## 1 Introduction  
The insulin-like growth factors, IGF-I and IGF-II, are mitogenic polypeptides with important roles in cell growth and differentiation [1]. The biological activities of IGF-I and IGF-II are mediated by the IGF-I receptor (IGF-IR) and are modulated by a family of IGF-binding proteins (IGFBPs) that control ligand bioavailability [2; 3; 4]. The IGF-IR is essential for normal cell cycle progression and has a crucial role in tumor growth and metastasis development. Ligand-mediated activation of the IGF-IR tyrosine kinase domain leads to mitogenic and antiapoptotic effects in multiple cells and cancer-derived cell lines [5]. In the specific context of breast tumorigenesis, the IGF-IR is overexpressed in most primary tumors [4]. In addition, and consistent with its potent antiapoptotic role, IGF-IR-null cells do not undergo transformation when exposed to different oncogenes [6; 7]. On the other hand, inhibition of IGF-IR signaling by IGF-IR antibodies, antisense oligonucleotides, IGF-BPs, or specific IGF-IR kinase inhibitors results in reduced cell proliferation and cancer progression _in vivo_ and _in vitro_[8; 9; 10; 11; 12; 13; 14]. Furthermore, epidemiological studies revealed that high levels of circulating IGF-I are linked to an increased risk of developing breast cancer in premenopausal women [15; 16].  
Regulation of IGF-IR gene expression is mainly attained at the transcription level. The IGF-IR promoter is a TATA-less, CCAAT-less, highly GC-rich, 'initiator'-type of promoter. IGF-IR gene transcription is dependent on a number of stimulatory zinc-finger nuclear proteins, including Sp1 [17] and KLF6 [18]. In addition, IGF-IR gene transcription is negatively regulated by several tumor suppressors, including BRCA1, p53/p63/p73, the von Hippel-Lindau protein (VHL), and the Wilms' protein-1 (WT1) [19-25]. Interactions between stimulatory and inhibitory transcription factors play an important role in IGF-IR gene regulation and, therefore, were postulated to have a major impact on the proliferative status of the cell. The molecular mechanisms and specific transcription factors responsible for regulating IGF-IR gene expression in breast cancer cells, however, have not yet been identified.  
The IGF-I and estrogen signaling systems were shown to act in a synergistic fashion in breast epithelial cells [25]. Estrogens regulate IGF-I signaling and the expression of several members of the IGF system [25; 26; 27; 28; 29; 30; 31]. Moreover, activation of estrogen receptor-\(\alpha\) (ER\(\alpha\)) by estrogens induces a physical interaction between ER\(\alpha\) and IGF-IR [32] that results in activation and phosphorylation of IGF-IR and downstream signaling molecules [33; 34; 35; 36]. The aim of this study was to identify the collections of IGF-IR promoter-binding transcription factors in ER-positive and ER-depleted breast cancer cells. Using DNA affinity chromatography, mass spectroscopy (MS), and Western blot analyses, we identified a series of known and previously unidentified transcription factors that specifically bind to the IGF-IR promoter in either cell type. The ability of selected proteins to bind and transactivate the IGF-IR promoter was confirmed by chromatin immunoprecipitation (ChIP) and transient transfection assays. In addition, we identified a number of non-DNA sequence-specific nuclear proteins that are probably involved in IGF-IR gene regulation. Identification of differentially expressed IGF-IR promoter-binding and non-binding transcription factors may help elucidate the mechanisms responsible for the differential expression of the IGF-IR gene in ER-positive and ER-depleted breast cancer cells.