AMPK signaling to promote radioresistance in nasopharyngeal carcinoma

ARTICLE IN PRESS Cancer Letters ■■ (2016) ■■–■■

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Q2 Original Articles

EBV-LMP1 suppresses the DNA damage response through DNA-PK/ AMPK signaling to promote radioresistance in nasopharyngeal carcinoma Q1 Jingchen Lu a,b,c, Hongde Li b,c, Zhijie Xu b,c, Min Tang b,c, Xinxian Weng b,c, Jiangjiang Li b,c,

Xinfang Yu d, Luqing Zhao e, Hongwei Liu f, Yongbin Hu e, Lifang Yang b,c,g, Meizuo Zhong a, Jian Zhou h, Jia Fan h, Ann M. Bode i, Wei Yi b,c, Jinghe Gao b,c, Lunquan Sun g, Ya Cao b,c,g,* a

Department of Medical Oncology, Xiangya Hospital, Central South University, Changsha, China Key Laboratory of Carcinogenesis of Chinese Ministry of Public Health, Xiangya School of Medicine, Central South University, Changsha, China c Key Laboratory of Chinese Ministry of Education, Cancer Research Institute, Xiangya School of Medicine, Central South University, Changsha, China d Hunan Cancer Hospital and The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, China e Department of Pathology, Xiangya Hospital, Central South University, Changsha, China f Department of Neurosurgery, Xiangya Hospital, Central South University, Changsha, China g Molecular Imaging Center, Central South University, Changsha, China h Key Laboratory of Chinese Ministry of Education, Zhongshan Hospital, Zhongshan Hospital, Shanghai, China i The Hormel Institute, University of Minnesota, Austin, MN, USA b

A R T I C L E

I N F O

Article history: Received 24 March 2016 Received in revised form 25 May 2016 Accepted 26 May 2016 Keywords: LMP1 DNA damage response DNA-PK AMPK Nasopharyngeal carcinoma

A B S T R A C T

We conducted this research to explore the role of latent membrane protein 1 (LMP1) encoded by the Epstein–Barr virus (EBV) in modulating the DNA damage response (DDR) and its regulatory mechanisms in radioresistance. Our results revealed that LMP1 repressed the repair of DNA double strand breaks (DSBs) by inhibiting DNA-dependent protein kinase (DNA-PK) phosphorylation and activity. Moreover, LMP1 reduced the phosphorylation of AMP-activated protein kinase (AMPK) and changed its subcellular location after irradiation, which appeared to occur through a disruption of the physical interaction between AMPK and DNA-PK. The decrease in AMPK activity was associated with LMP1-mediated glycolysis and resistance to apoptosis induced by irradiation. The reactivation of AMPK significantly promoted radiosensitivity both in vivo and in vitro. The AMPKα (Thr172) reduction was associated with a poorer clinical outcome of radiation therapy in NPC patients. Our data revealed a new mechanism of LMP1-mediated radioresistance and provided a mechanistic rationale in support of the use of AMPK activators for facilitating NPC radiotherapy. © 2016 Published by Elsevier Ireland Ltd.

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Introduction The Epstein–Barr virus (EBV) is an oncogenic virus implicated in the pathogenesis of a number of human malignancies of both lymphoid and epithelial origins and is associated with nearly 200,000 new malignancies each year worldwide [1–3]. Latent membrane protein 1 (LMP1) is a primary oncoprotein encoded by EBV that plays a key role in both the initiation and progression of nasopharyngeal carcinoma (NPC) [4]. Using newly developed support vector machine (SVM)-based methods, LMP1 was identified as a prognostic biomarker for NPC [5]. LMP1 activates several important oncogenic signaling pathways such as NF-κB, JNK, PI3-K/Akt, MAPK and JAK/ STAT and causes various downstream pathological changes in cell

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* Corresponding author. Tel.: +86 731 84805448; fax: +86 731 84470589. E-mail address: [email protected] (Y. Cao).

proliferation, apoptosis and metastasis [6]. Recently, we found that suppression of LMP1 expression by the LMP1-targeted DNAzyme, DZ1, enhanced radiosensitivity both in vivo and in vitro [7], which suggested an important role for LMP1 in the regulation of the radiosensitivity of NPC cells. The DNA damage response (DDR) is a cellular mechanism that protects against DNA damage induced by endogenous and exogenous factors, it includes changes in cellular processes such as cell cycle regulation, DNA damage repair, apoptosis and chromatin remodeling. In recent years, the DDR has been recognized as an important innate tumor suppressor pathway [8–11]. DNA tumor virus infections activate and modulate DDR signaling pathways through multiple independent mechanisms [12–14]. Recent evidence suggests that EBV oncoproteins target multiple aspects of DDR signaling, such as heightening DNA damage by increasing the cellular levels of reactive oxygen species (ROS), disrupting the mitotic checkpoint and suppressing DNA repair mechanisms [15].

http://dx.doi.org/10.1016/j.canlet.2016.05.032 0304-3835/© 2016 Published by Elsevier Ireland Ltd.

