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ESR1
Is Co-Expressed with Closely Adjacent
Uncharacterised Genes Spanning a Breast Cancer
Susceptibility Locus at 6q25.1
Anita K. Dunbier
1,2
*, Helen Anderson
1,2
, Zara Ghazoui
1,2
, Elena Lopez-Knowles
1,2
, Sunil Pancholi
2
,
Ricardo Ribas
2
, Suzanne Drury
1,2
, Kally Sidhu
1
, Alexandra Leary
1
, Lesley-Ann Martin
2
, Mitch Dowsett
1,2
1Royal Marsden Hospital, London, United Kingdom, 2Breakthrough Breast Cancer Research Centre, Institute of Cancer Research, London, United Kingdom
Abstract
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
27
). 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 ERaantagonist 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: ß2011 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
Introduction
Breast cancer is the most common malignancy in women,
accounting for more than 400,000 deaths per year worldwide [1].
Approximately 80% of human breast carcinomas present as
oestrogen receptor a-positive (ER+ve) disease and ER status is
arguably the most clinically important biological factor in all
oncology [2]. The major molecular features of breast cancer
segregate differentially between ER+ve and ER2ve tumours [3,4].
Tumours which express ERahave been termed luminal type [3,5]
and are associated with response to antioestrogen therapy and
improved survival, although the mechanisms by which oestrogen
receptor dictates tumour status are poorly understood.
Recent genome wide studies have identified SNPs around
C6ORF97, an open reading frame (ORF) immediately upstream of
the gene encoding ER (ESR1) to be associated with increased risk
of breast cancer. Zheng et al. found that heterozygosity at
rs2046210, a SNP in the region between C6ORF97 and ESR1,
increased breast cancer risk by an odds ratio of 1.59 in a Chinese
population and that this risk was also present in a European
population, albeit to a weaker extent [6]. Easton and colleagues
confirmed the risk associated with this SNP and reported an at
least partly independent risk associated with a second adjacent
SNP (rs3757318) in intron 7 of C6ORF97 [7]. Using ancestry-shift
refinement mapping, Stacey et al. closed in on the identification of
the pathogenic variant and found that the risk allele of a novel
SNP in this region (rs77275268), disrupts a partially methylated
CpG sequence within a known CTCF binding site [8]. More
recently, two further studies have confirmed an association with
the region [9,10]. Our studies have revealed unexpected
relationships in the expression patterns in breast carcinomas
between ESR1,C6ORF97 and the two genes immediately
upstream (C6ORF211 and C6ORF96 [RMND1]).
Oestrogenic ligands, predominantly oestradiol, are the key
mitogens for ER+ve breast cancer. In recent years, high
throughput genomic technologies have revealed significant
numbers of genes that are expressed in response to oestradiol
stimulation in vitro [11–13] and downregulated in response to
oestrogen deprivation in tumours [14–16]. Similarly, the tran-
scriptional targets of ERahave been characterised in detail using
PLoS Genetics | www.plosgenetics.org 1 April 2011 | Volume 7 | Issue 4 | e1001382
genome wide chromatin interaction mapping in MCF7 cells
[17,18]. Key oestrogen responsive genes such as TFF1 and GREB1
have been shown to be highly responsive to oestradiol stimulation
in cell culture models through the binding of ERato their
promoters [19,20]. Additional genes have been found in
hierarchical clustering analyses of ER+ve and ER2ve tumours
as part of the so-called ‘‘luminal epithelial’’ gene set characterized
by the expression of genes typically expressed in the cells that line
the ducts of normal mammary glands including GATA3 and
FOXA1 [12]. However, the correlates of ESR1 within an
exclusively ER+ve group and the inherent heterogeneity within
an exclusively ER+subgroup remain poorly defined.
Modern, non-steroidal aromatase inhibitors (AIs) are widely
used, effective treatments for ER+ve breast cancer [21,22] and are
also excellent pharmacological probes for oestrogen-dependent
processes in vivo because of their specificity and highly effective
suppression of oestrogen synthesis. In this study, we found that the
expression of genes in the region immediately upstream of ESR1
associate strongly with ESR1 expression in ER+ve primary breast
cancers before and after AI treatment and uncover evidence that
these associations might impact upon the biological and clinical
importance of ERa.
Results
ESR1 expression is correlated with three open reading
frames on chromosome 6 in tumours
To investigate correlates of ESR1, expression profiles were
derived from pairs of 14-guage core cut biopsies before and after 2
weeks’ treatment with 1 mg/d anastrozole, an AI, from 104
patients with ER+ve primary breast cancer [23]. Genes whose
expression correlated with expression of ESR1 levels pre-treatment
were identified (Spearman corrected for multiple testing at false
discovery rate ,1610
27
, Table 1 pre-treatment). The mRNA
species most highly correlated with ESR1 were chromosome 6
ORF 97 (C6ORF97, Rs = 0.67) (Figure 1a), followed by
C6ORF211. Other notable inclusions amongst the top 20 most
correlated genes included well-established ER-associated genes
such as FOXA1,MYB and GATA3, plus C6ORF96, also known as
RMND1 (Required for Meiotic Nuclear Division 1 homolog). The
mean pre-treatment expression of the three ORFs was highly
correlated with ESR1 (Rs = 0.70, Figure 1b). After 2 weeks’ AI
treatment, the top three genes correlating with ESR1 were
C6ORF96, C6ORF97 and C6ORF211 (Rs.0.7 for all, Table 1
two weeks post-treatment). These three ORFs are all located less
than 0.5 MB upstream of the ESR1 start site on the q arm of
chromosome 6 (Figure 1e). The expression of other genes located
within a 50 MB region surrounding ESR1 were not correlated
with ESR1 expression (Rs,0.25) (Table S1).
