Screening of polycomb Bmi1 targets for development of Cancer Stem Cell-targeted therapy for neuroblastoma

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We clarified the direct binding of MYCN to Bmi1 promoter and upregulation of Bmi1 transcription by MYCN. A correlation between MYCN and polycomb protein Bmi1 expression was observed in primary NB tumors. Expression of Bmi1 resulted in the acceleration of proliferation and colony formation in NB cells. Bmi1-related inhibition of NB cell differentiation was confirmed by neurite extension assay and analysis of differentiation marker molecules. Intriguingly, the above-mentioned Bmi1-related regulation of the NB cell phenotype seems not to be mediated only by p14ARF/p16INK4a in NB cells. Expression profiling analysis using a tumor-specific cDNA microarray addressed the Bmi1-dependent repression of KIF1Bb and TSLC1, which have important roles in predicting the prognosis of NB. Chromatin immunoprecipitation assay showed that KIF1Bb and TSLC1 are direct targets of Bmi1 in NB cells.Further comprehensive molecular analysis indicated that RUNX3 appears to be a target of Bmi1 and its transcription was suppressed by Bmi1 in several malignancies, including neuroblastoma.Analysis of the molecular mechanism of Bmi1-knockdown-induced apoptosis clarified that DNA damage induced by Bmi1 knockdown has an important role. This apoptosis is dependent on p53 and/or p73 and accompanied by production of reactive oxygen species. Furthermore, we studied the role of the other polycomb group molecules in the Bmi1-knockdown-induced apoptotic cell death.