Rory Stark

Differential oestrogen receptor binding is associated with clinical outcome in breast cancer

This paper, which maps ERα binding via ChIP-seq in tumour tissue from twenty ER+ breast cancer patients, relies on a concurrently developed Bioconductor package, DiffBind, which provides a framework for quantitative differential analysis of protein/DNA binding events. Here we use DiffBind to identify ERα sites significantly differentially bound between those found in tumours from patients with good prognosis vs. those with poor prognosis and metastases. Gene signatures that predict clinical outcome in ER+ disease, validated in publically available breast cancer gene expression datasets, are derived from these sites. These signatures are enriched for genes with relevant proximal cis-regulatory events. Statistical characterization of differentially bound ERα sites enables further downstream analysis, including identification of a differentially enriched motif for the transcription factor FoxA1. Further differential analysis in five ER+ breast cancer cell lines shows how ERα binding is extensively shifted in tamoxifen-resistance, with the FoxA1 motif enriched proximal to ERα binding sites differentially bound in cells resistant to treatment. Analysis of FoxA1 binding at mitogen-induced ERα sites demonstrates that the observed differential ER binding program is not due to the selection of a rare subpopulation of cells, but rather to the FoxA1-mediated reprogramming of ER binding on a rapid time scale. Focusing our analysis on differential binding in primary tumour material allows us to show the plasticity of ERα binding capacity, with distinct combinations of cis-regulatory elements linked with the different clinical outcomes. These techniques are applicable to other cancers (and indeed other diseases) where master transcription factor regulators are known.

The androgen receptor fuels prostate cancer by regulating central metabolism and biosynthesis

The androgen receptor (AR) is a key regulator of prostate growth and the principal drug target for the treatment of prostate cancer. Previous studies have mapped AR targets and identified some candidates which may contribute to cancer progression, but did not characterize AR biology in an integrated manner. In this study, we took an interdisciplinary approach, integrating detailed genomic studies with metabolomic profiling and identify an anabolic transcriptional network involving AR as the core regulator. Restricting flux through anabolic pathways is an attractive approach to deprive tumours of the building blocks needed to sustain tumour growth. Therefore, we searched for targets of the AR that may contribute to these anabolic processes and could be amenable to therapeutic intervention by virtue of differential expression in prostate tumours. This highlighted calcium/calmodulin‐dependent protein kinase kinase 2, which we show is overexpressed in prostate cancer and regulates cancer cell growth via its unexpected role as a hormone‐dependent modulator of anabolic metabolism. In conclusion, it is possible to progress from transcriptional studies to a promising therapeutic target by taking an unbiased interdisciplinary approach.

Piwi and piRNAs Act Upstream of an Endogenous siRNA Pathway to Suppress Tc3 Transposon Mobility in the Caenorhabditis elegans Germline

The Piwi proteins of the Argonaute superfamily are required for normal germline development in Drosophila, zebrafish, and mice and associate with 24-30 nucleotide RNAs termed piRNAs. We identify a class of 21 nucleotide RNAs, previously named 21U-RNAs, as the piRNAs of C. elegans. Piwi and piRNA expression is restricted to the male and female germline and independent of many proteins in other small-RNA pathways, including DCR-1. We show that Piwi is specifically required to silence Tc3, but not other Tc/mariner DNA transposons. Tc3 excision rates in the germline are increased at least 100-fold in piwi mutants as compared to wild-type. We find no evidence for a Ping-Pong model for piRNA amplification in C. elegans. Instead, we demonstrate that Piwi acts upstream of an endogenous siRNA pathway in Tc3 silencing. These data might suggest a link between piRNA and siRNA function.

The androgen receptor induces a distinct transcriptional program in castration-resistant prostate cancer in man.

