Extracellular SOD (EcSOD), encoded by the SOD3 gene, scavenges superoxide in the extracellular space. EcSOD is highly expressed in the lung where it comprises the majority of SOD activity in airway epithelial and vascular cells. The role of EcSOD in lung cancer, if any, has not yet been thoroughly studied. It has been reported, however, that EcSOD expression is lost in the lung cancer cell line A549 and that the transcription factors SP1/SP3 that regulate expression in MRC-5 cells could neither bind the promoter nor stimulate EcSOD expression in A549 cells. We hypothesized that EcSOD loss in lung cancer could be due to epigenetic events. To test this hypothesis we analyzed the epigenotype of the SOD3 promoter in three normal cell types (airway epithelial, MRC-5, HMEC) and two EcSOD negative lung cancer cell lines (A549, H1650) as well as in normal lung versus five human lung tumors. We also re-expressed EcSOD in the A549 cell line to assess possible changes in malignant phenotype. We now report that EcSOD expression is highly correlated with SOD3 promoter methylation. In the normal airway epithelial and MRC-5 lung cells that express EcSOD, the promoter has an overall cytosine methylation of <1% while in the non-expressing lung cancer cells and HMEC cells there was >86% methylation at the 18 CpG sites examined. Treatment of cells with 5-aza-dC induced SOD3 mRNA, indicating that DNA methylation was causal, at least in part, to the gene silencing. Moreover, A549 and H1650 cells displayed a ∼25-fold reduction in chromatin accessibility of the SOD3 promoter compared to MRC-5 cells. In the five lung tumors, EcSOD mRNA expression was significantly decreased and the SOD3 promoter showed a significantly more methylated state in the tumors compared to normal lung tissue. Re-expression of EcSOD attenuated the malignant phenotype of A549 cells by increasing doubling time by 25 hours, decreasing matrigel invasion and clonogenic capacity by 65% and 70% respectively. Taken together, our findings indicate that loss of EcSOD expression in human lung cancer is highly correlated with aberrant SOD3 promoter methylation and a repressive chromatin structure, and that re-acquisition of EcSOD attenuates the malignant phenotype of lung cancer cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2285.