Transcriptional implications of intragenic DNA methylation in the oestrogen receptor alpha gene in breast cancer cells and tissues.
BACKGROUND
DNA methylation variability regions (MVRs) across the oestrogen 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, tumour 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 by 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 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 tumours (n = 356 ER-pos, n = 109 ER-neg). DNA methylation was profiled in exfoliated breast epithelial cells from EBM using the Illumina 450 K (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 tumour 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 tumour cell lines.


CONCLUSIONS
DAC treatment inhibited ESR1 transcription in cells with an unmethylated ESR1 promoter and reduced intragenic DNA methylation. Intragenic methylation levels correlated with ESR1 expression in homogenous cell populations (cell lines and exfoliated primary breast epithelial cells), but not in heterogeneous tumour biopsies, highlighting the significant differences between the in vivo tumour microenvironment and individual homogenous cell types. These findings emphasise the need for care when choosing material for epigenetic research and highlights the presence of aberrant intragenic methylation levels in tumour tissue.
Department of Surgery and Cancer, Epigenetics Unit, Division of Cancer, Faculty of Medicine, Imperial College London, 4th Floor IRDB, Hammersmith Campus, Du Cane Road, London, W12 0NN, UK. natalie.shenker09@imperial.ac.uk.
Natalie S
NS
Shenker
Department of Surgery and Cancer, Epigenetics Unit, Division of Cancer, Faculty of Medicine, Imperial College London, 4th Floor IRDB, Hammersmith Campus, Du Cane Road, London, W12 0NN, UK. k.flower@imperial.ac.uk.
Kirsty J
KJ
Flower
Department of Surgery and Cancer, Epigenetics Unit, Division of Cancer, Faculty of Medicine, Imperial College London, 4th Floor IRDB, Hammersmith Campus, Du Cane Road, London, W12 0NN, UK. c.wilhelm-benartzi@imperial.ac.uk.
Charlotte S
CS
Wilhelm-Benartzi
Department of Surgery and Cancer, Epigenetics Unit, Division of Cancer, Faculty of Medicine, Imperial College London, 4th Floor IRDB, Hammersmith Campus, Du Cane Road, London, W12 0NN, UK. weidai2@hku.hk.|Current Address: The University of Hong Kong, Pokfulam, Hong Kong, P. R. China. weidai2@hku.hk.
Wei
W
Dai
Department of Surgery and Cancer, Epigenetics Unit, Division of Cancer, Faculty of Medicine, Imperial College London, 4th Floor IRDB, Hammersmith Campus, Du Cane Road, London, W12 0NN, UK. e.bell12@imperial.ac.uk.
Emma
E
Bell
Department of Surgery and Cancer, Epigenetics Unit, Division of Cancer, Faculty of Medicine, Imperial College London, 4th Floor IRDB, Hammersmith Campus, Du Cane Road, London, W12 0NN, UK. edmund.gore12@imperial.ac.uk.
Edmund
E
Gore
Department of Surgery and Cancer, Epigenetics Unit, Division of Cancer, Faculty of Medicine, Imperial College London, 4th Floor IRDB, Hammersmith Campus, Du Cane Road, London, W12 0NN, UK. m.elbahrawy@imperial.ac.uk.
Mona
M
El Bahrawy
Queen Charlotte and Chelsea Hospital Milk Bank, Du Cane Road, London, UK. gillian.weaver@imperial.nhs.uk.
Gillian
G
Weaver
Department of Surgery and Cancer, Epigenetics Unit, Division of Cancer, Faculty of Medicine, Imperial College London, 4th Floor IRDB, Hammersmith Campus, Du Cane Road, London, W12 0NN, UK. b.brown@imperial.ac.uk.
Robert
R
Brown
Department of Surgery and Cancer, Epigenetics Unit, Division of Cancer, Faculty of Medicine, Imperial College London, 4th Floor IRDB, Hammersmith Campus, Du Cane Road, London, W12 0NN, UK. j.flanagan@imperial.ac.uk.
James M
JM
Flanagan
D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism; D018270:Carcinoma, Ductal, Breast / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D047628:Estrogen Receptor alpha / Q000235:genetics* / Q000378:metabolism; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D042361:Mammary Glands, Human / Q000378:metabolism; D008895:Milk, Human / Q000166:cytology; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic
C506487:ESR1 protein, human; D047628:Estrogen Receptor alpha