Time-course analysis of genome-wide gene expression data from hormone-responsive human breast cancer cells

<p>Abstract</p> <p>Background</p> <p>Microarray experiments enable simultaneous measurement of the expression levels of virtually all transcripts present in cells, thereby providing a ‘molecular picture’ of the cell state. On the other hand, the genomic responses to a pharmacological or hormonal stimulus are dynamic molecular processes, where time influences gene activity and expression. The potential use of the statistical analysis of microarray data in time series has not been fully exploited so far, due to the fact that only few methods are available which take into proper account temporal relationships between samples.</p> <p>Results</p> <p>We compared here four different methods to analyze data derived from a time course mRNA expression profiling experiment which consisted in the study of the effects of estrogen on hormone-responsive human breast cancer cells. Gene expression was monitored with the innovative Illumina BeadArray platform, which includes an average of 30-40 replicates for each probe sequence randomly distributed on the chip surface. We present and discuss the results obtained by applying to these datasets different statistical methods for serial gene expression analysis. The influence of the normalization algorithm applied on data and of different parameter or threshold choices for the selection of differentially expressed transcripts has also been evaluated. In most cases, the selection was found fairly robust with respect to changes in parameters and type of normalization. We then identified which genes showed an expression profile significantly affected by the hormonal treatment over time. The final list of differentially expressed genes underwent cluster analysis of functional type, to identify groups of genes with similar regulation dynamics.</p> <p>Conclusions</p> <p>Several methods for processing time series gene expression data are presented, including evaluation of benefits and drawbacks of the different methods applied. The resulting protocol for data analysis was applied to characterization of the gene expression changes induced by estrogen in human breast cancer ZR-75.1 cells over an entire cell cycle.</p

Discovery of estrogen receptor α target genes and response elements in breast tumor cells

BACKGROUND: Estrogens and their receptors are important in human development, physiology and disease. In this study, we utilized an integrated genome-wide molecular and computational approach to characterize the interaction between the activated estrogen receptor (ER) and the regulatory elements of candidate target genes. RESULTS: Of around 19,000 genes surveyed in this study, we observed 137 ER-regulated genes in T-47D cells, of which only 89 were direct target genes. Meta-analysis of heterogeneous in vitro and in vivo datasets showed that the expression profiles in T-47D and MCF-7 cells are remarkably similar and overlap with genes differentially expressed between ER-positive and ER-negative tumors. Computational analysis revealed a significant enrichment of putative estrogen response elements (EREs) in the cis-regulatory regions of direct target genes. Chromatin immunoprecipitation confirmed ligand-dependent ER binding at the computationally predicted EREs in our highest ranked ER direct target genes, NRIP1, GREB1 and ABCA3. Wider examination of the cis-regulatory regions flanking the transcriptional start sites showed species conservation in mouse-human comparisons in only 6% of predicted EREs. CONCLUSIONS: Only a small core set of human genes, validated across experimental systems and closely associated with ER status in breast tumors, appear to be sufficient to induce ER effects in breast cancer cells. That cis-regulatory regions of these core ER target genes are poorly conserved suggests that different evolutionary mechanisms are operative at transcriptional control elements than at coding regions. These results predict that certain biological effects of estrogen signaling will differ between mouse and human to a larger extent than previously thought

Single-cell states in the estrogen response of breast cancer cell lines

Estrogen responsive breast cancer cell lines have been extensively studied to characterize transcriptional patterns in hormone-responsive tumors. Nevertheless, due to current technological limitations, genome-wide studies have typically been limited to population averaged data. Here we obtain, for the first time, a characterization at the single-cell level of the states and expression signatures of a hormone-starved MCF-7 cell system responding to estrogen. To do so, we employ a recently proposed model that allows for dissecting single-cell states from time-course microarray data. We show that within 32 hours following stimulation, MCF-7 cells traverse, most likely, six states, with a faster early response followed by a progressive deceleration. We also derive the genome-wide transcriptional profiles of such single-cell states and their functional characterization. Our results support a scenario where estrogen promotes cell cycle progression by controlling multiple, sequential regulatory steps, whose single-cell events are here identified. © 2014 Casale et al

