Epigenetic repression of the dopamine receptor D4 in pediatric tumors of the central nervous system

Abstract

Epigenetic alterations are common events in cancer. Using a genome wide methylation screen (Restriction Landmark Genomic Scanning-RLGS) we identified the gene for the dopamine receptor D4 (DRD4) as tumor-specific methylated. As DRD4 is involved in early brain development and may thus be involved in developmentally dependent tumors of the CNS in children epigenetic deregulation of DRD4 and its functional consequences were analyzed in vitro. CpG methylation of DRD4 was detected in 18/24 medulloblastomas, 23/29 ependymomas, 6/6 high-grade gliomas, 7/10 CNS PNET and 8/8 cell lines by qCOBRA and bisulfite sequencing. Real-time RT-PCR demonstrated a significantly inferior expression of DRD4 in primary tumors compared to cell lines and non-malignant control tissues. Epigenetic deregulation of DRD4 was analyzed in reexpression experiments and restoration of DRD4 was observed in medulloblastoma (MB) cells treated with 5-Aza-CdR. Reexpression was not accompanied by demethylation of the DRD4 promoter but by a significant decrease of H3K27me3 and of bound enhancer of zeste homologue 2 (EZH2). Knockdown of EZH2 demonstrated DRD4 as a direct target for inhibition by EZH2. Stimulation of reexpressed DRD4 resulted in an activation of ERK1/2. Our analyses thus disclose that DRD4 is epigenetically repressed in CNS tumors of childhood. DRD4 is a direct target of EZH2 in MB cell lines. EZH2 appears to dominate over aberrant DNA methylation in the epigenetic inhibition of DRD4, which eventually leads to inhibition of a DRD4-mediated stimulation of the ERK1/2 kinase pathway.

Gene Expression Analyses of the Spatio-Temporal Relationships of Human Medulloblastoma Subgroups during Early Human Neurogenesis

“…Over-expression of SNCA or SNCAIP in GPCs has been shown to promote GPC proliferation and protection from apoptosis [49] – [52] . Interestingly, a recent report describing epigenetic repression of the dopamine receptor family member, DRD4, in primary medulloblastoma adds further support for a significant role for deregulated dopamine signalling in medulloblastoma pathogenesis [53] . Additional novel findings from our analysis indicate that Group 4 medulloblastoma over-express semaphorin signalling molecules, including DPYSL3 and DPYSL4 , which regulate neuronal differentiation and apoptosis in the context of synaptic pruning [54] .…”

Inhibition of Dopamine Receptor D4 Impedes Autophagic Flux, Proliferation, and Survival of Glioblastoma Stem Cells

“…However, DRD4 antagonists showed better selectivity for GNS in our screen compared with DRD2 antagonists (data not shown). Although epigenetic suppression of DRD4 has been reported in pediatric CNS tumors (Unland et al, 2013), DRD4 is not methylated in TCGA GBM samples, and mutations in the DRD5 and DRD3 genes occur in a small fraction of GBM samples (Brennan et al, 2013). Finally, we note that dopamine receptor antagonists may have activity against other cancer stem cell types, including leukemia (Sachlos et al, 2012) and lung cancer (Yeh et al, 2012).…”

EZH2 (enhancer of zeste homolog 2 (Drosophila))

“…On the one hand, EZH2 is a histone methyltransferase, which plays an important role in tumor development (Santos-Rosa and Caldas, 2005). Moreover, this protein might also play essential roles in the control of central nervous systems by regulating the dopamine receptor D4 (Unland et al, 2014).…”

“…Their analyses suggested DRD4 as a direct target of EZH2. In particular, they showed that depletion of EZH2 is sufficient to induce re-expression of DRD4, suggesting the role of EZH2 for DNA hypermethylation in the epigenetic inhibition of DRD4 (Unland et al, 2014).…”