ESR 1 Is Co-Expressed with Closely Adjacent Uncharacterised Genes Spanning a Breast Cancer Susceptibility Locus at 6 q 25 . 1

Approximately 80% of human breast carcinomas present as oestrogen receptor a-positive (ER+ve) disease, and ER status is a critical factor in treatment decision-making. Recently, single nucleotide polymorphisms (SNPs) in the region immediately upstream of the ER gene (ESR1) on 6q25.1 have been associated with breast cancer risk. Our investigation of factors associated with the level of expression of ESR1 in ER+ve tumours has revealed unexpected associations between genes in this region and ESR1 expression that are important to consider in studies of the genetic causes of breast cancer risk. RNA from tumour biopsies taken from 104 postmenopausal women before and after 2 weeks treatment with an aromatase (oestrogen synthase) inhibitor was analyzed on Illumina 48K microarrays. Multiple-testing corrected Spearman correlation revealed that three previously uncharacterized open reading frames (ORFs) located immediately upstream of ESR1, C6ORF96, C6ORF97, and C6ORF211 were highly correlated with ESR1 (Rs = 0.67, 0.64, and 0.55 respectively, FDR,1610). Publicly available datasets confirmed this relationship in other groups of ER+ve tumours. DNA copy number changes did not account for the correlations. The correlations were maintained in cultured cells. An ERa antagonist did not affect the ORFs’ expression or their correlation with ESR1, suggesting their transcriptional co-activation is not directly mediated by ERa. siRNA inhibition of C6ORF211 suppressed proliferation in MCF7 cells, and C6ORF211 positively correlated with a proliferation metagene in tumours. In contrast, C6ORF97 expression correlated negatively with the metagene and predicted for improved disease-free survival in a tamoxifen-treated published dataset, independently of ESR1. Our observations suggest that some of the biological effects previously attributed to ER could be mediated and/or modified by these co-expressed genes. The co-expression and function of these genes may be important influences on the recently identified relationship between SNPs in this region and breast cancer risk. Citation: Dunbier AK, Anderson H, Ghazoui Z, Lopez-Knowles E, Pancholi S, et al. (2011) ESR1 Is Co-Expressed with Closely Adjacent Uncharacterised Genes Spanning a Breast Cancer Susceptibility Locus at 6q25.1. PLoS Genet 7(4): e1001382. doi:10.1371/journal.pgen. 1001382 Editor: Marshall S. Horwitz, University of Washington, United States of America Received September 15, 2010; Accepted March 25, 2011; Published April 28, 2011 Copyright: 2011 Dunbier et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by the Mary-Jean Mitchell Green Foundation, Breakthrough Breast Cancer, and NHS funding to the Royal Marsden NIHR Biomedical Research Centre. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: MD has received honoraria for advice and lectures for Astra Zeneca and has acted as an expert witness for them. * E-mail: anita.dunbier@icr.ac. uk