The androgen receptor (AR) regulates prostate cell growth in man, and prostate cancer is the commonest cancer in men in the UK. We present a comprehensive analysis of AR binding sites in human prostate cancer tissues, including castrate-resistant prostate cancer (CRPC). We identified thousands of AR binding sites in CRPC tissue, most of which were not identified in PC cell lines. Many adjacent genes showed AR regulation in xenografts but not in cultured LNCaPs, demonstrating an in-vivo-restricted set of AR-regulated genes. Functional studies support a model of altered signaling in vivo that directs AR binding. We identified a 16 gene signature that outperformed a larger in-vitro-derived signature in clinical data sets, showing the importance of persistent AR signaling in CRPC.

Cooperative interaction between retinoic acid receptor-α and estrogen receptor in breast cancer

Retinoic acid receptor-alpha (RAR alpha) is a known estrogen target gene in breast cancer cells. The consequence of RAR alpha induction by estrogen was previously unknown. We now show that RAR alpha is required for efficient estrogen receptor-alpha (ER)-mediated transcription and cell proliferation. RAR alpha can interact with ER-binding sites, but this occurs in an ER-dependent manner, providing a novel role for RAR alpha that is independent of its classic role. We show, on a genome-wide scale, that RAR alpha 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 RAR alpha. These findings implicate RAR alpha 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.

GATA3 acts upstream of FOXA1 in mediating ESR1 binding by shaping enhancer accessibility

Estrogen receptor (ESR1) drives growth in the majority of human breast cancers by binding to regulatory elements and inducing transcription events that promote tumor growth. Differences in enhancer occupancy by ESR1 contribute to the diverse expression profiles and clinical outcome observed in breast cancer patients. GATA3 is an ESR1-cooperating transcription factor mutated in breast tumors; however, its genomic properties are not fully defined. In order to investigate the composition of enhancers involved in estrogen-induced transcription and the potential role of GATA3, we performed extensive ChIP-sequencing in unstimulated breast cancer cells and following estrogen treatment. We find that GATA3 is pivotal in mediating enhancer accessibility at regulatory regions involved in ESR1-mediated transcription. GATA3 silencing resulted in a global redistribution of cofactors and active histone marks prior to estrogen stimulation. These global genomic changes altered the ESR1-binding profile that subsequently occurred following estrogen, with events exhibiting both loss and gain in binding affinity, implying a GATA3-mediated redistribution of ESR1 binding. The GATA3-mediated redistributed ESR1 profile correlated with changes in gene expression, suggestive of its functionality. Chromatin loops at the TFF locus involving ESR1-bound enhancers occurred independently of ESR1 when GATA3 was silenced, indicating that GATA3, when present on the chromatin, may serve as a licensing factor for estrogen-ESR1-mediated interactions between cis-regulatory elements. Together, these experiments suggest that GATA3 directly impacts ESR1 enhancer accessibility, and may potentially explain the contribution of mutant-GATA3 in the heterogeneity of ESR1+ breast cancer.

Independence of Repressive Histone Marks and Chromatin Compaction during Senescent Heterochromatic Layer Formation

The expansion of repressive epigenetic marks has been implicated in heterochromatin formation during embryonic development, but the general applicability of this mechanism is unclear. Here we show that nuclear rearrangement of repressive histone marks H3K9me3 and H3K27me3 into nonoverlapping structural layers characterizes senescence-associated heterochromatic foci (SAHF) formation in human fibroblasts. However, the global landscape of these repressive marks remains unchanged upon SAHF formation, suggesting that in somatic cells, heterochromatin can be formed through the spatial repositioning of pre-existing repressively marked histones. This model is reinforced by the correlation of presenescent replication timing with both the subsequent layered structure of SAHFs and the global landscape of the repressive marks, allowing us to integrate microscopic and genomic information. Furthermore, modulation of SAHF structure does not affect the occupancy of these repressive marks, nor vice versa. These experiments reveal that high-order heterochromatin formation and epigenetic remodeling of the genome can be discrete events.