The correlation was present in all of five published microarray
data sets of ER+ve breast cancer in which the C6orfs were
included on the array (Table 2). The expression of the three ORFs
was lower in ER2ve than ER+ve tumours in the Wang dataset
[24] (p = 0.002). No significant correlation was found in the
ER2ve subgroup of this dataset. This may be a characteristic of
ER2ve tumours or, alternatively, the measurement error
associated with low levels of ESR1 transcript could preclude
detection of a significant correlation in microarray data.
Correlation between ESR1 and the C6orfs is not explained
by amplification
Amplification of the ESR1 locus has been reported inconsis-
tently [25,26]. To determine whether the ESR1/C6ORFs correla-
tion may be the result of underlying genomic co-amplification or
deletion events, copy number (CN) status of ESR1 and the C6orfs
was examined using array CGH analysis (resolution 40–60 kb)
[27] on DNA from the 44 tumour samples from which adequate
further tissue was available. One tumour was shown to be
amplified and eight showed gains at ESR1,C6ORF96,C6ORF97
and C6ORF211, while four showed losses at all four loci. One was
measured as having loss of C6ORF96,C6ORF211 and part of
C6ORF97. While there was some correlation between CN and
transcription of the four genes (Figure S1), CN alterations did not
explain the correlation between ESR1 and the C6orfs. In fact,
when samples with identified CN changes were removed from the
dataset, the correlation between ESR1 and mean C6orf expression
levels strengthened rather than weakened (Rs = 0.83) (Figure 1c),
suggesting that transcriptional co-regulation rather than genomic
changes is more likely to underlie ESR1/C6ORF co-expression.
Change in ESR1 expression upon aromatase inhibitor
treatment is correlated with change in C6orf expression
To assess whether the correlation in ESR1/C6ORF expression
seen in pre-treatment biopsies is reflected in a concordant change in
expression of these genes upon treatment, the relationship between
the magnitude of change of each of these genes was investigated.
Change in expression of ESR1 induced by aromatase inhibitor
treatment over 2 weeks was strongly correlated with change in the
C6orfs (Rs = 0.70) (Figure 1d). Given that this short duration of
treatment, which has no measurable impact on cellularity or tumour
size, is unlikely to facilitate DNA copy number changes throughout
the sample this supports the probability that the co-regulation of
these genes is at a transcriptional level.
Expression of ESR1 and the C6orfs are correlated in MCF7
and BT-474 cells in vitro
To determine whether the ESR1/C6ORF correlations were
maintained in vitro, transcript levels of ERaand the three C6orfs
were measured in oestrogen-deprived MCF7 cells and lapatinib-
treated BT-474 cells over a 48- and 96-hour period, respectively.
These treatments are both known to have significant effects on the
Author Summary
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 arrange-
ment, clusters of genes which are regulated together have
been discovered. We have identified a previously unre-
ported transcriptional activity hub spanning ESR1, the
gene encoding the important breast cancer biomarker
oestrogen receptor. Genetic variants immediately up-
stream 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.
ESR1 Is Co-Expressed with Closely Adjacent Genes
PLoS Genetics | www.plosgenetics.org 2 April 2011 | Volume 7 | Issue 4 | e1001382
expression of ESR1. Lapatinib has been shown to increase ERain
BT-474 cells [28,29], potentially via loss of Akt and de-repression of
FOXO3a. This provides a useful model for manipulation to test the
correlation between ESR1 and the C6orfs in vitro. Conversely,
absence of oestradiol leads to a short-term reduction in ER
expression [30]. Expression of all four genes followed a similar time-
course of expression and was highly correlated (Figure 2a and 2b).
ICI 182,780 (ICI) is a steroidal pure anti-oestrogen which causes
ERaexpression to be suppressed and downregulated [31,32].
Treatment of MCF7 cells with ICI did not affect ORF expression
or their correlation with ESR1 (Figure 2c). To confirm that the
observed correlation was not being influenced by RNA transcribed
prior to the addition of ICI, we also measured newly synthesised
nascent RNA using PCR amplicons designed to cross an exon/
intron boundary [33]. This analysis revealed that nascent
transcripts for ESR1 and the C6orfs remained correlated in both
the presence and absence of ICI. The observation that
transcription of the genes remains strongly correlated in the
presence of ICI suggests that transcriptional regulation by ERais
not the main driver of the ESR1/C6ORF co-expression.
