Abstract 1063: Transcriptional implications of intragenic DNA methylation in the estrogen receptor alpha gene in breast cancer cells and tissues
Authors: NATALIE S. SHENKER, Kirsty J. Flower, Charlotte Wilhelm-Benartzi, Wei Dai, Emma Bell, Mona El Bahrawy, Gillian Weaver, James M. Flanagan, Robert Brown
Journal: Cancer Research (2015)

Abstract Introduction: DNA methylation variability regions (MVRs) across the estrogen receptor alpha (ESR1) gene have been identified in peripheral blood cells from breast cancer patients and healthy individuals. In contrast to promoter methylation, gene body methylation may be important in maintaining active transcription. This study aimed to assess MVRs in ESR1 in breast cancer cell lines, tumor biopsies and exfoliated epithelial cells from expressed breast milk (EBM) to determine their significance for ESR1 transcription. Methods: DNA methylation levels in eight MVRs across ESR1 were assessed in pyrosequencing bisulphite-converted DNA from three oestrogen receptor (ER)-positive and three ER-negative breast cancer cell lines. DNA methylation and expression were assessed following treatment with the demethylating agent, decitabine (DAC, 1 μM), or DMSO (controls). ESR1 methylation levels were also assayed in DNA from 155 invasive ductal carcinoma biopsies provided by the Breast Cancer Campaign Tissue Bank, and validated with DNA methylation profiles from the TCGA breast tumors (n = 356 ER-pos, n = 109 ER-neg). DNA methylation was profiled in exfoliated breast epithelial cells from EBM using the Illumina 450K (n = 36) and pyrosequencing in a further 53 donor samples. ESR1 mRNA levels were measured by qRT-PCR. Results: We show that ER-positive cell lines had unmethylated ESR1 promoter regions and highly methylated intragenic regions (median, 80.45%) while ER-negative cells had methylated promoters and lower intragenic methylation levels (median, 38.62%). DAC treatment increased ESR1 expression in ER-negative cells, but significantly reduced methylation and expression of ESR1 in ER-positive cells. The ESR1 promoter was unmethylated in breast tumor biopsies with high levels of intragenic methylation, independent of ER status. However, ESR1 methylation in the strongly ER-positive EBM DNA samples were very similar to ER-positive tumor cell lines. Conclusion: Intragenic methylation levels correlated with ESR1 expression in vitro, but with markedly different methylation patterns between homogenous cell populations and heterogeneous tumour biopsies. DAC treatment inhibited ESR1 transcription in cells with an unmethylated ESR1 promoter along with reduced levels of intragenic DNA methylation. These findings emphasize the need for care when choosing tissue types for epigenetic research and interpreting results from heterogenous tissue. Note: This abstract was not presented at the meeting. Citation Format: NATALIE S. SHENKER, Kirsty J. Flower, Charlotte Wilhelm-Benartzi, Wei Dai, Emma Bell, Mona El Bahrawy, Gillian Weaver, James M. Flanagan, Robert Brown. Transcriptional implications of intragenic DNA methylation in the estrogen receptor alpha gene in breast cancer cells and tissues. [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 1063. doi:10.1158/1538-7445.AM2015-1063