EBV-encoded miRNAs target ATM-mediated response in nasopharyngeal carcinoma

Raymond W-M Lung, Pok-Man Hau, Ken H-O Yu, Kevin Y Yip, Joanna H-M Tong, Wing-Po Chak, Anthony W-H Chan, Ka-Hei Lam, Angela Kwok-Fung Lo, Edith K-Y Tin, Shuk-Ling Chau, Jesse C-S Pang, Johnny S-H Kwan, Pierre Busson, Lawrence S Young, Lee-Fah Yap, Sai-Wah Tsao, Ka-Fai To, Kwok-Wai Lo, Raymond W-M Lung, Pok-Man Hau, Ken H-O Yu, Kevin Y Yip, Joanna H-M Tong, Wing-Po Chak, Anthony W-H Chan, Ka-Hei Lam, Angela Kwok-Fung Lo, Edith K-Y Tin, Shuk-Ling Chau, Jesse C-S Pang, Johnny S-H Kwan, Pierre Busson, Lawrence S Young, Lee-Fah Yap, Sai-Wah Tsao, Ka-Fai To, Kwok-Wai Lo

Abstract

Nasopharyngeal carcinoma (NPC) is a highly invasive epithelial malignancy that is prevalent in southern China and Southeast Asia. It is consistently associated with latent Epstein-Barr virus (EBV) infection. In NPC, miR-BARTs, the EBV-encoded miRNAs derived from BamH1-A rightward transcripts, are abundantly expressed and contribute to cancer development by targeting various cellular and viral genes. In this study, we establish a comprehensive transcriptional profile of EBV-encoded miRNAs in a panel of NPC patient-derived xenografts and an EBV-positive NPC cell line by small RNA sequencing. Among the 40 miR-BARTs, predominant expression of 22 miRNAs was consistently detected in these tumors. Among the abundantly expressed EBV-miRNAs, BART5-5p, BART7-3p, BART9-3p, and BART14-3p could negatively regulate the expression of a key DNA double-strand break (DSB) repair gene, ataxia telangiectasia mutated (ATM), by binding to multiple sites on its 3'-UTR. Notably, the expression of these four miR-BARTs represented more than 10% of all EBV-encoded miRNAs in tumor cells, while downregulation of ATM expression was commonly detected in all of our tested sequenced samples. In addition, downregulation of ATM was also observed in primary NPC tissues in both qRT-PCR (16 NP and 45 NPC cases) and immunohistochemical staining (35 NP and 46 NPC cases) analysis. Modulation of ATM expression by BART5-5p, BART7-3p, BART9-3p, and BART14-3p was demonstrated in the transient transfection assays. These findings suggest that EBV uses miRNA machinery as a key mechanism to control the ATM signaling pathway in NPC cells. By suppressing these endogenous miR-BARTs in EBV-positive NPC cells, we further demonstrated the novel function of miR-BARTs in inhibiting Zta-induced lytic reactivation. These findings imply that the four viral miRNAs work co-operatively to modulate ATM activity in response to DNA damage and to maintain viral latency, contributing to the tumorigenesis of NPC. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Keywords: ATM serine/threonine kinase (ATM); EBV-miRNAs; Epstein-Barr virus; nasopharyngeal carcinoma; transcriptome sequencing.

