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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA055677-03
Application #
2096790
Study Section
Metabolic Pathology Study Section (MEP)
Project Start
1992-09-01
Project End
1995-08-31
Budget Start
1994-09-01
Budget End
1995-08-31
Support Year
3
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Georgetown University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
049515844
City
Washington
State
DC
Country
United States
Zip Code
20057
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Wilson, Melissa A; Chrysogelos, Susan A (2002) Identification and characterization of a negative regulatory element within the epidermal growth factor receptor gene first intron in hormone-dependent breast cancer cells. J Cell Biochem 85:601-14
Yarden, R I; Wilson, M A; Chrysogelos, S A (2001) Estrogen suppression of EGFR expression in breast cancer cells: a possible mechanism to modulate growth. J Cell Biochem Suppl Suppl 36:232-46
McInerney, J M; Wilson, M A; Strand, K J et al. (2001) A strong intronic enhancer element of the EGFR gene is preferentially active in high EGFR expressing breast cancer cells. J Cell Biochem 80:538-49
Yarden, R I; Lauber, A H; El-Ashry, D et al. (1996) Bimodal regulation of epidermal growth factor receptor by estrogen in breast cancer cells. Endocrinology 137:2739-47
Murphey, J M; Chrysogelos, S A (1995) Direct measurement of transfection efficiency in transient transfection assays. Biotechniques 18:967-9
Chrysogelos, S A; Yarden, R I; Lauber, A H et al. (1994) Mechanisms of EGF receptor regulation in breast cancer cells. Breast Cancer Res Treat 31:227-36
Chrysogelos, S A; Dickson, R B (1994) EGF receptor expression, regulation, and function in breast cancer. Breast Cancer Res Treat 29:29-40
Chrysogelos, S A (1993) Chromatin structure of the EGFR gene suggests a role for intron 1 sequences in its regulation in breast cancer cells. Nucleic Acids Res 21:5736-41