Identification of insulin-like growth factor-I receptor (IGF-IR) gene promoter-binding proteins in estrogen receptor (ER)-positive and ER-depleted breast cancer cells

The insulin-like growth factor I receptor (IGF-IR) has been implicated in the etiology of breast cancer. Overexpression of the IGF-IR gene is a typical feature of most primary breast cancers, whereas low IGF-IR levels are seen at advanced stages. Hence, evaluation of IGF-IR levels might be important for assessing prognosis. In the present study, we employed a proteomic approach based on DNA affinity chromatography followed either by mass spectroscopy (MS) or Western blot analysis to identify transcription factors that may associate with the IGF-IR promoter in estrogen receptor (ER)-positive and ER-depleted breast cancer cells. A biotinylated IGF-IR promoter fragment was bound to streptavidin magnetic beads and incubated with nuclear extracts of breast cancer cells. IGF-IR promoter-binding proteins were eluted with high salt andanalyzed by MS and Western blots. Among the proteins that were found to bind to the IGF-IR promoter we identified zinc finger transcription factors Sp1 and KLF6, ER-α, p53, c-jun, and poly (ADP-ribosylation) polymerase. Furthermore, chromatin immuneprecipitation (ChIP) analysis confirmed the direct in vivo binding of some of these transcription factors to IGF-IR promoter DNA. The functional relevance of binding data was assessed by cotransfection experiments with specific expression vectors along with anIGF-IR promoter reporter. In summary, we identified nuclear proteins that are potentially responsible for the differential expression of the IGF-IR gene in ER-positive and ERdepleted breast cancer cells. © 2010 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland.