ChIP-seq of ER, PR, p300 in two ER+ breast cancer cell lines treated with and without estrogen and with and without progestins
ABSTRACT: Exploring effect of estrogen and progesterone/progestin treatment on ER and PR binding. Two cell lines, four conditions (Vehicle, E2, Progesterone, E2+Progesterone), three factors (ER, PR, p300), all with three replicates.
Project description:Exploring effect of progesterone/progestin treatment on ER and PR binding. Two cell lines, three conditions (Full Media with E2, E2+ Progesterone, Full Media + R5020 Progestin), three factors (ER, PR, p300), all with three replicates, each with a matched Input control.
Project description:Progesterone receptors (PR) are co-expressed in over half of estrogen receptor (ER) positive breast cancers and predict positive response to endocrine therapy. PR can directly and globally modify ER action to attenuate tumor growth. However, whether this suppression occurs solely through PR-ER interactions remains unknown. We assessed tumor growth in two highly ER and PR positive breast cancer patient-derived xenografts (PDX) and found that natural and synthetic progestins potently antagonize the mitogenic effects of estrogens. Here we probed the genome-wide mechanisms by which this occurs. Chronic progestin treatment reversed expression of up to half of estrogen up- and downregulated genes at the transcript level. However, fewer than a quarter of ER DNA binding events were altered by progesterone. The PR cistrome showed an interesting bimodal distribution. In the first group, more than half of PR binding sites were co-occupied by ER, with a propensity for both receptors to coordinately gain or lose binding in the presence of progesterone. In the second group, PR, but not ER, was associated with a large fraction of RNA polymerase III (Pol III)-transcribed tRNA genes regardless of hormone treatment. Furthermore, PR formed a physical association with the Pol III holoenzyme. Select tRNAs with colocalization of PR and POLR3A at their promoters were reduced in tumors grown with estrogen plus progestin compared to estrogen alone. These data uncover a mechanism in solid tumors by which PR modulates the bioavailability of translational molecules that are necessary for robust tumor growth, which could indirectly impede ER action. Overall design: Patient-derived luminal breast cancer xenografts from animals treated with placebo, estradiol (E2), E2+medroxyprogesterone acetate (MPA) ,E2+progesterone (P4), each in triplicate
Project description:Analysis of MCF-7 cells treated for 4h with Ethanol, Estradiol (E2), Dexamethasone (Dex), or Estradiol + Dexamethasone (E2 + Dex) In estrogen receptor (ER)-negative breast cancer (BC), high tumor glucocorticoid receptor (GR) expression has been associated with a relatively poor outcome. In contrast, using a meta-analysis of several genomic datasets, here we find that tumor GR mRNA expression is associated with improved ER+ relapse-free survival (RFS) (independently of progesterone receptor (PR) expression). To understand the mechanism by which GR expression is associated with a better ER+ BC outcome, the global effect of GR-mediated transcriptional activation in ER+ BC cells was studied. Analysis of GR chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) in ER+/GR+ MCF-7 cells revealed that upon co-activation of GR and ER, GR chromatin association became enriched at proximal promoter regions. Furthermore, following ER activation, increased association of GR was observed at ER, FOXO, and AP1 response elements. In addition, it was determined that ER associated with GR response elements, suggesting that ER and GR interact in a complex. Co-activation of GR and ER resulted in increased expression (relative to ER activation alone) of transcripts that encode proteins promoting cellular differentiation (e.g. KDM4B, VDR) and inhibiting Wnt-signaling (IGFBP4). Finally, expression of these individual pro-differentiation genes was associated with significantly improved RFS in ER+ BC patients. Together, these data demonstrate that the co-expression and subsequent activity of tumor cell GR and ER contribute to the less aggressive natural history of early-stage BC by coordinating the altered expression of genes favoring differentiation. Four treatment samples (Vehicle V, Dex D, E2, or Dex+E2). Three biological replicate experiments per sample. Vehicle sample is Ethanol control.
