Transcription profiling by array of LY2 endocrine resistant cell line treated with ICI 182780 to investigate ER-dependent gene expression
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ABSTRACT: ER-dependent gene expression was investigated in the LY2 endocrine resistant cell line by treatment with ICI 182780. Cells were steroid depleted for 3 days prior to treatment with ICI for 6 hours. Four biological replicates were processed and analysed.
Project description:The Estrogen Receptor cofactors SRC1 (NCOA1, KAT13A), SRC2 (NCOA2, GRIP1, TIF2, KAT13C) , SRC3 (NCOA3, AIB1, KAT13B, Rac3) , CBP and p300 are assessed for their genome-wide chromatin binding capacities in the breast cancer cell line MCF7. To determine the Estrogen Receptor dependency of interactions, experiments were performed in the absence of hormone and after Estradiol treatment. In addition, the data were compared with Estrogen Receptor ChIP-seq data from the same timepoint of Estradiol treatment.
Project description:MCF-7 TET Off cells (MCF-7 wt) were used to produce stable clones expressing ER-alpha tagged with TAP-tag at the C-term (C-TAP-ER-alpha). All cells were grown in Dulbecco's modified Eagle's medium (DMEM), starved by using DMEM w/o phenol red and 5% Dextran Coated Charcoal stripped serum (DCC-FBS) for 5 days. Cells were stimulated with 17 beta Estradiol (E2), 4-idrossi-tamoxifen (Tam), raloxifene (Ral) and Fulvestrant (ICI) at the concentration of 10-8M for 12 hours or with vehicle Ethanol. Biological replicate were lysed and RNA extracted were pooled. For mRNA expression profiling, 500 ng total RNA were reverse transcribed and used for synthesis of cDNA and biotinylated cRNA. Finally cRNA were hybridized for 19 hours on Illumina HumanHT-12 v4.0 BeadChips and after scanning, data analysis was performed.
Project description:MCF-7 breast cancer cells were treated with E2, ICI 182,789 (ICI), Raloxifene (Ral), and/or trans-hydroxytamoxifen (TOT) for 8 or 48 h. Duplicate samples are labeled set A or set B.
Project description:Understanding the complex molecular mechanisms underlying resistance to endocrine therapy is a major challenge in the treatment of estrogen receptor-positive (ER+) breast cancers. We have previously demonstrated that glial cell line-derived neurotrophic factor (GDNF) signaling via the receptor tyrosine kinase RET promotes estrogen independent activation of ER. Here we have addressed the relevance of GDNF-RET signaling in response to aromatase inhibitor treatment and explored the efficacy of using RET inhibitors in breast cancer models of aromatase inhibitor response and resistance. A GDNF-response gene set, identified from gene expression profiling, was demonstrated to be an independent prognostic marker of poor patient outcome and, importantly, to be predictive of poor response to aromatase inhibitor treatment and development of resistance. The relevance of these findings was validated first by demonstrating an association of RET protein expression in an independent cohort of aromatase inhibitor resistant patient samples. Second, in in vitro models, GDNF-mediated RET signaling was demonstrated to enhance the survival of aromatase inhibitor resistant cells and to increase resistance in aromatase inhibitor sensitive cells. These effects could be reversed by targeting GDNF/RET signaling with the RET selective inhibitor NVP-BBT594 thus identifying GDNF-RET signaling as a potential therapeutic target, particularly in breast cancers resistant to aromatase inhibitors.MCF7 cells were E2-deprived by culturing in phenol red-free RPMI 1640 supplemented with 10% DCC for 3 days and then serum-starved overnight in the presence or absence of fulvestrant (ICI182,780) (100 nM). The following day, cells were treated with GDNF (20 ng/ml) for 0, 4, 8, 24 and 48 hours in the presence or absence of fulvestrant (ICI182,780) (100 nM).
Project description:The estrogen receptor is the master transcriptional regulator of breast cancer phenotype and the archetype of a molecular therapeutic target. We mapped all estrogen receptor and RNA polymerase II binding sites on a genome-wide scale, identifying the authentic cis binding sites and target genes, in breast cancer cells. Combining this unique resource with gene expression data demonstrates distinct temporal mechanisms of estrogen-mediated gene regulation,particularly in the case of estrogen-suppressed genes. Furthermore, this resource has allowed the identification of cis-regulatory sites in previously unexplored regions of the genome and the cooperating transcription factors underlying estrogen signaling in breast cancer. Experiment Overall Design: This Series currently contains the gene expression data accompaning Carroll JS et al. Nature Genetics 38,1289-1297(2006). MCF7 cells were stimulated with 100 nM estrogen for 0, 3, 6, or 12h. All experiments were performed in triplicate.
Project description:Four vehicle-treated and four HhAntag-treated pancreatic xenograft tumors were profiled for gene expression changes using Affymetrix U133 Plus 2.0 and Affymetrix Mouse Genome 430 2.0 arrays. Keywords: comparative gene expression, hedgehog, hh A primary human pancreatic tumor xenograft (1051178-A) was established by direct implantation of surgical material into female CD1 nu/nu mice of 6-8 weeks of age. Tumors were serially passaged into larger cohorts of mice for efficacy testing and subsequently distributed into tumor volume-matched cohorts upon tumors reaching between 200 to 350 mm3. HhAntag was resuspended in 0.5% methyl-cellulose, 0.2% Tween-80 (MCT) and administered orally twice daily at 75 mg/kg from a 10 mg/ml suspension. MCT alone served as vehicle control. Tumor xenografts (4/group) were excised following 21 days of dosing and RNA was extracted. Preparation of complementary RNA, Human Genome U133 Plus 2.0 array and Mouse Genome 430 2.0 array hybridizations, and subsequent data analysis were carried out using Affymetrix protocols, with signal intensities being determined by the MAS5.0 algorithm.
Project description:Biopsies and surgical specimens were obtained from breast tumours treated and non-treated with celecoxib 400mg twice daily for 2 weeks