Abstract
Approximately 80% of human breast carcinomas present as oestrogen receptor α-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<1×10
−7
). 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 ERα antagonist did not affect the ORFs' expression or their correlation with
ESR1
, suggesting their transcriptional co-activation is not directly mediated by ERα. 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.
Recent genome-wide analysis has revealed that the way in which genes are arranged on chromosomes and the conformation of these chromosomes are crucial for the regulation of gene expression. Reflecting this arrangement, clusters of genes which are regulated together have been discovered. We have identified a previously unreported transcriptional activity hub spanning
ESR1
, the gene encoding the important breast cancer biomarker oestrogen receptor. Genetic variants immediately upstream of
ESR1
have recently been linked to breast cancer risk. We found that three open reading frames within this region are tightly co-expressed with
ESR1
. We investigated the function of these genes and discovered that one of these co-expressed genes,
C6ORF211
, affects proliferation in cultured cells and is correlated with proliferation in breast tumours. Another of the genes,
C6ORF97
, is negatively correlated with proliferation in breast tumours and predicts for outcome on the anti-oestrogen drug tamoxifen. These findings suggest that the genes could contribute to the phenotype associated with oestrogen-receptor positivity. In addition, they may be involved in the mechanism by which genetic variation in this region of the genome contributes to breast cancer susceptibility.