Lamers et al. BMC Cancer 2012, 12:285 http://www.biomedcentral.com/1471-2407/12/285 screening of chromosomal aberrations in Burkitt’s lymphoma showed that a region of 2p including the BIRC6 gene was gained in a few samples [10]. Addition- ally, high BIRC6 expression in colon cancer stem cells is associated with drug resistance [11]. Neuroblastoma are pediatric tumors that originate from the embryonal precursor cells of the sympathetic nervous system. High stage tumors have a poor progno- sis with 20 to 40% overall survival [12-14]. BIRC6 is located on chromosome 2p in the region which shows frequent gain in neuroblastoma [12]. This region includes both MYCN and ALK, two well characterized oncogenes in neuroblastoma. Amplification of MYCN occurs in 20–30% of neuroblastoma and strongly corre- lates with a poor prognosis [12-15]. ALK was recently found to be mutated in 6-10% of primary neuroblastoma [16-20]. MYCN amplification and ALK mutations seem independent of the gain of chromosome 2p [21] and therefore other additional tumor driving genes could be located on this frequently gained region. The apoptotic pathway has been widely investigated in neuroblastoma and only a few tumor driving events have been described. TP53 is mostly intact in primary neuro- blastoma although functional defects in the p53 pathway have been described [22]. Caspase 8 (CASP8) is hyper- methylated and thereby inactivated in some neuroblast- oma resulting in an inactive extrinsic apoptotic pathway [23]. The IAP BIRC5 (Survivin) is located on the chromosome 17q region which is frequently gained in neuroblastoma and high BIRC5 expression correlates with a poor prognosis [24-26]. Also, the anti-apoptotic mitochondrial BCL2 protein is highly expressed in neuroblastoma. Targeted inhibitors against BIRC5 [27-32] and BCL2 [33] are currently being tested for clinical implementation, however, the poor prognosis of high grade neuroblastoma makes the identification of add- itional targets for therapeutic intervention desirable. To identify patterns in aberrations of genes involved in intrinsic apoptotic signaling we combined high through- put analysis of DNA copy number and mRNA expres- sion of these genes in a dataset of 88 neuroblastoma tumors. We found BIRC5 and BIRC6 to be frequently gained and CASP9 often lost. Since BIRC6 was not pre- viously evaluated in a neuroblastoma model, we studied the potency of BIRC6 as a potential new target for neuroblastoma therapy. Silencing of BIRC6 induced apoptosis and up-regulation of DIABLO. We established BIRC6 to physically interact with DIABLO, indicating that BIRC6 can degrade DIABLO very effectively. Methods Patient samples We used a neuroblastoma tumor panel for Affymetrix Microarray analysis containing 88 primary neuroblastoma Page 2 of 10 tumor samples of untreated patients of which 87 neuro- blastoma tumor samples were also used for CGH ana- lysis and SNP array [34]. All neuroblastoma samples were residual material obtained during surgery for diag- nostic purposes and immediately frozen in liquid nitro- gen. Ethical approval of the Dutch Medical Ethical Committee was not needed for the use of surplus mate- rials. However, informed consent was taken from the parents of the patients for use of this material which is archived at the Academic Medical Center from the University of Amsterdam. The data were deposited in the NCBI Gene Expression Omnibus (http://www.ncbi. nlm.nih.gov/geo/) under accession number GSE16476 [35]. Public available neuroblastoma datasets we used were of Delattre [36] and Lastowska (geo ID: gse13136). Public available datasets were used for comparing neuroblastoma with normal tissues (Roth dataset, geo ID: gse3526) and adult tumors (EXPO dataset, geo ID: gse2109). Affymetrix mRNA expression analysis Total RNA of neuroblastoma tumors was extracted using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. RNA concentration and quality were determined using the RNA 6000 nano assay on the Agilent 2100 Bioanalyzer (Agilent Technologies). Fragmentation of cRNA, hybridization to hg-u133 plus 2.0 microarrays and scanning were performed according to the manufacturer’s protocol (Affymetrix inc). Array CGH analysis High-molecular-weight DNA was isolated from tumor tissue by a standard salt-chloroform extraction method [37]. For reference DNA we obtained healthy tissue. We used a custom 44 K Agilent aCGH chip, enriched for critical regions of loss/gain for neuroblastoma (10 kb resolution), miRNAs/T-UCRs (5 oligos/gene) and cancer gene census genes (5 oligos/gene) (Agilent Technologies). A total of 150 ng of tumor and refer- ence DNA was labeled with Cy3 and Cy5, respectively (BioPrime ArrayCGH Genomic Labeling System, Invi- trogen). Further processing was done according to the manufacturer’s guidelines. Features were extracted using the feature extraction v10.1.0.0.0 software pro- gram. Data were further analyzed using the R2 web application (see below). Circular binary segmentation was used for scoring the regions of gain, amplification and deletion. Whole-genome genotyping Tumor DNA was extracted as previously described, quantified with NanoDrop and the quality was deter- mined by the Abs 260/280 and 230/260 ratio. SNP arrays were processed for analysis of copy number