Changes in gene expression are central to the control of cellular processes such as proliferation, growth arrest, differentiation, and oncogenic transformation. Our research focuses on the CCAAT/enhancer binding protein (C/EBP) family of transcription factors and their involvement in regulating cell growth, survival and tumorigenesis. The C/EBP family is composed of five members - C/EBPalpha, beta, gamma, delta and epsilon - that are expressed in cell-specific patterns and regulate transcription by binding to target genes. We are using genetically engineered mice containing targeted deletions of individual c/ebp genes and cell lines derived from these animals to eluciate the functional roles of the various C/EBP family members. A major focus of our research is the involvement of C/EBPbeta in oncogenic transformation. C/EBPbeta is a widely expressed protein that is post-translationally regulated by the Ras signaling pathway. In collaboration with Drs. Robert Smart (North Carolina State University)and Esta Sterneck (CCR, NCI-Frederick), we previously observed that C/EBPbeta-deficient mice are completely resistant to the development of skin tumors induced by chemical carcinogens. Skin tumors that develop in wild-type mice occur as a result of mutations in the Ras proto-oncogene. Since C/EBPbeta is regulated by Ras signaling, our findings indicate that C/EBPbeta is an essential component of the Ras-dependent tumorigenesis pathway in skin cells. Because C/EBPbeta null animals displayed significantly increased numbers of apoptotic cells in the epidermis after carcinogen treatment, we propose that C/EBPbeta is involved in suppressing programmed cell death in pre-cancerous cells. If C/EBPbeta activity proves to be essential for survival in a variety of cancer cells, this protein may be an attractive target for anti-tumor drugs. In subsequent studies we found that C/EBPbeta is also required for transformation of macrophages infected with a Myc/Raf-expressing retrovirus (J2). In contrast to wild-type cells, C/EBPbeta-deficient bone marrow-derived macrophages infected with the J2 virus do not undergo a transition to growth factor independence. The mutant cells continue to require macrophage-colony stimulating factor (M-CSF), or similar cytokines, for survival, while wt J2-transformed cells are able to suppress apoptosis in the absence of an exogenous factor. Several lines of evidence show that the survival defect in C/EBPbeta null cells is due to impaired expression of IGF-I, a growth factor that has been shown to promote survival of many kinds of cancer cells but has not been implicated in myeloid tumorigenesis. Our results now demonstrate that C/EBPbeta-dependent expression of IGF-I provides a critical autocrine survival signal in macrophage tumor cells. We are currently attempting to determine whether IGF-I is a target of C/EBPbeta regulation in other kinds of tumors. Another aspect of this work involves a collaboration with Drs. Steve Hursting and Carl Barrett (CCR, NCI) to investigate the potential role of C/EBPbeta in regulating systemic levels of IGF-I. Circulating IGF-I levels are a risk factor for malignant progression of many cancers and can be modulated by environmental factors such as diet; therefore, we wish to determine whether C/EBPbeta mediates dietary influences on serum IGF-I expression. Another project in our laboratory involves determining the mechanism of C/EBPbeta activation by Ras signaling. We have observed that the DNA-binding activity of C/EBPbeta is autorepressed and becomes de-repressed in cells expressing oncogenic Ras. The sequences responsible for autorepression map to the N-terminal portion of the molecule and appear to involve at least three distinct regions. The structural basis for autorepression is currently being investigated in collaboration with Drs. Maria Miller and Alex Wlodawer (CCR, NCI-Frederick). We have also identified several sites of phosphorylation on C/EBPbeta that are induced by Ras signaling and are studying how these modifications affect C/EBPbeta activation.
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