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Title: MELK Leucine Zipper Kinase is a Key Differentiator between Glioblastoma Multiforme and Pilocytic Astrocytomas      
Transcriptome or Gene expression
Purpose: Gene expression analyses comparing tumors of different behaviors have been used for the identification of genes that have a role in the growth and maintenance of tumors, with the aim of both understanding the molecular basis of tumorigenesis and identifying novel potential therapeutic targets. We have here used this approach to identify the underlying gene expression differences between highly invasive glioblastoma multiforme and typically benign pilocytic astrocytomas. Experimental Design: We performed cDNA microarray analyses comparing astrocytomas of polar grade: grade I, pilocytic astrocytoma (PA), the non-invasive and most frequent pediatric tumor, with grade IV, the highly infiltrative glioblastoma multiforme (GBM) Results: Despite the significant clinical and pathological differences between the two tumor types, only 63 genes were found to exhibit two-fold or greater over-expression in GBM as compared with PA. A functional classification of the GBM over-expressed genes indicated that more than 50% are related to the regulation of the cell cycle and mitosis. Microarray data was validated by RQ-PCR testing six over-expressed genes in GBM related to PA: MELK, AUKB, ASPM, PRC1, IL13RA2 and KIAA0101. The differential expression was confirmed for all six genes, showing at least a 5 fold increase in the average expression levels in GBM compared to PA. Of the over expressed genes, that which exhibited the most statistically significant difference is Maternal Embryonic Leucine zipper Kinase (MELK). We undertook a more detailed investigation of the expression of this serine/threonine kinase gene in astrocytomas in the light of its role in the regulation of multipotent neural progenitor proliferation and previous suggestions that this may be a oncogenic target of general importance. In an examination of more than 100 tumors of the central nervous system we found progressively higher expression of MELK in GBM with astrocytoma grade and a noteworthy uniformity of high level expression in GBM. This latter feature probably explains the lack of observed association of MELK expression with survival of GBM patients. Over-expression at a similar level to GBM was also observed in medulloblastoma. We found neither gene methylation nor amplification to be a factor in MELK expression but were able to demonstrate that MELK knockdown in malignant astrocytoma cell lines caused a reduction in proliferation and anchorage-independent growth in in vitro assays. Conclusions: Our results indicate that GBM and PA differ by the expression of surprisingly few genes. Among them, MELK may play a key role in differentiating these two tumor types and represent an important therapeutic target for the management of the most frequent brain tumors in adult and children. Keywords: Comparizon of CNS tumors of different grades. Overall design: Nine primary astrocytomas (three samples of PA, mean age of 19 yo; and six samples of GBM, mean age of 56 yo, and mean survival time of 7.6 months), and a pool of three non-neoplastic brain tissues (mean age of 47 yo) were used for microarray analyses. Analyses were carried out with commercial oligonucleotide microarrays (CodeLink Bioarrays-Human , GE Healthcare, Piscataway, NJ), following the manufacturer’s protocol. Independent hybridizations for each sample were carried out in duplicate. Individual gene PA and GBM expression profiles were compared with each other, and with non-neoplastic brain tissue. Differentially expressed genes were identified by calculating the ratio of the mean normalized fluorescence values obtained from each duplicate GBM or PA sample and non-neoplastic brain tissue, and subsequently between duplicate GBM and PA sample. Results were expressed as fold variation, and genes displaying greater than 2-fold changes in transcript abundance and p<0.01 were selected.
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