Knockdown of C6ORF211 by siRNA induces a reduction in
proliferation in MCF7cells
The effect of reducing expression of each C6orf on cell
proliferation was determined by transfecting siRNA SMART-
POOLs directed against each ORF into MCF7 cells. In cells
grown in both E2-containing media and without E2, all three
siRNAs reduced transcript levels of their target ORF to ,30% of
levels in cells transfected with the control non-targeting siRNA
pool. Levels of ESR1, and the non-targeted ORFs were unaffected
by the SMARTpool’s (Figure S2) while ESR1-SMARTpool
siRNA led to a reduction in levels of all three C6orfs (Figure
S3). Immunoblotting with a polyclonal antibody raised against a
polypeptide of the predicted product of C6ORF211 showed an
86% reduction at the protein level (Figure S4). Cells transfected
with C6ORF211 siRNA showed a mean 36% reduction in cell
number (p,0.0001) over four separate repeat experiments
(Figure 3A). C6ORF211 knockdown had no effect on oestrogen-
dependent proliferation (Figure 3B). Deconvolution of the
SMARTPOOL showed that the four constituent siRNAs had a
reproducible anti-proliferative effect when compared with scram-
bled control siRNA (Figure S5). No consistent alteration in
proliferation was observed in cells transfected with siRNAs
directed against C6ORF96 or C6ORF97 (Figure 3A).
C6ORF211 correlates with proliferation and clinical
outcome in tumours
To determine whether the association between C6ORF211
expression and proliferation seen in cultured cells is reflected in
tumours, the relationship between C6ORF211 expression and a
metagene composed of known proliferation-associated genes [34]
was investigated. In baseline biopsies, levels of C6ORF211 but not
ESR1 correlated significantly with proliferation (C6ORF211,
Rs = 0.23, p = 0.04; ESR1,Rs=20.01, p = ns) (Figure 4a), suggest-
ing that C6ORF211 is more strongly associated with proliferation
than ESR1. Correlations were also observed with a number of well-
known proliferation-associated genes (Table S2). The relationship
with proliferation was validated in data from a set of 354 ER+ve
tumours [35] (Rs = 0.18, p = 0.0008) (Figure 4b) and the 209 ER+ve
tumours from the Wang dataset [24] (Rs = 0.21, p = 0.004).
Consistent with the findings in our own data, ESR1 was not
significantly correlated with the proliferation metagene in either of
the publicly available datasets (Loi, Rs = 20.03, p = ns; Wang, Rs =
0.02, p = ns). In contrast, C6ORF97 showed an independent,
reproducible negative correlation with proliferation, in our dataset
(Rs = 20.19, p = 0.05) and in the Loi (Rs = 20.22, p,0.0001)
(Figure 4c) and ER+ve Wang datasets (Rs = 20.24, p = 0.0007).
Table 1. Genes positively correlated with ESR1 gene
expression ranked according to Spearman correlation.
GB acc Gene symbol Cytoband
Correlation
coefficient
Pre-treatment
1NM_000125 ESR1 6q25.1 1
2NM_025059 C6orf97 6q25.1 0.672
3NM_024573 C6orf211 6q25.1 0.637
4NM_152437 ZNF664 12q24.31 0.608
5NM_019000 FLJ20152 5p15.1 0.562
6NM_015391 ANAPC13 3q22.1 0.552
7NM_018718 TSGA14 7q32 0.547
8NM_017909 C6orf96 6q25.1 0.546
9NM_021627 SENP2 3q27.2 0.545
10 NM_012319 SLC39A6 18q12.2 0.544
11 NM_004496 FOXA1 14q12-q13 0.537
12 NM_005001 NDUFA7 19p13.2 0.534
13 NM_207118 GTF2H5 6q25.3 0.532
14 NM_004703 RABEP1 17p13.2 0.528
15 NM_016058 TPRKB 2p24.3-p24.1 0.528
16 NM_005375 MYB 6q22-q23 0.527
17 NM_175924 ILDR1 3q13.33 0.526
18 NM_173079 RUNDC1 17q21.31 0.526
19 NM_032918 RERG 12p12.3 0.523
20 NM_002051 GATA3 10p15 0.523
2 weeks post-treatment
1NM_000125 ESR1 6q25.1 1
2NM_025059 C6orf97 6q25.1 0.741
3NM_017909 C6orf96 6q25.1 0.718
4NM_024573 C6orf211 6q25.1 0.705
5NM_004703 RABEP1 17p13.2 0.688
6NM_006452 PAICS 4q12 0.658
7NM_004496 FOXA1 14q12-q13 0.637
8NM_020784 KIAA1344 14q22.1 0.632
9NM_018199 EXDL2 14q24.1 0.629
10 NM_002222 ITPR1 3p26-p25 0.629
11 NM_181656 C17orf58 17q24.2 0.625
12 NM_002051 GATA3 10p15 0.623
13 NM_005080 XBP1 22q12.1|22q12 0.621
14 NM_012319 SLC39A6 18q12.2 0.62
15 NM_015575 TNRC15 2q37.1 0.619
16 NM_173079 RUNDC1 17q21.31 0.615
17 NM_015130 TBC1D9 4q31.21 0.608
18 NM_138809 LOC134147 5p15.2 0.598
19 NM_006405 TM9SF1 14q11.2 0.592
20 NM_152416 C8orf38 8q22.1 0.587
All genes shown have parametric p-value and false discovery rates ,1e-07.
doi:10.1371/journal.pgen.1001382.t001
ESR1 Is Co-Expressed with Closely Adjacent Genes
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To determine whether the relationship of the ORFs with
proliferation is related to clinical outcome, recurrence free survival
(RFS) in tamoxifen-treated patients was investigated for association
with C6ORF97 and C6ORF211 expression. Despite the fact that in
the Loi dataset ESR1 was not predictive of a significant difference in
survival over 5 years [36], the lowest quartile of C6ORF97 was
associated with significantly higher risk of recurrence (HR = 3.1,
p = 0.0014) (Figure 4d). A similar trend was observed in untreated
ER+ve tumours from the Wang dataset [24], although this was not
significant (HR = 1.6, p = 0.16) (Figure S6a). C6ORF211 was not
significantly associated with RFS (Figure S6b and S6c).
