Identification of FES As a Novel Radiosensitizing Target in Human Cancers

Published OnlineFirst October 1, 2019; DOI: 10.1158/1078-0432.CCR-19-0610 CLINICAL CANCER RESEARCH | TRANSLATIONAL CANCER MECHANISMS AND THERAPY Identification of FES as a Novel Radiosensitizing Target in Human Cancers Byoung Hyuck Kim1,2, Yong Joon Kim3,4, Myung-Ho Kim4, Yi Rang Na5, Daun Jung5, Seung Hyeok Seok5, Joon Kim4, and Hak Jae Kim1,6 ABSTRACT ◥ Purpose: The identification of novel targets for developing cell lines. In contrast, FES depletion alone did not significantly synergistic drug–radiation combinations would pave the way to affect cell proliferation without irradiation. An inducible RNAi overcome tumor radioresistance. We conducted cell-based screen- mouse xenograft model verified in vivo radiosensitizing effects. ing of a human kinome siRNA library to identify a radiation-specific FES-depleted cells showed increased apoptosis, DNA damage, – kinase that has a synergistic toxic effect with radiation upon G2 M phase arrest, and mitotic catastrophe after irradiation. FES inhibition and is not essential for cell survival in the absence of depletion promoted radiation-induced reactive oxygen species for- radiation. mation, which resulted in phosphorylation of S6K and MDM2. Experimental Design: Unbiased RNAi screening was performed The radiosensitizing effect of FES knockdown was partially reversed by transfecting A549 cells with a human kinome siRNA library by inhibition of S6K activity. Consistent with the increase in followed by irradiation. Radiosensitizing effects of a target gene and phosphorylated MDM2, an increase in nuclear p53 levels was involved mechanisms were examined. observed, which appears to contribute increased radiosensitivity of Results: We identified the nonreceptor protein tyrosine kinase FES-depleted cells. FES (FEline Sarcoma oncogene) as a radiosensitizing target. The Conclusions: We uncovered that inhibition of FES could expression of FES was increased in response to irradiation. Cell be a potential strategy for inducing radiosensitization in viability and clonogenic survival after irradiation were significantly cancer. Our results provide the basis for developing novel decreased by FES knockdown in lung and pancreatic cancer radiosensitizers. Introduction radiotherapy, but many of them are nonselective cytotoxic agents that are highly toxic to normal tissues (2). Hypoxic cell radiosensitizers or Radiotherapy is a cornerstone of cancer treatment, and its appli- hypoxic cytotoxins are also limited in use because of side effects (3, 4). cation and frequency are increasing with innovations in radiotherapy The ideal radiosensitizer should not affect the growth and survival of technology. However, the response rate is still unsatisfactory, and cells when they are not receiving radiotherapy and should be able to toxicity is substantial in several cancers, including locally advanced maximize cell death caused by radiotherapy. Although advanced non–small cell lung cancer or pancreatic cancer (1). Radioresistance of radiotherapy technology has enabled highly conformal therapy cancer cells has been the main obstacle, and there are unmet clinical that increases tumor radiation dose while reducing the dose to normal needs for rational approaches to drug–radiotherapy combinations. tissues, the biological efficacy of photon irradiation has not been Several chemotherapies are often used to increase the efficacy of improved (5). Thus, it is necessary to develop novel radiosensitizers that can increase the biological effectiveness of radiotherapy. An important task is to identify therapeutic targets for rational 1 Department of Radiation Oncology, Seoul National University College of radiosensitization. Medicine and Hospital, Seoul, Republic of Korea. 2Department of Radiation Protein kinases are one of the most widely studied therapeutic Oncology, Seoul Metropolitan Government Seoul National University Boramae Medical Center, Seoul, Republic of Korea. 3Department of Ophthalmology, targets because deregulation of kinase function occurs in most can- Institute of Vision Research, Severance Hospital, Yonsei University College of cers (6). In addition, the human kinome is a highly druggable class of Medicine, Seoul, Republic of Korea. 4Graduate School of Medical Science and proteins. However, there have been few efforts to investigate whole Engineering, KAIST, Daejeon, Republic of Korea. 5Department of Microbiology kinases (kinome) as a target for radiosensitization. Here, we present the and Immunology, Institute of Endemic Disease, Seoul National University result of cell-based screening of a human kinome siRNA library to Medical College, Seoul, Republic of Korea. 