API5 Confers Tumoral Immune Escape through FGF2 ......API5 Confers Tumoral Immune Escape through...

Therapeutics, Targets, and Chemical Biology

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

AbstractIdentifying immune escape mechanisms used by tumors may define strategies to sensitize them to immu-

notherapies to which they are otherwise resistant. In this study, we show that the antiapoptotic gene API5 acts asan immune escape gene in tumors by rendering them resistant to apoptosis triggered by tumor antigen-specific Tcells. Its RNAi-mediated silencing in tumor cells expressing high levels of API5 restored antigen-specific immunesensitivity. 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/ERKeffector pathway that triggered degradation of the proapoptotic molecule BIM. Blockade of FGF2, PKCd, or ERKphenocopied the effect of API5 silencing in tumor cells expressing high levels of API5 to either murine or humanantigen-specific T cells. Our results identify a novel mechanism of immune escape that can be inhibited topotentiate the efficacy of targeted active immunotherapies. Cancer Res; 74(13); 3556–66. �2014 AACR.

IntroductionDespite the presence of a competent immune system, tumor

cells may elude detection from host immune surveillancethrough a process of cancer immune editing. In this process,elimination of tumor cells sensitive to host immune attackleads to the selection and survival of immune-resistant cancercells. For this reason, immune-based strategies can engenderan initial response, but recurrences are common as immune-resistant tumor cell variants develop under immunoselectivepressure. Extrinsic mechanisms associated with upregulation

of immunosuppressive cytokines such as TGFb and IL10 andthe accumulation of regulatory cells (1–4) can exacerbate theimmune inhibitory milieu, whereas intrinsic genetic instabilitycan generate cells resistant to immune eradication (5). There-fore, successful anticancer therapies depend on the control oftumor cell growth and their microenvironment along withstrategies to overcome immune tolerance in patients. Howev-er, the current understanding of molecular mechanisms andsignaling pathways underlying tumor immune evasionremains nascent and calls for the identification of masterfactors governing immune escape.

In an effort to elucidate potential targetable pathways ofimmune resistance and restore immune sensitivity, we dis-sected the immune resistance phenotype with the prospect ofidentifying a master gene regulating tumor immune escape.Our studies in the murine model utilized a highly immune-resistant cervical tumor cell subline, TC-1/P3/A17, generatedby serial in vivo selection of its immune-susceptible parentalcell line TC-1/P0 expressing the CTL target antigen, HPV16/E7(6). This model allowed us to use E7-specific CTL to assessimmune sensitivity both in vitro and in vivo tumor models.Comparative microarray analysis revealed selective overex-pression of an antiapoptotic gene, apoptosis inhibitor 5 (API5),in the immune-resistant phenotype. Through a series of in vitroand in vivo assays assessing immune sensitivity, we found thatAPI5 plays a critical role as a master regulator of tumorimmune escape in mouse. We also validate the role of API5as an immune escape factor in human cancer cells by using aCTL clone generated from patients with melanoma thatrecognizes an endogenous tumor-associated antigen, MART-1. Furthermore, we define a new pathway involved in API5-induced immune resistance that is dependent on the secretionof FGF2 and downstream FGFR1 receptor signaling, which

Authors' Affiliations: 1Laboratory of Infection and Immunology, GraduateSchool of Medicine, Korea University; 2Global Research Lab, Division ofBrain Korea 21 Program for Biomedical Science and Department ofBiochemistry, Korea University College of Medicine, Seoul; 3Immunother-apy Research Center, Korea Research Institute of Bioscience & Biotech-nology, Daejeon, Korea; 4Department of Immunology, College ofMedicine,Konkuk University, Chungju, South Korea; 5Department of GynecologicOncology and 6Center for RNA Interference andNon-codingRNA; 7Depart-ment of MelanomaMedical Oncology and Immunology, U.T. MDAndersonCancer Center, Houston Texas; 8Department of Pathology, University ofChicago, Chicago, Illinois; and 9Clinical Research Division, Fred Hutch-inson Cancer Research Center, Seattle, Washington

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

K.H. Noh and S.-H. Kim contributed equally to this work.

