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

我们阐明了MYCN与Bmi1启动子的直接结合以及MYCN对Bmi1转录的上调作用。MYCN与多梳蛋白Bmi1在原发性神经母细胞瘤中的表达呈正相关。Bmi1基因的表达促进了NB细胞的增殖和集落形成。轴突延伸实验和分化标志物分子分析证实了BMI1对NB细胞分化的抑制作用。有趣的是，上述与Bmi1相关的对NB细胞表型的调节似乎不仅仅是由NB细胞中的p14ARF/p16INK4a介导的。使用肿瘤特异性基因芯片的表达谱分析解决了依赖于Bmi1的KIF1Bb和TSLC1的抑制，这两个基因在预测NB的预后方面具有重要作用。染色质免疫沉淀分析表明，KIF1Bb和TSLC1是Bmi1在NB细胞中的直接靶点。进一步的分子分析表明，RUNX3似乎是Bmi1的靶点，在包括神经母细胞瘤在内的多种恶性肿瘤中，RUNX3的转录被Bmi1抑制。通过对Bmi1基因敲除诱导细胞凋亡的分子机制的分析，阐明了Bmi1基因敲除诱导DNA损伤的重要作用。这种细胞凋亡依赖于P53和/或P73，并伴随着活性氧的产生。此外，我们还研究了其他多梳族分子在Bmi1基因敲除诱导的细胞死亡中的作用。

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.