ESR1 (Estrogen Specific Receptor 1) is a member of the nuclear hormone receptor (NHR) family. NHRs bind to steroid hormones in the cytoplasm, causing their translocation to the nucleus and increased affinity for transcriptional elements governing the expression of certain genes (1). NHRs activate genes leading to cell proliferation and differentiation, with the level of activity dependent on the target tissue. ESR1, also known as ER alpha, is up-regulated in Estrogen Receptor (ER)-positive breast cancers (2). Because steroid hormones can promote the growth of several types of malignancy, such as breast cancer, numerous drug companies have targeted ESR1 for development of antagonists. In this Application Note we examine ESR1 gene expression using Gene Logic's ASCENTA System and demonstrate how this tool can be used to reveal important expression characteristics of this drug target. This report not only confirms published gene expression results for female reproductive system and liver cancers, but also reveals novel findings of ESR1 expression in obesity and diabetes. These results both validate and expand the knowledgebase of ESR1 expression in human disease.
Materials and Methods
The ASCENTA System is a powerful and reliable gene expression database that contains over 8,000 well-characterized samples of normal and diseased tissues, expertly categorized into over 1300 sample sets. These sample sets have been created by Gene Logic's boardcertified pathologists. Samples undergo rigorous pathologic review to assure both their quality and the appropriateness of their inclusion in a particular sample set. Samples that meet pathologic criteria for inclusion in a sample set may still be excluded from that set, based on identification as statistical outliers using Gene Logic's detection algorithm. The mean and median number of samples in the curated sample sets are 18 and 8, respectively. Most samples are from human tissue, but samples from mouse and rat tissues are also included. Each sample is prepared and analyzed using the Affymetrix GeneChip array platform with strict standard operating procedures and rigorous pass/fail criteria for greater reproducibility.
The sample preparation methods include isolation of total RNA, then linear amplification and preparation of biotinylated cRNA, following the recommendations of the GeneChip Expression Analysis Technical Manual. Gene Logic's molecular biologists optimize the methods when necessary to ensure that high quality data is generated from each specimen. Biotin-labeled cRNA is fragmented prior to hybridization on the Affymetrix Human Genome U133 GeneChip array, the Murine Genome U74v2 Set, or the Rat Genome U34 Set. The arrays are hybridized for 24 hours at 45oC, washed and stained with Streptavidin Phycoerythrin (Molecular Probes) in Affymetrix fluidic stations, then detected by fluorometric scanning (Hewlett-Packard GeneArray Scanner). The quality of each microarray image is carefully examined for chip defects or abnormalities in hybridization signal prior to analysis with Affymetrix Microarray Suite (v. 5.0) and LIMS (v. 3.0). No data are entered into the database without passing more than 30 quality control metrics developed by Gene Logic.
For gene targets, the specificity of gene expression in human tissues is a common metric that is used to profile and qualify them as potential drug targets. ESR1 gene expression intensities were examined across a panel of 54 normal human tissues. Figure 1 shows a subset of the e-Northern report highlighting normal human tissue sample sets that display the highest expression levels of ESR1. Female reproductive tissues (uterus, fallopian tube, breast and ovary) have the highest degree of ESR1 expression, followed by liver, artery, prostate, skeletal muscle, skin and adipose tissues. ESR1 gene expression was notably absent, or below the limit of detection, in cells and tissues comprising the immune and central nervous systems (data not shown).
Knowledge of gene target expression levels in human cell lines is often used as a criterion to select specific cell types for further in vitro validation of genes as drug targets. For example, cell lines that highly express a gene target can be used for RNAi studies to determine the biological effect of "knock-down", whereas cell lines that do not express specific gene targets can be used for gene transfection studies to determine the impact of increased expression. We examined ESR1 gene expression levels in a panel of 60 commonly used human-derived cell lines (referred to as the "NCI-60 panel"), many of which are directly applicable to oncology studies. The ESR1 e-Northern report from the NCI-60 panel (data not shown) demonstrated that ESR1 was highly expressed in the MCF7 and T47D breast cancer cell lines and to a lesser yet significant level in the SK-OV-3 ovarian cancer cell line. ESR1 expression was not detected in several prostate, lung and colonderived cell lines.
The specificity and extent of expression regulation of a gene target with respect to human disease is another important metric that is used to select and prioritize genes as candidates for further development in drug discovery R&D pipelines. The ASCENTA System provides readily available gene expression regulation information in the form of a Diff/X report, which lists statistically significant regulation events in hundreds of human diseases with respect to control or normal morphologies, all contained within five major therapeutic areas: Oncology, Inflammation, Cardiovascular Disease, Central Nervous System Disorders and Metabolic Diseases. This information can be used to examine the specificity and scope of expression regulation to validate genes as priority drug targets for treatment of a specific disease or to uncover additional disease indications that may broaden the importance and applicability of gene targets as drug candidates.
The Diff/X report in Figure 2 shows that ESR1 was elevated (positive fold change) in ERpositive (ER+) infiltrating ductal carcinoma of the breast (the most common histologic variant), relative to normal and fibrocystic breast tissue and to ER-negative (ER-) carcinoma of the same histologic type. These data are in agreement with what has been reported in the literature (2).
Additional results in oncology revealed a significant decrease in ESR1 expression in a variety of conditions leading to cirrhosis of the liver and more marked downregulation in hepatocellular carcinoma (Figure 3). The presence of cirrhosis (a non-malignant condition) is known to significantly increase the risk of developing hepatocellular carcinoma. This correlation of down-regulated ESR1 expression with liver cirrhosis and hepatocellular carcinoma is an interesting finding, as ESR1 has recently been shown to be down regulated in hepatocellular carcinomas (3), but has not been reported in liver cirrhosis.