© 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Figures

Figure 1
Figure 1
Expression of viral miRNAs in EBV‐positive NPCs. (A) The number of EBV‐miRNA reads is indicated per 10 million of the total mapped mature miRNAs to normalize the sequencing depth in each library. The libraries of C666‐1, C17, and the average of four primary NPC‐derived xenografts (xeno‐C666, xeno‐2117, xeno‐1915, and C15) are shown as mean ± SD. (B) The distribution of the individual miR‐BARTs to the total viral miRNAs in the libraries is shown in the pie charts.
Figure 2
Figure 2
ATM is a potential target of several EBV‐encoded miRNAs. (A) Suggested putative miR‐BART recognition sites on the ATM 3'‐UTRs are shown. The seed‐binding regions of the miR‐BARTs are underlined and the bases mutated for the luciferase reporter analysis are marked in red. (B) The expression of miR‐BARTs of interest in primary NPC samples was examined using RT‐qPCR (n = 45). The expression of miR‐BARTs was normalized to EBNA1 for analysis. The low‐expression miR‐BARTs (BART21‐5p and BART20‐3p) and other high‐expression miR‐BARTs were included for comparison. The data shown are the mean ± SEM from the tested samples. (C) Immunoblotting of ATM protein in the NPC samples. Three immortalized normal NP cell lines (NP361, NP550, and NP69), C666‐1, and five NPC xenografts (xeno‐C666, xeno‐2117, xeno‐1915, C15, and C17) were analyzed. (D) ATM protein expression in FFPE specimens was analyzed by immunohistochemistry. NP‐1 and NP‐2 are examples of normal NP epithelia with strong ATM positive stain. NPC‐1 and NPC‐2 are examples of NPC cells that are negative for ATM expression. The infiltrated lymphocytes that served as internal controls were strongly positive. NPC‐3 is an example of NPC cells that are positive for ATM (original magnification × 400). (E) Expression of ATM in the primary NPC samples was demonstrated by RT‐qPCR (number of NPs = 16; number of NPCs = 45). (F) The scatter plot demonstrates the inverse correlation between the miR‐BART of interest and ATM mRNA expression levels in 25 NPC samples. (G) The direct interaction between ATM expression and miR‐BARTs was demonstrated in dual luciferase reporter assays. The pMIR‐REPORT vectors containing the wild‐type ATM binding sites (UTR‐wt) or the mutant binding sites (UTR‐mut) were tested against the corresponding miR‐BART mimics and inhibitors. The relative firefly luciferase activity was normalized to the Renilla luciferase control, and results were taken from at least three independent experiments. The data shown are the mean + SD. miR‐Ctl = miR‐BART mimic control; miR‐BART = miR‐BART mimic; Inh‐NEG = miRNA inhibitor negative control; Inh‐BART = miR‐BART inhibitor. **p < 0.01; ***p < 0.001.
Figure 3
Figure 3
Modulation of ATM expression by EBV‐encoded miRNAs. (A) Downregulation of ATM in NP69 and HeLa cells by ebv‐miRNAs from both clusters. Cells transfected with expression vectors containing BART‐Cluster 1 (miR‐BART‐C1) and BART‐Cluster 2 (miR‐BART‐C2) derived miRNAs or individual miR‐BARTs were analyzed by western blotting. The empty vector and irrelevant miRNA mimic were used as negative controls. (B, C) The ATM expression of BART‐Cluster 1 (C1) and BART‐Cluster 2 (C2) miRNA expressing cells was restored by the indicated miR‐BART inhibitors (Inh‐BARTs). Endogenous ATM expression was measured by western blot and the relative ATM expression is shown under the blots. (D) Endogenous ATM protein expression in C666‐1 cells was restored by the miR‐BART inhibitors. The inhibitors of BART5‐5p, BART7‐3p, BART9‐3p, and BART14‐3p were introduced, individually or together (All 4 Inh‐BARTs), into the C666‐1 cells and protein lysates were collected 48 h post‐transfection for western blot analysis.
Figure 4
Figure 4
EBV‐miRNAs enhance the ionizing radiation (IR) sensitivity of epithelial cells. The miR‐BART transfected cells were treated with different doses of IR and the cells were harvested at 30 min post‐irradiation for immunoblotting analysis. The expression of the basal ATM proteins, phospho‐ATM (p‐ATM), phospho‐CHK2 (p‐CHK2), and γ‐H2AX, was analyzed. Actin was probed as the loading control. ATM knockdown cells (siATM) were included as positive controls.
Figure 5
Figure 5
EBV‐miRNAs suppress the DNA damage response to ionizing radiation. (A) Representative images of the H2AX nuclear foci staining assay. Cells transfected with either miRNA mimics (miR‐NEG) or a combination of four miR‐BART mimics (All 4 miR‐BARTs) were treated with a single dose of 3 Gy irradiation, which was followed by immunostaining with the γ‐H2AXser139 antibody 1 h post‐irradiation. Cells containing more than five γ‐H2AX foci in the nucleus were considered positive and the percentage of γ‐H2AX‐positive cells was calculated (n = 100). The mean ± SD for three independent experiments are shown. (B) Comet assays of the DNA repair capacity were performed on NP69 and HeLa cells, which were treated with a single irradiation dose of 10 and 20 Gy, respectively. The tail moment of the irradiated cells at 6 h is shown in the bar charts; mean ± SEM. Student's t‐test was conducted compared with the miR‐NEG control. *p < 0.05; **p < 0.01; ***p < 0.001. (C, D) Clonogenic survival assays. Approximately 200–1600 transfected HeLa cells were seeded into a six‐well plate and treated with a single dose of 0.5, 1 or 2 Gy irradiation. The cells were stained and colonies containing more than 30 cells were counted. The survival fraction was calculated by dividing the plating efficiency of the irradiated cells by the plating efficiency of the untreated cultures. Statistical analyses using Student's t‐test were conducted and compared with the miR‐NEG control. *p < 0.05; **p < 0.01.
Figure 6
Figure 6
BZLF1‐induced virus reactivation is suppressed by miR‐BARTs. (A) The BZLF1‐expressing plasmid (1.25 μg) was co‐transfected with the indicated miR‐BART inhibitors (10 μm) into C666‐1 cells in a six‐well plate. Cells were harvested for immunoblotting analysis after 48 h. The expression of ATM, ATM downstream effectors (p‐ATM and γ‐H2AX), and the early viral lytic proteins (BRLF1 and BMRF1) was analyzed. (B) The synergistic effects of BZLF1 and ATM on p‐ATM, γ‐H2AX, BRLF1, and BMRF1 expression in C666‐1 cells. The cells were co‐transfected with ATM and BZLF1‐expressing plasmids and protein expression was examined by immunoblotting. The two isoforms of BMRF1 are indicated by arrows. The relative expression of the protein level was calculated for comparison.