Corresponding Authors: Tae Woo Kim, Laboratory of Infection and Immu-nology, Graduate School of Medicine, Korea University, 516 Gojan-1 Dong,Ansan-Si, Gyeonggi-Do 425-707, Republic of Korea. Phone: 82-31-412-6713; Fax: 82-31-412-6718; E-mail: [email protected]; Kyung-Mi Lee,E-mail: [email protected]; and Cassian Yee, Department of MelanomaMedicalOncology and Immunology, U.T.MDAndersonCancer Center, 1515Holcombe, Unit 904, Houston, TX 77030. Phone: 713-563-3750; Fax: 713-563-3424; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-13-3225

�2014 American Association for Cancer Research.

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

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triggers specific degradation of the proapoptotic molecule,BIM, by PKCd-dependent ERK activation. Therefore, our datauncover amajor axis of tumor immune resistance regulated byAPI5 and underline the necessity for combinatorial strategiesthat include targeting API5 to circumvent tumor immuneresistance in patients with cancer.

Materials and MethodsChemical kinase inhibitorsLY294002 (Calbiochem Corp) for PI3K, API-2 (Calbiochem

Corp) for AKT, SB203580 (Calbiochem Corp.) for p38, PD98059(Stressgen) for ERK, and rottlerin for PKCd (Sigma) were usedto specifically suppress the activity of indicated kinases.

Flow-cytometric analysis and CTL assaysFor in vitro CTL assays, 1� 105 E7-expressing or MART-1–

expressing/HLA-A2–restricted M27 peptide pulsed tumortarget cells were incubated with murine E7-specific CD8þ

T cells or MART-1–specific human CD8þ T cells, respec-tively, at 1:1 ratio for 4 hours. The percentages of activecaspase-3þ tumor cells were measured by flow cytometry todetermine the level of apoptotic cell death. All analysis wasperformed using a Becton Dickinson FACScan with CELL-Quest software (BD Biosciences).

Inhibition of BIM degradationTo measure the stability of BIM, MG132 (Calbiochem) was

dissolved in DMSO and then added to a final concentration of25 mmol/L for 3 hours to inhibit proteasome activity.

Real-time quantitative RT-PCRThe total RNAs of the cells were isolated using TRIzol

reagent (Invitrogen). First-strand synthesis were performedby using RT&Go Mastermix (MP Biomedicals) and real-timePCR was performed by a Lightcycler Fast start DNA SYBRGreen Master Mix (Roche) using specific primers with thefollowing sequences (rates normalized to the expression levelof actin): hAPI5, 5;-GGGCAAAAGAGAGCCAGTGA-30 (forward)and 50- AAAGTTGCCCAAATTGCTGCT-30 (reverse); hFGF2, 50-GGCTATGAAGGAAGATGGAAGATT-30 (forward) and 50-TGCCACATACCAACTGGTGTATTT-30 (reverse); b-actin, 50-CGACAGGATGCAGAAGGAGA-30 (forward) and 50-TAGAAG-CATTTGCGGTGGAC-30 (reverse).

FGF2 assessment of medium supernatantsThe cells were grown in 6-well plates and incubated with

0.1% FBS containing medium for 48 hours at 37�C in a 5% CO2incubator. Supernatants were collected and centrifuged toremove cell debris. FGF2 levels in the supernatant were deter-mined by following the eBioscience FlowCytomix detection kitinstructions. For Western blot analysis, supernatants werefurther concentrated in the 10� by Centricon Plus-70 centrif-ugal Filter Units-10 kDa (Millipore).

Statistical analysisAll data are representative of at least three separate experi-

ments. Nonparametric one-way or two-way ANOVA was per-

formed with SPSS version 12.0 software (SPSS), depending onthe data. Comparisons between individual data points wereanalyzed by Student t test.Where P valuewas less than 0.05, theresult was considered significant.

ResultsApi5 expression inmurine tumor cells is associated withimmune resistance to antigen-specific CD8þ T cells

To identify the master regulators governing the immune-resistance phenotype of A17 tumors, we analyzed a previouscomparativemicroarray data of parental TC-1/P0 and TC-1/P3(A17) cells (NCBI accession number GSE2774). Among severalcandidate genes, API5, an antiapoptotic factor expressed bothin humans and mice, was found to be highly overexpressed inA17 cells as compared with their parental P0 cells both at themRNA and protein levels (Supplementary Fig. S1; P < 0.05).Transfer of siRNA-targeting Api5 (siApi5) abolished proteinexpression of Api5 in A17 cells and led to a significant increasein active caspase-3þ A17 cell populations exposed to E7-specific CD8þ T cells (Fig. 1A). Conversely, ectopic expressionof Api5 in TC1/P0 parental cells rendered them resistant toCTL-mediated apoptosis (Fig. 1B). Api5was also overexpressedin murine cancer cells of skin and colorectal origin (B16 andCT26, respectively; Supplementary Fig. S2A). Downregulationof Api5 led to enhanced CTL-mediated killing, whereas forcedexpression in Api5-negative targets (EL-4) led to immuneresistance to antigen-specific lysis (Supplementary Fig. S2Band S2C).

To confirm the role of Api5 as an immune escape factor invivo, C57BL/6 mice were inoculated subcutaneously with A17cells and administered either siApi5- or siGFP-loaded chitosannanoparticles (CNP) starting 7 days after initial tumor chal-lenge followed by adoptive transfer of E7-specific CD8þ T cells(Fig. 1C). As expected, mice receiving E7-specific CTL dem-onstrated poor control of tumor growth; however, combina-tion with siApi5 restored the immune sensitivity, resulting insignificantly lower tumor volumes (P < 0.05). Conversely,forced expression of Api5 in parental TC-1/P0 cells led toimmune resistance and unchecked tumor growth in the pres-ence of antigen-specific CTL (Fig. 1D). These results demon-strate that Api5 directly controls immune resistance in tumorcells both in vitro and in vivo.

Api5-mediated immune resistance results from p-Erk–dependent degradation of the proapoptotic molecule,Bim

Because the resistance of Api5-expressing TC-1/P0 cells toCTLs could simply be due to inhibition of apoptosis orincreased cell survival, the expression of pro- and antiapoptoticmolecules was assessed byWestern blot analysis (Fig. 2A). Theprotein levels of Xiap, Bcl-2, and Bcl-XL antiapoptotic mole-cules and Bad, Bak, Bax, and Bid proapoptotic molecules werecomparable between TC-1/P0/Api5 and TC-1/P0/no insertcells. However, the expression of Bim, a proapoptotic protein,was substantially diminished in TC-1/P0/Api5 cells. Exposureto the proteasome inhibitor,MG 132, restored Bim levels in TC-1/P0/Api5 cells (Fig. 2B), indicating that Bim was undergoing

API5 Is a Novel Tumor Immune Escape Factor

www.aacrjournals.org Cancer Res; 74(13) July 1, 2014 3557




posttranscriptional degradation. Furthermore, silencing Bimusing siBim in TC-1/P0/no insert cells conferred resistance toCTL-mediated lysis (Fig. 2C). Because phosphorylation of Bimby MAPKs has been shown to be critical in the degradation ofBim via proteasomal pathway (7), we next investigated wheth-er Erk and other members of MAPKs were activated in TC-1/P0/Api5 cells. Expression of the active form of Erk, Thr202/Tyr204-pErk, was found to be highly elevated in TC-1/P0/Api5cells compared with TC-1/P0/no insert cells (Fig. 2D). Treat-ment with an Erk inhibitor, PD98059 (PD), reduced pErk andrestored levels of Bim (Fig. 2E), leading to enhanced killing ofTC-1/P0/Api5 cells by E7-specific CTL (Fig. 2F). Inhibition ofp38, Akt, or PI3K activity using their specific inhibitors did notexert significant effect on apoptotic death of TC-1/P0/Api5cells. To further confirm the roles of Erk in vivo, mice were