Whole-Genome Cartography of Estrogen Receptor α Binding Sites

Using a chromatin immunoprecipitation-paired end diTag cloning and sequencing strategy, we mapped estrogen receptor α (ERα) binding sites in MCF-7 breast cancer cells. We identified 1,234 high confidence binding clusters of which 94% are projected to be bona fide ERα binding regions. Only 5% of the mapped estrogen receptor binding sites are located within 5 kb upstream of the transcriptional start sites of adjacent genes, regions containing the proximal promoters, whereas vast majority of the sites are mapped to intronic or distal locations (>5 kb from 5′ and 3′ ends of adjacent transcript), suggesting transcriptional regulatory mechanisms over significant physical distances. Of all the identified sites, 71% harbored putative full estrogen response elements (EREs), 25% bore ERE half sites, and only 4% had no recognizable ERE sequences. Genes in the vicinity of ERα binding sites were enriched for regulation by estradiol in MCF-7 cells, and their expression profiles in patient samples segregate ERα-positive from ERα-negative breast tumors. The expression dynamics of the genes adjacent to ERα binding sites suggest a direct induction of gene expression through binding to ERE-like sequences, whereas transcriptional repression by ERα appears to be through indirect mechanisms. Our analysis also indicates a number of candidate transcription factor binding sites adjacent to occupied EREs at frequencies much greater than by chance, including the previously reported FOXA1 sites, and demonstrate the potential involvement of one such putative adjacent factor, Sp1, in the global regulation of ERα target genes. Unexpectedly, we found that only 22%–24% of the bona fide human ERα binding sites were overlapping conserved regions in whole genome vertebrate alignments, which suggest limited conservation of functional binding sites. Taken together, this genome-scale analysis suggests complex but definable rules governing ERα binding and gene regulation

Effects of Oestrogen on MicroRNA Expression in Hormone-Responsive Breast Cancer Cells

Oestrogen receptor alpha (ERα) is a ligand-dependent transcription factor that mediates oestrogen effects in hormone-responsive cells. Following oestrogenic activation, ERα directly regulates the transcription of target genes via DNA binding. MicroRNAs (miRNAs) represent a class of small noncoding RNAs that function as negative regulators of protein-coding gene expression. They are found aberrantly expressed or mutated in cancer, suggesting their crucial role as either oncogenes or tumour suppressor genes. Here, we analysed changes in miRNA expression in response to oestrogen in hormone-responsive breast cancer MCF-7 and ZR-75.1 cells by microarray-mediated expression profiling. This led to the identification of 172 miRNAs up- or down-regulated by ERα in response to 17β-oestradiol, of which 52 are similarly regulated by the hormone in the two cell models investigated. To identify mechanisms by which ERα exerts its effects on oestrogen-responsive miRNA genes, the oestrogen-dependent miRNA expression profiles were integrated with global in vivo ERα binding site mapping in the genome by ChIP-Seq. In addition, data from miRNA and messenger RNA (mRNA) expression profiles obtained under identical experimental conditions were compared to identify relevant miRNA target transcripts. Results show that miRNAs modulated by ERα represent a novel genomic pathway to impact oestrogen-dependent processes that affect hormone-responsive breast cancer cell behaviour. MiRNome analysis in tumour tissues from breast cancer patients confirmed a strong association between expression of these small RNAs and clinical outcome of the disease, although this appears to involve only marginally the oestrogen-regulated miRNAs identified in this study

Identification and characterization of estrogen receptor-regulated gene expression programs

