SummaryOestrogen receptor-α (ER) is the defining and driving transcription factor in the majority of breast cancers and its target genes dictate cell growth and endocrine response, yet genomic understanding of ER function has been restricted to model systems1-3. We now map genome-wide ER binding events, by chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq), in primary breast cancers from patients with different clinical outcome and in distant ER positive (ER+) metastases. We find that drug resistant cancers still have ER-chromatin occupancy, but that ER binding is a dynamic process, with the acquisition of unique ER binding regions in tumours from patients that are likely to relapse. The acquired, poor outcome ER regulatory regions observed in primary tumours reveal gene signatures that predict clinical outcome in ER+ disease exclusively. We find that the differential ER binding programme observed in tumours from patients with poor outcome is not due to the selection of a rare subpopulation of cells, but is due to the FoxA1-mediated reprogramming of ER binding on a rapid time scale. The parallel redistribution of ER and FoxA1 cis-regulatory elements in drug resistant cellular contexts is supported by histological co-expression of ER and FoxA1 in metastatic samples. By establishing transcription factor mapping in primary tumour material, we show that there is plasticity in ER binding capacity, with distinct combinations of cis-regulatory elements linked with the different clinical outcomes.

Estrogen Receptor-α (ER) is the key feature in the majority of breast cancers and ER binding to the genome correlates with the Forkhead protein FOXA1 (HNF3α). We now show that FOXA1 is a critical determinant that can influence differential ER-chromatin interactions. We show that almost all ER-chromatin interactions and gene expression changes are dependent on the presence of FOXA1 and that FOXA1 influences genome-wide chromatin accessibility. Furthermore, we show that CTCF is an upstream negative regulator of FOXA1-chromatin interactions. In ER responsive breast cancer cells, the dependency on FOXA1 for tamoxifen-ER activity is absolute and in tamoxifen resistant cells, ER binding occurs independently of ligand, but in a FOXA1 dependent manner. Importantly, expression of FOXA1 in non-breast cancer cells can alter ER binding and function. As such, FOXA1 is a major determinant of estrogen-ER activity and endocrine response in breast cancer cells.

Estrogen receptor-α (ER) is the driving transcription factor in most breast cancers, and its associated proteins can influence drug response, but direct methods for identifying interacting proteins have been limited. We purified endogenous ER using an approach termed RIME (rapid immunoprecipitation mass spectrometry of endogenous proteins) and discovered the interactome under agonist- and antagonist-liganded conditions in breast cancer cells, revealing transcriptional networks in breast cancer. The most estrogen-enriched ER interactor is GREB1, a potential clinical biomarker with no known function. GREB1 is shown to be a chromatin-bound ER coactivator and is essential for ER-mediated transcription, because it stabilizes interactions between ER and additional cofactors. We show a GREB1-ER interaction in three xenograft tumors, and using a directed protein-protein approach, we find GREB1-ER interactions in half of ER(+) primary breast cancers. This finding is supported by histological expression of GREB1, which shows that GREB1 is expressed in half of ER(+) cancers, and predicts good clinical outcome. These findings reveal an unexpected role for GREB1 as an estrogen-specific ER cofactor that is expressed in drug-sensitive contexts.

Cross talk between the Estrogen Receptor (ER) and ErbB2/HER-2 pathways have long been implicated in breast cancer aetiology and drug response 1 , yet no direct connection at a transcriptional level has been shown. We now show that estrogen-ER and tamoxifen-ER complexes directly repress ErbB2 transcription via a cis-regulatory element within the ERBB2 gene. We implicate the Paired Box 2 gene product (Pax2), in a novel role, as a crucial mediator of ER repression of ErbB2 by the anti-cancer drug tamoxifen. We show that Pax2 and the ER coactivator AIB-1/SRC-3 compete for binding and regulation of ErbB2 transcription, the outcome of which determines tamoxifen response in breast cancer cells. The repression of ErbB2 by ER-Pax2 links these two important breast cancer subtypes and suggests that aggressive ErbB2 positive tumours can originate from ER positive luminal tumours by circumventing this repressive mechanism. These data provide mechanistic insight into the molecular basis of endocrine resistance in breast cancer.The genomic mapping of Estrogen Receptor binding sites has revealed insight into how ER functions in breast cancer cells, including the finding that ER rarely binds to promoter regions and that ER loading on the chromatin requires the presence of pioneer factors, such as FoxA1 2-4 . We have replicated genome-wide ER Chromatin Immunoprecipitation (ChIP)-on-chip analyses in ER positive MCF-7 cells. Identification of the ER binding sites using a false discovery rate of 5% revealed 8,525 ER sites, with excellent representation (86%) of the published ER binding profile 2 (Supplementary data 2). Included within the new, more extensive list, was an ER binding site within the intron of the ERBB2/HER-2 genomic region ( Figure 1a). Sequence analysis of all 8,525 ER binding sites revealed a statistical enrichment (p-value < 0.0001) for the Paired Box (Pax) transcription factor motif (GTCANGN(A/G)T) ( Figure 1b). Little is known about the role that Pax proteins play in hormone signalling, however, Pax2 was shown to be expressed in a subset of breast cancers and was recently identified as a tamoxifen-regulated effector in endometrial cancer cells 5,6 .5 To whom correspondence should be addressed (jason.carroll@cancer.org.uk). NIH Public Access Author ManuscriptNature. Author manuscript; available in PMC 2010 August 11. Tamoxifen is one of the most successful and effective therapies in the treatment of breast cancer, but tamoxifen resistance is inevitable 7 . Tamoxifen resistant breast tumours are characterised by elevated ErbB2 levels 8 and ER positive cell line models over expressing ErbB2 acquire resistance to tamoxifen 9 . We assessed Pax2 binding to a select number of ER binding sites adjacent to important estrogen regulated genes, including the newly identified binding site within the ERBB2 gene. Pax2 was generally recruited only after tamoxifen treatment, with the exception of the ER binding site within ERBB2 (Figure 1c), where Pax2 was recruited to the ER binding site after both estrogen and tamoxifen...

Retinoic acid receptor-a (RARa) is a known estrogen target gene in breast cancer cells. The consequence of RARa induction by estrogen was previously unknown. We now show that RARa is required for efficient estrogen receptor-a (ER)-mediated transcription and cell proliferation. RARa can interact with ER-binding sites, but this occurs in an ER-dependent manner, providing a novel role for RARa that is independent of its classic role. We show, on a genome-wide scale, that RARa and ER can co-occupy regulatory regions together within the chromatin. This transcriptionally active co-occupancy and dependency occurs when exposed to the predominant breast cancer hormone, estrogen-an interaction that is promoted by the estrogen-ER induction of RARa. These findings implicate RARa as an essential component of the ER complex, potentially by maintaining ER-cofactor interactions, and suggest that different nuclear receptors can cooperate for effective transcriptional activity in breast cancer cells.[Keywords: Breast cancer; estrogen receptor; retinoic acid receptor-a; transcription; chromatin] Supplemental material is available at http://www.genesdev.org.