Project description:Exploring effect of progesterone/progestin treatment on gene expression Overall design: Two cell lines, three conditions (Full Media with E2, E2+ Progesterone, Full Media + R5020 Progestin)
Project description:Progesterone and estrogen are important drivers of breast cancer proliferation. Herein, we probed estrogen receptor-α (ER) and progesterone receptor (PR) cross-talk in breast cancer models. Stable expression of PR-B in PR-low/ER+ MCF7 cells increased cellular sensitivity to estradiol and insulin-like growth factor 1 (IGF1), as measured in growth assays performed in the absence of exogenous progestin; similar results were obtained in PR-null/ER+ T47D cells stably expressing PR-B. Genome-wide microarray analyses revealed that unliganded PR-B induced robust expression of a subset of estradiol-responsive ER target genes, including cathepsin-D (CTSD). Estradiol-treated MCF7 cells stably expressing PR-B exhibited enhanced ER Ser167 phosphorylation and recruitment of ER, PR and the proline-, glutamate- and leucine-rich protein 1 (PELP1) to an estrogen response element in the CTSD distal promoter; this complex co-immunoprecipitated with IGF1 receptor (IGFR1) in whole-cell lysates. Importantly, ER/PR/PELP1 complexes were also detected in human breast cancer samples. Inhibition of IGF1R or phosphoinositide 3-kinase blocked PR-B-dependent CTSD mRNA upregulation in response to estradiol. Similarly, inhibition of IGF1R or PR significantly reduced ER recruitment to the CTSD promoter. Stable knockdown of endogenous PR or onapristone treatment of multiple unmodified breast cancer cell lines blocked estradiol-mediated CTSD induction, inhibited growth in soft agar and partially restored tamoxifen sensitivity of resistant cells. Further, combination treatment of breast cancer cells with both onapristone and IGF1R tyrosine kinase inhibitor AEW541 was more effective than either agent alone. In summary, unliganded PR-B enhanced proliferative responses to estradiol and IGF1 via scaffolding of ER-α/PELP1/IGF1R-containing complexes. Our data provide a strong rationale for targeting PR in combination with ER and IGF1R in patients with luminal breast cancer. Overall design: The study contains 4 different sample groups measured in triplicate, for a total of 12 individual samples (12 arrays). From parental MCF7 human breast cancer cell lines, we created two stable clones expressing either (1) an empty vector (pSG5) or (2) the wild type progesterone receptor isoform B (pSG5-PR-B). These two cell lines were treated with either (1) vehicle control (ethanol) or (2) estradiol 10e-7 M (E2) for 6 hours before total RNA harvest. Thus, the experiment contains two cell lines, and two treatments (4 sample groups) treated and analyzed in triplicate (12 microarrays). Standard Illumina HT-12v4 chip controls were used during hybridization.
Project description:Major roadblocks to developing effective progesterone receptor (PR)-targeted therapies in breast cancer include the lack of highly-specific PR modulators, a poor understanding of the pro- or anti-tumorigenic networks for PR isoforms and ligands, and an incomplete understanding of the cross talk between PR and estrogen receptor (ER) signaling. Through genomic analyses of xenografts treated with various clinically-relevant ER and PR-targeting drugs, we describe how the activation or inhibition of PR differentially reprograms estrogen signaling, resulting in the segregation of transcriptomes into separate PR agonist and antagonist-mediated groups. These findings address an ongoing controversy regarding the clinical utility of PR agonists and antagonists, alone or in combination with tamoxifen, for breast cancer management. Additionally, the two PR isoforms PRA and PRB, bind distinct genomic sites and interact with different sets of co-regulators to differentially modulate estrogen signaling to be either pro- or anti-tumorigenic. Of the two isoforms, PRA inhibited gene expression and ER chromatin binding significantly more than PRB. Differential gene expression was observed in PRA and PRB-rich patient tumors and importantly, PRA-rich gene signatures had poorer survival outcomes. In support of antiprogestin responsiveness of PRA-rich tumors, gene signatures associated with PR antagonists, but not PR agonists, predicted better survival outcomes. This better patient survival associated with PR antagonists versus PR agonists treatments was further reflected in the higher anti-tumor activity of combination therapies of tamoxifen with PR antagonists and modulators. The study suggests that distinguishing common effects observed due to concomitant interaction of another receptor with its ligand (agonist or antagonist) from unique isoform and ligand-specific effects will guide the development of biomarkers for patient selection and translation of PR-targeted therapies to the clinic. Overall design: For the xenograft studies involving PR agonists (progesterone, MPA and R5020), PR antagonists (CDB4124, CDB4453 and EC313) and SERMs (tamoxifen, raloxifene, bazedoxifene and fulvestrant), mice harboring T47D xenografts were injected intra-peritoneal for three weeks with 10 mg/kg/day of the respective drugs. Five injections per week were administered. At the end of the duration of the experiment, mice were sacrificed; tumors were excised, weighed, fixed and snap-frozen for later analyses. RNA-seq was performed on these harvested xenografts.
Project description:Immuno-precipitation followed by MS was performed using the RIME protocol in this study. Estrogen Receptor (ER) and Progestorone Receptor (PR) were targetted using antibodies. The experiments were performed in a quantitative manner using SILAC labelling. Cells grown in complete serum (estrogenic) conditions were compared against an cells in similar complete media, however supplemented with Progesterone (PG) or R5020 for 4 hours. Experiments were performed in MCF7 and T47D cell lines
Project description:We performed ChIP seq experiment in MDA-MB-134 cell line in order to map the estrogen receptor alpha (ER) binding sites following the estrogen treatment in an ILC model. We have characterized the genome wide recruit of ER and scaned the binding sites for the presence of cofactor motifs. The binding peaks were also correlated to E2 regulated genes in this ILC model. Four samples were subjected to high throughput sequencing: E-ER (estrogen treated followed by ER IP), E-IgG (estrogen treated followed by IgG), V-ER (EtOH treated followed by ER IP) and Input (MCF7 genomic DNA)
Project description:Disruption of Estrogen Signaling Enhances Invasiveness of Breast Cancer Cells by Attenuating a HER2-independent Gene Repression Program Overall design: In ER+ breast cancer cells, genes that were directly or indirectly repressed by E2, but not E2-activated genes, overlapped the gene overexpression signature of clinical progression of ductal carcinoma in situ to invasive ductal carcinoma. They also showed a strong collective functional bias toward tumor progression. In MCF-7 (ER+/PR+), ZR-75-1 (ER+/PR+) and BT474 (ER+/PR+/HER2 amplified) cells hormone depletion or tamoxifen treatment restored gene expression and invasiveness that were inhibited by E2.