Discussion
Our observation of a previously unreported transcriptional
activity hub in the ESR1/C6ORF region of 6q25.1 has implications
for recently identified associations between SNPs in the ESR1
region and breast cancer risk, as well as broader implications for
the biological and clinical importance of ERain established breast
cancer. A number of SNPs, including rs3757318 within intron 7 of
C6ORF97 [7], have been associated with breast cancer risk but the
causative variant and mechanism remain undefined [6–10]. In an
attempt to identify the pathogenic variant, Stacey and colleagues
recently reported that GG homozygotes at rs9397435, located
immediately downstream of C6ORF97, may express higher mean
levels of ESR1 and that the rs9397435 [G] allele conferred
significant risk of both hormone receptor positive and hormone
receptor negative breast cancer in European and Taiwanese
patients [8]. The association of a SNP in this region with ER
expression is consistent with findings from our own group which
have revealed that the variant genotype of SNP rs2046210 is
associated with increased ERaexpression as measured by
Figure 1. Correlation of
ESR1
expression and oestrogen-responsive gene expression. a. Scatterplot of relationship between expression of
ESR1 and C6ORF97 in baseline biopsies. b. Correlation between expression of ESR1 and the mean of C6ORF96,C6ORF97 and C6ORF211 in baseline
biopsies. c. Correlation between ESR1 and the mean of C6ORF96,C6ORF97 and C6ORF211 with samples with measured copy number variations shown
omitted. d. Scatterplot of relationship between change in ESR1 and the mean change in C6ORF96,C6ORF97 and C6ORF211 e. Location of open
reading frames, ESR1 and breast cancer associated SNPs on chromosome 6q25.1.
doi:10.1371/journal.pgen.1001382.g001
ESR1 Is Co-Expressed with Closely Adjacent Genes
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immunohistochemistry [37]. The findings reported in this paper
suggest that, due to their high degree of correlation with ESR1,
levels of C6ORF97,C6ORF96 and C6ORF211 are also likely to
correlate with the rs2046210 and rs9397435 genotype. Conse-
quently, these genes may be involved in the pathogenesis of the
variant SNPs and could explain the apparent anomaly noted by
Stacey and colleagues in that the SNPs predispose to both
hormone receptor positive and negative disease.
To date, analysis of ESR1 co-expressed genes has focussed on
genes which are also downstream targets of the oestradiol-
activated transcription factor activity of ERasuch as FOXA1,
TFF1 and GATA3. High throughput technologies have identified
numerous classical and novel ERa-dependent targets of oestradiol
[11,17]. This association with the expression of ORFs has,
however, not been reported other than by ourselves in abstract
form [38].
The transcriptional correlation between ESR1 and these ORFs
is highly statistically significant in our dataset, and in all of the
publicly available datasets we examined. In our own patient
cohort, we showed that two weeks’ treatment with anastrozole
induces a concomitant change in ESR1 and the C6orfs and a yet
stronger correlation in their expression. Genomic amplification
does not account for the correlations. This suggests that
transcriptional co-regulation rather than major genomic rear-
rangement is likely to underlie their co-expression. To our
knowledge, a transcriptional activity hub surrounding a major
cancer related gene has not previously been identified.
The observation that the four transcripts remain correlated over
a short timecourse in MCF7 and BT474 cells further supports the
idea that the co-regulation of these genes is likely to occur at a
transcriptional level. Given that ERacan autoregulate its own
transcription by binding to an oestrogen responsive element (ERE)
in its promoter [17,39], the possibility that ERacould co-regulate
itself and the C6orfs provides an attractive potential explanation
for the correlation. We tested this hypothesis by treating MCF7
cells with the ERaantagonist ICI in the absence of E2. Our
finding that the nascent transcripts of ESR1 and the three C6orfs
remain correlated in the presence of ICI (Figure 2c) suggests that
this co-regulation is not dependent on ERatranscriptional
activation.
Regulation of the steady-state level of ERain breast cancer cells
is a complex phenomenon that includes transcriptional and post-
transcriptional mechanisms [40–42]. C6ORF96 is transcribed off
the opposite DNA strand to ESR1 (Figure 1e), therefore excluding
the possibility that ESR1 and the ORFs are transcribed as a single
polycistronic mRNA. Recent genome-wide mapping experiments
have revealed the importance of chromatin organisation for gene
expression [18,43] suggesting that 3-D chromatin arrangement
could represent a potential explanation for C6ORF/ESR1 co-
expression. However, analysis of the data produced by Fullwood
and colleagues [18] shows that C6ORF96,C6ORF97 and
C6ORF211 are not encompassed by an ERa-bound long-range
chromatin loop. Nevertheless, it remains possible that a loop
driven by an alternative transcription factor could explain the
transcriptional activity in this area.
