RANK ( TNFRSF 11 A ) Is Epigenetically Inactivated and Induces Apoptosis in Gliomas 1 , 2

Alterations of DNA methylation play an important role in gliomas. In a genome-wide screen, we identified a CpG-rich fragment within the 5′ region of the tumor necrosis factor receptor superfamily, member 11A gene (TNFRSF11A) that showedde novomethylation in gliomas. TNFRSF11A, also knownas receptor activator of NF-κB (RANK), activates several signaling pathways, such as NF-κB, JNK, ERK, p38α, and Akt/PKB. Using pyrosequencing, we detected RANK/ TNFRSF11A promoter methylation in 8 (57.1%) of 14 diffuse astrocytomas, 17 (77.3%) of 22 anaplastic astrocytomas, 101 (84.2%) of 120 glioblastomas, 6 (100%) of 6 glioma cell lines, and 7 (100%) of 7 glioma stem cell–enriched glioblastoma primary cultures but not in four normal white matter tissue samples. Treatment of glioma cell lines with the demethylating agent 5-aza-2′-deoxycytidine significantly reduced the methylation level and resulted in increased RANK/ TNFRSF11AmRNA expression. Overexpression of RANK/TNFRSF11A in glioblastoma cell lines leads to a significant reduction in focus formation and elevated apoptotic activity after flow cytometric analysis. Reporter assay studies of transfected glioma cells supported these results by showing the activation of signaling pathways associated with regulation of apoptosis. We conclude that RANK/TNFRSF11A is a novel and frequent target for de novo methylation in gliomas, which affects apoptotic activity and focus formation thereby contributing to the molecular pathogenesis of gliomas. Neoplasia (2012) 14, 526–534 Address all correspondence to: Andreas Waha, PhD, Department of Neuropathology, University of Bonn Medical Center, Sigmund-Freud-Str. 25, D-53105 Bonn, Germany. E-mail: awaha@uni-bonn.de This study was supported by National Genome Research Network “NGFN,” Brain Tumor Network plus (grant 01GS08187, SP8) and the BONFOR program of the Medical Faculty of the University of Bonn. B.S. is supported by the Lichtenberg program of the VW Foundation. No potential conflicts of interest are disclosed. This article refers to supplementary materials, which are designated by Table W1 and Figures W1 to W4 and are available online at www.neoplasia.com. A. von dem Knesebeck and J. Felsberg contributed equally to this work. Received 15 February 2012; Revised 27 April 2012; Accepted 30 April 2012 Copyright © 2012 Neoplasia Press, Inc. All rights reserved 1522-8002/12/$25.00 DOI 10.1596/neo.12360 www.neoplasia.com Volume 14 Number 6 June 2012 pp. 526–534 526