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Mechanism of action of a novel subtype specific estrogen related receptor alpha (ERRalpha) antagonist and evidence for the important role of this orphan nuclear receptor in breast cancer

by
Michael Chisamore
M.S., 2002
University of Pennsylvania

Thesis Advisor: J. Don Chen, Ph.D.
Graduate Program in Cellular and Molecular Pharmacology

RWJMS - Research Tower - 4th floor
Pharmacology Conference Room

Thursday, September 11, 2008
10:00 am


Abstract

The estrogen receptor alpha (ERalpha) is a clinically proven target for treating breast cancer. Yet it is clear that there continues to be an unmet medical need for treatments that target estrogen signaling via alternate pathways. Estrogen related receptor alpha (ERRalpha) is an orphan member of the nuclear receptor superfamily. Excluding estrogen related receptors (ERR) beta and gamma, ERRalpha exhibits the greatest homology to ERalpha. Although estradiol is not a ligand for ERRalpha, ERalpha and ERRalpha share many commonalities and overlapping pathways involving estrogen and ERRalpha signaling. While an endogenous ligand has yet to be identified, a novel synthetic antagonist that is highly specific for the ligand binding domain of ERRalpha (termed Compound A) has been identified and studied in preclinical breast cancer models. Compound A inhibited ERRalpha transcriptional activity in MCF-7 breast cancer cells by luciferase assay but did not affect ERR mRNA levels measured by real-time RT-PCR. Further, ERalpha mRNA levels were not affected by treatment with the ERRalpha antagonist, but other ERRalpha target genes such as pS2 (TFF1), osteopontin (SPP1), and aromatase (CYP19A1) mRNA levels decreased upon treatment. The ERRalpha antagonist prevented the constitutive interaction between ERRalpha and nuclear receptor coactivators in vitro. Western blot analysis revealed that ERRalpha protein degradation was increased by the ERRalpha subtype specific antagonist through the ubiquitin proteasome pathway. ChIP was utilized to demonstrate the interaction between ACADM, ERRalpha, or pS2 endogenous gene promoters (ERRalpha target genes) and the decrease in ERRalpha protein in the presence of ligand. Compound A inhibited cell proliferation in both ERalpha positive (MCF-7 and T47D) and ERalpha negative (BT-20 and MDA-MD-231) breast cancer cell lines. The differential expression of 226 genes involved in ERRalpha signaling in MCF-7 and BT-20 cell lines were examined and Ingenuity Pathway Analysis software identified ERRalpha signaling networks in breast cancer. To validate the proposed ERRalpha signaling networks, phospho- mitogen-activated-protein kinase (MAPK) arrays were utilized to study the phosphorylation status of MAPKs in vivo, the ERRalpha ligand inhibited tumor growth in both the MCF-7 and BT-20 mouse xenograft models and demonstrated antagonistic effects on the uterus. Subsequently, ERRalpha protein down-modulation was observed by immunohistochemistry (IHC) in both MCF-7 and BT-20 mouse xenograft tumors that were treated with the ERRalpha antagonist. A subset of genes also were evaluated and confirmed in vivo, by analyzing the uterine gene expression profile in mice from the xenograft studies. Lastly, we confirmed that antagonizing/ down-modulating ERRalpha by Compound A treatment is specifically mediated through ERRalpha by establishing stable ERRalpha knock-down cell lines (MCF-7 / shRNA ERRalpha) and studying both gene expression and proliferation rates.

Based on these studies, we have demonstrated that 1) Compound A, an ERRalpha selective ligand is a useful tool to study a role of ERRalpha in breast cancer; 2) Compound A antagonizes ERRalpha activity by interfering with nuclear receptor cofactor association and directing the receptor to the ubiquitin proteasome pathway; 3) ERRalpha is a novel drug target for the treatment of breast cancer.


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