EPIG-11THE IMPLICATION OF CpG island AND SPINT2 HYPERMETHYLATION IN BOTH IDH1 WILD-TYPE AND MUTANT ADULT DIFFUSE GLIOMAS

Abstract

BACKGROUND: IDH1/2 mutation and associated glioma CpG island methylator phenotype (G-CIMP) is associated with glioma prognosis. G-CIMP is defined as CpG islands hypermethylated in IDH1/2 mutant (IDH1/2MUT) gliomas. G-CIMP thus excludes hypermethylated CpG islands shared between IDH1/2MUT and IDH1/2 wildtype (IDH1/2WT) gliomas. These shared hypermethylated CpG islands may be important in gliomagenesis. We aimed to determine whether a set of CpG islands are hypermethylated in both IDH1/2MUT and IDH1/2WT gliomas and its functional implications. METHODS: Methylation profiling was performed using reduced representation bisulfite sequencing. Unsupervised clustering identified a novel sets of hypermethylated CpG islands shared between IDH1/2MUT and IDH1/2WT tumors. Targeted bisulfite sequencing of selected genes was used to confirm this novel set of hypermethylated CpG islands. SPINT2 was identified as a candidate hypermethylated tumor suppressor gene. SPINT2 DNA methylation and gene expression were measured in glioma samples. To assess regulation of SPINT2 expression by methylation, DNMT1 was knockdown and SPINT2 promoter activity was measured by luciferase assay. SPINT2 tumor suppressive function was investigated in vitro. RESULTS: A group of CpG islands were found hypermethylated in both IDH1/2MUT and IDH1/2WT gliomas compared with normal brain (designated as Glioma Total CpG island Methylator, GT-CIM). Nine candidate genes within GT-CIM were confirmed by target bisulfite sequencing. Within GT-CIM, the cell motility gene SPINT2 mRNA level was significantly lower in the hypermethylated glioblastomas as compared to the hypomethylated ones. DNMT1 knockdown restored SPINT2 expression. In the luciferase assay, SPINT2 promoter activity was reduced by DNA methylation. Over-expression of SPINT2 inhibited the glioma cells migration and invasion in vitro. CONCLUSIONS: We identified a set of CpG islands hypermethylated in all gliomas, termed GT-CIM. Within GT-CIM, we found that SPINT2 is regulated by DNA methylation, and its overexpression suppresses tumor cell migration and invasion. Further studies on GT-CIM and its related genes are warranted.