challenged with TC-1/P0/Api5 cells and intratumorallyinjected with PD98059-loadedchitosan hydrogel along withE7-specific CTL. Delivery of PD98059 almost completelyrestored immune sensitivity of TC-1/P0/Api5 to E7-specificCTL (Fig. 2G). Notably, PD98059 alone seemed to slightlyreduce the tumor volume, but was not found to be statisticallysignificant (P < 0.12). Taken together, these data corroboratethe essential role of Erk in the degradation of Bim and thedevelopment of immune resistance in vivo.

API5 regulates immune resistance in human tumor cellsTo explore the physiologic relevance of API5 in the devel-

opment of immune resistance in patients with cancer, theexpression of human API5 (hAPI5) was investigated in severalhuman tumor cell lines arising from various tissue types

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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 weretreated with siGFP (control) or siApi5 and the levels of Api5 protein analyzed. Right, the percentages of active caspase-3þ A17 cells were treated with siGFPor siApi5 generated during CTL assay in response to E7-specific CTL. Data presented are representative of three independent experiments. B, left,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-3þ TC-1/P0/no insert orTC-1/P0/Api5 cells generated during CTL assay. Data presented are representative of three independent experiments (mean � SD). C, schematicrepresentation illustrating in vivo challenge of A17 tumors and subsequent treatment protocol. Left, Western blot analysis of Api5 and b-actin in siGFP- orsiApi5-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 orabsence 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-specificCTL following in vivo challenge of TC-1/P0/no insert or TC-1/P0/Api5 tumors. Tumor volumewasmeasured over thecourse of 25 days after tumor challenge. Each group contained 5 mice and all data shown are representative of three independent experiments.

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Cancer Res; 74(13) July 1, 2014 Cancer Research3558




(Fig. 3A). Among those tested, API5 was found to be markedlyelevated in HeLa, PC-3, MCF-7, HCT116, and 526mel, while theexpression of API5 in other tumor lines was comparable withthat of nontumorigenic HEK293 cells (Fig. 3A).When theexpression of hAPI5 was silenced in HeLa, PC3, HCT116, and526mel cells using siAPI5-loaded CNP (Fig. 3B), pERK level wassignificantly reduced with concomitant elevation of cellularBIM, confirming the role of API5 in regulating the activity ofERK and BIM degradation in human cancer cells. To furtherexamine the immune sensitivity of API5-silenced human can-cer cells, we chose two API5-expressing tumor lines: HeLa cellsand 526mel. HeLa cells, expressing the highest levels of API5,

were engineered to express single-chain trimer (SCT) of MHCclass I (H-2Db) linked to an HPV-16 E7 immunodominant CTLepitope (aa 49–57; HeLa/SCT-E7) for recognition bymurineE7-specific CTL (Fig. 3C; ref. 8). The human melanoma cell line526mel was chosen for high endogenous expression of thetumor-associated antigen, MART-1, and presentation of itsHLA-A2–restricted epitope for recognition by MART-1–spe-cific CTL (clone KKM; Supplementary Fig. S3). When HeLa/SCT-E7 and 526mel cells were transfected with siAPI5,enhanced killing by E7-specific and MART-1–specific CTLwas observed as compared with siGFP controls (Fig. 3C).Similar enhancement was noted when the cellular activity of