Please cite this article in press as: Jingchen Lu, et al., EBV-LMP1 suppresses the DNA damage response through DNA-PK/AMPK signaling to promote radioresistance in nasopharyngeal carcinoma, Cancer Letters (2016), doi: 10.1016/j.canlet.2016.05.032

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DNA double strand breaks (DSBs) are considered to be the most lethal form of DNA damage induced by DNA-damaging agents, such as ionizing irradiation. The ability to repair DSBs is related not only to cancer susceptibility but also to sensitivity of cells to radiotherapy and chemotherapy [16]. The DNA-dependent protein kinase (DNA-PK) is a critical component of the DSB repair machinery [17–20]. It is a serine/threonine kinase complex composed of a heterodimer of the catalytic subunit DNA-PKcs and the regulator subunit Ku proteins (Ku70/Ku86). In response to DSB formation, DNAPKcs is recruited to DSBs by the DNA end-binding Ku70/86 heterodimer and is rapidly phosphorylated at multiple serine and threonine residues. DNA-PKcs phosphorylation at the Thr2609 cluster region is particularly significant because it is critical for DSB repair [21,22]. In addition to its classical role in DSB repair, recent findings demonstrate damage-independent functions of DNA-PK that affect a variety of critical cellular processes associated with malignancy [23,24]. Specifically, a recent report defines a general role for DNA-PK in metabolic regulation [25–27]. AMP-activated protein kinase (AMPK) is a crucial energy sensor that helps maintain cellular energy homeostasis and especially as a negative regulator of glycolysis [28,29]. It is a serine/threonine protein kinase composed of a heterotrimeric complex, including α, β and γ subunits. The α-subunit of AMPK has catalytic activity and phosphorylation of AMPKα at Thr172 is necessary for full enzyme activity [30,31]. Besides acting as an energy sensor, recent work describes a novel function for AMPK as a sensor of genomic stress and a participant in the DDR pathway [32–34]. In this study, we report that in NPC cells, EBV-encoded LMP1 repressed DSB repair by inhibiting DNA-PK phosphorylation and activity. Moreover, LMP1 disrupted the physical interaction between DNA-PK and AMPK and reduced the phosphorylation of AMPK by disturbing the subcellular location of the protein after DNA damage. Reactivation of AMPK markedly inhibited glycolysis and promoted apoptosis induced by DNA damage, and enhanced radiosensitivity both in vivo and in vitro. This study revealed a new mechanism of LMP1-induced radioresistance occurring through the modulation of DDR signaling pathways and provided a mechanistic rationale supporting the use of AMPK activators for facilitating NPC radiotherapy.

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Cell lines and culture

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CNE1-LMP1 and HNE2-LMP1 cells are NPC cell lines that were constructed from LMP1-negative NPC cell lines, CNE1 and HNE2, respectively, which were stably transfected with an LMP1 plasmid. Cells were grown in RPMI 1640 (Gibco BRL, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA).

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NU7026, metformin, DAPI (4′,6′-diamidino-2-phenylindole) and the antibody against β-actin were purchased from Sigma-Aldrich (St. Louis, MO, USA). The antibody against LMP1 was from DAKO (Glostrup, Denmark). The antibodies against DNAPKcs, α-tubulin, Cy3-conjugated anti-rabbit IgG, Alexa 488-conjugated anti-mouse IgG and Alexa 488-conjugated anti-goat IgG for immunofluorescence were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against phosphorylated DNAPKcs (Thr2609) and histone H3 were from Abcam (Cambridge, UK). Antibodies against AMPKα, phosphorylated AMPKα (Thr172) and phosphorylated histone H2AX (Ser139) were from Cell Signaling Technologies (Danvers, MA, USA). The Annexin-V FITC Apoptosis Kit was purchased from KeyGEN Biotech (Nanjing, Jiangsu, P.R. China) and Lipofectamine 2000 was purchased from Invitrogen (Carlsbad, CA, USA).

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Protein samples (50–100 μg) were separated by 6–12% SDS-PAGE, transferred onto a nylon membrane and immunoblotted with primary antibodies. Binding of primary antibodies was detected using peroxidase-conjugated secondary antibodies and developed with an enhanced chemiluminescence reagent. Visualization of

Materials and methods

Reagents and antibodies

Western blot analysis

proteins was performed using the ChemiDoc XRS system with Image Lab software (Bio-Rad, Hercules, CA).

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Co-immunoprecipitation (Co-IP)

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Cells were disrupted with IP lysis buffer containing protease inhibitor cocktails (Roche Diagnostics, Mannheim, Germany). Aliquots of 2000 μg of proteins from each sample were pre-cleared by incubation with 20 μl of Dynabeads protein G (Invitrogen) for 1 h at 4 °C. Pre-cleared samples were incubated with a DNA-PKcs antibody (2 μg/ sample) overnight at 4 °C. Then 20 μl Dynabeads protein G was added into each sample and incubated for 2 h at 4 °C. The beads were then washed 3 times with cold lysis buffer, boiled, separated by 6–12% SDS–PAGE, and transferred onto a nylon membrane followed by Western blot analysis.

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Immunofluorescence analysis

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The cells were fixed and permeabilized with −20 °C cold methyl alcohol for 10 min, then blocked in 5% donkey serum in PBS for 1 h and then incubated with a primary antibody in PBS containing 1% BSA at 4 °C overnight. The cells were washed 3 times with PBS, and incubated for 30 min with a secondary antibody for 30 min. The cells were washed with PBS and stained with DAPI for 10 min and viewed by a confocal laser scanning microscope (LSM 510 META, Carl Zeiss, Germany).

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Immunohistochemistry

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Immunohistochemistry (IHC) was performed using a Histomouse SP Broad Spectrum DAB kit (Invitrogen-Zymed, Carlsbad, CA, USA). Paraffin sections were immunostained using a streptavidin–peroxidase procedure after microwave antigen retrieval. The signal was detected using a diaminobenzidine solution. The stained sections were independently examined by two pathologists and the staining intensity of each protein was scored.