6Cancer Research Institute, Seoul identify a radiation-specific kinase that has a synergistic toxic effect National University, Seoul, Republic of Korea. with radiation upon inhibition and is not essential for cell survival in Note: Supplementary data for this article are available at Clinical Cancer the absence of radiation. We identified FES as a novel radiosensitizing Research Online (http://clincancerres.aacrjournals.org/). target. FES (FEline Sarcoma oncogene) is known to be an oncogene B.H. Kim and Y.J. Kim contributed equally to this article. that encodes a nonreceptor protein tyrosine kinase and regulates Corresponding Authors: Hak Jae Kim, Seoul National University College of cytoskeletal rearrangements, cell-to-cell interaction, inflammation, Medicine and Hospital, 101 Daehak-ro, Jongno-gu, Seoul 03080, Republic of and innate immunity (7). However, the potential role of radiosensi- Korea. Phone: 82-2-2072-2520; Fax: 82-2-765-3317; E-mail: [email protected]; tization of this orphan kinase is totally unknown to date. and Joon Kim, Graduate School of Medical Science and Engineering, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea. Phone: 82-42-350- 4242; E-mail: [email protected] Materials and Methods – Clin Cancer Res 2020;26:265 73 Cell culture doi: 10.1158/1078-0432.CCR-19-0610 Human pancreatic cancer cell lines MiaPaCa2 and Panc-1, lung Ó2019 American Association for Cancer Research. cancer cell line A549, and human embryonic kidney 293 cells AACRJournals.org | 265 Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst October 1, 2019; DOI: 10.1158/1078-0432.CCR-19-0610 Kim et al. Translational Relevance Results Kinome siRNA library screening identified FES as a novel We identified a novel radiosensitizing target FES using an radiosensitizing target unbiased phenotype-based screening strategy. Our findings shed Unbiased RNAi screening was performed by transfecting A549 new light on the potential strategy for inducing radiosensitization cells with a human kinome siRNA library (715 siRNA pools). Two in human cancer. populations of cells were either nonirradiated or irradiated at 4 Gy, and cell viability was measured after 4 days (Supplementary Fig. S1A). Targets of siRNA pools with a Z-score of surviving fraction at 4 Gy (SF4; cell viability at 4 Gy/cell viability at 0 Gy) of (HEK293T) were purchased from the ATCC. Normal human astrocyte less than -2 were considered as potential hits (Fig. 1A; Supplemen- (NHA) was purchased from the Korean Cell Line Bank. The cells tary Table S1). We then proceeded to secondary screening using fi were cultured at 37 Cina5%CO2 humidi ed chamber. Cells were three or four individual siRNAs for each target and two cancer cell maintained in either DMEM (Welgene) or RPMI1640 medium lines. FES was identified as a radiosensitizing target because two (Welgene) supplemented with 10% FBS and 1% penicillin/strepto- independent FES siRNAs induced a significant reduction in SF4 in mycin, depending on the cell line. both A549 and MiaPaCa2 cells (Supplementary Fig. S1B). Accord- ing to the Human Protein Atlas, most human cancer cells showed RNA interference and chemical reagents moderate to strong cytoplasmic positivity with additional nuclear Cells were transfected with FES-specific siRNAs (CGAGGAUC- positivity of anti-FES immunostaining (8). Several signaling path- CUGAAGCAGUA or GAAAGUGGAUGGCCCAGCG) or nonspe- ways are known to be affected by FES, but, to our knowledge, its role cific negative control siRNA (AllStars Negative Control siRNA, Qia- in radiosensitization has never been addressed. We observed an gen) during 48 hours at 20 nmol/L using Lipofectamine RNAiMAX increase in the total level of FES after irradiation in both A549 and transfection reagent (Invitrogen) in reduced serum medium (Opti- MiaPaCa2 cells, having a peak around 6 hours, which suggests that MEM, Gibco), according to the manufacturer's reverse transfection FES may be functionally related to cellular response to radiation protocol. TAE684 was purchased from Selleckchem. PF-4708671 was (Fig. 1B; Supplementary Fig. S1C). purchased from Sigma-Aldrich. Knockdown of FES increases cancer cell radiosensitivity Kinome-wide siRNA library screening To characterize the effect of FES knockdown on radiosensitivity of Akinome-widesiRNAlibraryfor715humankinaseswas cancer cells, A549, MiaPaCa2, and Panc1 cells were transfected purchased from Dharmacon; the library consists of pools of four with two FES-specific siRNAs or a nonspecific control siRNA. different siRNAs per gene. Polystyrene flat-bottomed 384-well The CCK-8 assay showed that cell viability after irradiation at various plates (Greiner) were spotted with 3 mLof0.25mmol/L siRNA doses was significantly decreased by each FES-specific siRNA in the library pools (62.5 nmol/L for each siRNA) using the BioTek FX three cell

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