Target Gene Expression In Response Research Articles

Abstract 2792: Development of pharmacodynamic assays for quantifying SMARCA2 protein degradation and target gene expression in response to a SMARCA2 degrader (PRT3789)

Abstract The SWI/SNF complex plays an important role in controlling gene expression via chromatin remodeling. One of its catalytic subunits, SMARCA4, is frequently mutated in multiple tumor types, and SMARCA4-deficient cells are highly dependent on the other catalytic subunit, SMARCA2, for survival. Therefore, targeting SMARCA2 with selective protein degraders has therapeutic potential in SMARCA4 deficient human cancers. We have previously described the development of a potent and selective SMARCA2 targeted degrader, PRT3789, that demonstrates robust degradation of SMARCA2 protein and excellent preclinical efficacy in SMARCA4-del models (Hulse, et al. 2022). PRT3789 will be evaluated in a Phase 1 clinical trial in patients with SMARCA4-mutant cancer. In order to assess target engagement following clinical administration of PRT3789, we developed two pharmacodynamic (PD) assays to measure SMARCA2 protein degradation and changes in SMARCA2 target gene expression in human peripheral mononuclear cells (PBMCs). We used the MSD® S-PLEX platform for the development of a sensitive and quantitative, plate-based immunoassay for determining the protein concentration of SMARCA2 in human PBMC lysates. This assay exhibits a wide dynamic range of detection for SMARCA2 with an LLOD at 1.5 pg/mL, as well as exhibiting acceptable spike recovery and minimal cross-reactivity to SMARCA4. In addition, we optimized the PBMC isolation protocol and lysis conditions to enable sensitive detection of SMARCA2 protein. In order to demonstrate downstream gene expression changes as a consequence of SMARCA2 protein degradation, we developed a secondary qPCR assay. PBMCs from healthy human donors were treated ex vivo with PRT3789 for 24 - 48 hours, followed by RNA-sequencing to identify differentially expressed genes. We refined an 8-gene panel that showed robust expression in PBMCs, as well as consistent and dose-dependent changes in response to PRT3789 treatment. We further calculated a differential gene expression (DGE) score based on expression of this gene panel that correlates with SMARCA2 protein degradation. In summary, we describe the development and characterization of two independent clinically relevant PD assays that can be used to evaluate target engagement in blood samples following PRT3789 administration. Citation Format: Andrew Moore, Carly Bachner, Lalitha Srinivasan, Pradeep Kurup, Alex Grego, Caroline Vitkovitsky, Koichi Ito, Neha Bhagwat, Peggy Scherle. Development of pharmacodynamic assays for quantifying SMARCA2 protein degradation and target gene expression in response to a SMARCA2 degrader (PRT3789) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2792.

CzcR Is Essential for Swimming Motility in Pseudomonas aeruginosa during Zinc Stress.

Two-component system (TCS) plays a vital role in modulating target gene expression in response to the changing environments. Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen that can survive under diverse stress conditions. The great adaptability of P. aeruginosa relies heavily on the abundant TCSs encoded by its genome. However, most TCSs in P. aeruginosa have not been well-characterized. CzcS/CzcR is a metal responsive TCS which displays multiple regulatory functions associated with metal hemostasis, quorum sensing activity and antibiotic resistance. In this study, we found that swimming motility of P. aeruginosa was completely abolished during zinc (Zn2+) stress when the czcR gene from the TCS CzcS/CzcR was deleted. Noticeably, CzcR was dispensable for swimming without the stress of Zn2+ excess. CzcR was shown to be activated by Zn2+ stress possibly through inducing its expression level and triggering its phosphorylation to positively regulate swimming which was abolished by Zn2+ stress in a CzcR-independent manner. Further TEM analyses and promoter activity examinations revealed that CzcR was required for the expression of genes involved in flagellar biosynthesis during Zn2+ stress. In vitro protein-DNA interaction assay showed that CzcR was capable of specifically recognizing and binding to the promoters of operons flgBCDE, flgFGHIJK, and PA1442/FliMNOPQR/flhB. Together, this study demonstrated a novel function of CzcR in regulating flagellar gene expression and motility in P. aeruginosa when the pathogen encounters Zn2+ stress conditions. IMPORTANCE The fitness of bacterial cells depends largely on their ability to sense and respond quickly to the changing environments. P. aeruginosa expresses a great number of signal sensing and transduction systems that enable the pathogen to grow and survive under diverse stress conditions and cause serious infections at different sites in many hosts. In addition to the previously characterized functions to regulate metal homeostasis, quorum sensing activity, and antibiotic resistance, here we report that CzcR is a novel regulator essential for flagellar gene expression and swimming motility in P. aeruginosa during Zn2+ stress. Since swimming motility is important for the virulence of P. aeruginosa, findings in this study might provide a new target for the treatment of P. aeruginosa infections with Zn2+-based antimicrobial agents in the future.

