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Pioneer factors are a special class of transcription factor that can associate with compacted chromatin to facilitate the binding of additional transcription factors. This Progress article discusses the importance of pioneer factors in breast cancer and prostate cancer. Pioneer factors are a special class of transcription factor that can associate with compacted chromatin to facilitate the binding of additional transcription factors. The function of pioneer factors was originally described during development; more recently, they have been implicated in hormone-dependent cancers, such as oestrogen receptor-positive breast cancer and androgen receptor-positive prostate cancer. We discuss the importance of pioneer factors in these specific cancers, the discovery of new putative pioneer factors and the interplay between these proteins in mediating nuclear receptor function in cancer.

This protocol describes affinity purification of endogenous protein complexes for mass spectrometry analysis. Optimized to study formaldehyde-crosslinked proteins isolated by chromatin immunoprecipitation, it can be adapted to study other protein complexes. Rapid immunoprecipitation mass spectrometry of endogenous protein (RIME) is a method that allows the study of protein complexes, in particular chromatin and transcription factor complexes, in a rapid and robust manner by mass spectrometry (MS). The method can be used in parallel with chromatin immunoprecipitation–sequencing (ChIP-seq) experiments to provide information on both the cistrome and interactome for a given protein. The method uses formaldehyde fixation to stabilize protein complexes. By using antibodies against the endogenous target, the cross-linked complex is immunoprecipitated, rigorously washed, and then digested into peptides while avoiding antibody contamination (on-bead digestion). By using this method, MS identification of the target protein and several dozen interacting proteins is possible using a 100-min LC-MS/MS run. The protocol does not require substantial proteomics expertise, and it typically takes 2–3 d from the collection of material to results.

This Opinion article discusses many controversial issues surrounding the connections of progestogens, which stimulate the progesterone receptor, to breast cancer risk and their possible therapeutic use in breast cancer. Most breast cancers are driven by oestrogen receptor-α. Anti-oestrogenic drugs are the standard treatment for these breast cancers; however, treatment resistance is common, necessitating new therapeutic strategies. Recent preclinical and historical clinical studies support the use of progestogens to activate the progesterone receptor (PR) in breast cancers. However, widespread controversy exists regarding the role of progestogens in this disease, hindering the clinical implementation of PR-targeted therapies. Herein, we present and discuss data at the root of this controversy and clarify the confusion and misinterpretations that have consequently arisen. We then present our view on how progestogens may be safely and effectively used in treating breast cancer.

Jason Carroll and colleagues report that the forkhead protein FOXA1 is an important determinant of estrogen receptor binding and show that expression of FOXA1 in non–breast cancer cells is sufficient to confer estrogen receptor binding and response to endocrine treatment. Estrogen receptor-α (ER) is the key feature of most breast cancers and binding of ER to the genome correlates with expression of the Forkhead protein FOXA1 (also called HNF3α). Here we show that FOXA1 is a key determinant that can influence differential interactions between ER and chromatin. Almost all ER-chromatin interactions and gene expression changes depended on the presence of FOXA1 and FOXA1 influenced genome-wide chromatin accessibility. Furthermore, we found that CTCF was an upstream negative regulator of FOXA1-chromatin interactions. In estrogen-responsive breast cancer cells, the dependency on FOXA1 for tamoxifen-ER activity was absolute; in tamoxifen-resistant cells, ER binding was independent of ligand but depended on FOXA1. 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.

BET inhibitors that target bromodomain chromatin readers such as BRD4 are being explored as potential therapeutics in cancer; here triple-negative breast cancer cell lines are shown to respond to BET inhibitors and resistance seems to be associated with transcriptional changes rather than drug efflux and mutations, opening potential avenues to improve clinical responses to BET inhibitors. The roots of resistance to BET inhibitors BET inhibitors that target bromodomain chromatin readers such as BRD4 are being explored as potential therapeutics in cancer. Here Kornelia Polyak and colleagues investigate the response of breast cancer cell lines and xenograft mouse models to BET inhibitors. They find that triple-negative breast cancer cell lines respond to BET inhibitors. Resistance can emerge, but there is no evidence for mechanisms involving drug efflux or mutations in the bromodomain genes or known driver genes. Instead, there are transcriptional changes and increased recruitment of BRD4 to chromatin independent of its bromodomain, concomitant with its increased phosphorylation. Together with two recent Nature publications from the laboratories of Mark Dawson and Johannes Zuber dealing with different cancers, the study suggests potential avenues to improve clinical responses to BET inhibitors. Jeff Settleman discusses all three papers in News & Views. Triple-negative breast cancer (TNBC) is a heterogeneous and clinically aggressive disease for which there is no targeted therapy 1 , 2 , 3 . BET bromodomain inhibitors, which have shown efficacy in several models of cancer 4 , 5 , 6 , have not been evaluated in TNBC. These inhibitors displace BET bromodomain proteins such as BRD4 from chromatin by competing with their acetyl-lysine recognition modules, leading to inhibition of oncogenic transcriptional programs 7 , 8 , 9 . Here we report the preferential sensitivity of TNBCs to BET bromodomain inhibition in vitro and in vivo , establishing a rationale for clinical investigation and further motivation to understand mechanisms of resistance. In paired cell lines selected for acquired resistance to BET inhibition from previously sensitive TNBCs, we failed to identify gatekeeper mutations, new driver events or drug pump activation. BET-resistant TNBC cells remain dependent on wild-type BRD4, which supports transcription and cell proliferation in a bromodomain-independent manner. Proteomic studies of resistant TNBC identify strong association with MED1 and hyper-phosphorylation of BRD4 attributable to decreased activity of PP2A, identified here as a principal BRD4 serine phosphatase. Together, these studies provide a rationale for BET inhibition in TNBC and present mechanism-based combination strategies to anticipate clinical drug resistance.

