Guanine nucleotide-binding protein 1 is one of the key molecules contributing to cancer cell radioresistance Motoi Fukumoto,1 Tatsuya Amanuma,1 Yoshikazu Kuwahara,1 Tsutomu Shimura,1 Masatoshi Suzuki,1 Shiro Mori,2 Hiroyuki Kumamoto,3 Yohei Saito,4 Yasuhito Ohkubo,4 Zhenfeng Duan,5 Kenji Sano,6 Tomohiro Oguchi,7 Kazuyuki Kainuma,7 Shinichi Usami,7 Kengo Kinoshita,8 Inchul Lee9 and Manabu Fukumoto1 1Department of Pathology, Institute of Development, Aging and Cancer, Tohoku University, Sendai; 2Department of Oral and Maxillofacial Surgery, Tohoku University, Sendai; 3Division of Oral Pathology, Department of Oral Medicine and Surgery, Graduate School of Dentistry, Tohoku University, Sendai; 4Department of Radiopharmacy, Tohoku Pharmaceutical University, Sendai, Japan; 5Department of Hematology/Oncology, Massachusetts General Hospital, Boston, Massachusetts, USA; 6Department of Pathology, School of Medicine, Shinshu University, Matsumoto; 7Department of Otorhinolaryngology, School of Medicine, Shinshu University, Matsumoto, Japan; 8Graduate School of Information Sciences, Tohoku University, Sendai; 9Department of Pathology, Asan Medical Center, Seoul, Korea Key words Standard fractionated radiotherapy for the treatment of cancer consists of daily GTP-binding proteins, head and neck neoplasms, irradiation of 2-Gy X-rays, 5 days a week for 5–8 weeks. To understand the char- neoplasms, radiation, radiation oncology acteristics of radioresistant cancer cells and to develop more effective radiother- Correspondence apy, we established a series of novel, clinically relevant radioresistant (CRR) cells Manabu Fukumoto, Department of Pathology, IDAC, that continue to proliferate with 2-Gy X-ray exposure every 24 h for more than Tohoku University, 4-1 Seiryou-machi, Aoba-ku, Sendai, 30 days in vitro. We studied three human and one murine cell line, and their CRR Japan. derivatives. Guanine nucleotide-binding protein 1 (GBP1) gene expression was Tel: +81-22-717-8507; Fax: +81-22-717-8512; higher in all CRR cells than their corresponding parental cells. GBP1 knockdown E-mail: [email protected] by siRNA cancelled radioresistance of CRR cells in vitro and in xenotransplanted Funding Information tumor tissues in nude mice. The clinical relevance of GBP1 was immunohistochem- Ministry of Education, Culture, Sports, Science and Tech- ically assessed in 45 cases of head and neck cancer tissues. Patients with GBP1- nology of Japan; Ministry of Health, Labor and Welfare positive cancer tended to show poorer response to radiotherapy. We recently of Japan; Japan Science and Technology Agency. reported that low dose long-term fractionated radiation concentrates cancer stem Received January 29, 2014; Revised July 18, 2014; cells (CSCs). Immunofluorescence staining of GBP1 was stronger in CRR cells than Accepted July 22, 2014 in corresponding parental cells. The frequency of Oct4-positive CSCs was higher in CRR cells than in parental cells, however, was not as common as GBP1-positive Cancer Sci 105 (2014) 1351–1359 cells. GBP1-positive cells were radioresistant, but radioresistant cells were not nec- doi: 10.1111/cas.12489 essarily CSCs. We concluded that GBP1 overexpression is necessary for the radiore- sistant phenotype in CRR cells, and that targeting GBP1-positive cancer cells is a more efficient method in conquering cancer than targeting CSCs. gene cluster located on chromosome 1.(5) GBP1 is one of the adiotherapy is one of the major therapeutic modalities for genes most strongly induced by interferons.(6) GBP1 is highly R eradicating malignant tumors. The existence of radioresis- expressed in endothelial cells, where it inhibits the proliferation tant cells remains one of the major obstacles in radiotherapy and invasion of endothelial cells in response to c-interferon and – and chemoradiotherapy. Ordinary radiotherapy for cancers is is activated by inflammatory cytokines in vitro and in vivo.(7 9) composed of fractionated radiation (FR), with approximately Downregulation of GBP1 by siRNA resulted in higher levels of 2 Gy of X-ray irradiation once a day, 5 days a week, over a hepatitis C virus replication in a human hepatoma cell line, 5–8-week period.(1) In order to develop more effective tumor Huh-7.(10) In addition to the GTPase activity and its involvement radiotherapies, we established three human and one murine clin- in viral infections, GBP1 overexpression also contributes to cell ically relevant radioresistant (CRR) cell lines independently. survival by inhibiting apoptosis in human umbilical vein These cells continue to proliferate under exposure to 2 Gy ⁄ day endothelial cells after growth factor and serum depletion.(11) for more than 30 days in vitro.(2,3) The total dose to these CRR Ovarian cancer cases with GBP1 protein overexpression are cells over the whole process added up to more than 1500 Gy. resistant to paclitaxel, leading to poor prognoses.(12) GBP1 We carried out cDNA microarray analyses of differential gene overexpression is directly associated with moderate levels of expression in association with the CRR phenotype. We found paclitaxel resistance in ovarian cancer cell lines.