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. 2012 Jun;14(6):526-34.
doi: 10.1596/neo.12360.

RANK (TNFRSF11A) is epigenetically inactivated and induces apoptosis in gliomas

Anna von dem Knesebeck  1 , Jörg Felsberg, Anke Waha, Wolfgang Hartmann, Björn Scheffler, Martin Glas, Jennifer Hammes, Thomas Mikeska, Pearlly S Yan, Elmar Endl, Matthias Simon, Guido Reifenberger, Torsten Pietsch, Andreas Waha
Affiliations

RANK (TNFRSF11A) is epigenetically inactivated and induces apoptosis in gliomas

Anna von dem Knesebeck et al. Neoplasia. 2012 Jun.

Abstract

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 showed de novo methylation in gliomas. TNFRSF11A, also known as 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/TNFRSF11A mRNA 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.

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Figures

Figure 1
Figure 1
Localization of the investigated DNA fragment within the CpG island at chromosome 18q21.33 (59991900-59992679). The sequence investigated by pyrosequencing, and the ATG start codon is indicated. Below, details of two representative pyrograms within the 5′ upstream region of RANK/TNFRSF11A are shown. The glioblastoma sample GBM042 (A) shows strong methylation, whereas low methylation levels were found in normal white matter tissue (B).
Figure 2
Figure 2
Average methylation level of 156 primary glioma tissues, glioblastoma cell lines, stem cell-enriched primary glioblastoma cell cultures, and normal white matter tissues within the investigated region of RANK/TNFRSF11A (A). Heat map of quantitative methylation data of the 14 investigated CpG sites within the RANK/TNFRSF11A CpG island in three patients, for which tissue from the initial low-grade glioma and the high-grade relapse tumor were available. The color code shows the methylation range (0%–60%) (B).
Figure 3
Figure 3
Relative transcript levels of RANK/TNFRSF11A (RANK/TNFRSF11A/ARF1) analyzed by real-time RT-PCR in gliomas, glioblastoma cell lines, and normal white matter tissue (A). Relative transcript levels of RANK/TNFRSF11A in glioblatoma cell lines with (□) or without (■) 1 and 5 µM 5-aza-2′-deoxycytidine treatment (B).
Figure 4
Figure 4
Focus formation assay and flow cytometric analysis of U373MG glioblastoma cells transfected with RANK/TNFRSF11A or control vector pcDNA4/myc-His. Representative image of a focus assay of U373MG and A172 cells. Number of foci formed after transfection with RANK/TNFRSF11A (■) or pcDNA4/myc-His control (□); t test, * U373MG, P <.0001; **A172, P = .0193 (A). Representative image and summarized results of all flow cytometric analysis showing significant induction of apoptosis (t test, P <.0001) as measured by detection of cleaved PARP and analysis of phospho(Ser10)-histone H3 in U373MG cells transiently cotransfected with RANK-pcDNA4/myc-His plasmid and pMAX GFP or pcDNA4/myc-His control vector and pMAX GFP (B).
Figure 5
Figure 5
Reporter array analysis of 45 signaling pathways in U373MG glioblastoma cells transfected with RANK-pcDNA4/myc-His plasmid and pcDNA4/myc-His control vector. The diagram depicts the fold change of reporter activity indicative for specific cancer-associated signaling pathways. *Signaling pathways showing significant up-regulation or down-regulation.
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