MOLECULAR DISSECTION AND FUNCTIONAL DEFINITION OF ESTROGEN-RELATED RECEPTOR ALPHA SIGNALING PATHWAY

The estrogen-related receptor alpha (ERRα) is an orphan nuclear receptor (NR) with no natural ligand identified. Recent studies report that ERRα expression and activity correlate with poor prognosis in breast cancer. It is also suggested that ERRα is involved in tumor growth and progression, thus this receptor may be a therapeutic target in the treatment of breast cancer. However, the specific role of ERRα in breast cancer is not fully understood. Similar to other nuclear receptors, ERR has been suggested to regulate target gene transcription through both classical (direct DNA binding) and non-canonical (tethering mechanisms) to effect various aspects of tumor pathogenesis, such as angiogenesis, regulation of hypoxic response, tumor growth, and migration. Thus, the objective of this dissertation research is to explore the roles of ERRα in breast cancer by (a) identifying novel ERRα target genes important for tumor pathogenesis, (b) characterizing the molecular mechanism of non-canonical actions of ERRα-mediated gene transcription, and (c) examining the structure basis of ERRα antagonism for future pharmaceutical exploitation. First, we identified an ERRα target gene, ECM1, which is relevant to breast cancer angiogenesis. The role of ECM1 in angiogenesis was confirmed by endothelial tube formation assay. We further showed that knocking down ECM1 has a dramatic inhibitory effect on tumor xenograft growth. This result, for the first time, directly demonstrates the role of ECM1 in tumor environment and further sheds light on the significance of ERR&alpha-regulated genes in tumors angiogenesis. Next, we explored the molecular mechanism of ERRα non-canonical pathways using transcriptional reporter assay and ERRα DNA-binding domain (DBD) mutants. We discovered that the expression of carbonic anhydrase 9 (CA9), a target gene of one of the ERRα tethering partner hypoxia inducible factor-1 (HIF-1), does not require direct binding of ERRα to DNA but its DBD is indispensible. These results reflect on the importance of ERRα DBD even in the non-canonical signaling of ERRα, which brings challenges to dissecting ERRα canonical/non-canonical pathways in the future. Finally, to determine the molecular mechanisms underlying ERRα antagonism, we probed the conformations of ERRα upon antagonist treatments. M13 phage display was used to screen for ERRα-interacting peptides. We identified peptides that interact with ERRα in the activation function 2 (AF2) domain, some of which are able to distinguish the binding of different classes of ERRα antagonists. Cumulatively, these studies have explored the biological functions of ERRα and the molecular basis ERRα-mediated signaling pathways.