Abstract LB-152: A new role for ERα: Gene silencing via DNA methylation
Authors: Ariazi Eric A. , Taylor John C. , Nicolas Emmanuelle , Slifker Michael J. , Boyd Jeff 
Published: August 2015

As a master transcription factor, estrogen receptor-α (ERα) regulates expression of a multitude of genes. We hypothesized that ERα may also regulate gene expression via DNA methylation since methylation of specific CpG sites associates with ERα-positive status in human breast cancer. We tested this hypothesis by identifying genes normally silenced in ERα-positive T47D and MCF-7 luminal breast cancer cell lines but which became expressed (or derepressed) upon exposure to the demethylating agent decitabine and antagonizing ERα activity. These genes were identified using a combination of Agilent gene expression microarrays and assessed according to the Significance Analysis of Microarrays (SAM) method at a 2-fold change and 5% FDR. Selected genes were validated by pyrosequencing to quantitate CpG methylation, lentiviral re-introduction of ERα, real-time PCR and immunoblotting. In a short-term study, T47D cells were treated with decitabine and the ER antagonist fulvestrant (FUL) for 96 h. Array analysis identified 87 genes that were derepressed by decitabine and FUL, of which 31 (36%) were markers of the basal subtype of breast cancer. In long-term studies (8 - 36 weeks), MCF-7 and T47D cells were FUL-treated or estrogen-deprived (ED) to derive multiple antihormone-resistant cell lines (MCF-7/FUL, T47D/FUL, and 2 independent T47D/ED), all of which lost ≥ 95% of ERα. Comparing all of the resistant cell lines and decitabine-treated T47D cells by array analysis revealed that 58 genes were up-regulated by both loss of ERα and decitabine treatment, 35 (60%) of which were basal markers. Two basal breast cancer subtype markers, LCN2 and IFI27 which are involved in epithelial-mesenchymal transition and interferon signaling, respectively, were selected for verification. First, LCN2 and IFI27 expression increased ∼132- and 343-fold, respectively, while their CpG methylation levels significantly decreased upon ERα loss in all T47D antihormone-resistant cell lines. Second, LCN2 and IFI27 expression decreased while their methylation levels increased upon estrogen re-exposure or lentiviral ERα re-introduction in the T47D/ED cell lines. Moreover, high CpG methylation levels of LCN2 and IFI27 directly associated with ERα-positive status but inversely correlated with their expression levels across a panel of 12 breast cancer cell lines. Therefore, ERα targets specific genes, such as basal markers, for DNA methylation-mediated silencing. Mechanistically, ERα-dependent DNA methylation may occur as a consequence of long-term transcriptional repression. Although not well appreciated, estrogen-bound ERα represses transcription of more genes than it activates. Thus, ERα may direct DNA methylation by interacting with multicomponent corepressor complexes, which could then recruit DNA methyltransferases to target promoters. Moreover, ERα-dependent silencing of basal markers may promote the prognostically more favorable luminal breast cancer subtype.

Citation Format: Eric A. Ariazi, John C. Taylor, Emmanuelle Nicolas, Michael J. Slifker, Jeff Boyd. A new role for ERα: Gene silencing via DNA methylation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-152. doi:10.1158/1538-7445.AM2015-LB-152