DNA methylation‐mediated silencing of nonsteroidal anti‐inflammatory drug‐activated gene (NAG‐1/GDF15) in glioma cell lines

Abstract

Nonsteroidal anti-inflammatory drug-activated gene, NAG-1, a transforming growth factor-β member, is involved in tumor progression and development. The association between NAG-1 expression and development and progression of glioma has not been well defined. Glioblastoma cell lines have lower basal expression of NAG-1 than other gliomas and normal astrocytes. Most primary human gliomas have very low levels of NAG-1 expression. NAG-1 basal expression appeared to inversely correlate with tumor grade in glioma. Aberrant promoter hypermethylation is a common mechanism for silencing of tumor suppressor genes in cancer cells. In glioblastoma cell lines, NAG-1 expression was increased by the demethylating agent, 5-aza-2’-deoxycytidine. To investigate whether the NAG-1 gene was silenced by hypermethylation in glioblastoma, we examined DNA methylation status using genomic bisulfite sequencing. The NAG-1 promoter was densely methylated in several glioblastoma cell lines as well as in primary oligodendroglioma tumor samples, which have low basal expression of NAG-1. DNA methylation at two specific sites (−53 and +55 CpG sites) in the NAG-1 promoter was strongly associated with low NAG-1 expression. The methylation of the NAG-1 promoter at the −53 site blocks Egr-1 binding and thereby suppresses Nag-1 induction. Treatment of cells with low basal NAG-1 expression with NAG-1 inducer also did not increase NAG-1. Incubation with a demethylation chemical increased Nag-1 basal expression and subsequent incubation with a NAG-1 inducer increased NAG-1 expression. We concluded from these data that methylation of specific promoter sequences causes transcriptional silencing of the NAG-1 locus in glioma and may ultimately contribute to tumor progression.

Pathophysiological mechanisms regulated by cytokines in gliomas

“…DR4 (death receptor 4) promoter methylation in glioblastomas is associated with TRAIL resistance [98,99]. Hypermethylation of NAG-1 gene promoter (a member of TGF-b family) also appears in glioblastoma cell lines [100]. Further interaction of cytokines with tumorigenesis-related epigenetic alterations remains to be elucidated.…”

RNPC1, an RNA-binding Protein and a p53 Target, Regulates Macrophage Inhibitory Cytokine-1 (MIC-1) Expression through mRNA Stability

“…Generally, within early stages MIC-1 tends to display anti-cancer properties, but this effect is altered in later stage cancers (7). MIC-1 expression has also been found to be subject to epigenetic regulation in some tumor cell lines (17,18).…”

COX inhibitors directly alter gene expression: role in cancer prevention?

“…TSA upregulates expression and acts synergistically with 5-AZA-dC to induce NAG-1 expression. TSA induces NAG-1 promoter activity as well as NAG-1 mRNA and protein expression [112]. SiRNA experiments link NAG-1 expression to apoptosis induced by TSA.…”

“…Reporter gene assays, specific inhibition by small interfering RNA (siRNA) transfections, and ChIP assays indicate that EGR-1/Sp1 transcription factors mediate TSA-induced NAG-1 expression. TSA also increases the stability of NAG-1 mRNA [112]. The mechanism of TSA-induced NAG-1 expression involves not only the interaction with Sp1 and EGR-1 but also multiple regulations at the transcriptional and posttranscriptional levels.…”