The Mettl3 epitranscriptomic writer amplifies p53 stress responses

The p53 transcription factor drives anti-proliferative gene expression programs in response to diverse stressors, including DNA damage and oncogenic signaling. Here, we seek to uncover new mechanisms through which p53 regulates gene expression using tandem affinity purification/mass spectrometry to identify p53-interacting proteins. This approach identified METTL3, an m6A RNA-methyltransferase complex (MTC) constituent, as a p53 interactor. We find that METTL3 promotes p53 protein stabilization and target gene expression in response to DNA damage and oncogenic signals, by both catalytic activity-dependent and independent mechanisms. METTL3 also enhances p53 tumor suppressor activity in invivo mouse cancer models and human cancer cells. Notably, METTL3 only promotes tumor suppression in the context of intact p53. Analysis of human cancer genome data further supports the notion that the MTC reinforces p53 function in human cancer. Together, these studies reveal a fundamental role for METTL3 in amplifying p53 signaling in response to cellular stress.

Development of picomolar liver receptor homolog‐1 agonist

Nuclear receptors are ligand‐regulated transcription factors that alter target gene expression in response to the binding of lipophilic signaling molecules. Liver receptor homolog‐1 (LRH‐1) is a nuclear receptor that has shown promise as an anti‐diabetic therapeutic in murine studies and regulates bile acid biosynthesis, steroidogenesis, and glucose homeostasis, making it an attractive target for treating a variety of metabolic diseases. Although phospholipids are the putative endogenous ligands for LRH‐1, they are insoluble, readily remodeled and hydrolyzed, and are only efficacious at very high concentrations, making development of synthetic agonists critical for targeting LRH‐1 in clinical and laboratory contexts. Using structural data that delineated the binding pose of the only class of synthetic agonists known for LRH‐1, we have conducted structure‐activity relationship studies to enhance potency, efficacy, and solubility of small molecules. Specifically, we targeted residues deep within the binding pocket that radically enhance potency, which has led to the first low‐nanomolar LRH‐1 agonist (“6N”). We have also targeted residues contacted by the headgroups of activating phospholipids as a complementary strategy for activation. These modifications resulted in our most efficacious small molecule (“10CA”). To capture the characteristics of both compounds (6N and 10CA), we have synthesized a chimeric small molecule that includes the key moieties of 6N and 10CA responsible for high affinity and efficacy, respectively. Excitingly, this new hybrid compound binds with picomolar affinity, is more efficacious than 10CA, and elicits LRH‐1 target gene expression to a greater degree than either 6N or 10CA alone. The hybrid molecule is our most effective agonist to date, providing a critical tool for both studying LRH‐1 biology and treating a range of metabolic disorders.Support or Funding InformationNIH‐5T32GM008367‐29 (BCDB Training Grant) | NIH‐5R01DK115213‐02 | Emory Catalyst ProgramBinding mode and residue contacts of leading agonists.Figure 1Binding and in‐cell reporter data demonstrating affinity, potency, and efficacy of the hybrid compound relative to our two leading agonists.Figure 2

Alteration of microRNA 340-5p and Arginase-1 Expression in Peripheral Blood Cells during Acute Ischemic Stroke.