The physiological effects of natural and synthetic estrogens are mediated by estrogen receptor alpha (ER alpha), and estrogen receptor beta (ER beta). Within the nucleus of target cells, ER alpha and ER beta serve as ligand-activated transcription factors to stimulate or repress the transcription of estrogen receptor regulated genes. ER alpha and ER beta may be co-expressed in estrogen-responsive cells, but may also be differentially expressed in a cell- and tissue-specific manner. In addition, within a given context these two receptors have different ligand binding and transcriptional activities. Taken together, these attributes underlie differences in target gene regulation, and overall, different physiological actions by ER subtypes. The work described here is an attempt to understand the roles of ER alpha and ER beta in target tissues (e.g. bone, breast, uterus) including the gene networks and cell signaling pathways under ER regulation. We have also characterized the regulation of one of the ER-regulated genes, Carbonic Anhydrase XII, and examined its regulation by ER alpha through use of a conserved distal enhancer. The work described here reports the characterization of individual gene regulatory actions of ER alpha and ER beta. To investigate the individual actions of ER alpha or ER beta, we utilized Affymetrix oligonucleotide arrays to profile transcripts regulated by 17beta-estradiol (E2) in U2OS-ER alpha and U2OS-ER beta cells. These cell lines were constructed by stable integration of ER alpha or ER beta into human osteoblast-like U2OS osteosarcoma cells and initially characterized for ER subtype expression, E2-binding, and cellular responses to E2, including proliferation, motility, and adhesion. Cells expressing apo-ER alpha or apo-ER beta did not show significant alteration in adhesion or proliferation after addition of E2, however there was a significant stimulation of migration in E2-treated ER beta-expressing cells. U2OS-ER alpha, and U2OS-ER beta cells were treated with 10 nM E2 for 0, 4, 8, 24, and 48 hours and total RNA was collected and hybridized to Affymetryx U95Av2 GeneChips and subjected to a Confidence Score to determine E2-regulated RNAs. Of the ca. 100 stimulated or repressed genes identified, some were stimulated by E2 equally through ER alpha and ER beta, whereas others were selectively stimulated via ER alpha or ER beta. The E2-regulated genes showed three distinct temporal patterns of expression over the 48 hour time course studied. Among stimulated genes, ER alpha-containing cells exhibited a greater number of regulated transcripts, and overall magnitude of stimulation was increased as compared those regulated by ER beta. Of the functional categories of the E2-regulated genes, most numerous were those encoding cytokines and factors associated with immune response, signal transduction, and cell migration and cytoskeleton regulation, indicating that E2 can exert effects on multiple pathways in these osteoblast-like cell lines. Of note, E2 up-regulated several genes associated with cell motility selectively via ER beta, in keeping with the selective E2 enhancement of the motility of ER beta-containing cells. On genes regulated equally by E2 via ER alpha or ER beta, the phytoestrogen genistein preferentially stimulated gene expression via ER beta. These studies indicate both common as well as distinct target genes for these two ERs, and identify many novel genes not previously known to be under estrogen regulation. We have examined the ER regulation of the Carbonic Anhydrase XII (CA12) gene, a gene identified as E2-regulated in the studies described above. We investigated the expression of CA12 and its and regulation of by 17beta-estradiol and selective estrogen receptor modulators in breast cancer cells, and characterize the ER usage of a distal enhancer necessary for CA12 gene regulation. We find that CA12 expression is highly correlated with ER alpha expression in human breast tumors. We demonstrate that E2 and SERMS increase CA12 mRNA and protein in multiple breast cancer cell types expressing ER alpha, and that CA12 regulation by estrogen is a primary transcriptional response mediated by ER alpha. By genome-wide chromatin immunoprecipitation (ChIP) and ChIP scanning of the CA12 locus, we find E2-occupied ER alpha is recruited to a distal region 6.1 kb upstream of the CA12 transcription start site (TSS) in vivo. We find that E2 treatment results in recruitment of RNA polymerase II and steroid receptor coactivators SRC-2 and SRC-3 to the CA12 genomic locus and is correlated with increased histone H4 acetylation. Mutagenesis of an imperfect estrogen-responsive element within this -6.1kb distal enhancer region abolishes estrogen-dependent heterologous reporter activity. Chromosome conformation capture (3C) and chromatin immunoprecipitation assays demonstrate that this distal enhancer communicates with the transcriptional start site of the CA12 gene via intra-chromosomal looping upon hormone treatment. This distal enhancer element is observed in the homologous mouse genomic sequence, and the expression of the mouse homolog, Car12, is rapidly and robustly stimulated by estradiol in the mouse uterus in vivo, suggesting that the ER regulation of CA12 is mechanistically and evolutionarily conserved. Our findings highlight the crucial role of ER in regulation of the CA12 gene, and provide insight into the transcriptional regulatory mechanism that accounts for the strong association of CA12 and ER in human breast cancers. In addition, our findings imply that involvement of long distance enhancers in regulation of estrogen-responsive genes in breast cancer may be more frequent than previously appreciated