References

    1. Chan AT, Felip E, Group EGW . Nasopharyngeal cancer: ESMO clinical recommendations for diagnosis, treatment and follow‐up. Ann Oncol 2009; 20(suppl 4): 123–125.
    1. Raab‐Traub N. Epstein–Barr virus in the pathogenesis of NPC. Semin Cancer Biol 2002; 12 : 431–441.
    1. Lo KW, Chung GT, To KF. Deciphering the molecular genetic basis of NPC through molecular, cytogenetic, and epigenetic approaches. Semin Cancer Biol 2012; 22 : 79–86.
    1. Lo AK, Dawson CW, Young LS, et al Activation of the FGFR1 signalling pathway by the Epstein–Barr virus‐encoded LMP1 promotes aerobic glycolysis and transformation of human nasopharyngeal epithelial cells. J Pathol 2015; 237 : 238–248.
    1. Raab‐Traub N. Nasopharyngeal carcinoma: an evolving role for the Epstein–Barr virus. Curr Top Microbiol Immunol 2015; 390 : 339–363.
    1. Lee SP. Nasopharyngeal carcinoma and the EBV‐specific T cell response: prospects for immunotherapy. Semin Cancer Biol 2002; 12 : 463–471.
    1. Lung RW, Tong JH, To KF. Emerging roles of small Epstein–Barr virus derived non‐coding RNAs in epithelial malignancy. Int J Mol Sci 2013; 14 : 17378–17409.
    1. Jung YJ, Choi H, Kim H, et al MicroRNA miR‐BART20‐5p stabilizes Epstein–Barr virus latency by directly targeting BZLF1 and BRLF1. J Virol 2014; 88 : 9027–9037.
    1. Qiu J, Thorley‐Lawson DA. EBV microRNA BART 18‐5p targets MAP3K2 to facilitate persistence in vivo by inhibiting viral replication in B cells. Proc Natl Acad Sci U S A 2014; 111 : 11157–11162.
    1. Barth S, Pfuhl T, Mamiani A, et al Epstein–Barr virus‐encoded microRNA miR‐BART2 down‐regulates the viral DNA polymerase BALF5. Nucleic Acids Res 2008; 36 : 666–675.
    1. Lung RW, Tong JH, Sung YM, et al Modulation of LMP2A expression by a newly identified Epstein–Barr virus‐encoded microRNA miR‐BART22. Neoplasia 2009; 11 : 1174–1184.
    1. Lo AK, To KF , Lo KW, et al Modulation of LMP1 protein expression by EBV‐encoded microRNAs. Proc Natl Acad Sci U S A 2007; 104 : 16164–16169.
    1. Nachmani D, Stern‐Ginossar N, Sarid R, et al Diverse herpesvirus microRNAs target the stress‐induced immune ligand MICB to escape recognition by natural killer cells. Cell Host Microbe 2009; 5 : 376–385.
    1. Kim H, Choi H, Lee SK. Epstein–Barr virus miR‐BART20‐5p regulates cell proliferation and apoptosis by targeting BAD. Cancer Lett 2015; 356 : 733–742.
    1. Lin TC, Liu TY, Hsu SM, et al Epstein–Barr virus‐encoded miR‐BART20‐5p inhibits T‐bet translation with secondary suppression of p53 in invasive nasal NK/T‐cell lymphoma. Am J Pathol 2013; 182 : 1865–1875.
    1. Kang D, Skalsky RL, Cullen BR. EBV BART microRNAs target multiple pro‐apoptotic cellular genes to promote epithelial cell survival. PLoS Pathog 2015; 11 : e1004979.
    1. Choy EY, Siu KL, Kok KH, et al An Epstein–Barr virus‐encoded microRNA targets PUMA to promote host cell survival. J Exp Med 2008; 205 : 2551–2560.
    1. Cai LM, Lyu XM, Luo WR, et al EBV‐miR‐BART7‐3p promotes the EMT and metastasis of nasopharyngeal carcinoma cells by suppressing the tumor suppressor PTEN. Oncogene 2015; 34 : 2156–2166.