At the nucleotide level, all three ORFs show some homology
with ESR1, suggesting they may have arisen from gene duplication
events [44]. C6ORF97 encodes a 715 amino acid coiled-coil
domain-containing protein that is conserved across 11 species [45]
while C6ORF211 is a member of the UPF0364 protein family of
unknown function and is also conserved across multiple species
[45]. Confocal analysis revealed that the protein encoded by
C6ORF211 was expressed mainly in the cytoplasm and did not co-
localize with ER (Figure S7). In a proteomic screen it has been
found to interact with SAP18, a Sin3A-associated cell growth
inhibiting protein [46].
This reported interaction with a growth inhibitory protein could
explain our observation that knockdown of C6ORF211 induces
suppression of proliferation in cultured cells. This association is
mirrored in tumours, where a proliferation metagene correlates
significantly with C6ORF211. Conversely, C6ORF97 expression
correlates negatively with expression of the proliferation metagene
and high C6ORF97 predicts for improved disease-free survival in a
tamoxifen-treated published dataset, independently of ESR1
(Figure 4d). As high ESR1 has previously been shown to be
associated with improved outcome on endocrine therapy [47], this
raises the possibility that, given the observed correlation of
C6ORF97 with ESR1, some of this association with outcome could
be attributable to C6ORF97.
The high degree of correlation between ESR1 and the C6orfs
has significant potential implications for our interpretation of ER
levels and therapy of ER+ve breast cancers. As a transducer of
mitogenic oestrogen signalling, disruption of ER represents a key
target of therapies for ER+ve breast cancer, including tamoxifen
and fulvestrant. Our data shows that C6ORF211 and C6ORF97
may contribute to the proliferative phenotype of ER+ve tumours,
yet these proteins are unlikely to be affected by therapies targeted
directly at ERa. Consequently, these proteins may represent
potential targets for synergistic therapies in patients with high
levels of C6orf expression or targets for breast cancer prevention.
In addition, along with further research these relationships could
shed light on recent associations between breast cancer risk and
SNPs in the region.
Materials and Methods
Patient samples
Core-cut tumor biopsies (14-gauge) were obtained from 112
postmenopausal women with stage I to IIIB ER+early breast
cancer before and after two-weeks’ anastrozole treatment in a
neoadjuvant trial [23]. This study received approval from an
institutional review board at each site and was conducted in
accordance with the 1964 Declaration of Helsinki [48] and
International Conference on Harmonization/Good Clinical
Practice guidelines. Written informed consent was obtained from
each patient before participation. Tissue was stored in RNAlater
at 220uC. Two 4 mm sections from the core were stained with
Table 2. Correlations in other breast cancer datasets.
Study Number of samples
C6ORF96 C6ORF97 C6ORF211
TransBig [52] 198 breast tumours 0.607 0.776 0.656
Wang – All
tumours [24]
286 breast tumours 0.524 0.558 0.769
-ER+ve 209 breast tumours 0.388 0.418 0.608
-ER2ve 65 breast tumours 0.056 0.189 0.087
Loi [35] 354 breast tumours 0.468 0.555 0.588
Huang [53] 23 primary cell lines 0.759 0.759 0.878
Miller [51] 251 breast tumours 0.623 0.547 0.674
Data from five large, publicly available breast cancer datasets performed on
Affymetrix U133A arrays which contained probes for ESR1,C6ORF96,C6ORF97,
and C6ORF211 were examined. The mean of all probes for ESR1 was correlated
with each of the three C6ORFs. Correlation co-efficients for each of the genes
versus ESR1 is shown.
doi:10.1371/journal.pgen.1001382.t002
ESR1 Is Co-Expressed with Closely Adjacent Genes
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hematoxylin and eosin to confirm the presence of cancerous tissue
and the histopathology and six 8 mm sections were retained for
microarray CGH analysis (see below). Total RNA was extracted
using RNeasy Mini kits (Qiagen, Sussex, UK). RNA quality was
checked using an Agilent Bioanalyser (Santa Clara, CA, USA):
samples with RNA integrity values of less than 5 were excluded
from further analysis. ER status and Ki67 values by immunohis-
tochemistry were already available [23].
Gene expression analysis and data pre-processing
RNA amplification, labelling and hybridization on HumanWG-
6 v2 Expression BeadChips were performed according to the
manufacturer’s instructions (http://www.illumina.com) at a single
Illumina BeadStation facility. Tumor RNA of sufficient quality
and quantity was available to generate expression data from 104
pre-treatment biopsies. Data was extracted using BeadStudio
software and normalized with variance-stabilizing transformation
(VST) and Robust Spline Normalisation method (RSN) in the
Lumi package [49]. Probes that were not detected in any samples
(detection p value .1%) were discarded from further analysis.
Data analysis
Multiple correlation analysis was performed in BRB-Array
Tools (http://linus.nci.nih.gov/BRB-ArrayTools.html). A statisti-
cal significance level for each gene for testing the hypothesis that
the Spearman’s correlation between expression of ESR1 and other
genes was zero was calculated and p-values were then used in a
multivariate permutation test [50] from which false discovery rates
were computed. Other statistical analyses were performed in SPSS
for Windows (SPSS Inc., Chicago, IL), S-PLUS (TIBCO Software
Inc., Palo Alto, CA) and Graphpad Prism (Graphpad Software
Inc., La Jolla, CA).
Multivariable analysis was performed in a forward stepwise
fashion, the most significant additional variable (satisfying p,0.05)
being added at each stage. Cases with missing values for any of the
variables in the model were excluded from analysis.