Acute stroke alters the systemic immune response as can be observed in peripheral blood; however, the molecular mechanism by which microRNA (miRNA) regulates target gene expression in response to acute stroke is unknown. We performed a miRNA microarray on the peripheral blood of 10 patients with acute ischemic stroke and 11 control subjects. Selected miRNAs were quantified using a TaqMan assay. After searching for putative targets from the selected miRNAs using bioinformatic analysis, functional studies including binding capacity and protein expression of the targets of the selected miRNAs were performed. The results reveal a total of 30 miRNAs that were differentially expressed (16 miRNAs were upregulated and 14 miRNAs were downregulated) during the acute phase of stroke. Using prediction analysis, we found that miR-340-5p was predicted to bind to the 3'-untranslated region of the arginase-1 (ARG1) gene; a luciferase reporter assay confirmed the binding of miR-340-5p to ARG1. miR-340-5p was downregulated whereas ARG1 mRNA was upregulated in peripheral blood in patients experiencing acute stroke. Overexpression of miR-340-5p in human neutrophil and mouse macrophage cell lines induced downregulation of the ARG1 protein. Transfection with miR-340-5p increased nitric oxide production after LPS treatment in a mouse macrophage cell line. Our results suggest that several miRNAs are dynamically altered in the peripheral blood during the acute phase of ischemic stroke, including miR-340-5p. Acute stroke induces the downregulation of miR-340-5p, which subsequently upregulates ARG1 protein expression.

Revisiting progesterone receptor (PR) actions in breast cancer: Insights into PR repressive functions

Progesterone receptor (PR) is a master regulator in female reproductive tissues that controls developmental processes and proliferation and differentiation during the reproductive cycle and pregnancy. PR also plays a role in progression of endocrine-dependent breast cancer. As a member of the nuclear receptor family of ligand-dependent transcription factors, the main action of PR is to regulate networks of target gene expression in response to binding its cognate steroid hormone, progesterone. Liganded-PR transcriptional activation has been thoroughly studied and associated mechanisms have been described while progesterone-mediated repression has remained less explored. The present work summarizes recent advances in the understanding of how PR-mediated repression is accomplished in breast cancer cells and highlights the significance of fully understanding the determinants of context-dependent PR action.

MiR430: the novel heat-responsive microRNA identified from miRNome analysis in wheat (Triticum aestivum L.)

miRNAs are 21–24 nt non-coding RNAs involved in negative regulation of the target gene expression in response to stress and developmental cues. Wheat, the major staple food crop, is highly sensitive to heat stress (HS). Even moderate high temperatures during critical stages causes reduction in yield and quality of the grains. Here, we identified a novel miRNA (candidate miR430) from wheat using de novo assembly, and cloned it from wheat cv. HD2985 using universal adaptor. The identified miRNA was mapped on to the Chromosome 3B and predicted to have more than 30 target genes. Most of the targets identified were associated with heat shock proteins. Expression analysis of miR430 in contrasting wheat cultivars by quantitative real-time PCR revealed significant genotypic variations under HS. Tissue specific expression analysis showed relatively low expression in leaves, as compared with stem and root under HS. A negative correlation was found between the expression of miR430 and their respective target genes under HS. MiR430 can be used to manipulate the expression of target genes under HS towards enhancing HS tolerance for the development of ‘climate-smart’ wheat crop.

Type I Interferon Supports Inducible Nitric Oxide Synthase in Murine Hepatoma Cells and Hepatocytes and during Experimental Acetaminophen-Induced Liver Damage.

Cytokine regulation of high-output nitric oxide (NO) derived from inducible NO synthase (iNOS) is critically involved in inflammation biology and host defense. Herein, we set out to characterize the role of type I interferon (IFN) as potential regulator of hepatic iNOS in vitro and in vivo. In this regard, we identified in murine Hepa1-6 hepatoma cells a potent synergism between pro-inflammatory interleukin-β/tumor necrosis factor-α and immunoregulatory IFNβ as detected by analysis of iNOS expression and nitrite release. Upregulation of iNOS by IFNβ coincided with enhanced binding of signal transducer and activator of transcription-1 to a regulatory region at the murine iNOS promoter known to support target gene expression in response to this signaling pathway. Synergistic iNOS induction under the influence of IFNβ was confirmed in alternate murine Hepa56.1D hepatoma cells and primary hepatocytes. To assess iNOS regulation by type I IFN in vivo, murine acetaminophen (APAP)-induced sterile liver inflammation was investigated. In this model of acute liver injury, excessive necroinflammation drives iNOS expression in diverse liver cell types, among others hepatocytes. Herein, we demonstrate impaired iNOS expression in type I IFN receptor-deficient mice which associated with diminished APAP-induced liver damage. Data presented indicate a vital role of type I IFN within the inflamed liver for fine-tuning pathological processes such as overt iNOS expression.

