The progression of human breast cancer from hormone-responsiveness to a more aggressive, estrogen-independent state may involve the development of the ability to constitutively produce growth factors or their receptors which permit a tumor to bypass the need for estrogen. Clinically, this more malignant phenotype is often characterized by the loss of estrogen receptor (ER) and the acquisition of epidermal growth factor receptor (EGFR). EGFR is an indicator of poor response to endocrine therapy, and its presence overrides the beneficial effects of ER in coexpressors. Because of its prognostic value and the strong transcriptional component to its expression, this project focuses on the regulatory mechanisms by which transcription of the EGFR gene is controlled in human breast cancer. Preliminary studies indicate that there are structural differences (as revealed by DNase I sensitivity) in the promoter, first exon, and intron I of the EGFR gene which correlate with its expression in human breast cancer cell lines. Specifically, a site around the exon I/intron I boundary disappears in high expressors, while a group of sites in intron I appears in these cell lines. Additionally, a region in the promoter shows .changes in both the level of sensitivity and the extent of the area which is susceptible. These regions will be further analyzed to precisely localize sites of protein-DNA interaction whose presence or absence are associated with EGFR expression. This will be done using human breast cancer cell lines (both ER+ and ER-) with a wide range of EGFR expression. Protein binding sites will be mapped sequentially by native genomic blotting gel retardation assays, and in vitro footprinting. In this way, the in vivo significance of the protein binding sites will be established first, before pinpointing them at single-nucleotide resolution. This strategy will also be used to identify the cis-regulatory elements involved in the induction of EGFR by estrogen in hormone-dependent cell lines. The functionality of the various sequence elements identified by these methods as protein binding sites will be assessed in transient transfection assays using CAT expression vectors transfected into human breast cancer cell lines expressing high and low levels of EGFR. In the long term, sequences which are shown to be involved in the regulation of EGFR levels will then be used to isolate the protein factors responsible for controlling EGFR expression. It is hoped that by understanding these regulatory mechanisms and the role they play in the evolution of more aggressive forms of human breast cancer, new opportunities will arise for developing effective treatments against the disease.