The ASCENTA System also identified additional novel therapeutic indications for ESR1 (Figure 4) in metabolic diseases. ESR1 expression was down-regulated in morphologically normal liver tissue from diabetic patients as compared to tissue from nondiabetic patients; and in skeletal muscle from obese and/or diabetic patients as compared to non-obese and/or non-diabetic patients. It is known that ESR1 knock-out mice show adipocyte hyperplasia and hypertrophy (i.e. obesity), as well as insulin resistance (a feature of diabetes) (4). These mouse data, combined with the findings in the ASCENTA System, indicate that regulation of ESR1 in obesity and diabetes is worth further investigation and may provide clues about additional disease application areas for this drug target. The ESR1 expression changes found by the ASCENTA System in obese and diabetic human liver and skeletal muscle tissue samples are indeed novel findings.
For many gene targets, it is often appropriate to investigate their expression metrics within the broadened context of a gene family or a biological pathway to which they belong. Such investigations can often reveal novel expression relationships between members of gene families or pathway interactors which may provide more insight into the mechanism of human disease and reveal additional related targets to be considered as biomarkers or drug candidates themselves.
Gene Logic has curated and annotated members of "drug-able" gene families and key regulatory biological pathways and provides these as compiled collections of genes in the ASCENTA System. The gene families include many commonly investigated groups such as G-protein coupled receptors and protein kinases. Biological pathways are from the Kyoto Encyclopedia of Genes and Genomes (KEGG) and BioCarta. Since ESR1 is a member of the Nuclear Hormone Receptor (NHR) family, one can select the NHR family in the Gene Family Browser, quickly review detailed information about NHRs with links to other information sources, view a compiled list of curated family members, and obtain a Diff/X report of differential gene expression of NHRs in all or any one of the five major therapeutic areas (Figure 5, left panel). Detailed information about two pathways (of which ESR1 is a member) is easily accessed through the Pathway Browser (Figure 5, right panel). These two pathways are titled "CARM1 and Regulation of the Estrogen Receptor" and "Pelp1 Modulation of Estrogen Receptor Activity". CARM1 (coactivator-associated arginine methyltransferase) methylates many proteins that modulate transcriptional regulation and is a coactivator of gene transcription for ESR1. Pelp1 (Proline-, glutamic acid-, leucine-rich protein 1) is a scaffold protein that may mediate the rapid cellular proliferation response transduced by ESR1 in response to estrogen binding. These pathways can be used as gene sets in the ASCENTA System to explore how they are regulated in the aforementioned oncology and metabolic disease therapeutic areas that display ESR1 regulation. These two pathway reports may provide new insight into the disease-specific gene expression modulation of ESR1 and related gene targets for followup studies.
These findings, combined with the pathway analysis for ESR1 seen in Figure 5, provide a global picture of ESR1 function in both normal biology and in human disease. This gene is clearly involved in cellular proliferation, transcriptional activation, and growth in response to steroid hormones. Regulation of this gene or other members of the cellular machinery with which it interacts can lead to abnormal growth conditions such as cancer. A broader understanding of the biological networks related to ESR1 function is easily obtained using the ASCENTA System. This global perspective may further facilitate the identification of additional pathways or gene families that could serve as alternative targets for novel disease therapeutics.
This Application Note demonstrates how the ASCENTA System can be used to quickly and easily examine expression levels of target genes, such as ESR1, across pre-selected tissue sample sets to determine where they are differentially regulated in hundreds of disease morphologies. In addition, the advanced analysis features of the ASCENTA System, such as pathway analysis, clearly demonstrate how a global genomic perspective of target regulation can broaden the scope of discovery and lead to alternative, complementary, or possibly better targets for drug design. In aggregate, these data can be used to prioritize and validate gene targets for further therapeutic development.
ESR1 gene expression was observed in several normal human tissues, but to a much higher extent in female reproductive tissues. Disease-specific regulation of ESR1 gene expression was shown in breast and liver cancer, which largely confirms published observations. We extended ESR1 gene expression findings by demonstrating significant regulation in liver cirrhosis and in metabolic diseases (obesity and diabetes) in liver and skeletal muscle tissue. These data may be useful not only to confirm and validate ESR1 as a viable gene target in oncology, but also to consider ESR1 as a useful target or biological marker in other disease and therapeutic areas. The ASCENTA System provides a unique opportunity to gain access to comprehensive gene expression data which leads Figure 5 - Gene Family and Pathway Reports. These reports contain comprehensive information on how members of gene families and pathways are differentially regulated in human diseases. Reports contain descriptions, lists of annotated members as well as an aggregated Diff/X report which reveals gene regulation data for all members of the data set. to both an in-depth and broader understanding of how drug targets are expressed and regulated with respect to human disease.
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2. Cancer Res. (1991) 51: 817-1822.
3. Liver Int. (2003) 23: 63-69.
4. Proc. Natl. Acad. Sci. USA (2000) 97:12729-12734.
For more information about Gene Logic's ASCENTA System, visit Gene Logic's web site at www.genelogic.com/solutions/ascenta and access the ASCENTA System overview and animated presentations on "ASCENTA in Action". To have a Gene Logic representative contact you, please email firstname.lastname@example.org or call 1-800-GENELOGIC.