Lamers et al. BMC Cancer 2012, 12:285 http://www.biomedcentral.com/1471-2407/12/285 R E S E A R C H A R T I C L E Open Access Identification of BIRC6 as a novel intervention target for neuroblastoma therapy Fieke Lamers1, Linda Schild1, Jan Koster1, Frank Speleman2, Ingrid Rogier Versteeg1, Huib N Caron4 and Jan J Molenaar1,5* ra3, Ellen M Westerhout1, Peter van Sluis1, Abstract Background: Neuroblastoma are pediatric tumors of the sympathetic nervous system with a poor prognosis. Apoptosis is often deregulated in cancer cells, but only a few defects in apoptotic routes have been identified in neuroblastoma. Methods: Here we investigated genomic aberrations affecting genes of the intrinsic apoptotic pathway in neuroblastoma. We analyzed DNA profiling data (CGH and SNP arrays) and mRNA expression data of 31 genes of the intrinsic apoptotic pathway in a dataset of 88 neuroblastoma tumors using the R2 bioinformatic platform (http://r2.amc.nl). BIRC6 was selected for further analysis as a tumor driving gene. Knockdown experiments were performed using BIRC6 lentiviral shRNA and phenotype responses were analyzed by Western blot and MTT-assays. In addition, DIABLO levels and interactions were investigated with immunofluorescence and co-immunoprecipitation. Results: We observed frequent gain of the BIRC6 gene on chromosome 2, which resulted in increased mRNA expression. BIRC6 is an inhibitor of apoptosis protein (IAP), that can bind and degrade the cytoplasmic fraction of the pro-apoptotic protein DIABLO. DIABLO mRNA expression was exceptionally high in neuroblastoma but the protein was only detected in the mitochondria. Upon silencing of BIRC6 by shRNA, DIABLO protein levels increased and cells went into apoptosis. Co-immunoprecipitation confirmed direct interaction between DIABLO and BIRC6 in neuroblastoma cell lines. Conclusion: Our findings indicate that BIRC6 may have a potential oncogenic role in neuroblastoma by inactivating cytoplasmic DIABLO. BIRC6 inhibition may therefore provide a means for therapeutic intervention in neuroblastoma. Keywords: Neuroblastoma, BIRC6, DIABLO, Apoptosis, Cancer Background BIRC6 (also known as BRUCE or APOLLON) is a cyto- plasmic protein with a dual role. Firstly, BIRC6 has an anti-apoptotic function in the intrinsic apoptotic path- way by antagonizing the pro-apoptotic DIABLO protein. BIRC6 can bind the cytoplasmic DIABLO fraction and induce ubiquitination and proteasomal degradation of this protein [1,2]. BIRC6 thereby protects against the * Correspondence: j.j.molenaar@amc.uva.nl 1Department of Oncogenomics, Academic Medical Center, University of Amsterdam, Meibergdreef 15, PO box 22700, Amsterdam, AZ 1105, The Netherlands 5Department of Oncogenomics, M1-132, Academic Medical Center, University of Amsterdam, Meibergdreef 9, Amsterdam, AZ 1105, The Netherlands Full list of author information is available at the end of the article pro-apoptotic function of DIABLO. DIABLO is a mito- chondrial protein which is released into the cytoplasm upon an apoptotic stimulus. This release is regulated by the levels of the BH3 family proteins, which induce pore formation in the mitochondrial membrane [3-5]. Cyto- plasmic DIABLO can bind to the BIR domains of BIRC2 (cIAP1), BIRC3 (cIAP2) and BIRC4 (XIAP), thereby inhibiting the anti-apoptotic function of these proteins [6]. A second function of BIRC6 has been shown in re- cent studies where BIRC6 was required for abscission and membrane delivery during the midbody ring forma- tion during cell division [7,8]. BIRC6 is highly expressed in several types of cancer. BIRC6 over-expression in acute myeloid leukemia is correlated with a poor outcome [9]. A genome wide © 2012 Lamers et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.