Lamers et al. BMC Cancer 2012, 12:285 http://www.biomedcentral.com/1471-2407/12/285 triggered apoptosis. As expected, BIRC6 directly inter- acted with DIABLO proteins. The apoptotic response in neuroblastoma cells upon BIRC6 silencing occurs in a background of surprisingly high DIABLO expression levels. High cytoplasmic levels of DIABLO can be tumor inhibitory through binding of IAPs in the cytoplasm. We suggest two mechanisms why high DIABLO expression does not induce apoptosis in neuroblastoma. Firstly, we show that the major fraction of DIABLO protein has a mitochondrial localization. This mitochondrial sequestration is probably mediated by the exceptionally high levels of BCL2, which occurs in most neuroblastoma tumors [33]. Targeted inhibition of BCL2 caused an increase in cytoplasmic DIABLO levels, suggesting that sequestration of DIABLO in the mitochondria occurs by inhibition of pore formation through BCL2. Secondly, we showed that DIABLO is ef- fectively bound by BIRC6 in neuroblastoma and that DIABLO levels increase upon silencing of BIRC6. This suggests an effective degradation of cytoplasmic DIA- BLO by BIRC6. We did not find a correlation between BIRC6 expression and prognosis or prognostic markers. We also did not find a correlation between BIRC6 and BCL2 or DIABLO mRNA expression (data not shown). This can be explained by the observation that the ex- pression of both BCL2 and DIABLO is generally high in all neuroblastoma tumor samples and not in just a sub- set [33]. The function of high mitochondrial DIABLO levels in neuroblastoma remains elusive. Other pro- apoptotic proteins in the mitochondria, like cytochrome C, have shown to be involved in energy metabolism [44] but no such mechanism has been found for DIABLO yet. The interaction of BIRC6 with CASP9, which has been reported previously [1,2,45], has not been validated in this paper. Since CASP9 has been shown to be function- ally active in neuroblastoma despite its location on a fre- quently lost region [46], it would be interesting to investigate whether there is an additional inhibition of this protein by BIRC6 in neuroblastoma cells. Release of the strongly increased mitochondrial DIABLO levels would offer a therapeutic potential in neuroblastoma. This suggests that the proteins that impair the pro-apoptotic function of DIABLO could be effective drug targets. The previously shown efficacy of targeted BCL2 inhibition could relate to high DIABLO levels [33]. Moreover, direct BIRC6 inhibition would also increase the cytoplasmic pro-apototic function of DIABLO. BIRC6 inhibitors are not available at this moment, but targeted drug development might be worth considering. BIRC6 is not located on the SRO of 2p, but it is frequently gained and functionally active and we therefore consider BIRC6 as a potentially im- portant player in the dysfunction of apoptosis in neuro- blastoma. The BIRC6 gene is validated as a novel Page 9 of 10 therapeutic target. If BIRC6 inhibitors are developed, a combined inhibition of BIRC6 and BCL2 could yield synergistic effects. Competing interests The authors declare that they have no competing interests. Authors’ contributions All authors have made substantial contributions to conception and design, acquisition of data, analysis and interpretation of data. All authors have been involved in drafting the manuscript or revising it critically for important intellectual content and have given final approval of the version to be published. Acknowledgements This research was supported by grants from KIKA foundation, SKK and Netherlands Cancer Foundation. Author details 1Department of Oncogenomics, Academic Medical Center, University of Amsterdam, Meibergdreef 15, PO box 22700, Amsterdam, AZ 1105, The Netherlands. 2Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium. 3Department of Pediatric Oncology, Skåne University Hospital, Lund University, Lund, Sweden. 4Department of Pediatric Oncology, Emma Kinderziekenhuis, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. 5Department of Oncogenomics, M1-132, Academic Medical Center, University of Amsterdam, Meibergdreef 9, Amsterdam, AZ 1105, The Netherlands. Received: 27 February 2012 Accepted: 26 June 2012 Published: 12 July 2012 References 1. Hao YY, Sekine K, Kawabata A, Nakamura H, Ishioka T, Ohata H, Katayama R, Hashimoto C, Zhang XD, Noda T, et al: Apollon ubiquitinates SMAC and caspase-9, and has an essential cytoprotection function. Nat Cell Biol 2004, 6:849–860. 2. 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