Abstract 5368: Epigenetic changes in breast cancer cells associated with acquired Tamoxifen resistance.
Authors: Williams Kristin E. , Anderton Douglas L. , Lee Maxwell P. , Pentecost Brian T. , Arcaro Kathleen F. 
Published: August 2014

Estrogen receptor (ER) status remains one of the most important breast cancer diagnostic and prognostic biomarkers. Roughly two-thirds of all breast cancers are ER-positive and can be treated with the anti-estrogen, Tamoxifen, however resistance occurs in 33% of women who take the drug for more than 5 years.

MCF-7 breast cancer cells were previously cultured with 1 uM Tamoxifen, and after 6 months the clonal lines TMX2-11 and TMX2-28 with acquired resistance were isolated. TMX2-28 lost ER-α gene expression while a second clonal line, TMX2-11, did not. TMX2-28 cells are highly invasive in vitro, in contrast to TMX2-11 and the parent cell line, MCF-7.

Studies of endocrine resistance and global gene expression in Tamoxifen-resistant cell cultures and human tumors have detected alterations in numerous pathways including ER-signaling, growth factor receptor signaling, cell cycle, and apoptosis. Aberrant DNA promoter methylation is a major epigenetic mechanism affecting gene expression in cancer. Our focus is on its role in Tamoxifen resistance. Hypermethylation of normally unmethylated cytosines in CpG-rich promoter regions frequently results in gene silencing, while hypomethylation in CpGs outside promoter regions leads to genetic instability.

To develop a broad understanding of Tamoxifen resistance and to identify novel pathways and targets of aberrant methylation, DNA from MCF-7, TMX2-11, and TMX2-28 was analyzed using the Illumina HumanMethylation450 BeadChip. The three cell lines have similar hypermethylation patterns in 306,000 CpG sites and similar hypomethylation patterns in 132,000 CpG sites across the genome. A subset of CpG sites was identified where the two Tamoxifen-selected lines share a methylation pattern distinct from the MCF-7 parent line. When normalized to MCF-7, TMX2-11 and TMX2-28 share 722 hypermethylated and 65 hypomethylated CpG sites in the promoter region encompassing 539 and 55 genes respectively. Pathway analysis indicates the hypermethylated genes are involved in processes relevant to acquired Tamoxifen resistance. These pathways include cell signaling, adhesion, transcriptional activation and repression, differentiation, proliferation, and apoptosis.

Ongoing studies on a subset of the genes found differentially methylated between parental and Tamoxifen-selected cell lines are aimed at confirming methylation findings and examining gene expression. Additionally, the cells will be treated with the demethylating agent 5-aza-deoxycitidine to determine whether reversal of the hypermethylation restores Tamoxifen sensitivity. Greater knowledge of the molecular modifications accompanying Tamoxifen-resistance is needed, as it will lead to discovery of new therapeutic targets and improved treatment, possibly allowing prediction of resistance from biopsy DNA.

Citation Format: Kristin E. Williams, Douglas L. Anderton, Maxwell P. Lee, Brian T. Pentecost, Kathleen F. Arcaro. Epigenetic changes in breast cancer cells associated with acquired Tamoxifen resistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5368. doi:10.1158/1538-7445.AM2013-5368