Figure 2. Correlation of C6orf expression
in vitro
.a. Timecourse of expression of ESR1,C6ORF96,C6ORF97 and C6ORF211 in MCF7 cells cultured
in the absence of oestradiol. Each gene is normalized to the mean of two housekeeping genes, TBP and FKBP15. b. Timecourse of expression of ESR1,
C6ORF96,C6ORF97 and C6ORF211 in BT-474 cells after addition of lapatinib. c. Timecourse of expression of ESR1,C6ORF96,C6ORF97 and C6ORF211 in
MCF7 cells cultured with the addition of ICI 182,780. d. Analysis of expression of nascent ESR1,C6ORF96,C6ORF97 and C6ORF211 in MCF7 cells.
e. Analysis of expression of nascent ESR1,C6ORF96,C6ORF97 and C6ORF211 in MCF7 cells treated with ICI. Points represent the mean of three
triplicate samples 6SEM.
doi:10.1371/journal.pgen.1001382.g002
ESR1 Is Co-Expressed with Closely Adjacent Genes
PLoS Genetics | www.plosgenetics.org 6 April 2011 | Volume 7 | Issue 4 | e1001382
Figure 3. Exploration of the function of the C6orfs in MCF7 cells. a. Wild type-MCF7 cells were stripped of steroid for 48 hours then
transfected with either control siRNA, siRNA SMARTpool for C6ORF96,C6ORF97 or C6ORF211. b. Stripped MCF7 cells were transfected with C6ORF211
siRNA SMARTpool and 48 hours post transfection these were treated with increasing concentrations of oestradiol. After 6 days, proliferation in
response to siRNA knockdown was established by change in cell number using a Coulter counter. Bars represent the mean 6SEM of four separate
repeats of the experiment. Oestradiol-dependent proliferation is shown as fold change relative to cells with no added oestradiol.
doi:10.1371/journal.pgen.1001382.g003
ESR1 Is Co-Expressed with Closely Adjacent Genes
PLoS Genetics | www.plosgenetics.org 7 April 2011 | Volume 7 | Issue 4 | e1001382
Analysis of publicly available datasets
For analysis of the breast cancer datasets from public resources
the publicly available normalised gene expression data and clinical
data were retrieved from Gene Expression Omnibus (http://www.
ncbi.nlm.nih.gov/geo/) (‘Wang’ dataset [24], n = 286;GEO,
accession number GSE2034) or obtained from the authors (‘Loi’
dataset [35], n = 354 tamoxifen-treated tumours composed of
GEO, accession numbers GSE9195, GSE6532 and GSE2990;
combined normalised dataset received courtesy of Dr Christos
Sotiriou). Correlations between ESR1 and the C6orfs in the
‘Miller’ [51] (n = 251), ‘TransBig’ (n = 198) [52] and Huang [53]
(n = 23 cell lines) were calculated using the correlation analysis tool
in Oncomine (http://www.oncomine.org).
Data from the 72 genes comprising the proliferation metagene
was retrieved from tumours from the Wang and Loi datasets and
proliferation metagene scores were calculated as described
previously [54]. Spearman correlation between the proliferation
metagene and ESR1 and the C6orfs was calculated in Graphpad
Prism. Survival analysis was carried out in these datasets using the
quartiled expression of the C6orfs and the endpoints of recurrence
free survival or time to relapse, according to the original publication.
DNA extraction
Five 8 mm sections from frozen core biopsies were mounted
onto Superfrost glass slides, stained with nuclear fast red, and
microdissected with a sterile needle under a stereomicroscope to
obtain a percentage of tumor cells .75% as described previously
[55]. Genomic DNA was extracted as described previously [55].
The concentration of the DNA was measured with Picogreen
according to the manufacturer’s instructions (Invitrogen).
Array CGH analysis
The 32K bacterial artificial chromosome (BAC) re-array
collection (CHORI) tiling path aCGH platform used for this study
was constructed in the Breakthrough Breast Cancer Research
Centre [55]. DNA labelling, array hybridisations, image acquisition
and filtering were performed as described in Natrajan et al. [56].
Data were smoothed using the circular binary segmentation (cbs)
algorithm [27]. A categorical analysis was applied to the BACs after
classifying them as representing gain, loss or no-change according to
their smoothed Log2 ratio values as defined [56].
Cell culture
MCF7 cells were routinely maintained in phenol red free
RPMI1640 (Invitrogen, Paisley, UK) supplemented with 10%
foetal bovine serum and oestradiol (1 nM). Cells were passaged
weekly and medium replenished every 48–72 hours. In the case of
BT474, cell monolayers were cultured in phenol red containing
medium supplemented with 10% foetal bovine serum. Cell lines
were shown to be free of mycoplasma by routine testing.
Figure 4. Association between C6orf expression, proliferation, and outcome in tumours. a. Relationship between C6ORF211 expression
and expression of proliferation metagene in 104 breast cancers. b. Relationship between C6ORF211 expression and expression of proliferation
metagene in 354 breast cancers from the Loi dataset. c. Relationship between C6ORF97 expression and expression of proliferation metagene in the
Loi dataset. d. Kaplan–Meier curve representing the fraction relapse-free survival comparing the lowest quartile of C6ORF97 expression with the
highest in the Loi dataset.
doi:10.1371/journal.pgen.1001382.g004
ESR1 Is Co-Expressed with Closely Adjacent Genes
PLoS Genetics | www.plosgenetics.org 8 April 2011 | Volume 7 | Issue 4 | e1001382
Real-time quantitative PCR
Total RNA from treated MCF7 and BT-474 cells was extracted
using the RNeasy Mini Kit (Qiagen) according to the manufactur-
er’s instructions. All RNA quantification was performed using the
Agilent 2100 Bioanalyzer with RNA Nano LabChip Kits (Agilent
Technologies, Wokingham, Berkshire, UK). RNA was reverse
transcribed using SuperScript III (Invitrogen), and random primers.
