Ht has been gained into the relevance and function of histone methylation-dependent epigenetic events in Group 3 and Group 4 MB, much less is known about lysine acetylation- (or HDAC-) dependent epigenetic aberrations in MB at a chromatin-wide level. The zinc-dependent HDAC1 through HDAC11 comprise 11 members grouped into four classes (I, IIa, IIb, and IV) [18]. In SHH MBs, SHH-induced HDAC activity is required for continued proliferation of cerebellar granule precursor cells [19]. We and others have previously shown that HDACi treatment exerts anti-tumoral effects in MB in vitro and in vivo [20-24]. Our group has shown that distinct HDAC family members control specific oncogenic functions in pediatric neuronal cancer models including differentiation, cell cycle regulation, apoptosis, autophagy, chemotherapy resistance [25,26], and alterations in tumor suppressor pathways [27,28]. We have further demonstrated that specific HDAC isoforms are differentially expressed in MB [29,30], andfound that expression of class IIa HDACs 5 and 9 correlates with cytogenetic aberrations and poor clinical outcome in the entire HS-173 web cohort of MB tumors, and high HDAC2 expression in group 3 MBs PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28506461 [30]. With the recent advent of class-selective HDAC inhibitors (HDACis), such as the class IIa-selective HDACis MAZ1863 and MAZ1866 [31] and selective substrates has opened the possibility of class-selective exploration of HDAC biology. The aim of the presented study is to investigate the selective targeting of HDAC family members in a MB subgroup specific manner, and to elucidate the translational consequences.Materials and methodsPatients and clinical samplesMaterial from patients of tissue microarray (TMA) set (paraffin embedded medulloblastoma samples) were randomly collected at the Department of Neuropathology, Burdenko Neurosurgical Institute (Moscow, Russia) between 1993 and 2011. Approval to link laboratory data to clinical data was obtained by the Institutional Review Board. Two neuropathologists confirmed the diagnoses according to the 2000 WHO classification. None of the patients had received irradiation or chemotherapy before collection of specimens. Metastatic state (M stage) was determined by magnetic resonance imaging and cerebro- spinal fluid cytopathology at diagnosis. Clinical and histopathologic data are summarized in Additional file 1: Table S1.Cell lines, cell culture and siRNA-mediated knockdownCell lines and cell culture conditions have been described previously: MED8A, UW228-2, ONS76 and DAOY in [29], HD-MB03 in [24], D458 in [32]. All cell lines had their identity confirmed and proven to be free of contamination by mycoplasma or viral contamination using the Multiplex cell Contamination Test (McCT) service [33]. MYC status of all cell lines was confirmed by fluorescent in-situ hybridization (see below). siRNA transfection was performed as reported previously [29]. siRNA reagents were purchased from Qiagen (Hilden, Germany) (see Additional file 2: Table S2).RNA-isolation, cDNA synthesis, quantitative reverse transcription real-time PCR (qPCR) and gene expression analysisRNA extraction, cDNA synthesis, quantitative real-time PCR, and software analysis was performed as reported previously [29]. Primers were purchased from Qiagen (see Additional file 3: Table S3). Normal cerebellum RNA was purchased from Clontech (Mountain View, CA, USA). The database analysis tool R2 (http://r2.amc.nl) was used to investigate HDAC1, 2, and 3 mRNA expression in brai.
