Emerging evidence suggests that neural stem cells and brain tumors regulate their proliferation via similar pathways. In a previous study, we demonstrated that maternal embryonic leucine zipper kinase (Melk) is highly expressed in murine neural stem cells and regulates their proliferation. Here we describe how MELK expression is correlated with pathologic grade of brain tumors, and its expression levels are significantly correlated with shorter survival, particularly in younger glioblastoma patients. In normal human astrocytes, MELK is only faintly expressed, and MELK knockdown does not significantly influence their growth, whereas Ras and Akt overexpressing astrocytes have up-regulated MELK expression, and the effect of MELK knockdown is more prominent in these transformed astrocytes. In primary cultures from human glioblastoma and medulloblastoma, MELK knockdown by siRNA results in inhibition of the proliferation and survival of these tumors. Furthermore, we show that MELK siRNA dramatically inhibits proliferation and, to some extent, survival of stem cells isolated from glioblastoma in vitro. These results demonstrate a critical role for MELK in the proliferation of brain tumors, including their stem cells, and suggest that MELK may be a compelling molecular target for treatment of high-grade brain tumors.

To identify target genes for the hemizygous deletions of chromosome 13 that are recurrently observed in malignant gliomas, we performed genome-wide DNA copy-number analysis using array-based comparative genomic hybridization and gene expression analysis using an oligonucleotide-array. The response gene to complement 32 (RGC32) at 13q14.11 was identified as a deletion target, and its expression was frequently silenced in glioma cell lines compared with normal brain. Levels of RGC32 mRNA tended to decrease toward higher grades of primary astrocytomas, especially in tumors with mutations of p53. Expression of RGC32 mRNA was dramatically increased by exogenous p53 in a p53-mutant glioma cell line, and also by endogenous p53 in response to DNA damage in p53 +/+ colon-cancer cells, but not in isogenic p53 À/À cells. Chromatin immunoprecipitation and reporter assays demonstrated binding of endogenous p53 protein to the promoter region of the RGC32 gene, implying p53-dependent transcriptional activity. Transiently and stably overexpressed RGC32 suppressed the growth of glioma cells, probably owing to induction of G2/M arrest. Immunocytochemical analysis revealed a concentration of RGC32 protein at the centrosome during mitosis. RGC32 formed a protein complex with polo-like kinase 1 and was phosphorylated in vitro. These observations implied a novel mechanism by which p53 might negatively regulate cell-cycle progression by way of this newly identified transcriptional target. Our results provide the first evidence that RGC32 might be a possible tumorsuppressor for glioma, that it is directly induced by p53, and that it mediates the arrest of mitotic progression.

Glioblastoma multiforme (GBM) is a devastating disease, and the current therapies have only palliative effect. Evidence is mounting to indicate that brain tumor stem cells (BTSCs) are a minority of tumor cells that are responsible for cancer initiation, propagation, and maintenance. Therapies that fail to eradicate BTSCs may ultimately lead to regrowth of residual BTSCs. However, BTSCs are relatively resistant to the current treatments. Development of novel therapeutic strategies that effectively eradicate BTSC are, therefore, essential. In a previous study, we used patient-derived GBM sphere cells (stemlike GBM cells) to enrich for BTSC and identified maternal embryonic leucine-zipper kinase (MELK) as a key regulator of survival of stemlike GBM cells in vitro. Here, we demonstrate that a thiazole antibiotic, siomycin A, potently reduced MELK expression and inhibited tumor growth in vivo. Treatment of stemlike GBM cells with siomycin A resulted in arrested self-renewal, decreased invasion, and induced apoptosis but had little effect on growth of the nonstem cells of matched tumors or normal neural stem/progenitor cells. MELK overexpression partially rescued the phenotype of siomycin A-treated stemlike GBM cells. In vivo, siomycin A pretreatment abraded the sizes of stemlike GBM cell-derived tumors in immunodeficient mice. Treatment with siomycin A of mice harboring intracranial tumors significantly prolonged their survival period compared with the control mice. Together, this study may be the first model to partially target stemlike GBM cells through a MELK-mediated pathway with siomycin A to pave the way for effective treatment of GBM.