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Figure 2. Identification of BIM as a proapoptotic molecule downregulated by API5. A, Western blot analysis characterizing the expression of pro- andantiapoptotic 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 wasassessed byWestern blot analysis in cells treated with or without MG132. C, TC-1/P0 cells were transfected with siGFP or siBim and exposed to E7-specificCTL. Fractions of apoptotic tumor cells induced by CTL killing are represented by the percentage of activated caspase-3þ cells. D, putative MEKkinase intermediates upstream of Bim were analyzed by Western blot analysis. Expression of pAkt, Akt pp38 MAP kinase, p38 MAP kinase, pErk, Erk, andb-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 12hours 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 toE7-CTL. G, C57BL/6 mice were inoculated subcutaneously with 1 � 105 TC-1/P0/Api5 cells/mouse and chitosan hydrogel loaded with either PD98059 orDMSOwas intratumorally administered at day 7. One day later, mice were adoptively transferredwith E7-CTL. Bar graphs represent tumor volumes at day 18from TC-1/P0/Api5-challenged mice treated with or without PD98059-loaded chitosan hydrogel in the presence or absence of E7 CTL. The data arerepresentative of three separate experiments and bar graphs represent the tumor volume of 5 mice in each group (mean � SD).






ERK was suppressed by treatment with PD98059 (Fig. 3D).Thus, inhibition of ERK activity led to reduced BIM degra-dation and restored immune sensitivity to antigen-specificCTL in human.

To further demonstrate the role of hAPI5 in immune resis-tance, API5-negative HEK293Db cells andA375melanoma cellswere retrovirally transduced with hAPI5 and their immunesensitivity monitored. As expected, overexpression of API5 inboth cell types caused elevation of pERK (>4 fold), and down-regulation of BIM (�80% reduction) compared with no-insertcontrols (Fig. 3E). Furthermore, expression of API5 inHEK293Db cells pulsed with E7-peptides or A375 melanomacells pulsed with MART-1-peptides mounted resistance toapoptotic killing by their cognate antigen-specific CTL (Fig.3F), whereas inhibition of ERK by PD98059 restored immunesensitivity against API5þ target cells (Fig. 3G). The immuneresistance byAPI5 overexpressionwasneither due to the defect

in antigen processing through the MHC class I pathway andactivation of T cells nor triggering of T-cell death (Supplemen-tary Fig. S4). Taken together, our data indicate that API5represents a shared immune escape factor in human cancercells and endogenous overexpression of API5 confers immuneresistance through an ERK-dependent mechanism while genesilencing of API5 restores immune sensitivity to antigen-spe-cific CTL.

API5 activates ERK via the FGF2/FGFR1 pathwayAs a transcription factor, the ability of API5 to deliver an

activation signal to ERK is not well defined. Because multiplereceptor tyrosine kinases (RTK) can mediate ERK signalingin tumor cells, an antiphosphotyrosine receptor antibodyarray was performed to identify an upstream RTK, whichmight have been involved in API5-mediated ERK activation(Supplementary Fig. S5A). These arrays detected increased

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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 byWestern 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 transfectedwith siGFP or siAPI5were subject to CTL assays usingmurine E7-specific or humanMART-1–specific CD8þ T cells, respectively. D, HeLa/SCT-E7 or 526meltumor cells treated with DMSO or PD98059 were subject to CTL assay using murine E7-specific or human MART-1–specific CD8þ T cells. E, expressionof API5, pERK, ERK, BIM, and b-actin in HEK293Db and A375 cells. F, left, HEK293Db/no insert and HEK293Db/API5 cells were subjected to CTLassays using E7 CTL. Right, A375 tumor cells transfected with empty vector or API5 were subject to CTL assays. G, HEK293Db/API5 or A375 tumorcells treated with DMSO or PD98059 were subject to CTL assays. The data are representative of three separate experiments (mean � SD).

Noh et al.