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Clonogenic survival assay

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Cells were counted and seeded in six-well plates in triplicate at densities of 1 × 103, 1 × 104 and 1 × 105 per well and exposed to 0, 4, 6 or 8 Gy doses of irradiation according to the density of the cells. After 7–14 days, cells were fixed with methanol, stained with crystal violet, and colonies containing more than 50 cells were counted using Image J. Results are plotted as the mean surviving fraction ± S.E.M. The survival curves were drawn using the GraphPad Prism 5 software program (GraphPad Software, La Jolla, CA, USA).

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Comet assay

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A neutral comet assay was performed using the comet assay kit (Trevigen, Gaithersburg, MD, USA) according to the manufacturer’s protocol.

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DNA-PK activity assay

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A kinase activity assay for DNA-PK was performed using the SignaTECT® DNADependent Protein Kinase Assay System kit (Promega) according to the manufacturer’s protocol. The radioactivity was determined in a liquid scintillation counter (TriCarb® 2810, PerkinElmer, Waltham, MA, USA).

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Irradiation of cells

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Cells were irradiated at a dose rate of 2 Gy/min at room temperature using an electron linear accelerator (Siemens Primus, E. Germany) as an X-ray source and were allowed to recover at 37 °C under 5% CO2 for the times indicated.

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Animal experiments

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All mice were maintained and manipulated according to strict guidelines established by the Medical Research Animal Ethics Committee, Central South University, China. Six-week-old female athymic nude mice (BALB/C) were injected with 5 × 106 CNE1-LMP1 cells. Tumor volumes were calculated using the formula: volume = length × width2/2. When the tumor volume reached 60–100 mm3, the animals were treated with intraperitoneal injection of metformin (250 mg/kg daily) or water every day [35]. In groups of mice subjected to irradiation treatment, local irradiation of 5 Gy was administered once a week for three weeks. Ten days after the last irradiation, the animals were sacrificed and the tumors were removed.

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Statistical analysis

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Data analysis was conducted by the SPSS software (version 21.0; SPSS). Statistical differences were determined using Student’s t-test or one-way ANOVA. The Kaplan–Meier method was used to estimate overall survival and the log-rank test was used to evaluate differences between survival curves. All values are expressed as mean values ± S.E. of 3 individual experiments. A value of p < 0.05 was considered statistically significant.

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Please cite this article in press as: Jingchen Lu, et al., EBV-LMP1 suppresses the DNA damage response through DNA-PK/AMPK signaling to promote radioresistance in nasopharyngeal carcinoma, Cancer Letters (2016), doi: 10.1016/j.canlet.2016.05.032

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Results LMP1 represses DSB repair in NPC cells Once DSBs are induced in cells, serine 139 of histone H2AX is quickly phosphorylated around the DSB site and is then referred to as γH2AX, which is a sensitive marker of DSBs [36]. To determine the effect of LMP1 expression on the ability of NPC cells to repair DSBs, we first measured γH2AX foci formation by immunofluorescence staining intensity after irradiation. Cells included LMP1-positive cells, CNE1-LMP1 and HNE2-LMP1, and LMP1-negative cells, CNE1 and HNE2. In non-irradiated NPC cells, the percentage of γH2AX-positive cells was very low. No difference was observed in either LMP1-positive or -negative cells. Irradiation induced a robust formation of γH2AX foci and the percentage of γH2AX-positive NPC cells increased by 8-fold (p < 0.05) at 0.5 h post-irradiation. At 24 h post-irradiation, a time point that most DSBs should be repaired, the percentage of γH2AX-positive cells was 12.7% in CNE1 cells compared to 29.7% in CNE1-LMP1 cells (p < 0.05; Fig. 1A) and was 11.2% and 28.6% in HNE2 and HNE2-LMP1 cells, respectively (p < 0.01; Fig. 1B). Then we used Western blot analysis to detect the protein level of γH2AX in NPC cells. In non-irradiated cells, the protein level of γH2AX was not different in CNE1 or CNE1-LMP1 cells, but was higher in HNE2-LMP1 cells compared to HNE2 cells (Fig. 1C, D). After exposure to irradiation, γH2AX levels in both CNE1-LMP1 and HNE2LMP1 cells were higher compared with those observed in CNE1 or HNE2 cells at different time points (Fig. 1C, D). We also used a neutral comet assay to investigate DSB repair capacity. Irradiation significantly increased comet formation in NPC cells and the comet tails were gradually eliminated as the DSBs were repaired (Fig. 1E, F). The length of the comet tails in LMP1-positive cells was significantly longer than LMP1-negative cells at either 1 or 4 h after irradiation. Specifically, CNE1-LMP1 cells exhibited tails of 200.945 nm vs. 151.699 nm in CNE1 cells at 1 h (p < 0.05) and 253.625 nm vs. 100.294 nm at 4 h (p < 0.01; Fig. 1G). Compared to HNE2 cells, HNE2LMP1 cells also retained longer comet tails of 167.852 nm vs. 116.297 nm at 1 h (p < 0.001) and 89.318 nm vs. 49.655 nm (p < 0.001) at 4 h after irradiation (Fig. 1H). These data indicated that LMP1 repressed the ability of NPC cells to perform DSB repair. LMP1 inhibits DSB repair by attenuating DNA-PK phosphorylation and activity Because DNA-PK plays a dominant role in DSB repair, we performed Western blot analysis to detect DNA-PKcs in LMP1-positive and -negative NPC cell lines. Results indicated that total DNAPKcs protein levels were not significantly different between CNE1LMP1 cells and CNE1 cells (Fig. 2A), but decreased in HNE2-LMP1 cells compared to levels observed in HNE2 cells (Fig. 2B). We then measured the mRNA level of DNA-PKcs and found no difference between LMP1-positive or -negative NPC cell lines (Fig. 2C). DNAPKcs phosphorylation at the Thr2609 cluster region is essential for its activity, we found that exposure to irradiation induced a robust phosphorylation of DNA-PKcs at Thr2609, and LMP1 substantially inhibited this phosphorylation in NPC cells (Fig. 2D, E). Accordingly, the DNA-PK activity decreased in LMP1-positive NPC cell lines (p < 0.001; Fig. 2F, G). After knockdown of LMP1 (Fig. 2H), the phosphorylation level of DNA-PKcs at Thr2609 increased (Fig. 2I, J). These data indicated that LMP1 inhibited the phosphorylation and activity of DNA-PK, but had an unsettling effect on the total protein level of DNA-PKcs and no effect on the mRNA level of DNA-PKcs. LMP1 inhibits AMPK phosphorylation through disrupting its subcellular location and the interaction between AMPK and DNA-PK DSB repair is accompanied by increased energy consumption. As AMPK is not just an energy sensor, but also a potential genomic

