ABSTRACT
Elevated expression of the orphan nuclear receptor estrogen-related receptor α (ERRα) has been associated with a negative outcome in several cancers, although the mechanism(s) by which this receptor influences the pathophysiology of this disease and how its activity is regulated remain unknown. Using a chemical biology approach, it was determined that compounds, previously shown to inhibit canonical Wnt signaling, also inhibited the transcriptional activity of ERRα. The significance of this association was revealed in a series of biochemical and genetic experiments that show that (a) ERRα, ß-catenin (ß-cat), and lymphoid enhancer-binding factor-1 form macromolecular complexes in cells, (b) ERRα transcriptional activity is enhanced by ß-cat expression and vice versa, and (c) there is a high level of overlap among genes previously shown to be regulated by ERRα or ß-cat. Furthermore, silencing of ERRα and ß-cat expression individually or together dramatically reduced the migratory capacity of breast, prostate, and colon cancer cells in vitro. This increased migration could be attributed to the ERRα/ß-cat-dependent induction of WNT11. Specifically, using (a) conditioned medium from cells overexpressing recombinant WNT11 or (b) WNT11 neutralizing antibodies, we were able to show that this protein was the key mediator of the promigratory activities of ERRα/ß-cat. Together, these data provide evidence for an autocrine regulatory loop involving transcriptional upregulation of WNT11 by ERRα and ß-cat that influences the migratory capacity of cancer cells.
Subject(s)
Cell Movement/physiology , Receptors, Estrogen/metabolism , Wnt Proteins/metabolism , beta Catenin/metabolism , Autocrine Communication/physiology , Blotting, Western , Cadherins/genetics , Cadherins/metabolism , Cell Line, Tumor , Cell Movement/drug effects , Cell Survival/drug effects , Gene Expression Regulation, Neoplastic , HCT116 Cells , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Humans , MSX1 Transcription Factor/genetics , MSX1 Transcription Factor/metabolism , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathology , Nitriles/pharmacology , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha , RNA Interference , Receptors, Estrogen/genetics , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/physiology , Thiazoles/pharmacology , Transcription Factors/genetics , Transcription Factors/metabolism , Transcriptional Activation , Wnt Proteins/genetics , beta Catenin/genetics , ERRalpha Estrogen-Related ReceptorABSTRACT
An analysis of mRNA expression in T47D breast cancer cells treated with the synthetic progestin R5020 revealed a subset of progesterone receptor (PR) target genes that are enriched for E2F binding sites. Following up on this observation, we determined that PR-B acts in both direct and indirect manners to positively upregulate E2F1 expression in T47D cells. The direct effects of PR on E2F1 expression were confirmed by chromatin immunoprecipitation (ChIP) analysis, which indicated that the agonist-bound receptor was recruited to several enhancer elements proximal to the E2F1 transcript. However, we also noted that cycloheximide partially inhibits R5020 induction of E2F1 expression, indicating that the ligand-dependent actions of PR on this gene may involve additional indirect regulatory pathways. In support of this hypothesis, we demonstrated that treatment with R5020 significantly increases both hyperphosphorylation of Rb and recruitment of E2F1 to its own promoter, thus activating a positive feedback loop that further amplifies its transcription. Furthermore, we established that PR-mediated induction of Krüppel-like factor 15 (KLF15), which can bind to GC-rich DNA within the E2F1 promoter, is required for maximal induction of E2F1 expression by progestins. Taken together, these results suggest a new paradigm for multimodal regulation of target gene expression by PR.