Abstract P5-05-07: The Loss of EcSOD Expression in Mammary Epithelial Cells Is Associated with Aberrant DNA Methylation When Mammary Tissue Organization Is Disrupted

Abstract

Abstract Loss of tissue organization is one of the earliest manifestations of tumorigenic progression in breast cancer. It is well known that during structural and functional differentiation, mammary epithelial cells form polarized and organized structure called acini, which can be recapitulated in 3D culture system. Interestingly, we have recently found that gene expression of the extracellular superoxide dismutase (EcSOD) is differentially regulated in normal mammary epithelial cells (HMEC), in the 2D culture compared to the 3D culture system. We have previously shown that overexpression of EcSOD in EcSOD-null breast cancer cells resulted in inhibition of growth and invasion. Since heightened oxidative stress has been widely implicated in the etiology of human cancers and EcSOD is the predominant enzyme that modulates extracellular oxidative stress, understanding gene regulation of EcSOD is important in developing pharmalogical interventions for breast cancer. The expression of EcSOD in mammary epithelial cells was not recognized until recently. In fact, EcSOD mRNA and protein expression were not detectable in commercially available normal mammary epithelial cells which were cultured in 2Dcondition. However, our immuno-histological studies revealed a definitive positive staining for EcSOD in normal breast tissues specifically in mammary epithelium of the lobule and duct. Based on these observations, we hypothesized that EcSOD gene expression is lost when mammary tissue organization is disrupted. Therefore, we examined whether culturing HMEC in a 3D context, in the presence of extracellular matrix signaling and with the proper tissue organization, will reactivate EcSOD gene expression. Indeed, EcSOD gene expression was activated in HMEC after 4 days of 3D culture. We have further shown that the 3D cultured HMEC formed organized and polarized acinar structures by using immuno-fluorescence microscopy. Intriguingly, the expression of EcSOD dramatically decreased in a time dependent manner after passing the 10-day acini back into the 2D culture. Moreover, we observed a marked reduction of EcSOD immunoreactivity in ductal hyperplasia and ductal carcinoma in situ (DCIS) compared to normal mammary tissues. In addition, more than 87% of clinical mammary adenocarcinoma samples showed decreased EcSOD mRNA expression levels compared to their normal counterparts. We have further shown that EcSOD mRNA expression was strongly induced in 2D cultured HMEC after treatment with a DNA methyl transferase inhibitor and to a lesser extend with a histone deacetylase inhibitor. Furthermore, the diminished expression of EcSOD in 2D cultured HMEC is tightly associated with CpG hypermethylation of the EcSOD promoter. These results suggest that epigenetic mechanisms play an important role in regulating EcSOD gene expression and the loss of EcSOD expression in mammary adenocarcinomas could be due to aberrant CpG methylation of the EcSOD promoter. Taken together, our study implies that mammary tissue organization is required for EcSOD expression and that restoring EcSOD expression in mammary adenocarcinomas could be an effective strategy for breast cancer treatment. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-05-07.