    1. Metheetrairut C, Slack FJ. MicroRNAs in the ionizing radiation response and in radiotherapy. Curr Opin Genet Dev 2013; 23 : 12–19.
    1. Yan D, Ng WL, Zhang X, et al Targeting DNA‐PKcs and ATM with miR‐101 sensitizes tumors to radiation. PLoS One 2010; 5 : e11397.
    1. Hu H, Du L, Nagabayashi G, et al ATM is down‐regulated by N‐Myc‐regulated microRNA‐421. Proc Natl Acad Sci U S A 2010; 107 : 1506–1511.
    1. Liu X, Liao W, Peng H, et al miR‐181a promotes G1/S transition and cell proliferation in pediatric acute myeloid leukemia by targeting ATM. J Cancer Res Clin Oncol 2016; 142 : 77–87.
    1. Bose S, Yap LF, Fung M, et al The ATM tumour suppressor gene is down‐regulated in EBV‐associated nasopharyngeal carcinoma. J Pathol 2009; 217 : 345–352.
    1. Hau PM, Deng W, Jia L, et al Role of ATM in the formation of the replication compartment during lytic replication of Epstein–Barr virus in nasopharyngeal epithelial cells. J Virol 2015; 89 : 652–668.
    1. Chung GT, Lung RW, Hui AB, et al Identification of a recurrent transforming UBR5–ZNF423 fusion gene in EBV‐associated nasopharyngeal carcinoma. J Pathol 2013; 231 : 158–167.
    1. Yip YL, Pang PS, Deng W, et al Efficient immortalization of primary nasopharyngeal epithelial cells for EBV infection study. PLoS One 2013; 8 : e78395.
    1. Langmead B, Salzberg SL. Fast gapped‐read alignment with Bowtie 2. Nat Methods 2012; 9 : 357–359.
    1. Li H, Handsaker B, Wysoker A , et al The Sequence Alignment/Map format and SAMtools. Bioinformatics 2009; 25 : 2078–2079.
    1. Canman CE, Lim DS, Cimprich KA, et al Activation of the ATM kinase by ionizing radiation and phosphorylation of p53. Science 1998; 281 : 1677–1679.
    1. To KF , Tong JH, Yeung KS, et al Detection of ALK rearrangement by immunohistochemistry in lung adenocarcinoma and the identification of a novel EML4–ALK variant. J Thorac Oncol 2013; 8 : 883–891.
    1. Wong QW, Lung RW, Law PT, et al MicroRNA‐223 is commonly repressed in hepatocellular carcinoma and potentiates expression of Stathmin1 . Gastroenterology 2008; 135 : 257–269.
    1. Lung RW, Wang X, Tong JH, et al A single nucleotide polymorphism in microRNA‐146a is associated with the risk for nasopharyngeal carcinoma. Mol Carcinog 2013; 52(suppl 1): E28–E38.
    1. Chow JP, Man WY, Mao M, et al PARP1 is overexpressed in nasopharyngeal carcinoma and its inhibition enhances radiotherapy. Mol Cancer Ther 2013; 12 : 2517–2528.
    1. Munshi A, Hobbs M, Meyn RE. Clonogenic cell survival assay. Methods Mol Med 2005; 110 : 21–28.
    1. Bollen KA, Jackman RW. Regression diagnostics: an expository treatment of outliers and influential cases. Sociol Methods Res 1985; 13 : 510–542.
    1. Cheung ST, Huang DP, Hui AB, et al Nasopharyngeal carcinoma cell line (C666‐1) consistently harbouring Epstein‐Barr virus. Int J Cancer 1999; 83 : 121–126.
    1. Huang DP, Ho JH, Chan WK , et al Cytogenetics of undifferentiated nasopharyngeal carcinoma xenografts from southern Chinese. Int J Cancer 1989; 43 : 936–939.
    1. Busson P, Ganem G, Flores P, et al Establishment and characterization of three transplantable EBV‐containing nasopharyngeal carcinomas. Int J Cancer 1988; 42 : 599–606.