phosphorylation of FGFR1 and EGFR in HEK293/hAPI5 cellscompared with HEK293/no insert cells. Because both API5and FGF2 have been found to be upregulated in some malig-nancies (Supplementary Fig. S5B; ref. 9), we postulated thatthe FGF2/FGFR1 signaling pathway may be involved in deli-vering API5 signals downstream to ERK and initiating thesubsequent antiapoptotic cascade by transcriptionally reg-ulating FGF2 production. As expected, HEK293/hAPI5 cellsexpressed higher levels of phosphorylated FGFR1 (4.2-fold)and accompanying downstream signals (p-PKCd, p-ERK)compared with HEK293/no insert cells (Fig. 4A; refs. 10, 11).We also found that both mRNA and protein levels of FGF2were elevated among API5-overexpressing cells (Fig. 4B–D).Knockdown or neutralization of secretable 18 kDa FGF2by its specific siRNA (Fig. 4E) or mAbs (10 mg/mL of anti-FGF2; Fig. 4F), respectively, led to a significant decrease inphosphorylated FGFR1 and downstream phosphorylationintermediates (p-PKCd, p-ERK) with concomitant elevationof cellular BIM, suggesting a direct role for FGF2 in the API5-induced antiapoptotic axis (9, 12). Functionally, anti-FGF2mAbtreatment of HKE293/hAPI5 cells led to increased sensitizationto immune-mediated apoptosis when exposed to antigen-specific CTL (Fig. 4G).To further validate the significance of FGF2 in API5-

mediated immune resistance, we assessed for a correlationin protein expression levels between FGF2 and API5 invarious tumor cell lines (Fig. 4H). A highly significant near1:1 correlation was observed between API5 and FGF2 level,suggesting close relationship between these two proteins inall cell lines tested (Fig. 4H). Consistent with these findings,we observed increased mRNA and protein levels of FGF2among the A375 cells engineered to express hAPI5, anddecreased levels of FGF2 when API5-expressing tumor cells(HeLa and 526mel) were treated with siAPI5 (Fig. 4I–K).Furthermore, antibody blockade of FGF2 led to a decrease inp-FGFR1, p-PKCd, and pERK, and reciprocal increase in BIMexpression in API5-positive A375/hAPI5, HeLa, and 526melcells (Fig. 4L). These intracellular signaling events, occurringin the presence of neutralizing anti-FGF2 Abs, were accom-panied by increased sensitization of API5þ tumor targets toantigen-specific CTL-mediated lysis (Fig. 4L). Taken togeth-er, these results provide strong evidence for FGF2 in medi-ating API5-induced immune resistance and degradation ofBIM via the FGFR1/PKCd/ERK axis.

Silencing of PKCd leads to reconstitution of immunesensitivity in API5-expressing tumor cellsWe next determined whether silencing of PKCd, a down-

stream molecule of FGFR1 signaling proximal to the ERK/MAPK (13), was sufficient to inhibit API5-induced tumorimmune resistance. Exposure of HeLa/SCT-E7 and526mel torottlerin, an inhibitor of PKCd, resulted in a dose-dependentdecrease in pERK levels and accompanying increase in BIMexpression (Fig. 5A). Furthermore, rottlerin treatmentrestored immune sensitivity of HeLa/SCT-E7 or 526mel cellsto antigen-specific CTLs in vitro (Fig. 5B). No effect ofrottlerin was observed on API5low HeLa/SCT-E7 or 526melcells treated with siAPI5 (Fig. 5B). To exclude potential off-

target effects of rottlerin, we performed the same experi-ment using siRNA targeting PKCd and observed similarresults (left panels, Fig. 5C and D). Consistent with thisresult, siPKCd-treated HeLa/SCT/E7 cells and 526mel cellswere more susceptible to CTL-mediated killing (rightpanels, Fig. 5C and D). Likewise, the in vivo tumor growthof 526mel-bearing NOD/SCID mice, in vivo siPKCd treatmentusing CNP before the adoptive transfer of MART-1–specificCD8þ T cells (Fig. 5E), demonstrated significantly lowertumor volume compared with those receiving control siGFPtreatment (Fig. 5F). Together, these data reveal that theactivation of PKCd by FGF2/FGFR1 pathways in API5-over-expressing human tumor cells can lead to ERK activationand BIM degradation, hence controlling API5-mediatedimmune resistance both in vitro and in vivo.

