Original Article HDAC4 and HDAC6 sustain DNA double strand break repair and stem-like phenotype by promoting radioresistance in glioblastoma cells Q4 Q3 Francesco Marampon a, *, 1 , Francesca Megiorni b, 1 , Simona Camero b, c , Clara Crescioli d , Heather P. McDowell b, e , Roberta Sferra a , Antonella Vetuschi a , Simona Pompili a , Luca Ventura f , Francesca De Felice g , Vincenzo Tombolini g , Carlo Dominici b , Roberto Maggio a , Claudio Festuccia a, 1 , Giovanni Luca Gravina a, 1 a Department of Biotechnological and Applied Clinical Sciences, University of L'Aquila, Coppito, L'Aquila, Italy b Department of Pediatrics and Infantile Neuropsychiatry, Sapienza University of Rome, Rome, Italy c Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy d Department of Movement, Human and Health Sciences, University of Rome Foro Italico, Rome, Italy e Department of Oncology, Alder Hey Children's NHS Foundation Trust, Liverpool, United Kingdom f Department of Pathology, San Salvatore Hospital, Coppito, L'Aquila, Italy g Department of Radiological, Oncological and Pathological Sciences, Sapienza University of Rome, Rome, Italy article info Article history: Received 17 January 2017 Received in revised form 15 March 2017 Accepted 15 March 2017 Keywords: HDAC4 HDAC6 Glioblastoma Radiotherapy Radioresistance abstract The role of histone deacetylase (HDAC) 4 and 6 in glioblastoma (GBM) radioresistance was investigated. We found that tumor samples from 31 GBM patients, who underwent temozolomide and radiotherapy combined treatment, showed HDAC4 and HDAC6 expression in 93.5% and 96.7% of cases, respectively. Retrospective clinical data analysis demonstrated that high-intensity HDAC4 and/or HDAC6 immunostaining was predictive of poor clinical outcome. In vitro experiments revealed that short hairpin RNA-mediated silencing of HDAC4 or HDAC6 radiosensitized U87MG and U251MG GBM cell lines by promoting DNA double-strand break (DSBs) accumulation and by affecting DSBs repair molecular machinery. We found that HDAC6 knock-down predisposes to radiation therapy-induced U251MG apoptosis- and U87MG autophagy-mediated cell death. HDAC4 silencing promoted radiation therapy-induced senescence, independently by the cellular context. Finally, we showed that p53 WT expression contributed to the radiotherapy lethal effects and that HDAC4 or HDAC6 sustained GBM stem-like radioresistant phenotype. Altogether, these observations suggest that HDAC4 and HDAC6 are guardians of irradiation-induced DNA damages and stemness, thus promoting radioresistance, and may represent potential prognostic markers and therapeutic targets in GBM. © 2017 Elsevier B.V. All rights reserved. Introduction Epigenetic mechanisms, including enzymatic modifications to histones and DNA methylation, contribute to tumor development, progression, chemoresistance, and radioresistance [1,2]. Because of their reversible nature and their role in gene expression, epigenetic alterations, such as histone acetylation, are a current focus for therapeutic targeting in clinical research. Histone acetyl- transferases (HATs) and histone deacetylases (HDACs) control a wide array of biological processes by transferring and removing acetyl groups to or from histones/proteins, respectively [3e5]. The aberrant expression and activity of HDACs, which result in the Abbreviations: GBM, glioblastoma; TMZ, temozolomide; RT, radiotherapy; HDACs, Histone deacetylases; HATs, Histone acetyltransferases; DSBs, DNA double- strand breaks; OS, Overall survival; shRNA, short hairpin RNAs; NHEJ, non-ho- mologous end-joining; HR, homologous recombination; HCN-2, human cortical neuronal; NHA, normal human astrocytes; siRNA, small interfering RNAs; p53, Tumor Protein p53; ATM, Ataxia-telangiectasia mutated; DNAP-PKcs, DNA-depen- dent protein kinase, catalytic subunit; H2AX, H2A histone family, member X. * Corresponding author. Department of Biotechnological and Applied Clinical Sciences, University of L'Aquila, Via Vetoio 1, 67100 Coppito, L'Aquila, Italy. Fax: þ39 0862 433523. E-mail address: f.marampon@gmail.com (F. Marampon). 1 Equal contribution. Contents lists available at ScienceDirect Cancer Letters journal homepage: www.elsevier.com/locate/canlet http://dx.doi.org/10.1016/j.canlet.2017.03.028 0304-3835/© 2017 Elsevier B.V. All rights reserved. Cancer Letters xxx (2017) 1e11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 CAN13292_proof ■ 27 March 2017 ■ 1/11 Please cite this article inpress as: F. Marampon, et al., HDAC4 and HDAC6 sustain DNA double strand break repair and stem-like phenotype by promoting radioresistance in glioblastoma cells, Cancer Letters (2017), http://dx.doi.org/10.1016/j.canlet.2017.03.028