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

Laboratory of Molecular Carcinogenesis, NIEHS, National Institutes of Health, Research Triangle Park, NC 27709, USA.
International Journal of Cancer (Impact Factor: 5.09). 01/2012; 130(2):267-77. DOI: 10.1002/ijc.26082
Source: PubMed


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.

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Available from: Paul A Wade, May 20, 2014
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    • "Thus, NAG-1 basal expression appears to inversely correlate with tumor grade in glioma. NAG-1 overexpression inhibits the colony-forming capacity and induces apoptosis in glioblastoma cells [28]. Increased expression of NAG-1 induced by histone deacetylase inhibitor trichostatin A (TSA) may mediate, in part, TSA-induced apoptosis [17]. "
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    ABSTRACT: Nonsteroidal anti-inflammatory drug (NSAID) activated gene-1 (NAG-1) is a divergent member of the transforming growth factor-beta (TGF-β) superfamily. NAG-1 plays remarkable multifunctional roles in controlling diverse physiological and pathological processes including cancer. Like other TGF-β family members, NAG-1 can play dual roles during cancer development and progression by negatively or positively modulating cancer cell behaviors. In glioblastoma brain tumors, NAG-1 appears to act as a tumor suppressor gene; however, the precise underlying mechanisms have not been well elucidated. In the present study, we discovered that overexpression of NAG-1 induced apoptosis in U87 MG, U118 MG, U251 MG, and T98G cell lines via the intrinsic mitochondrial pathway, but not in A172 and LN-229 cell lines. NAG-1 could induce the phosphorylation of PI3K/Akt and Smad2/3 in all six tested glioblastoma cell lines, except Smad3 phosphorylation in A172 and LN-229 cell lines. In fact, Smad3 expression and its phosphorylation were almost undetectable in A172 and LN-229 cells. The PI3K inhibitors promoted NAG-1-induced glioblastoma cell apoptosis, while siRNAs to Smad2 and Smad3 decreased the apoptosis rate. NAG-1 also stimulated the direct interaction between Akt and Smad3 in glioblastoma cells. Elevating the level of Smad3 restored the sensitivity to NAG-1-induced apoptosis in A172 and LN-229 cells. In conclusion, our results suggest that PI3K/Akt and Smad-dependent signaling pathways display opposing effects in NAG-1-induced glioblastoma cell apoptosis.
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    ABSTRACT: Nonsteroidal anti-inflammatory drug (NSAID) activated gene-1, NAG-1, is a divergent member of the transforming growth factor-beta (TGF-β) superfamily that plays a complex but poorly understood role in several human diseases including cancer. NAG-1 expression is substantially increased during cancer development and progression especially in gastrointestinal, prostate, pancreatic, colorectal, breast, melanoma, and glioblastoma brain tumors. Aberrant increases in the serum levels of secreted NAG-1 correlate with poor prognosis and patient survival rates in some cancers. In contrast, the expression of NAG-1 is up-regulated by several tumor suppressor pathways including p53, GSK-3β, and EGR-1. NAG-1 expression is also induced by many drugs and dietary compounds which are documented to prevent the development and progression of cancer in mouse models. Studies with transgenic mice expressing human NAG-1 demonstrated that the expression of NAG-1 inhibits the development of intestinal tumors and prostate tumors in animal models. Laboratory and clinical evidence suggest that NAG-1, like other TGF-β family members, may have different or pleiotropic functions in the early and late stages of carcinogenesis. Upon understanding the molecular mechanism and function of NAG-1 during carcinogenesis, NAG-1 may serve as a potential biomarker for the diagnosis and prognosis of cancer and a therapeutic target for the inhibition and treatment of cancer development and progression.
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