Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of human MCF7 cells were infected with adenovirus overexpressing one of the four proteins (PGC1 WT, PGC1 2x9, PGC1 L2L3 and BGAL) reveals cross-talk between Estrogen Receptor-related Receptor alpha (ERRalpha) and Estrogen Receptor (ERalpha) pathways.

ABSTRACT: Orphan nuclear receptor estrogen-related receptor alpha (ERRÎ±) has recently been shown to carry negative prognostic significance in breast and ovarian cancers. The specific role of ERRÎ± in tumor growth and progression, however, is yet to be fully understood. The significant homology between estrogen receptor alpha (ERÎ±) and ERRÎ± initially suggested that these receptors may share similar transcriptional targets. Using the well-characterized ERÎ±-positive MCF-7 breast cancer cell line, we sought to gain a genome-wide picture of ERÎ±-ERRÎ± cross-talk using an unbiased microarray approach. Since a small molecule ligand has not been identified for ERRÎ±, its transcriptional activity in these studies was induced using its known coactivator PGC-1Î± (peroxisome proliferator-activated receptor-Î³ coactivator-1Î±) as a protein ligand. Both wild-type PGC1, as well as ERRa-specific variant (PGC1 2x9) were used to activate ERRa. Non-NR-dependent activities of PGC-1Î± were controlled for using a variant PGC-1Î± in which the leucines within the NR-interacting domain had been mutated to alanines (L2L3M). Beta-galactosidase (BGAL) overexpression was used as a non-specific background control. Activation of endogenous ER was achieved by treatment with 10 nM estadiol. Experiment Overall Design: MCF7 cells were infected with adenovirus overexpressing one of the four proteins (PGC1 WT, PGC1 2x9, PGC1 L2L3 and BGAL); Twelve hours post infection cells were then treated with either 10 nM estradiol (E2)or ethanol vehicle for 12 hours. Each combination of infection and treatment was was repeated three times in three independent experiments. A total of 23 samples are presented. Two replicates instead of three are shown for PGC1 WT E2 for technical reasons.

ORGANISM(S): Homo sapiens

SUBMITTER: Dmitri Kazmin

PROVIDER: E-GEOD-11266 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:This dataset is part of the manuscript titled "The metabolic regulator ERRalpha, a downstream target of HER2/IGF1, as a therapeutic target in breast cancer" (in review). The expression data obtained in human mammary epithelial cells were used to generate a list of ERRalpha-regulated genes that was later refined in clinical breast cancer datasets to generate a clinically relevant signature of ERalpha activity (referred to as Cluster 3 signature). Using this signature of the estrogen-related receptor alpha (ERRa) to profile more than eight-hundred breast tumors, we found that patients with tumors exhibiting higher ERRa activity were predicted to have shorter disease free survival. Further, the ability of an ERRa antagonist, XCT790, to inhibit breast cancer cell proliferation correlates with the cellâs intrinsic ERRa activity. These findings highlight the potential of using the ERRa signature and antagonists in targeted therapy for breast cancer. Using a chemical genomic approach we determined that activation of the HER2/IGF1 signaling pathways upregulates the expression of PGC-1b, an obligate cofactor for ERRa activity. Knockdown of PGC-1b in HER2 positive breast cancer cells impaired ERRa signaling and reduced cell proliferation, implicating a functional role of PGC1b/ERRa in the pathogenesis HER2 positive breast cancer. Primary human mammary epithelial cells were a gift from Dr. J. Marks (Duke University, Durham, NC) and cultured in MEBM (Cambrex, East Rutherford, NJ) with MEGM bullet kit and supplemented with 5mg/ml transferrin and 10-5M isoproterenol. To generate ERR-alpha signature, hMECs were serum starved for 36h followed by infection with MOI=150 of adenoviruses expressing two variants of PGC1alpha, a protein ligand for ERRalpha: PGC-1alpha2x9 or PGC-1alpha L2L3M. PGC-1-2x9 is specific to ERRalpha, while PGC-1-L2L3M lacks the NR box and does not interact with ERRalpha or other nuclear receptors. The generation and purification of variant PGC-1alpha viruses were described previously (Gaillard et al., Molecular Cell 24:5, 2006). Comparable expression levels of the two PGC-1alpha variants were verified by Western immonoblot analysis (data not shown). RNA was collect 16h after infection and purified using RNeasy mini kit (Qiagen, Valencia, CA). Ten independent biological replicates from each virus infection were collected.

Dataset Information

Transcription profiling of human MCF7 cells were infected with adenovirus overexpressing one of the four proteins (PGC1 WT, PGC1 2x9, PGC1 L2L3 and BGAL) reveals cross-talk between Estrogen Receptor-related Receptor alpha (ERRalpha) and Estrogen Receptor (ERalpha) pathways.

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