Identification of BIRC6 as a novel intervention target for neuroblastoma therapy.
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.


CONCLUSIONS
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.
Department of Oncogenomics, Academic Medical Center, University of Amsterdam, Meibergdreef 15, PO box 22700, Amsterdam, AZ 1105, The Netherlands.
Fieke
F
Lamers
Linda
L
Schild
Jan
J
Koster
Frank
F
Speleman
Ingrid
I
Øra
Ellen M
EM
Westerhout
Peter
P
van Sluis
Rogier
R
Versteeg
Huib N
HN
Caron
Jan J
JJ
Molenaar
D017209:Apoptosis / Q000235:genetics; D051017:Apoptosis Regulatory Proteins; D053453:Caspase 9 / Q000235:genetics; D055028:Comparative Genomic Hybridization; D003593:Cytoplasm / Q000378:metabolism; D020869:Gene Expression Profiling; D055785:Gene Knockdown Techniques; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000235:genetics* / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism; D058990:Molecular Targeted Therapy / Q000379:methods; D009447:Neuroblastoma / Q000235:genetics* / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide; D034741:RNA, Small Interfering / Q000235:genetics; D000077022:Survivin
D051017:Apoptosis Regulatory Proteins; C107489:BIRC5 protein, human; C401541:BIRC6 protein, human; C412403:DIABLO protein, human; D051034:Inhibitor of Apoptosis Proteins; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; D034741:RNA, Small Interfering; D000077022:Survivin; C506433:CASP9 protein, human; D053453:Caspase 9