    1. Moss WN, Lee N, Pimienta G, et al RNA families in Epstein–Barr virus. RNA Biol 2014; 11 : 10–17.
    1. Marquitz AR, Mathur A, Chugh PE, et al Expression profile of microRNAs in Epstein–Barr virus‐infected AGS gastric carcinoma cells. J Virol 2014; 88 : 1389–1393.
    1. Hooykaas MJ, Kruse E, Wiertz EJ, et al Comprehensive profiling of functional Epstein–Barr virus miRNA expression in human cell lines. BMC Genomics 2016; 17 : 644.
    1. Hagemeier SR, Barlow EA, Meng Q, et al The cellular ataxia telangiectasia‐mutated kinase promotes Epstein–Barr virus lytic reactivation in response to multiple different types of lytic reactivation‐inducing stimuli. J Virol 2012; 86 : 13360–13370.
    1. Cai X, Schafer A, Lu S, et al Epstein–Barr virus microRNAs are evolutionarily conserved and differentially expressed. PLoS Pathog 2006; 2 : e23.
    1. Qiu J, Cosmopoulos K, Pegtel M, et al A novel persistence associated EBV miRNA expression profile is disrupted in neoplasia. PLoS Pathog 2011; 7 : e1002193.
    1. Gottwein E, Corcoran DL, Mukherjee N, et al Viral microRNA targetome of KSHV‐infected primary effusion lymphoma cell lines. Cell Host Microbe 2011; 10 : 515–526.
    1. Skalsky RL, Corcoran DL, Gottwein E, et al The viral and cellular microRNA targetome in lymphoblastoid cell lines. PLoS Pathog 2012; 8 : e1002484.
    1. Lu J, He ML, Wang L, et al MiR‐26a inhibits cell growth and tumorigenesis of nasopharyngeal carcinoma through repression of EZH2. Cancer Res 2011; 71 : 225–233.
    1. Lin X, Tsai MH, Shumilov A, et al The Epstein–Barr virus BART miRNA cluster of the M81 strain modulates multiple functions in primary B cells. PLoS Pathog 2015; 11 : e1005344.
    1. Ames BN, Gold LS. Endogenous mutagens and the causes of aging and cancer. Mutat Res 1991; 250 : 3–16.
    1. Ashworth A. A synthetic lethal therapeutic approach: poly(ADP) ribose polymerase inhibitors for the treatment of cancers deficient in DNA double‐strand break repair. J Clin Oncol 2008; 26 : 3785–3790.
    1. Jasin M, Rothstein R. Repair of strand breaks by homologous recombination. Cold Spring Harb Perspect Biol 2013; 5 : a012740.
    1. Haber JE. DNA repair. Gatekeepers of recombination. Nature 1999; 398 : 665–667.
    1. Fong PC, Yap TA, Boss DS, et al Poly(ADP)‐ribose polymerase inhibition: frequent durable responses in BRCA carrier ovarian cancer correlating with platinum‐free interval. J Clin Oncol 2010; 28 : 2512–2519.
    1. Gelmon KA, Tischkowitz M, Mackay H, et al Olaparib in patients with recurrent high‐grade serous or poorly differentiated ovarian carcinoma or triple‐negative breast cancer: a phase 2, multicentre, open‐label, non‐randomised study. Lancet Oncol 2011; 12 : 852–861.
    1. Bryant HE, Helleday T. Inhibition of poly (ADP‐ribose) polymerase activates ATM which is required for subsequent homologous recombination repair. Nucleic Acids Res 2006; 34 : 1685–1691.
    1. McCabe N, Turner NC, Lord CJ, et al Deficiency in the repair of DNA damage by homologous recombination and sensitivity to poly(ADP‐ribose) polymerase inhibition. Cancer Res 2006; 66 : 8109–8115.

Source: PubMed

Sponsorzy i współpracownicy

Warunki medyczne

Interwencje lekowe

3
Subskrybuj