Tumorigenesis is often the result of an imbalance between proliferation, differentiation and programmed cell death. The transcription factor, CCAAT/Enhancer Binding Protein B (C/EBPB) is a key regulator of these processes and thus plays an important role in the tumorigenesis of many tissues. Encoded by an intronless gene, C/EBPB is expressed as several distinct protein isoforms (LAP1, LAP2 and LIP) whose expression is tightly regulated by the differential use of a number of in-frame translation start sites. The LAP isoforms are normally associated with differentiation, while LIP expression is commonly observed in proliferating tissues and in breast, ovarian and colorectal tumors. Thus the LAP/LIP ratio is critical for the maintenance of normal growth and development and deceases in this ratio may lead to tumorigenesis. A detailed understanding of the mechanisms regulating the LAP/LIP ratio is lacking. To address this issue, we have demonstrated that epidermal growth factor receptor (EGFR) signaling selectively regulates expression of the LIP isoform in mammary epithelial cells. Furthermore, EGFR-mediated-p44/42 MAPK signaling phosphorylates and activates the RNA binding protein, CUGBP1, which then binds to C/EBPB mRNA to regulate translation of the LIP isoform. Based on this data, we hypothesize that the C/EBPB isoforms are important in mediating the mitogenic and tumorigenic effects of receptor tyrosine kinase signaling in the breast. We will address this hypothesis through the following aims: 1) To determine the functional importance of LAP1, LAP2 or LIP expression on growth, development and tumorigenesis of mammary epithelial cells and to determine how alterations in the ratio of these isoforms may lead to tumorigenesis. We have developed a C/EBPB-null, mammary epithelial cell line, and specific retroviral expression constructs as unique reagents to study the individual functions of these isoforms. Each C/EBPB isoform will be expressed as homodimers or as heterodimers (LAP1/LAP2, LAP1/LIP, LAP2/LIP) in the C/EBPB-nulls cells to address their effects on morphogenesis in in vitro 2D/3D assays and in vivo transplantation studies. These assays will test whether a decrease in the LAP/LIP ratio leads to proliferation and tumorigenesis. 2) To determine the molecular mechanisms by which receptor tvrosine kinase signaling regulates the expression of the C/EBPB-LAP1. -LAP2 and -LIP isoforms and thereby controls the LAP/LIP ratio. By using knock-down strategies as well as dominant-negative and constitutively active p44/42 MAPK constructs we will determine whether CUGBP1 activity is required for the EGFR-mediated regulation of LIP expression, is dependant upon the activity of p44/42 MAPK and is associated with a specific ErbB signaling cascade. Mass spectrometry and site-directed mutagenesis will also be used to identify the CUGBP1 residues that are phosphorylated by EGFR-p44/42 MAPK signaling and are necessary for binding to C/EBPB mRNA. Functional characterization of the C/EBPB isoforms and the receptor tyrosine kinase pathways that regulate their expression will further our understanding of the molecular basis of mammary development and tumorigenesis.
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