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stress sensor participating in the DDR, we examined the impact of LMP1 on AMPK expression and phosphorylation. The total protein level of AMPKα was not different between LMP1-positive and -negative NPC cells. However, the phosphorylation of AMPKα at Thr172 was substantially decreased in LMP1-positive cells compared to LMP1-negative cells (Fig. 3A), suggesting that LMP1 influences AMPK phosphorylation without affecting the total levels of protein expression. In CNE1 cells, AMPKα was localized in both cytoplasm and nucleus (Fig. 3B), but after irradiation, the nuclear AMPKα shifted to the cytoplasm, resulting in an increase in cytoplasmic AMPKα protein expression. However, in CNE1-LMP1 cells, AMPKα was mainly distributed in the cytoplasm, and no substantial change in cytoplasmic AMPKα protein level was observed after irradiation. Consistent with the results of total protein expression, phosphorylated AMPKα (Thr172) localized in the cytoplasm was higher in CNE1 cells compared to CNE1-LMP1 cells (Fig. 3C). These results suggest that LMP1 might inhibit the phosphorylation of AMPKα by regulating its subcellular localization. A recent study suggests that DNA-PK is an important regulator of AMPK activation in response to energy depletion [25]. We hypothesize that LMP1 might regulate the phosphorylation of AMPK by modulating DNA-PK. In LMP1-positive cells, phosphorylation of AMPKα at Thr172 was suppressed by NU7026, a specific DNA-PK inhibitor. However, the phosphorylation was not affected in LMP1-negative cells (Fig. 3D). We next determined whether DNA-PK could physically interact with AMPK after irradiation and whether this association was affected by LMP1. DNA-PKcs was immunoprecipitated from NPC cells and the presence of the AMPKα subunit was detected by immunoblotting. DNA-PKcs coimmunoprecipitated with AMPKα in both LMP1-negative and -positive cells. However, greater amounts of the AMPKα protein co-immunoprecipitated with DNA-PKcs in LMP1-negative cells (Fig. 3E, F), indicating that the interaction of DNA-PKcs with AMPKα was disrupted by LMP1. To further expand these results, we performed an immunofluorescence assay. Irradiation induced the co-localization of DNA-PKcs and AMPKα in the cytoplasm of LMP1-negative cells; however, co-localization was almost undetectable in LMP1-positive cells (Fig. 3G, H). These data indicated that LMP1 could possibly inhibit the phosphorylation of AMPKα (Thr172) directly through DNA-PK or indirectly by disrupting the interaction of AMPKα with DNA-PKcs.

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Reactivation of AMPK reverses LMP1-mediated NPC cell radioresistance Recent findings suggest that activation of AMPK could radiosensitize tumor cells. To determine whether LMP1-mediated AMPK inactivation confers radioresistance to NPC cells, we treated cells with metformin (5 μM), which is a pharmacological drug that can activate AMPK [28], and then examined the colony formation. Reactivation of AMPK significantly reduced colony formation of LMP1-positive NPC cells (Fig. 4A, B) and resulted in a lower survival rate after irradiation compared with LMP1-negative NPC cells (p < 0.01; Fig. 4C, D). To further investigate whether reactivation of AMPK could reverse LMP1-induced radioresistance in vivo, we treated athymic nude mice bearing CNE1-LMP1 xenografts with metformin alone, irradiation alone, or both metformin and irradiation. Treatment with metformin alone had no effect on tumor growth and irradiation alone significantly suppressed tumor growth, whereas metformin combined with irradiation therapy further inhibited tumor growth (p < 0.01; Fig. 4E, F). These data indicated that reactivation of AMPK could reverse LMP1-induced cellular radioresistance in NPC cells.