(13) Higher that the Guanine nucleotide-binding protein 1 (GBP1) gene was GBP1 levels are associated with higher pathological stages, overexpressed in all of the CRR cells examined. positive perineural invasion, and poorer prognosis of patients GBP1 is a member of the large GTPase family.(4) The human with oral squamous cell carcinoma.(14) In this study we found large GTPase family consists of seven members, encoded by a that GBP1 is necessary but not sufficient for cellular radiore- © 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd Cancer Sci | October 2014 | vol. 105 | no. 10 | 1351–1359 on behalf of Japanese Cancer Association. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non- commercial and no modifications or adaptations are made. Original Article GBP1-contributes to cancer cell redioresistance www.wileyonlinelibrary.com/journal/cas sistance in vitro. We carried out immunohistochemical studies out using the primer pair 50-CTGCACAGGCTTCAGCAAAA- on clinicopathological specimens from head and neck cancers 30 and 50-AAGGCTCTGGTCTTTAGCTT-30.(13) Reverse tran- (HNC) to confirm the relevance of GBP1 in cancer treatment. scription–PCR of ISG20 was carried out using the primer set This study revealed that GBP1 is one of the key molecules 50-ATCTCTGAGGGTCCCCAAG-30 and 50-TTCAGTCTG contributing to radioresistance. ACACAGCCAGG-30.(17) The RT-PCR was carried out using TB SYBR gPCR Mix (Toyobo, Osaka, Japan). The PCR con- ditions were: 95°C for 1 min, followed by 60 cycles of 95°C Materials and Methods for 15 s, and 60°C for 30 s using the Thermal Cycler Dice Cell culture and drugs. Human cancer cell lines SAS, HepG2, Real Time System (Takara, Shiga, Japan). and KB and a mouse breast cancer cell line, MM102, were RNA interference. Lipofectamine 2000 was used for transfec- obtained from the Cell Resource Center for Biomedical tion. GBP1 siRNA (Hs_GBP1_8 and Hs_GBP1_9) and All- Research (IDAC, Tohoku University, Sendai, Japan). We Stars Negative Control siRNA were purchased from Qiagen. established CRR cell lines SAS-R1, HepG2-8960-R, and KB-R Apoptosis assay. Apoptotic cells were quantified using an by exposing these parental cells to FR of X-rays for more than annexin V–FITC apoptosis detection kit (BioVision, Mountain 5 years.(3) For the maintenance of the CRR phenotype, FR at View, CA, USA). Cells (5 9 105) were collected 48 h after 2 Gy was carried out every 24 h. All cells used in this study irradiation and were analyzed by a FACScan (Cytomics were maintained in RPMI-1640 medium (Nacalai Tesque, FC500; Becton Dickinson, Mountain View, CA, USA). Kyoto, Japan) and supplemented with 5% FBS (Invitrogen, Immunofluorescence staining of culture cells. Immunofluores- Carlsbad, CA, USA) in a humidified atmosphere at 37°C in air cence staining was carried out as previously described.(18) with 5% CO2. Acute exposure experiments were carried out Images were randomly captured in a fluorescence microscope with cells in the exponential growth phase, 24 h after the last (BZ-8000; Keyence, Osaka, Japan). We scored cH2AX foci maintenance irradiation. We introduced pIRES GBP1 expres- and Oct4-positive cells by counting 50 cells in total. sion vectors(13) into CRR cells by Lipofectamine 2000 (Invitro- Animal experiments. This study was approved by Regulations gen) and selected colonies resistant to 100 lg ⁄ mL G418 for Animal Experiments and Related Activities, Tohoku Uni- (Geneticin, Grand Island, NY, USA). versity, and carried out as described previously.(16) Atelo Gene Irradiation. X-ray irradiation was carried out in a 150-KVp (Koken, Tokyo, Japan) was used to deliver siRNA into animal X-ray generator (MBR-1520R; Hitachi, Tokyo, Japan) with a tissues according to the manufacturer’s protocol. total filtration of 0.5 mm aluminum plus 0.1 mm copper filter, Immunohistochemistry. Tumor tissues were fixed in 10% for- at a dose rate of 1.0 Gy ⁄ min. malin and immunohistochemical staining was carried out as Cell survival assay after irradiation. Cell survival was deter- described previously.(19) mined by the modified high density survival (MHDS) assay.(15) In situ hybridization. GBP1 (NM_002053) gene expression in Microarray analysis. Genome-wide expression arrays (Illu- tumor tissues was visualized using RNAscope and the HybEZ mina, San Diego, CA, USA) were used for the analysis of system according to the manufacturer’s protocol (Advanced SAS (human WG6, version 3, 48 803 genes) and HepG2 Cell Diagnostics, Hayward, CA, USA). (human WG6, version 1, 47 296 genes). Data were analyzed Demographic analysis. This study was approved by the com- by TransGenic (Kumamoto, Japan). For MM102, a 3D-Gene mittees of medical ethics, Shinshu University School of Medi- mouse Oligo chip 24k (23 522 genes; Toray Industries, Tokyo, cine (No.354; Matsumoto, Japan) and Tohoku University, Japan) was used and the data were analyzed by Toray Indus- Graduate School of Medicine (No.