kD P < 0.07 P < 0.05 1 1.1 0.1 0.1 98 kD pFGFR1 SSC-H 8 1 1.0 0.5 0.5 98 kD FGFR1 P 6 < 0.12 1 1.0 1.0 1.0 4 CFSE+ T cell pPKCδ 70 kD P < 0.3 1 0.7 0.2 0.3 4 PKCδ 70 kD 3 1 1.0 1.0 1.0 45 kD 2 pERK 2 1 1.1 0.1 0.1 45 kD ERK 0 1 1.0 1.0 1.0 1 BIM 22 kD CTL – + – + 1 1.0 3.2 3.0 cells apoptotic tumor of %

% of CFSE+ T cell % 0 β 45 kD -Actin siGFP siAPI5 siGFP siAPI5 1 1.0 1.0 1.0

Figure 6. Inhibition of API5 renders tumor susceptible to immune-mediated control. A, schematic of the therapy regimen in mice implanted with 526mel melanoma cancer cells. NOD/SCID mice were inoculated subcutaneously with 1 106 526mel cells/mouse. Seven days following tumor challenge, siRNA CNP targeting either GFP or API5 (5 mg/mouse) was injected intravenously for three consecutive days. Approaching the tenth day, mice received adoptive transfer with 1 107 MART-1–specific CTL. This treatment regimen was repeated for six cycles. B, tumor growth of mice inoculated with 526mel. C, tumor weight of mice at 49 days after challenge. D, Western blot analysis of expression in tumor mass. E, flow-cytometric analysis of the frequency of CFSE-labeled, MART-1–specific CTL in the tumors of mice that received adoptive transfer. F, the frequency of apoptotic cells in the tumors of siGFP-or siAPI5-treated mice, with or without adoptive transfer of MART-1–specific CTL.

to enhance the cytotoxic effect of chemotherapeutic drugs not only antiapoptotic BCL-2 family members, such as (18), presumably by facilitating apoptosis of tumor cells in BCL-XL and BCL-2, but also BAK and BAX, was shown to vivo. Notably, the proapoptotic factor, BIM, which can bind be critical for paclitaxel-mediated cell death (19). These

E7-specific mCTL MART1-specific hCTL FGF2 Figure 7. Schematic interpretation

P P of API5-mediated immune FGFRI resistance. Overexpression of API5 ERK upregulates FGF2 and FGFR1 P signaling. Subsequent activation of δ Caspase-3 API5 PKC BIM Apoptosis PKCd leads to ERK P activation P phosphorylation and facilitates ERK ubiquitin-dependent degradation P of the proapoptotic molecule, BIM. BIM

Ubiquitin-dependent BIM degradation

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API5 Is a Novel Tumor Immune Escape Factor

observations, together with our findings presented here, Authors' Contributions suggest that BIM is likely a key mediator of cell death Conception and design: K.H. Noh, S.-H. Kim, K.-H. Song, V. Kumar, K.-M. Lee, T.W. Kim induced not only by chemotherapeutic agents, but also by Development of methodology: K.H. Noh, T.H. Kang, H.-D. Han, A.K. Sood, antigen-specific T cells. Thus, controlling the cellular level of C. Yee, K.-M. Lee, T.W. Kim fi Acquisition of data (provided animals, acquired and managed patients, BIM by API5 seems to be an ef cient means of conferring provided facilities, etc.): S.-H. Kim, K.-H. Song, Y.-H. Lee, T.H. Kang, A.K. Sood, immune resistance in tumors. J. Ng, C.H. Sonn The impact of API5 overexpression in tumor immune eva- Analysis and interpretation of data (e.g., statistical analysis, biostatistics, fi computational analysis): K.H. Noh, S.-H. Kim, C. Yee, T.W. Kim sion has signi cant therapeutic implications. Although several Writing, review, and or revision of the manuscript: K.H. Noh, S.-H. Kim, A.K. clinical trials demonstrated tumor regression following anti- Sood, K. Kim, C. Yee, K.-M. Lee, T.W. Kim gen-specific vaccination or adoptive cellular therapy, a sub- Administrative, technical, or material support (i.e., reporting or orga- nizing data, constructing databases): K.H. Noh, J.H. Kim, Y.-H. Lee stantial proportion of patients experience partial responses Study supervision: C. Yee, K.-M. Lee, T.W. Kim and subsequent relapse (20, 21). Altered expression of apoptosis-regulating molecules, such as BIM, represents one possible Acknowledgments mechanism of immune resistance, which may be exacerbated The authors thank Dr. T.-C. Wu for providing TC-1 P0 and TC-1 P3(A17) cell line and Dr. Ju-hong Jun for providing E7-specific antibody. by prior treatment with chemotherapy or irradiation (22–24), both of which can induce upregulation of API5 and inhibition Grant Support of apoptotic death pathways in tumor cells. The increasing This work was supported by a grant of the Korea Healthcare Technology availability of clinic-ready pharmacologic and biologic R&D Project, Ministry for Health, Welfare and Family Affairs, Republic of Korea reagents that target and silence API5 or downstream elements (A080816), and the National Research Foundation of Korea (NRF) funded by d the Korea government (MEST; 2012R1A2A2A01007527, 2013M3A9D3045881 including anti-FGF2, FGFR, ERK, and PKC provides a rich and 2013M3A9D3045719). K.-M. Lee and her group were supported by a grant opportunity for developing broadly applicable combinational from KICOS (K20703001994-12A0500-03610) and MEST (2012K001404). strategies designed to control immune-resistant and recurrent The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement human malignancies. in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Disclosure of Potential Conﬂicts of Interest Received November 11, 2013; revised March 11, 2014; accepted March 26, 2014; No potential conﬂicts of interest were disclosed. published OnlineFirst April 25, 2014.

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3566 Cancer Res; 74(13) July 1, 2014 Cancer Research

API5 Confers Tumoral Immune Escape through FGF2-Dependent Cell Survival Pathway

Kyung Hee Noh, Seok-Ho Kim, Jin Hee Kim, et al.

Cancer Res 2014;74:3556-3566. Published OnlineFirst April 25, 2014.

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