X-linked Dystonia-Parkinsonism patient cells exhibit altered signaling via nuclear factor-kappa B

X-linked Dystonia-Parkinsonism (XDP) is a progressive neurodegenerative disease involving the loss of medium spiny neurons within the striatum. An XDP-specific haplotype has been identified, consisting of seven sequence variants which cluster around the human TAF1 gene, but a direct relationship between any of these variants and disease pathogenesis has not yet been demonstrated. Because the pathogenic gene lesion remains unclear, it has been difficult to predict cellular pathways which are affected in XDP cells. To address that issue, we assayed expression of defined gene sets in XDP vs. control fibroblasts to identify networks of functionally-related transcripts which may be dysregulated in XDP patient cells. That analysis derived a 51-gene signature distinguishing XDP vs. control fibroblasts which mapped strongly to nuclear factor-kappa B (NFκB), a transcription factor pathway also implicated in the pathogenesis of other neurodegenerative diseases, including Parkinson's (PD) and Huntington's disease (HD). Constitutive and TNFα-evoked NFκB signaling was further evaluated in XDP vs. control fibroblasts based on luciferase reporter activity, DNA binding of NFκB subunits, and endogenous target gene transcription. Compared to control cells, XDP fibroblasts exhibited decreased basal NFκB activity and decreased levels of the active NFκB p50 subunit, but increased target gene expression in response to TNFα. NFκB signaling was further examined in neural stem cells differentiated from XDP and control induced pluripotent stem cell (iPSC) lines, revealing a similar pattern of increased TNFα responses in the patient lines compared to controls. These data indicate that an NFκB signaling phenotype is present in both patient fibroblasts and neural stem cells, suggesting this pathway as a site of dysfunction in XDP.

ΔN-ASPP2, a novel isoform of the ASPP2 tumor suppressor, promotes cellular survival

ASPP2 is a tumor suppressor that works, at least in part, through enhancing p53-dependent apoptosis. We now describe a new ASPP2 isoform, ΔN-ASPP2, generated from an internal transcription start site that encodes an N-terminally truncated protein missing a predicted 254 amino acids. ΔN-ASPP2 suppresses p53 target gene transactivation, promoter occupancy, and endogenous p53 target gene expression in response to DNA damage. Moreover, ΔN-ASPP2 promotes progression through the cell cycle, as well as resistance to genotoxic stress-induced growth inhibition and apoptosis. Additionally, we found that ΔN-ASPP2 expression is increased in human breast tumors as compared to adjacent normal breast tissue; in contrast, ASPP2 is suppressed in the majority of these breast tumors. Together, our results provide insight into how this new ASPP2 isoform may play a role in regulating the ASPP2-p53 axis.

Auxin-dependent compositional change in Mediator in ARF7- and ARF19-mediated transcription

Mediator is a multiprotein complex that integrates the signals from transcription factors binding to the promoter and transmits them to achieve gene transcription. The subunits of Mediator complex reside in four modules: the head, middle, tail, and dissociable CDK8 kinase module (CKM). The head, middle, and tail modules form the core Mediator complex, and the association of CKM can modify the function of Mediator in transcription. Here, we show genetic and biochemical evidence that CKM-associated Mediator transmits auxin-dependent transcriptional repression in lateral root (LR) formation. The AUXIN/INDOLE 3-ACETIC ACID 14 (Aux/IAA14) transcriptional repressor inhibits the transcriptional activity of its binding partners AUXIN RESPONSE FACTOR 7 (ARF7) and ARF19 by making a complex with the CKM-associated Mediator. In addition, TOPLESS (TPL), a transcriptional corepressor, forms a bridge between IAA14 and the CKM component MED13 through the physical interaction. ChIP assays show that auxin induces the dissociation of MED13 but not the tail module component MED25 from the ARF7 binding region upstream of its target gene. These findings indicate that auxin-induced degradation of IAA14 changes the module composition of Mediator interacting with ARF7 and ARF19 in the upstream region of their target genes involved in LR formation. We suggest that this regulation leads to a quick switch of signal transmission from ARFs to target gene expression in response to auxin.

Editorial: Recent Advances of Epigenetics in Crop Biotechnology.

Editorial: Recent Advances of Epigenetics in Crop Biotechnology.

