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Medulloblastoma is a highly malignant embryonal tumor of the cerebellum that accounts for 20% to 25% of all primary brain tumors in children. Despite aggressive multimodal therapy, including surgery, irradiation, and chemotherapy, the 5-year survival rate is only 50% to 60%. The underlying genetic and epigenetic events that give rise to the majority of medulloblastomas are not known.

As part of a larger study to define genetic abnormalities in medulloblastoma, we identified in a single, primary medulloblastoma a discrete area of homozygous deletion on 9q31, a locus that shows frequent loss of heterozygosity in medulloblastoma. This 2.03 Mb deletion encompasses 8 known genes: ZNF462, RAD23B, KLF4, ACTL7B, ACTL7A, IKBKAP, C9orf6, and CTNNAL1. Exon re-sequencing of RAD23B (DNA repair)and KLF4 (known tumor suppressor gene) in a series of medulloblastomas with loss of heterozygosity on chromosome 9q did not reveal any mutations. However, treatment of medulloblastoma cell lines with 5-aza-deoxycytidine (5-Aza-C) resulted in greatly increased expression of KLF4 in 7/9 cell lines, suggesting that KLF4 is silenced by promotor methylation, and may be acting as a tumour suppressor gene. KLF4 is a zinc-finger transcription factor that is expressed primarily in post-mitotic, terminally differentiated epithelial cells of the skin, lungs, and gastrointestinal tract. KLF4, a known tumor suppressor gene regulates the expression of many genes, including those involved in differentiation and cell-cycle arrest. Recently, KLF4 has been shown to be epigenetically inactivated by promoter methylation in gastrointestinal cancer.

The transcription level of KLF4 was restored in 7/9 medulloblastoma cell lines after treatment with 5-Aza-C, as shown by real time RT-PCR. Bisulfite sequencing of multiple clones for each cell line demonstrated methylation of CpGs in the promoter region of KLF4, including 25 CpG sites (at -2168 to -1712) in those cell lines whose expression rose with 5-Aza-C; but no methylation was detected in cell lines where 5-Aza-C had no effect. Bisulfite sequencing of normal fetal and adult human cerebellum did not demonstrate any methylation of the KLF4 promotor, highly suggesting that methylation of KLF4 seen in medulloblastoma is abnormal. We examined the DNA methylation status of KLF4 in 45 primary pediatric medulloblastomas using methylation-specific PCR (MSP) and determined that 18% of primary medulloblastomas (7 of 45) are methylated at the KLF4 promoter region.

Our data suggest that the KLF4 tumour suppressor gene is inactivated by either genetic or epigenetic mechanisms in a significant subset of medulloblastoma. KLF4 may represent a novel target for the therapy of medulloblastoma.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA