Frequent promoter hypermethylation of Wnt pathway inhibitor genes in malignant astrocytic gliomas

Aberrant activation of wingless (Wnt) signaling is involved in the pathogenesis of various cancers. Recent studies suggested a role of Wnt signaling in gliomas, the most common primary brain tumors. We investigated 70 gliomas of different malignancy grades for promoter hypermethylation in 8 genes encoding members of the secreted frizzled-related protein (SFRP1, SFRP2, SFRP4, SFRP5), dickkopf (DKK1, DKK3) and naked (NKD1, NKD2) families of Wnt pathway inhibitors. All tumors were additionally analyzed for mutations in exon 3 of the beta-catenin gene (CTNNB1). While none of the tumors carried CTNNB1 mutations, we found frequent promoter hypermethylation of Wnt pathway inhibitor genes, with at least one of these genes being hypermethylated in 6 of 16 diffuse astrocytomas (38%), 4 of 14 anaplastic astrocytomas (29%), 7 of 10 secondary glioblastomas (70%) and 23 of 30 primary glioblastomas (77%). Glioblastomas often demonstrated hypermethylation of 2 or more analyzed genes. Hypermethylation of SFRP1, SFRP2 and NKD2 each occurred in more than 40% of the primary glioblastomas, while DKK1 hypermethylation was found in 50% of secondary glioblastomas. Treatment of SFRP1-, SFRP5-, DKK1-, DKK3-, NKD1- and NKD2-hypermethylated U87-MG glioblastoma cells with 5-aza-2'-deoxycytidine and trichostatin A resulted in increased expression of each gene. Furthermore, SFRP1-hypermethylated gliomas showed significantly lower expression of the respective transcripts when compared with unmethylated tumors. Taken together, our results suggest an important role of epigenetic silencing of Wnt pathway inhibitor genes in astrocytic gliomas, in particular, in glioblastomas, with distinct patterns of hypermethylated genes distinguishing primary from secondary glioblastomas.