Please cite this article in press as: Jingchen Lu, et al., EBV-LMP1 suppresses the DNA damage response through DNA-PK/AMPK signaling to promote radioresistance in nasopharyngeal carcinoma, Cancer Letters (2016), doi: 10.1016/j.canlet.2016.05.032

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Fig. 1. LMP1 represses double-strand break repair of nasopharyngeal carcinoma cells. (A) Analysis of H2AX foci at different times after 4 Gy 6 MV X-ray irradiation. Immunofluorescence staining for γ-H2AX followed by confocal microscopy was performed. Cells displaying 10 or more foci were counted as positive and 300–500 cells were counted. Representative images of γH2AX foci and the percentage of CNE1 and CNE1-LMP1 cells displaying γH2AX foci are shown. (B) Representative images of γH2AX foci and the percentage of HNE2 and HNE2-LMP1 cells displaying γH2AX foci are shown. (C, D) Detection of γH2AX in CNE1/CNE1-LMP1 or HNE2/HNE2-LMP1 cells at different times after irradiation. Total proteins were or were not exposed to 4 Gy irradiation and prepared at 0.5, 1, 4 or 24 h after irradiation. The γH2AX protein level was determined by Western blot analysis using specific antibodies to detect γH2AX and β-actin. (E and F) Neutral comet assay of CNE1/CNE1-LMP1 and HNE2/HNE2-LMP1 cells. Comet images of DSBs detected by neutral single cell gel electrophoresis at 0.5, 1, 4 and 24 h after 4 Gy X-ray irradiation are shown. (G and H) The repair kinetics of 4 Gy-induced DNA DSBs were detected by comet assay. The tail moment was used as the endpoint of DSBs. One hundred individual comets were counted per time point for each experiment. The tail moments were measured and data are represented as the mean tail moment from 3 independent experiments. Data are shown as mean values ± S.D. from 3 independent experiments. For A, B, G, and H, the asterisks indicate a significant (*p < 0.05, **p < 0.01 and ***p < 0.001) difference.

Please cite this article in press as: Jingchen Lu, et al., EBV-LMP1 suppresses the DNA damage response through DNA-PK/AMPK signaling to promote radioresistance in nasopharyngeal carcinoma, Cancer Letters (2016), doi: 10.1016/j.canlet.2016.05.032

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Fig. 2. LMP1 inhibits double-strand break repair by inhibiting DNA-PK phosphorylation and activity. (A, B) CNE1/CNE1-LMP1 and HNE2/HNE2-LMP1 cells were or were not exposed to 4 Gy X-ray irradiation. After 24 h, cells were harvested and analyzed for DNA-PKcs protein levels. (C) Evaluation of DNA-PKcs mRNA levels by real-time RT-PCR analysis in CNE1/CNE1-LMP1 and HNE2/HNE2-LMP1 cells at 24 h after irradiation at a dose of 4 Gy. (D, E) CNE1/CNE1-LMP1 and HNE2/HNE2-LMP1 cells were exposed to 4 Gy irradiation. After 30 min, cells were harvested and analyzed for DNA-PKcs phosphorylation at Thr2609. (F, G) CNE1/CNE1-LMP1 and HNE2/HNE2-LMP1 cells were exposed to 4 Gy irradiation. After 30 min, whole-cell extracts (25–50 μg) prepared from NPC cells were assayed for DNA-PK activity as described in Materials and Methods. (H) The knockdown effect of shLMP1 in CNE1-LMP1 cells. CNE1-LMP1 cells were transfected with shLMP1 and at 48 h after transfection, cells were harvested and analyzed for LMP1 protein levels. (I) CNE1-LMP1 cells were transfected with shLMP1 and at 48 h after transfection, CNE1-LMP1 and CNE1 cells were exposed to 4 Gy irradiation. After 30 min, cells were harvested and analyzed for DNA-PKcs phosphorylation at Thr2609. (J) HNE2-LMP1 cells were transfected with shLMP1 and at 48 h after transfection, HNE2LMP1 and HNE2 cells were exposed to 4 Gy irradiation. After 30 min, cells were harvested and analyzed for DNA-PKcs phosphorylation at Thr2609.Data are shown as mean values ± S.D. from 3 independent experiments and for F, the asterisks indicate a significant (***p < 0.001) difference.

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Reactivation of AMPK inhibits LMP1-induced glycolysis and promotes apoptosis of NPC cells after DNA damage Next we investigated the possible mechanisms of activation of AMPK in promoting radiosensitivity. Emerging evidence indicates that AMPK is a negative regulator of aerobic glycolysis (i.e., the Warburg effect) in cancer cells and suppresses tumor growth in vivo. Thus, we examined the level of glucose consumption and lactate production in NPC cells after irradiation. Compared with LMP1negative cells, LMP1-positive NPC cells displayed increased glucose consumption and lactate production. After treatment with metformin (Fig. 5A), glucose consumption and lactate production decreased dramatically in LMP1-positive NPC cells (Fig. 5B-E). These findings indicated that LMP1 might promote glycolysis by inhibiting AMPK. Apoptosis is a major form of cell death induced by irradiation. To gain insight into the role of AMPK in apoptosis induced by DNA

damage in NPC cells, we performed fluorescence-activated cell sorting (FACS) to assess apoptosis in LMP1-positive and -negative NPC cells. Results indicated that LMP1-negative NPC cells underwent substantial apoptosis induced by irradiation, whereas LMP1positive cells were relatively resistant (Fig. 5F, G). However, treatment with metformin significantly increased apoptosis in LMP1-positive cells. These results suggested that LMP1 might inhibit DNA damageinduced apoptosis by inhibiting AMPK. Decreased phosphorylation of DNA-PK and AMPK corresponds with overall survival of NPC patients Because decreased phosphorylation of AMPKα (Thr172) plays an important role in LMP1-mediated radioresistance, we determined whether reduction of DNA-PK and AMPK phosphorylation was associated with the prognosis of NPC patients. We used IHC staining