Maize miRNA and target regulation in response to hormone depletion and light exposure during somatic embryogenesis.

Maize somatic embryogenesis (SE) is induced from the immature zygotic embryo in darkness and under the appropriate hormones' levels. Small RNA expression is reprogrammed and certain miRNAs become particularly enriched during induction while others, characteristic to the zygotic embryo, decrease. To explore the impact of different environmental cues on miRNA regulation in maize SE, we tested specific miRNA abundance and their target gene expression in response to photoperiod and hormone depletion for two different maize cultivars (VS-535 and H-565). The expression levels of miR156, miR159, miR164, miR168, miR397, miR398, miR408, miR528, and some predicted targets (SBP23, GA-MYB, CUC2, AGO1c, LAC2, SOD9, GR1, SOD1A, PLC) were examined upon staged hormone depletion in the presence of light photoperiod or darkness. Almost all examined miRNA, except miR159, increased upon hormone depletion, regardless photoperiod absence/presence. miR528, miR408, and miR398 changed the most. On the other hand, expression of miRNA target genes was strongly regulated by the photoperiod exposure. Stress-related miRNA targets showed greater differences between cultivars than development-related targets. miRNA/target inverse relationship was more frequently observed in darkness than light. Interestingly, miR528, but not miR159, miR168 or miR398, was located on polyribosome fractions suggesting a role for this miRNA at the level of translation. Overall our results demonstrate that hormone depletion exerts a great influence on specific miRNA expression during plant regeneration independently of light. However, their targets are additionally influenced by the presence of photoperiod. The reproducibility or differences observed for particular miRNA-target regulation between two different highly embryogenic genotypes provide clues for conserved miRNA roles within the SE process.

Rational Re-engineering of a Transcriptional Silencing PreQ1 Riboswitch.

Re-engineered riboswitches that no longer respond to cellular metabolites, but that instead can be controlled by synthetic molecules, are potentially useful gene regulatory tools for use in synthetic biology and biotechnology fields. Previously, extensive genetic selection and screening approaches were employed to re-engineer a natural adenine riboswitch to create orthogonal ON-switches, enabling translational control of target gene expression in response to synthetic ligands. Here, we describe how a rational targeted approach was used to re-engineer the PreQ1 riboswitch from Bacillus subtilis into an orthogonal OFF-switch. In this case, the evaluation of just six synthetic compounds with seven riboswitch mutants led to the identification of an orthogonal riboswitch-ligand pairing that effectively repressed the transcription of selected genes in B. subtilis. The streamlining of the re-engineering approach, and its extension to a second class of riboswitches, provides a methodological platform for the creation of new orthogonal regulatory components for biotechnological applications including gene functional analysis and antimicrobial target validation and screening.

Specific phosphorylation of histone demethylase KDM3A determines target gene expression in response to heat shock.

Author SummaryHistone methylation regulates gene expression and can have drastic consequences for health if the process is defective. Histone lysine demethylases (KDMs) counteract the activity of methyl-transferases and remove methyl group(s) from histones. KDM3A is a H3K9me2/1 demethylase that performs diverse functions via the regulation of its target genes, which are involved in spermatogenesis, metabolism, and cell differentiation. However, the mechanisms underlying KDM3A regulation of specific genes at specific times are largely unknown. Here we found that a physiological stress—elevated temperature—induces KDM3A phosphorylation in human cells via the MSK1 kinase. This phosphorylated form of KDM3A directly interacts with the transcription factor Stat1, which enables Stat1 to recruit KDM3A to Stat1-binding sequences at the promoters of specific target genes. KDM3A then acts to demethylate H3K9me2/1 at these targets, thereby causing specific gene expression in response to the thermal stress. We conclude that heat shock can affect the expression of many genes in human cells via a novel activation mechanism that is centered around the phosphorylation of KDM3A.

Abstract IA04: EWS-FLI1, polycomb proteins and stem cells: Clues to the developmental origins of Ewing sarcoma

Abstract IA04: EWS-FLI1, polycomb proteins and stem cells: Clues to the developmental origins of Ewing sarcoma

Discovery of Pantoea rodasii strain ND03 that produces N-(3-Oxo-hexanoyl)-L-homoserine lactone.