Twenty nanograms of resulting cDNA of each sample was analyzed
in triplicates by qRT-PCR using the ABI Perkin-Elmer Prism
7900HT Sequence detection system (Applied Biosystems). Taqman
gene expression assays (Applied Biosystems) were used to quantitate
processed transcripts of ESR1 (Hs01046818_m1), C6ORF96
(Hs00215537_m1), C6ORF97 (Hs01563344_m1), C6ORF211
(Hs00226188_m1), which were normalized to two housekeeping
genes, FKBP15 (Hs00391480_m1) and TBP (Hs00427620_m1).
These housekeepers were selected from a previously published list of
appropriate reference genes for breast cancer [57]. Custom assays
using primers designed to span intron-exon boundaries were used to
measure nascent RNA (Table S3). Gene expression was quantified
using a standard curve generated from serial dilutions of reference
cDNA from a pooled breast cancer cell line RNA.
Immunoblots
Cell monolayers were washed with cold PBS twice and collected by
scraping. Cell pellets were lysed in extraction buffer, resolved by SDS-
PAGE and transferred to nitrocellulose membranes as described
previously [30]. Membranes were blocked and probed with a
polyclonal antibody directed against the predicted peptide (amino
acids 368–382) of C6orf211 (Eurogentec, Southampton, UK) and anti
b-actin (Sigma-Aldrich, Poole, UK) using the methods described
previously [58]. Quantification of immunoblots was performed using
the NIH ImageJ software, and immunoblots were normalized to actin.
Immunofluorescence and confocal studies
Cells were grown on glass coverslips in standard growth
medium. Cells were fixed and incubated in the presence of
primary antibodies as described previously [58]. Coverslips were
washed with PBS and cells were incubated in the presence of
appropriate Alexa Fluor 555 (red) or Alexa Fluor 488 (green)-
labeled secondary antibodies (Molecular Probes, Invitrogen,
Paisley, UK) diluted 1:1000 for 1 hr. Cells were washed in PBS
and nuclei (DNA) were counterstained with 4,6-diamidino-2-
phenylindole (DAPI; Invitrogen) diluted 1:10000. Coverslips were
mounted onto glass slides using Vectashield mounting medium
(Vector Laboratories, Peterborough, UK). Images were collected
sequentially in three channels on a Zeiss LSM710 (Carl Zeiss Ltd,
Welwyn Garden City, UK) laser scanning confocal microscope at
the same magnification (663 oil immersion objective).
Cell proliferation assays
Cell lines were depleted of steroids for 3 days by culturing in DCC-
medium [59], seeded into 12-well plates at a density of 1610
4
cells/
well for MCF7 and 4610
4
cells per well for BT474, monolayers were
allowed to acclimatize for 24 h before treatment with drug
combinations indicated for 6 d with daily changes. Cell number
was determined using a Z1 Coulter Counter (Beckman Coulter).
Results were confirmed in a minimum of three independent
experiments, and each experiment was performed in triplicate.
Effect of oestradiol and ICI182780 on ORF RNA
expression
Wt-MCF7 cells were stripped of steroid for 3 days as described
above. Cells were subsequently seeded into 12 well plates at a
density of 1610
5
cells/well. After 24 hours monolayers were treated
with vehicle (0.01% v/v ethanol), oestradiol (1 nM) or ICI182780
(10 nM) for the time intervals indicated. RNA was extracted using
RNeasy Mini kit (Qiagen) and subjected to qRT-PCR as described.
SiRNA knockdown of ORFs
Wt-MCF7 cells were stripped of steroid for 24 hours in DCC-
medium. Stripped cells were subsequently seeded into 12 well
plates at a density of 2610
4
cells/well for proliferation assays or
1610
5
cells/well for RNA expression analysis. After 24 hours
monolayers were transfected with 100 nM of either siRNA against
C6ORF96,C6ORF97,C6ORF211 or control siRNA using
DharmaFECT 1 reagent (Dharmacon, Thermo Fisher Scientific,
UK). Medium was then replenished the following day and cells
were allowed to acclimatise for a further 24 hours. After 24 hours
samples were taken for RNA expression analysis. For analysis of
oestrogen-dependent proliferation, the monolayers were treated
with increasing concentrations of oestradiol (0.01, 0.1 or 1 nM)
48 hours post transfection. The remaining plates were treated
daily with the treatments indicated for 6 days before carrying out
cell counts as described above.
Supporting Information
Figure S1 Correlation between ESR1 and the mean of
C6ORF96,C6ORF97, and C6ORF211 showing tumours with
measured copy number variations shown in colour.
Found at: doi:10.1371/journal.pgen.1001382.s001 (0.18 MB
DOC)
Figure S2 Validation of C6ORF gene silencing by siRNA. MCF7
cells were grown in either media containing stripped serum or
stripped serum plus 1 nM oestradiol and transfected with siRNA.