Please cite this article in press as: Jingchen Lu, et al., EBV-LMP1 suppresses the DNA damage response through DNA-PK/AMPK signaling to promote radioresistance in nasopharyngeal carcinoma, Cancer Letters (2016), doi: 10.1016/j.canlet.2016.05.032

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Fig. 3. LMP1 inhibits AMPK phosphorylation through disrupting its subcellular localization and the interaction between AMPK and DNA-PK. (A) CNE1/CNE1-LMP1 and HNE2/ HNE2-LMP1 cells were exposed to 4 Gy irradiation. After 30 min, cells were harvested and analyzed for total AMPKα protein levels and phosphorylation at Thr172. (B) CNE1 and CNE1-LMP1 cells were either left untreated or treated with 4Gy irradiation. After 30 min, cells were fractionated into cytoplasmic or nuclear extracts as described in Materials and Methods. Protein (50 μg) was separated by SDS-PAGE and probed for AMPKα, histone H3 and tubulin as indicated. (C) CNE1 and CNE1-LMP1 cells were either left untreated or treated with 4 Gy irradiation. After 30 min, cells were fractionated into cytoplasmic or nuclear extracts. Protein (50 μg) was separated by SDS-PAGE and probed for AMPKα (Thr172), histone H3 and tubulin as indicated. (D) CNE1/CNE1-LMP1 and HNE2/HNE2-LMP1 cells were irradiated in the presence or absence of NU7026 (10 μM, 1 h before irradiation). After 30 min, cells were harvested and analyzed for AMPKα phosphorylation at Thr172. (E, F) CNE1/CNE1-LMP1 and HNE2/HNE2-LMP1 cells were exposed to 4 Gy of irradiation and then whole-cell lysates were prepared. Proteins were immunoprecipitated from the lysates with a DNA-PKcs antibody and the immunoprecipitates were subjected to immunoblot analysis with an antibody specific for AMPKα or control immunoglobulin (normal rabbit IgG). Representative blots from 3 independent experiments are shown. (G, H) CNE1/CNE1-LMP1 and HNE2/HNE2-LMP1 cells were mock-treated or exposed to 4 Gy of irradiation and allowed to recover for 30 min before fixation and staining with monoclonal antibodies to detect DNA-PKcs (green) and AMPKα (red). The images were acquired using confocal microscopy. Representative images from 3 independent experiments are shown. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

407 408 to examine the expression status of LMP1, DNA-PKcs (Thr2609), 409 AMPKα (Thr172) and γH2AX in a commercial NPC tissue array. 410 Clinical–pathological characteristics of 60 patients are listed in the 411 Supplementary data (Table S1). Results showed that the expres412 sion of LMP1 was significantly higher in NPC tumors (p = 0.001). 413 Strong or moderate expression of phosphorylated DNA-PKcs 414 (Thr2609) and AMPKα (Thr172) was found in normal nasopharyn415 geal epithelium, but a low level of these 2 phosphorylated proteins 416 was common in primary NPC specimens (p < 0.05) and correlated 417 significantly with the expression of LMP1 (Fig. 6A). The correla418 tion coefficient between LMP1 and phosphorylated DNA-PKcs 419 Q4 (Thr2609) was 0.389 (p < 0.01) or phosphorylated AMPKα (Thr172) 420 was 0.353 (p < 0.05; Fig. 6B). In addition, the correlation coeffi421 cient between phosphorylated DNA-PKcs (Thr2609) and 422 phosphorylated AMPKα (Thr172) was 0.458 (p < 0.001; Fig. 6B). These 423 findings indicated that DNA-PKcs and AMPKα inactivation corre424 lates with LMP1 expression in NPC. 425 In contrast, the expression of γH2AX was significantly higher in 426 NPC tumors compared to expression in normal nasopharyngeal ep427 ithelium (p < 0.001). Strong or moderate expression was observed 428 in NPC tumors, particularly in tumors with positive LMP1 stain-

ing. The correlation coefficient between LMP1 and γH2AX was 0.566 (p < 0.01) and the correlation between phosphorylated DNA-PKcs (Thr2609) and γH2AX was 0.476 (p < 0.01). In contrast, no significant correlation was observed between phosphorylated AMPKα (Thr172) and γH2AX (0.064, p > 0.05). These data indicated that LMP1 inhibits DSB repair in NPC cells at least partly through DNA-PK inactivation. We divided NPC patients into two groups based on the expression level of phosphorylated DNA-PKcs (Thr2609) and phosphorylated AMPKα (Thr172) and analyzed the correlation between their expression level and the 5-year overall survival (OS) rate. Results of a univariate analysis indicated that the expression of DNA-PKcs (Thr2609) was not associated with the 5-year OS rate, which was 66.67% in the DNA-PKcs (Thr2609)negative group compared to 70.37% in the DNA-PKcs (Thr2609)positive group (p = 0.757; Fig. 6C). However, the 5-year OS rate was significantly lower in AMPKα (Thr172)-negative patients compared to AMPKα (Thr172)-positive patients (53.57% vs. 78.26%, p = 0.037; Fig. 6D). These results suggested that AMPKα (Thr172) reduction was associated with a poorer clinical outcome of radiation therapy in NPC patients.