Proteobacteria use quorum sensing to regulate target gene expression in response to population density. Quorum sensing (QS) is achieved via so-called signalling molecules and the best-studied QS signalling system uses N-acyl homoserine lactones (AHLs). This study aimed to identify and characterize the production of AHLs by a bacterium ND03 isolated from a Malaysian tropical rainforest waterfall. Molecular identification showed that ND03 is a Pantoea sp. closely related to Pantoea rodasii. We used Chromobacterium violaceum CV026, an AHL biosensor for preliminary AHL production screening and then used high resolution triple quadrupole liquid chromatography-mass spectrometry, to confirm that P. rodasii strain ND03 produced N-(3-oxo-hexanoyl)-l-homoserine lactone (3-oxo-C6-HSL). To the best of our knowledge, this is the first report for such a discovery in P. rodasii strain ND03.

G Protein–Coupled Receptors Revisited: Therapeutic Applications Inspired by Synthetic Biology

G protein-coupled receptors (GPCRs) mediate the majority of cellular responses to hormones and neurotransmitters within the human body. They have much potential in the emerging field of synthetic biology, which is the rational, systematic design of biological systems with desired functionality. The responsiveness of GPCRs to a plethora of endogenous and exogenous ligands and stimuli make them ideal sensory receptor modules of synthetic gene networks. Such networks can activate target gene expression in response to a specific stimulus. Additionally, because GPCRs are important pharmacological targets of various human diseases, genes encoding their protein/peptide ligands can also be incorporated as target genes of the response output elements of synthetic gene networks. This review aims to critically examine the potential role of GPCRs in constructing therapeutic synthetic gene networks and to discuss various challenges in utilizing GPCRs for synthetic biology applications.

MondoA senses adenine nucleotides: transcriptional induction of thioredoxin-interacting protein

The MondoA-Mlx transcription complex plays a pivotal role in glucose homoeostasis by activating target gene expression in response to G6P (glucose 6-phosphate), the first reaction intermediate in glycolysis. TXNIP (thioredoxin-interacting protein) is a direct and glucose-responsive target of MondoA that triggers a negative-feedback loop by restricting glucose uptake when G6P levels increase. We show in the present study that TXNIP expression is also activated by AICAR (5-amino-4-imidazolecarboxamide ribofuranoside) and adenosine. Using pharmacological inhibitors and genetic knockdowns of purine metabolic enzymes, we establish that TXNIP induction by AICAR and adenosine requires their cellular uptake and metabolism to adenine nucleotides. AICAR induction of TXNIP depended on MondoA, but was independent of AMPK (AMP-activated protein kinase) activation and calcium. The findings of the present study have two important implications. First, in addition to activating AMPK, AICAR may have AMPK-independent effects on gene expression by regulating MondoA-Mlx activity following its flux into the adenine nucleotide pool. Secondly, MondoA-Mlx complexes sense elevated levels of G6P and adenine nucleotides to trigger a TXNIP-dependent feedback inhibition of glycolysis. We propose that this mechanism serves as a checkpoint to restore metabolic homoeostasis.

Differential gene expression in ERα-positive and ERα-negative breast cancer cells upon leptin stimulation

In postmenopausal women, adipositas represents a serious risk factor for cancer development and progression. White adipose tissue secretes the 16 kDa hormone leptin which plays a key role in the regulation of appetite and metabolism. An increasing number of reports indicate that leptin also interferes with signal transduction pathways implicated in the development of breast cancer. In our previous study, we identified the estrogen receptor alpha (ERα) as a relevant enhancer of leptin-induced signal transduction leading to transactivation of signal transducer and activator of transcription 3 (Stat3). The purpose of this study is the investigation of specific target gene expression in response to leptin-mediated Stat3 signaling. We performed a comprehensive microarray analysis of ERα-positive and ERα-negative MDA-MB-231 cells upon leptin treatment and identified 49 genes which showed a significant ERα-dependent regulation in leptin-treated MDA-MB-231 cells. There was no intersection with genes which were merely up- or downregulated by ERα expression and only 9 and 11 genes overlapping targets which were regulated by leptin stimulation either in ERα-expressing or ERα-negative MDA-MB-231 cells, respectively. To demonstrate the specificity, expression of three target genes was validated by quantitative real-time PCR. In conclusion, these data imply that leptin can induce a different set of target genes dependent on ERα expression, which might contribute to the development and progression of cancer diseases.