After 48 h, RNA was extracted from cells and complementary DNA
synthesized using standard methods. Using Assay-on-Demand
primer/probe sets (Applied Biosystems, UK), we performed real-
time quantitative PCR. Gene expression was calculated relative to
expression of TBP and FKBP15 and adjusted relative to expression
in cells transfected with a non-targeting siRNA (siControl). Error
bars represent the standard error of the mean (SEM). MCF7 cells
were transfected with siRNA against C6ORF96,C6ORF97,
C6ORF211 or control siRNA in A. DCC or B. 1 nM oestradiol.
Found at: doi:10.1371/journal.pgen.1001382.s002 (0.78 MB
DOC)
Figure S3 Validation of C6ORF211 gene silencing in deconvolu-
tion of siRNA SMARTPOOL. MCF7 cells were grown in media
containing stripped serum and transfected with individual siRNAs.
After 48 h, RNA was extracted from cells and complementary DNA
synthesized using standard methods. Using Assay-on-Demand
primer/probe sets (Applied Biosystems, UK), we performed real-
time quantitative PCR. Gene expression was calculated relative to
expression of TBP and FKBP15 and adjusted relative to expression
in cells transfected with a non-targeting siRNA (siRNA Control).
Error bars represent the standard error of the mean (SEM).
Found at: doi:10.1371/journal.pgen.1001382.s003 (0.84 MB
DOC)
Figure S4 Validation of C6ORF protein knockdown by siRNA.
MCF7 cells were transfected with siRNA against C6ORF97,
C6ORF211 or control siRNA. 72 h after siRNA transfection, cell
lysates were generated and immunoblotted using a. a polyclonal
antibody generated against C6orf211 and b. anti-b-actin as a
loading control.
Found at: doi:10.1371/journal.pgen.1001382.s004 (0.06 MB
DOC)
ESR1 Is Co-Expressed with Closely Adjacent Genes
PLoS Genetics | www.plosgenetics.org 9 April 2011 | Volume 7 | Issue 4 | e1001382
Figure S5 Validation of proliferation changes induced by
individual siRNAs. WT-MCF7 cells were stripped of steroid for
24 hours in DCC-medium. Stripped cells were seeded into 12 well
plates at a density of 20,000 cells/well for proliferation assays or
100,000 cells/well for RNA expression analysis. After 24 hours
monolayers were transfected with 100 nM of single siRNAs against
C6ORF211 or control siRNA (SMARTPool). Medium was
replenished the following day and cells were allowed to acclimatise
for a further 24 hours. Monolayers were subsequently treated with
fresh DCC medium. The remaining plates were treated with DCC
medium for 6 days. Proliferation in response to individual siRNA
knockdown were established by change in cell number using a
coulter counter (Beckman Scientific UK). Data presented is
expressed as absolute cell number or fold change over siControl
(SMARTpool). All data is from triplicate wells, each well read twice.
Found at: doi:10.1371/journal.pgen.1001382.s005 (0.27 MB
DOC)
Figure S6 a. Kaplan-Meier curve comparing proportion relapse-
free survival in the lowest quartile of C6ORF97 expression versus the
highest in 142 untreated ER+ve tumours from the Wang dataset. b.
Kaplan-Meier curve comparing the proportion relapse-free survival
in the lowest quartile of C6ORF211 expression versus the highest in
345 tamoxifen-treated ER+ve tumours from the Loi dataset. c.
Kaplan-Meier curve comparing the proportion relapse-free survival
in the lowest quartile of C6ORF211 expression versus the highest in
142 untreated ER+ve tumours from the Wang dataset.
Found at: doi:10.1371/journal.pgen.1001382.s006 (0.69 MB
DOC)
Figure S7 Confocal analysis of C6orf211 localisation. To
determine the subcellular localization of C6orf211 protein,
confocal analysis was carried out using a polyclonal antibody
directed against the predicted peptide (amino acids 368–381).
MCF-7 cells were plated onto coverslips and stained. a. Nuclei
were visualized using DAPI and stained with antibodies against
C6ORF211 (b) and oestrogen receptor (c). An overlay of all three
images is shown in (d).
Found at: doi:10.1371/journal.pgen.1001382.s007 (0.07 MB
DOC)
Table S1 Correlation of expression of genes in the region of
amplification surrounding ESR1 as defined by Reis-Filho et al.
(2008) [26] with expression of ESR1 in baseline biopsies from 104
patients with ER+ve breast cancer.
Found at: doi:10.1371/journal.pgen.1001382.s008 (0.06 MB
DOC)
Table S2 Correlation expression of the C6ORFs and ESR1 with
expression of well-known proliferation genes. Correlations signif-
icant at p,0.05 are indicated with an asterisk.
Found at: doi:10.1371/journal.pgen.1001382.s009 (0.04 MB
DOC)
Table S3 Custom assays designed to measure nascent RNA.
Found at: doi:10.1371/journal.pgen.1001382.s010 (0.04 MB
DOC)
Author Contributions
Conceived and designed the experiments: AKD HA LAM MD. Performed
the experiments: AKD HA ELK SP RR SD KS AL LAM. Analyzed the
data: AKD HA ZG ELK SP SD LAM. Contributed reagents/materials/
analysis tools: AL. Wrote the paper: AKD MD.
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