Progesterone receptor modulates ERα action in breast cancer

Progesterone receptor (PR) expression is used as a biomarker of oestrogen receptor-α (ERα) function and breast cancer prognosis. Here we show that PR is not merely an ERα-induced gene target, but is also an ERα-associated protein that modulates its behaviour. In the presence of agonist ligands, PR associates with ERα to direct ERα chromatin binding events within breast cancer cells, resulting in a unique gene expression programme that is associated with good clinical outcome. Progesterone inhibited oestrogen-mediated growth of ERα+ cell line xenografts and primary ERα+ breast tumour explants, and had increased anti-proliferative effects when coupled with an ERα antagonist. Copy number loss of PGR, the gene coding for PR, is a common feature in ERα+ breast cancers, explaining lower PR levels in a subset of cases. Our findings indicate that PR functions as a molecular rheostat to control ERα chromatin binding and transcriptional activity, which has important implications for prognosis and therapeutic interventions.

BayesPeak: Bayesian analysis of ChIP-seq data

BackgroundHigh-throughput sequencing technology has become popular and widely used to study protein and DNA interactions. Chromatin immunoprecipitation, followed by sequencing of the resulting samples, produces large amounts of data that can be used to map genomic features such as transcription factor binding sites and histone modifications.MethodsOur proposed statistical algorithm, BayesPeak, uses a fully Bayesian hidden Markov model to detect enriched locations in the genome. The structure accommodates the natural features of the Solexa/Illumina sequencing data and allows for overdispersion in the abundance of reads in different regions. Moreover, a control sample can be incorporated in the analysis to account for experimental and sequence biases. Markov chain Monte Carlo algorithms are applied to estimate the posterior distributions of the model parameters, and posterior probabilities are used to detect the sites of interest.ConclusionWe have presented a flexible approach for identifying peaks from ChIP-seq reads, suitable for use on both transcription factor binding and histone modification data. Our method estimates probabilities of enrichment that can be used in downstream analysis. The method is assessed using experimentally verified data and is shown to provide high-confidence calls with low false positive rates.

Choline Kinase Alpha as an Androgen Receptor Chaperone and Prostate Cancer Therapeutic Target

Background: The androgen receptor (AR) is a major drug target in prostate cancer (PCa). We profiled the AR-regulated kinome to identify clinically relevant and druggable effectors of AR signaling. Methods: Using genome-wide approaches, we interrogated all AR regulated kinases. Among these, choline kinase alpha (CHKA) expression was evaluated in benign (n = 195), prostatic intraepithelial neoplasia (PIN) (n = 153) and prostate cancer (PCa) lesions (n = 359). We interrogated how CHKA regulates AR signaling using biochemical assays and investigated androgen regulation of CHKA expression in men with PCa, both untreated (n = 20) and treated with an androgen biosynthesis inhibitor degarelix (n = 27). We studied the effect of CHKA inhibition on the PCa transcriptome using RNA sequencing and tested the effect of CHKA inhibition on cell growth, clonogenic survival and invasion. Tumor xenografts (n = 6 per group) were generated in mice using genetically engineered prostate cancer cells with inducible CHKA knockdown. Data were analyzed with χ2 tests, Cox regression analysis, and Kaplan-Meier methods. All statistical tests were two-sided. Results: CHKA expression was shown to be androgen regulated in cell lines, xenografts, and human tissue (log fold change from 6.75 to 6.59, P = .002) and was positively associated with tumor stage. CHKA binds directly to the ligand-binding domain (LBD) of AR, enhancing its stability. As such, CHKA is the first kinase identified as an AR chaperone. Inhibition of CHKA repressed the AR transcriptional program including pathways enriched for regulation of protein folding, decreased AR protein levels, and inhibited the growth of PCa cell lines, human PCa explants, and tumor xenografts. Conclusions: CHKA can act as an AR chaperone, providing, to our knowledge, the first evidence for kinases as molecular chaperones, making CHKA both a marker of tumor progression and a potential therapeutic target for PCa.