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Fig. 4. Reactivation of AMPK reverses LMP1-induced NPC cell radioresistance. (A) Sensitization of radioresistant LMP1-positive NPC cells by activation of AMPK. Colony formation of CNE1 and CNE1-LMP1 cells treated or not treated with metformin (5 μM) for 1 h before irradiation was evaluated 10 days after treatment with a single dose of 0, 4, 6 or 8 Gy irradiation. (B) Colony formation of HNE2 and HNE2-LMP1 cells treated or not treated with metformin (5 μM 1 h before irradiation) was evaluated 7 days after treatment with a single dose of 0, 4, 6 or 8 Gy irradiation. (C) Survival curve of CNE1 and CNE1-LMP1 cells treated or not treated with metformin. Surviving fractions were calculated by comparing the colony number of each treatment group with untreated groups (0 Gy). Results are plotted as the mean surviving fraction ± S.E.M. The survival curves were drawn using the GraphPad Prism 5 software program. (D) Survival curve of HNE2 and HNE2-LMP1 cells treated or not treated with metformin. Surviving fractions were calculated by comparing the colony number of each treatment group with untreated groups (0 Gy). Results are plotted as the mean surviving fraction ± S.E.M. The survival curves were drawn using the GraphPad Prism 5 software program, the asterisks indicate a significant (**p < 0.01 and ***p < 0.001) difference. (E) Representative images of xenografts from different treatment groups. The nasopharyngeal carcinoma xenograft model was established using CNE1-LMP1 cells. When the tumor volume reached 60–100 mm3, the animals were irradiated with a single dose of 5 Gy per week for 3 weeks alone or combined with intraperitoneal injection of metformin (250 mg/ kg daily). The tumor volume was measured every 2 days. Ten days after the last irradiation, the animals were sacrificed and the tumors were removed. (F) The tumor growth curve of different treatment groups in a CNE1-LMP1 cell xenograft model, the asterisks indicate a significant (**p < 0.01 and ***p < 0.001) difference.

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Fig. 5. Repression of AMPK activity by LMP1 promotes glycolysis and inhibits apoptosis of NPC cells after DNA damage. (A) CNE1 and CNE1-LMP1 cells were treated with metformin (5 μM) for 1 h. Then the cells were harvested and analyzed for phosphorylation at AMPKα (Thr172). (B, C) CNE1 and CNE1-LMP1 cells were treated with 4 Gy of irradiation in the presence or absence of metformin (5 μM, 1 h before irradiation). The relative levels of glucose consumption and lactate production rate at the indicated times were examined in these cell lines using the Automatic Biochemical Analyzer. (D, E) HNE2 and HNE2-LMP1 cells were irradiated with 4 Gy of irradiation in the presence or absence of metformin (5 μM, 1 h before irradiation). The relative levels of glucose consumption and lactate production rate at the indicated times were examined. (F, G) FACS analyses of apoptosis in CNE1 and CNE1-LMP1 cells and HNE2 and HNE2-LMP1 cells. Cells were irradiated in the presence or absence of metformin (5 μM, 1 h before irradiation). After 24 h cells were harvested and stained with Annexin-V and propidium iodide, and then analyzed using a FACScan. The percentages of Annexin-Vpositive apoptotic cells are shown as mean values ± S.D. from 3 independent experiments. For B–G, the asterisks indicate a significant (* p < 0.05; **p < 0.01; ***p < 0.001) difference.

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Discussion The global burden of mortality from EBV-attributed malignancies accounts for 1.8% of all cancer deaths in 2010, in which NPC and gastric cancer accounted for 92% of all EBV-attributed cancer deaths [37,38]. Human DNA tumor viruses often impinge on the DDR in order to establish the necessary cellular milieu to replicate viral DNA. Studies have shown that EBV-encoded LMP1 can modulate the DDR by inhibiting DNA repair through different mechanisms, such as repressing the PI3-K/Akt/FOXO3a pathway or p53-mediated DNA repair and transcriptional activity [39,40]. In this study, we demonstrated that LMP1 could repress DSB repair in NPC cells, at least partly by inhibiting the phosphorylation and activity of DNA-PK, a central component of DSB repair. Corresponding with this, γH2AX, a marker for DSBs, was significantly higher in NPC tumors, especially in LMP1-positive tumors, suggesting that LMP1 impaired the ability to repair DSBs in NPC and might produce genetic instability, which is a cause of cancer. AMPK is a critical regulator of cellular energy homeostasis. Recent work describes a novel function for AMPK as a sensor of genomic stress and as a participant in the DDR pathway. Herein, we found that phosphorylated AMPKα (Thr172) mainly localized in the cy-

toplasm after DNA damage and was substantially decreased in CNE1LMP1 cells compared to CNE1 cells. After exposure to irradiation, AMPKα localization in the nucleus shifted to the cytoplasm in CNE1 cells, but not in CNE1-LMP1 cells. These findings suggested that the decrease in phosphorylation of AMPKα (Thr172) in LMP1-positive NPC cells was partly due to the decrease in total protein levels of AMPKα in the cytoplasm. Thus, LMP1 might participate in regulating the subcellular location of AMPKα after DNA damage. A recent study revealed that in human glioma cells, DNA-PKcs interacted with the regulatory γ subunit of AMPK and positively regulated AMPK phosphorylation and activation under glucose-deprived conditions. This suggested that DNA-PK is an important regulator of AMPK activation in response to energy depletion [25]. To elucidate the role of DNA-PK in regulating AMPK activation in response to irradiation, we treated LMP1-positive cells with NU7026, a specific DNA-PK inhibitor, and found that the phosphorylation of AMPKα (Thr172) was further decreased, suggesting that DNA-PK is a positive regulator of AMPK activation in response to DNA damage. We also observed that the catalytic subunit of DNA-PK physically interacted with the α-subunit of AMPK under DNA damage conditions, whereas LMP1 disrupted this interaction. These data indicated that the interaction between AMPK and DNA-PK promotes the phos-