Endocrine therapy resistant ESR1 variants revealed by genomic characterization of breast cancer derived xenografts

To characterize patient-derived xenografts (PDXs) for functional studies, we made whole-genome comparisons with originating breast cancers representative of the major intrinsic subtypes. Structural and copy number aberrations were found to be retained with high fidelity. However, at the single-nucleotide level, variable numbers of PDX-specific somatic events were documented, although they were only rarely functionally significant. Variant allele frequencies were often preserved in the PDXs, demonstrating that clonal representation can be transplantable. Estrogen-receptor-positive PDXs were associated with ESR1 ligand-binding-domain mutations, gene amplification, or an ESR1/YAP1 translocation. These events produced different endocrine-therapy-response phenotypes in human, cell line, and PDX endocrine-response studies. Hence, deeply sequenced PDX models are an important resource for the search for genome-forward treatment options and capture endocrine-drug-resistance etiologies that are not observed in standard cell lines. The originating tumor genome provides a benchmark for assessing genetic drift and clonal representation after transplantation

Selective Activation of Alternative MYC Core Promoters by Wnt-Responsive Enhancers.

In Metazoans, transcription of most genes is driven by the use of multiple alternative promoters. Although the precise regulation of alternative promoters is important for proper gene expression, the mechanisms that mediates their differential utilization remains unclear. Here, we investigate how the two alternative promoters (P1, P2) that drive MYC expression are regulated. We find that P1 and P2 can be differentially regulated across cell-types and that their selective usage is largely mediated by distal regulatory sequences. Moreover, we show that in colon carcinoma cells, Wnt-responsive enhancers preferentially upregulate transcription from the P1 promoter using reporter assays and in the context of the endogenous Wnt induction. In addition, multiple enhancer deletions using CRISPR/Cas9 corroborate the regulatory specificity of P1. Finally, we show that preferential activation between Wnt-responsive enhancers and the P1 promoter is influenced by the distinct core promoter elements that are present in the MYC promoters. Taken together, our results provide new insight into how enhancers can specifically target alternative promoters and suggest that formation of these selective interactions could allow more precise combinatorial regulation of transcription initiation

Biopsy confirmation of metastatic sites in breast cancer patients:clinical impact and future perspectives

Determination of hormone receptor (estrogen receptor and progesterone receptor) and human epidermal growth factor receptor 2 status in the primary tumor is clinically relevant to define breast cancer subtypes, clinical outcome,and the choice of therapy. Retrospective and prospective studies suggest that there is substantial discordance in receptor status between primary and recurrent breast cancer. Despite this evidence and current recommendations,the acquisition of tissue from metastatic deposits is not routine practice. As a consequence, therapeutic decisions for treatment in the metastatic setting are based on the features of the primary tumor. Reasons for this attitude include the invasiveness of the procedure and the unreliable outcome of biopsy, in particular for biopsies of lesions at complex visceral sites. Improvements in interventional radiology techniques mean that most metastatic sites are now accessible by minimally invasive methods, including surgery. In our opinion, since biopsies are diagnostic and changes in biological features between the primary and secondary tumors can occur, the routine biopsy of metastatic disease needs to be performed. In this review, we discuss the rationale for biopsy of suspected breast cancer metastases, review issues and caveats surrounding discordance of biomarker status between primary and metastatic tumors, and provide insights for deciding when to perform biopsy of suspected metastases and which one (s) to biopsy. We also speculate on the future translational implications for biopsy of suspected metastatic lesions in the context of clinical trials and the establishment of bio-banks of biopsy material taken from metastatic sites. We believe that such bio-banks will be important for exploring mechanisms of metastasis. In the future,advances in targeted therapy will depend on the availability of metastatic tissue