The objective of the proposed research program is to investigate the role of the IGF-1R in the growth of the human breast cancer cell line MCF-7. Synergistic action of two factors: estrogens and the IGFs (insulin like- growth factors) greatly enhances growth of these cells in vitro. It has been shown that one of the functions of estrogens in MCF-7 cells is to sensitize these cells to IGF-I stimulation. The mechanism of this phenomenon involves elevation of the IGF-1R mRNA levels and a parallel increase of IGF-I binding sites. These findings are the basis for the question posed in the first aim of this project, which is to investigate the mechanisms by which estrogens regulate the expression of the IGF-1R, mRNA, and to identify regulatory elements in the IGF-1R gene promoter that are responsible for the expression of the IGF-1R gene in response to estrogens. Transient expression assays, gel-shift analysis, and DNase foot-printing will be used in this aim. The second and third aims of this project will explore the possibility that overexpression of the IGF-1R can result in an estrogen-independent phenotype nb MCF-7 cells. To this end cell lines expressing different numbers of the IGF-1R will be generated, and their ability to proliferate in anchorage-dependent or -independent growth conditions will be assessed in the absence or reduced concentrations of estrogens. I will also generate cell lines overexpressing various mutants of the IGF-1R, and cell overexpressing IRS-1, in an attempt to study which elements of the IGF-1R signaling pathway are necessary to overcome estrogen-dependence in MCF-7 cells. The cellular density and biological activity of receptors will be assayed by Scatchard analysis and Western blotting. The stimulation of the IGF-1R signaling in generated cell lines will be measured by the analysis of IRS-1 phosphorylation, P-I-3 kinase activation, Ras-GTP/Ras- GDP ratio, and activities of MEK and MAP kinases.
In aim #4 I will test growth inhibitory abilities of antisense oligonucleotides or antisense expression vectors designed to inactivate either the IGF-1R mRNA or mRNA encoding a target protein or the IGF-1R signaling pathway; IGF-1 peptide analogues will also be used in attempts to block the IGF-1R function in MCF-7 cells.