FOXA1 is a key pioneer factor in androgen-receptor activity but has been an elusive drug target. A new study shows that inhibition of the associated cofactor LSD1 modifies the methylation state of FOXA1, thus resulting in chromatin dissociation and tumor inhibition, even in models of treatment-resistant prostate cancer.

Understanding the dynamics of endogenous protein–protein interactions in complex networks is pivotal in deciphering disease mechanisms. To enable the in-depth analysis of protein interactions in chromatin-associated protein complexes, we have previously developed a method termed RIME (Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins). Here, we present a quantitative multiplexed method (qPLEX-RIME), which integrates RIME with isobaric labelling and tribrid mass spectrometry for the study of protein interactome dynamics in a quantitative fashion with increased sensitivity. Using the qPLEX-RIME method, we delineate the temporal changes of the Estrogen Receptor alpha (ERα) interactome in breast cancer cells treated with 4-hydroxytamoxifen. Furthermore, we identify endogenous ERα-associated proteins in human Patient-Derived Xenograft tumours and in primary human breast cancer clinical tissue. Our results demonstrate that the combination of RIME with isobaric labelling offers a powerful tool for the in-depth and quantitative characterisation of protein interactome dynamics, which is applicable to clinical samples. Chromatin-associated protein complexes play a critical role in the regulation of gene expression in health and disease. Here, the authors describe a sensitive mass spectrometry-based method to monitor the dynamic interactions of endogenous chromatin-associated protein complexes in clinical samples.

HER2 is a transmembrane receptor tyrosine kinase, which plays a key role in breast cancer due to a common genomic amplification. It is used as a marker to stratify patients in the clinic and is targeted by a number of drugs including Trastuzumab and Lapatinib. HER2 has previously been shown to translocate to the nucleus. In this study, we have explored the properties of nuclear HER2 by analysing the binding of this protein to the chromatin in two breast cancer cell lines. We find genome-wide re-programming of HER2 binding after treatment with the growth factor EGF and have identified a de novo motif at HER2 binding sites. Over 2,000 HER2 binding sites are found in both breast cancer cell lines after EGF treatment, and according to pathway analysis, these binding sites were enriched near genes involved in protein kinase activity and signal transduction. HER2 was shown to co-localise at a small subset of regions demarcated by H3K4me1, a hallmark of functional enhancer elements and HER2/H3K4me1 co-bound regions were enriched near EGF regulated genes providing evidence for their functional role as regulatory elements. A chromatin bound role for HER2 was verified by independent methods, including Proximity Ligation Assay (PLA), which confirmed a close association between HER2 and H3K4me1. Mass spectrometry analysis of the chromatin bound HER2 complex identified EGFR and STAT3 as interacting partners in the nucleus. These findings reveal a global role for HER2 as a chromatin-associated factor that binds to enhancer elements to elicit direct gene expression events in breast cancer cells.

Using genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screens to understand endocrine drug resistance, we discovered ARID1A and other SWI/SNF complex components as the factors most critically required for response to two classes of estrogen receptor-alpha (ER) antagonists. In this context, SWI/SNF-specific gene deletion resulted in drug resistance. Unexpectedly, ARID1A was also the top candidate in regard to response to the bromodomain and extraterminal domain inhibitor JQ1, but in the opposite direction, with loss of ARID1A sensitizing breast cancer cells to bromodomain and extraterminal domain inhibition. We show that ARID1A is a repressor that binds chromatin at ER cis -regulatory elements. However, ARID1A elicits repressive activity in an enhancer-specific, but forkhead box A1-dependent and active, ER-independent manner. Deletion of ARID1A resulted in loss of histone deacetylase 1 binding, increased histone 4 lysine acetylation and subsequent BRD4-driven transcription and growth. ARID1A mutations are more frequent in treatment-resistant disease, and our findings provide mechanistic insight into this process while revealing rational treatment strategies for these patients. ARID1A and other SWI/SNF components are critical for response to estrogen-receptor antagonists in breast cancer. SWI/SNF-specific gene knockouts lead to drug resistance and ARID1A mutations are more frequent in resistant tumors from patients.

Genome-wide mapping of oestrogen receptor-α binding sites in primary breast cancer tissues shows that oestrogen receptor binding is dynamically regulated and that the expression of genes near differentially bound regulatory regions is associated with clinical outcome. Clinical outcome in ER + tumours Most breast tumours express the oestrogen receptor-α (ER), and this transcription factor is seen as an important drug target for the treatment of breast cancer. Jason Carroll and colleagues have mapped ER binding sites in solid primary cancers for the first time, and find distinct ER-binding profiles that are mediated in part by the FoxA1 DNA-binding protein and are associated with better or worse clinical outcome. As well as providing a potentially useful method of prognosis, this work may help to answer one of the major questions in breast cancer biology: how some ER + tumours become drug resistant. Oestrogen 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 systems 1 , 2 , 3 . Here we 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 outcomes and in distant ER-positive metastases. We find that drug-resistant cancers still recruit ER to the chromatin, 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 ER regulatory regions associated with poor clinical outcome observed in primary tumours reveal gene signatures that predict clinical outcome in ER-positive 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 timescale. The parallel redistribution of ER and FOXA1 binding events 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.