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

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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 immu- notherapies 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. Ó2014 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 Ó2014 American Association for Cancer Research. of FGF2 and downstream FGFR1 receptor signaling, which 3556 Cancer Res; 74(13) July 1, 2014 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst April 25, 2014; DOI: 10.1158/0008-5472.CAN-13-3225 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 Â 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
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