The epidermal growth factor receptor (EGFR) plays an important role in normal cell growth and development. Activation of the EGFR signaling pathway has many effects including increased proliferation and angiogenesis, and decreased apoptosis. EGFRs are inappropriately expressed or highly expressed in a variety of human tumors including breast, gastric, ovarian, non-small cell lung cancer, head and neck cancers, prostate and others. Cancer cell lines derived from the tumors also overexpress the receptor. Increased levels of transcription cause the increase in a variety of tumors in the absence of gene amplification. High-level EGFR expression has been associated with advanced tumor stage, resistance to standard therapies (hormonal therapy, chemotherapy and radiation) and, in some tumors, with poor patient prognosis. The critical role of EGFR in both normal and abnormal cell growth and development necessitates a clear understanding of the regulation of its' expression. We hypothesize that changes in transcription factors interacting with the EGFR promoter leads to increased expression. Our research focuses on two projects seeking to understand the factors that regulate EGFR expression during normal cell growth and in cancer. Our approach is to determine the transcription factors involved in EGFR gene regulation and to examine their role in cancer. We further seek to understand how GC-binding factor 2 (GCF2), a repressor of EGFR expression, decreases the activity of cellular and viral promoters. Our laboratory and others initially characterized the EGFR promoter region as a GC-rich, TATA-less regulatory region with multiple transcription initiation sites and specificity protein 1 (Sp1) binding sites. Many DNA-binding factors have been identified that interact with the promoter region including p53, activator protein 2 (AP2) and interferon regulated factor 1 (IRF-1). We have determined the roles of activator protein 1 (AP-1), p63 and early growth response gene 1 (egr-1) in regulating EGFR expression. AP-1 was determined to bind to at least seven sites in the promoter and mediate phorbol ester induced EGFR expression. The p53 homologue, p63, was shown to repress EGFR expression through protein-protein interactions with Sp1, an activator of EGFR expression. Egr-1 was determined to increase endogenous EGFR expression. Furthermore, we were able to show that upregulation of egr-1 during hypoxia leads to increased EGFR expression. Additionally, we examined the role of nuclear factor kappa B (NF-kB) in regulating EGFR expression but could not find evidence of an effect by NF-kB. Another family of factors that regulate EGFR expression is the p53 family. The tumor suppressor p53 homologue, p73, has a high degree of homology and possibly similar functions. Analysis of p73 in ovarian carcinoma cell lines has revealed that 71% of invasive tumors and 92% of borderline tumor tissues express elevated levels of p73 transcript and protein. This increased level of p73 expression and the association of EGFR with a more invasive and metastatic phenotype has prompted us to determine if p73 regulates EGFR expression in ovarian cancer cells. We hypothesize that increased p73 expression in ovarian cancer leads to increased EGFR expression. This project is a collaborative effort with Dr. Hirotaka Nishi, affiliated with Tokyo Medical University. Using transient transfection experiments, we determined that increased p73-alpha expression resulted in a 4 - 5 fold increase in EGFR promoter activity. Consistent with this finding was a similar increase in the endogenous EGFR level. We further determined that p73-alpha binds to the previously defined EGFR p53 response element. Mutation of this element abrogates the p73 induction. We have initiated characterization of GCF2, a transcriptional repressor identified by our laboratory. We have determined that the region of GCF2 that binds to the EGFR promoter and that GCF2 can partially inhibit AP2 activation of EGFR promoter activity. In addition, we have shown that GCF2 was able to bind to a highly structured RNA element TAR (transactivation response element) that is located at the 5' end of nascent HIV transcripts. GCF2 inhibits activation of the HIV-1 LTR by a small regulatory protein, Tat, which is required for efficient transcription of genes linked to the HIV-1 LTR. We determined that the amino terminal region of GCF2 was required to repress the Tat-mediated enhancement of the HIV-1 LTR and that GCF2 can also decrease basal HIV-1 LTR activity.
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