Gliomas are the most common central nervous system tumor in both adults and children. Although in vivo
and in vitro
studies have shed new light on the mechanism of tumorigenesis of neuroepithelial tumors, current treatments do not result in an improvement in the very poor prognosis of this disease. However, epigenetic studies have indicated possible strategies for tumor specific and customized therapy 32–35
. The restoration of silenced tumor suppressor genes is an attractive therapeutic approach that could result in improvements in prognosis.
In a previous study 27
, we have investigated epigenetic modifications, including histone deacetylation and DNA methylation, ultimately identifying the NAG-1
gene as being epigenetically regulated and possibly silenced in gliomas. The histone deacetylase inhibitor, trichostatin A induces NAG-1 expression in several glioma cell lines including T98G cells. Trichostatin A induces apoptosis in T98G cells and the increase in expression of NAG-1 plays an important role in trichostatin A-mediated apoptosis, suggesting histone acetylation regulates the expression of NAG-1. However, 5-AZA-dC did not induce NAG-1 expression in T98G cells suggesting methylation was not important in regulation of NAG-1 expression in these cells. In this report we examined in more detail the methylation status of the NAG-1
promoter in glioblastoma cells and in human glimoma tumors. The promoter region of NAG-1
was poorly methylated in cells that have high basal expression of NAG-1and hypermethylated in cells that have low basal expression of NAG-1. In glioblastoma cell lines, an inverse relationship between the basal expression and promoter hyper-methylation is clearly apparent with hypermethylation observed in the high grade cells. The study of methylation status and basal NAG-1 expression was continued by examination of the basal NAG-1expression and promoter methylation in human glioma samples. In general, we observed a very low basal NAG-1 expression in the tumors and a high level of promoter methylation. 5-AZA-dC induced NAG-1 expression in glioblastoma cell lines with low basal expression of NAG-1 and a highly methylated promoter but was not effective in cells like T98G that have high basal expression and a poorly methylated NAG-1
promoter. Thus we conclude that NAG-1 is epigenetically regulated and silenced in glioblastomas by methylation.
In the human tumors samples the relationship between hypermethylation and basal NAG-1 expression is not as clear. In cell lines where the NAG-1
promoter is extensively methylated, clusters of DNA methylation were identified that correlate with known transcription factor binding sites responsible for the regulation of basal expression. NAG-1 expression is down-regulated in greater than 90% of the tumors. However, hypermethylation at the sites identified in cell lines (the −118 to −53 regions) was not observed in all human glioblastoma samples. Our observation is similar to results reported for methylation of other genes in glioblastoma 36
. As previously suggested 36
, the decreased levels of methylation observed in tumors versus cell lines may reflect differences in cellularity (tumors have multiple cell types), differences in the signaling environment in vivo versus in culture, or that DNA methylation in cell lines may reflect a growth advantage in culture not evident in tumors.
NAG-1 expression is regulated by several transcriptional factors and by posttranscriptional mechanisms, indicating a diverse regulation by anti-tumorigenic compounds. From our studies on the regulation of NAG-1 expression by tumor prevention drugs reveals NAG-1 as an important downstream target of three tumor suppressor pathways, p53 30
, Egr-1 37
, and AKT/GSK-3β38
. These pathways involve specific sites located in the −133 to +55 region of NAG-1
promoter, which we analyzed for the methylation status. The Egr-1/Sp-1 binding sites (−67 and −53 respectively) regions, are pivotal regulation sites for increasing NAG-1 expression by COX inhibitors, Troglitazone, and TSA. In addition, these sites are critical in the regulation of the basal expression of NAG-1 23
. Our current study indicated that Egr-1/Sp-1 binding site was highly methylated in glioblastoma cell lines and primary oligodendroglioma samples which have low basal expression of NAG-1. In addition, our methylation status assay showed that +55 region located close to p53 binding site, was also highly methylated. The p53 sites play an important role in the regulation of NAG-1 expression and are pivotal in induction of NAG-1 expression by dietary compounds, for example DADS and resveratrol 30
. Thus, the −128 to −53 region and +55 region of NAG-1
promoter are likely involved in silencing of the tumor suppressor gene in tumorigenesis of glioma.
Methylation of the Egr-1/SP-1 site decreases the basal expression of NAG-1 but also prevents an increase in expression after treatment with sulindac sulfide or TSA. Sulindac sulfide increased NAG-1 expression in T98G cells with a poorly methylated NAG-1 promoter while treatment of U-118 cells that have a highly methylated promoter did not increase NAG-1 expression. After removal of the methyl group by 5-AZA-dC treatment, sulindac sulfide increased NAG-1 expression in the U-118 cells. Although sulindac sulfide increased in both the T98G and U-118 cells the expression of Egr-1, a transcription factor required for the increase in transcriptional activity by sulindac sulfide, the binding of Egr-1 to the promoter site was blocked on the methylated promoter in U-118 cells as determined by the CHIP assay. Thus in glioma hypermethylation of NAG-1 promoter silenced basal expression and blocked drug induced expression, a finding that may provide insight into why gliomas are resistant to many drug therapies.
The role for NAG-1 in development of tumors is highly complex. Studies in a number of tumor cells, colorectal 21
, breast 18
and now glioblastoma all indicate the expression of NAG-1 induced apoptosis, inhibited the growth on soft agar and altered the cell cycle. Other studies in nude mice show the expression of NAG-1 inhibits the growth of tumors in xenographs 21
. Studies with NAG-1 transgenic mouse expressing the human NAG-1 are resistant to both chemical and genetically induced intestinal tumors 22
. Our current studies support the hypothesis that NAG-1 is a potential tumor suppressor gene and results in this investigation confirm that its expression can be regulated by gene hypermethylation in glioblastoma. Further studies are needed to determine whether NAG-1 transgenic mice are resistant to development of glioblastoma and if hypermethylation silences NAG-1 expression in other cancers.