Abstract
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 and analyzed 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 immune-precipitation (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 an IGF-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 ER-depleted breast cancer cells.
Highlights
The insulin-like growth factors, IGF-I and IGF-II, are mitogenic polypeptides with important roles in cell growth and differentiation [1]
Using DNA affinity chromatography, mass spectroscopy (MS), and Western blot analyses, we identified a series of known and previously unidentified transcription factors that bind to the IGF-I receptor (IGF-IR) promoter in either cell type
To address the potential regulation of IGF-IR expression during breast cancer progression, we measured in initial experiments the levels of total and phospho-IGF-IR in syngeneic cell lines representing different stages of the disease
Summary
The insulin-like growth factors, IGF-I and IGF-II, are mitogenic polypeptides with important roles in cell growth and differentiation [1]. 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]. Consistent with its potent antiapoptotic role, IGF-IR-null cells do not undergo transformation when exposed to different oncogenes [6,7]. 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]
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