Silencing of hAPI5 renders the tumor susceptible toimmune-mediated control in a preclinical humanmelanoma model

To demonstrate the potential translational relevance ofAPI5-targeting and its downstream FGF2/FGFR1 signalingpathways in human tumor immunity, the efficacy of anti-gen-specific CTL recognizing MART-1 was tested in NOD/SCID mice bearing established API5þ human tumors. Micereceiving MART-1–specific CTL together with siAPI5, accord-ing to the schedule described in Fig. 6A, experienced signifi-cantly lower tumor burden at 49 days after tumor challengecompared with siAPI5 alone or CTL with control siRNA (Fig.6B). Tumors excised on day 49 were also substantially smallerby weight and size among mice receiving both MART-1–specific CTL and siAPI5 compared with either treatment alone(Fig. 6C).Western blot analysis of ex vivo isolated tumors at day49 after challenge demonstrated decreased FGF2, pFGFR1,pPKCd, and pERK and a concurrent increase in BIM proteinsamong those mice receiving siAPI5 treatment (Fig. 6D), dem-onstrating that in vivo delivery of siAPI5 to the tumor andmodulation of the tumor immune resistance pathway wassuccessfully achieved. Treatment with siAPI5 resulted in anincreased percentage of apoptotic tumor cells (Fig. 6F), not as aresult of increased CTL infiltration at the tumor site (Fig. 6E),but rather due to enhanced lytic capacity of infiltrating CTL.Taken together, our data demonstrate that targeting andsilencing of API5 or any of its downstream elements representsan attractive strategy for restoring immune sensitivity toresistant tumor cells.

DiscussionIn this study, we identify API5 as a novel shared immune

escape gene that plays a significant role in controlling immuneresistance to antigen-specific T cells both inmouse and humancancer cells. Using murine TC-1/P3 (A17) lung cancer andhuman 526mel tumor cells that endogenously overexpressedAPI5, the role of API5 in controlling immune resistance wasvalidated both in vitro and in vivo tumormodels receiving theirantigen-specific T-cell adoptive transfer. Specific knockdownof API5 in API5-positive tumor cells restored antigen-specificimmune sensitivity, whereas the introduction of API5 into






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Noh et al.





API5-null tumor cells rendered tumors resistant to immune-mediated cytotoxicity. At themolecular level, we report, for thefirst time, that the tumor immune resistance conferred by API5is attributable to upregulation of FGF2 and activation of adownstreampathway involving, FGFR1/PKCd/ERK, ultimatelyleading to the ubiquitin-dependent degradation of the proa-poptotic molecule BIM (Fig. 7).

API5 has previously been shown to be expressed inmultiple cancers and contribute to tumor invasion andmetastases (14, 15); however, its precise molecular mecha-nism of action remained unclear. Recent studies have shownthat API5 caused suppression of apoptosis by inhibitingcaspase-3–mediated DNA fragmentation (16) and in anE2F-dependent manner (17). Depletion of API5 was shown

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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 tumorcells was analyzed in the treatment of 0, 10, 100, or 1,000 nmol/L of rottlerin. B, the percentage of caspase-3þ apoptotic cells in siGFP- or siAPI5-transfectedHeLa/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,protein expression was assessed in HeLa/SCT-E7 cells silenced with siGFP or siPKCd. Right, levels of caspase-3þ cells in HeLa/SCT-E7 cells silencedwith siGFP or siPKCd following exposure to E7-CTL. D, left, protein expression was assessed in 526mel cells silenced with siGFP or siPKCd. Right, levelsof caspase-3þ cells in 526mel cells silenced with siGFP or siPKCd following exposure to MART-1–specific CD8þ T cells. E, schematic representationillustrating in vivo xenogeneic challenge of 526mel tumors and subsequent treatment protocol. F, tumor volume was analyzed at day 31. The data arerepresentative of three separate experiments.

Figure 4. API5 activates ERK through the FGF2/FGFR1 pathway. A, FGFR1 signaling following API5 expression is evaluated by Western blot analysis ofpFGFR1, 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 FGF2secreted into themediawasmeasuredby flowcytomix. E,Western blot resultswere shown in siGFP- or siFGF2-transfectedHEK293Db/API5 cells. F,Westernblot 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,mRNAexpressionofFGF2 (I), protein expressionofAPI5 and internal FGF2 (as surrogates for all FGF2; J), andsecretion of FGF2weremonitored in A375 cells transfectedwith no insert or API5 aswell asHeLa and 526mel tumor cells silencedwith either siGFP- or siAPI5(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.






to enhance the cytotoxic effect of chemotherapeutic drugs(18), presumably by facilitating apoptosis of tumor cells invivo. Notably, the proapoptotic factor, BIM, which can bind

not only antiapoptotic BCL-2 family members, such asBCL-XL and BCL-2, but also BAK and BAX, was shown tobe critical for paclitaxel-mediated cell death (19). These

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Figure 6. Inhibition of API5 renders tumor susceptible to immune-mediated control. A, schematic of the therapy regimen in mice implanted with 526melmelanoma 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 receivedadoptive 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 frequencyof 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- orsiAPI5-treated mice, with or without adoptive transfer of MART-1–specific CTL.

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Noh et al.




observations, together with our findings presented here,suggest that BIM is likely a key mediator of cell deathinduced not only by chemotherapeutic agents, but also byantigen-specific T cells. Thus, controlling the cellular level ofBIM by API5 seems to be an efficient means of conferringimmune resistance in tumors.The impact of API5 overexpression in tumor immune eva-

sion has significant therapeutic implications. Although severalclinical trials demonstrated tumor regression following anti-gen-specific vaccination or adoptive cellular therapy, a sub-stantial proportion of patients experience partial responsesand subsequent relapse (20, 21). Altered expression of apopto-sis-regulating molecules, such as BIM, represents one possiblemechanism of immune resistance, which may be exacerbatedby prior treatment with chemotherapy or irradiation (22–24),both of which can induce upregulation of API5 and inhibitionof apoptotic death pathways in tumor cells. The increasingavailability of clinic-ready pharmacologic and biologicreagents that target and silence API5 or downstream elementsincluding anti-FGF2, FGFR, ERK, and PKCd provides a richopportunity for developing broadly applicable combinationalstrategies designed to control immune-resistant and recurrenthuman malignancies.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: K.H. Noh, S.-H. Kim, K.-H. Song, V. Kumar, K.-M. Lee,T.W. KimDevelopment of methodology: K.H. Noh, T.H. Kang, H.-D. Han, A.K. Sood,C. Yee, K.-M. Lee, T.W. KimAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S.-H. Kim, K.-H. Song, Y.-H. Lee, T.H. Kang, A.K. Sood,J. Ng, C.H. SonnAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K.H. Noh, S.-H. Kim, C. Yee, T.W. KimWriting, review, andor revision of themanuscript:K.H. Noh, S.-H. Kim, A.K.Sood, K. Kim, C. Yee, K.-M. Lee, T.W. KimAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): K.H. Noh, J.H. Kim, Y.-H. LeeStudy supervision: C. Yee, K.-M. Lee, T.W. Kim

AcknowledgmentsThe 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.

Grant SupportThis work was supported by a grant of the Korea Healthcare Technology

R&D Project, Ministry for Health, Welfare and Family Affairs, Republic of Korea(A080816), and the National Research Foundation of Korea (NRF) funded bythe Korea government (MEST; 2012R1A2A2A01007527, 2013M3A9D3045881and 2013M3A9D3045719). K.-M. Lee and her group were supported by a grantfrom KICOS (K20703001994-12A0500-03610) and MEST (2012K001404).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received November 11, 2013; revisedMarch 11, 2014; acceptedMarch 26, 2014;published OnlineFirst April 25, 2014.

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Noh et al.




2014;74:3556-3566. Published OnlineFirst April 25, 2014.Cancer Res Kyung Hee Noh, Seok-Ho Kim, Jin Hee Kim, et al. Cell Survival PathwayAPI5 Confers Tumoral Immune Escape through FGF2-Dependent

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