Please cite this article in press as: Jingchen Lu, et al., EBV-LMP1 suppresses the DNA damage response through DNA-PK/AMPK signaling to promote radioresistance in nasopharyngeal carcinoma, Cancer Letters (2016), doi: 10.1016/j.canlet.2016.05.032

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Fig. 6. Decreased phosphorylation of DNA-PK and AMPK corresponds with overall survival of NPC patients. (A) Immunohistochemistry analysis was used to examine the expression of LMP1, DNA-PKcs (Thr2609), AMPKα (Thr172) and γH2AX in an NPC tissue array. Case 1 was a biopsy from a normal nasopharyngeal epithelium and Cases 2 and 3 were both biopsies from NPC tumors. (B) Correlation between LMP1 and phosphorylated DNA-PKcs (Thr2609), AMPKα (Thr172) and γH2AX expression in the NPC tissue array. (C) Five-year overall survival rates of NPC patients with DNA-PKcs (Thr2609)-negative or -positive expression were estimated with the Kaplan–Meier method by log-rank test (p = 0.757). (D) Five-year overall survival rates of NPC patients with AMPKα (Thr172)-negative or -positive expression were estimated with the Kaplan– Meier method by log-rank test (p = 0.037).

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phorylation of AMPKα at Thr172 and LMP1 might affect the phosphorylation by disrupting this interaction. Recently, a series of our studies confirmed that LMP1 contributes to the radioresistance of NPC. LMP1 might promote radioresistance by regulating tumor angiogenesis through the JNKs/ HIF-1 pathway or glycolysis through the up-regulation of hexokinase 2 (HK2), a rate-limiting enzyme of glycolysis, or by inhibiting telomerase activity in NPC cells [41–43]. Other studies showed that LMP1-induced cancer stem cell (CSC)-like properties might contribute to radioresistance in NPC cells [44]. Here we found that reactivation of AMPK by metformin could substantially reverse radioresistance of LMP1-positive NPC cells both in vitro and in vivo, suggesting that AMPK plays an important role in regulating LMP1mediated radioresistance. AMPK is considered a negative regulator of aerobic glycolysis and recent findings revealed that the lactate concentration was significantly correlated with tumor response to fractionated irradiation, probably due to its anti-oxidative capacity [45–47]. We found that LMP1-positive NPC cells displayed dramatically increased glucose consumption and lactate production after irradiation, a metabolic signature consistent with the Warburg effect. When AMPK was activated with metformin, glucose consumption and lactate production in LMP1-positive NPC cells dropped back to a similar level as observed in LMP1-negative NPC cells. Apoptosis is a primary means of death after DNA damage. We found that LMP1 profoundly inhibited irradiation-induced apoptosis, but apoptosis increased dramatically in LMP1-positive cells after reactivation of AMPK. Together these findings suggested that high concentrations of lactate and resistance to apoptosis may contribute to radioresistance in LMP1-positive NPC cells, whereas AMPK reactivation reversed ra-

dioresistance by negatively regulating aerobic glycolysis and promoting apoptosis. A low level of phosphorylated AMPK is common in primary NPC specimens and significantly correlates with the expression of LMP1 [48]. Our data revealed that a lower level of phosphorylated AMPKα (Thr172) predicted a poorer 5-year overall survival rate in NPC patients. A previous study showed that DNA-PKcs overexpression was observed in 36.8% of NPC tumor specimens and was highly correlated to advanced clinical stages and poor survival [49]. Another study revealed that negative expression of DNA-PKcs was detected in 35 of 87 (40.2%) NPC patients and was significantly associated with poor patient survival [50]. A separate study by Lee et al., however, showed no association between DNA-PKcs overexpression and the clinical outcome of NPC [51]. Herein, we found no significant relationship between phosphorylated DNA-PKcs (Thr2609) expression and the 5-year overall survival rate of NPC patients, which might possibly be due to the small sample size. Overall, this study demonstrated that EBV-encoded LMP1 impaired DSB repair induced by irradiation, possibly by inhibiting DNAPK phosphorylation and activity. Moreover, LMP1 reduced the phosphorylation of AMPK and disturbed its subcellular localization after irradiation, possibly by disrupting the physical interaction between AMPKα and DNA-PKcs. The decrease in AMPK activity was involved in LMP1-mediated glycolysis and resistance to apoptosis induced by irradiation, which might confer radioresistance to NPC cells. Our data revealed a novel mechanism for LMP1-mediated radioresistance by suppressing the DDR through DNA-PK/AMPK signaling. This could provide a mechanistic rationale supporting the use of AMPK activators, such as metformin, for enhancing NPC radiotherapy.

Please cite this article in press as: Jingchen Lu, et al., EBV-LMP1 suppresses the DNA damage response through DNA-PK/AMPK signaling to promote radioresistance in nasopharyngeal carcinoma, Cancer Letters (2016), doi: 10.1016/j.canlet.2016.05.032

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Acknowledgments This work was supported by the National High Technology Research and Development Program of China (2009AA02Z403, 2012AA02A501) and the National Key Basic Research Program of China (2011CB504300, 2011CB504305).

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Conflict of interest None.

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Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.canlet.2016.05.032.

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