Cancer arises when the normal controls on cell survival and proliferation are disrupted as a result of genetic mutations. Recent work has implicated the CCAAT/enhancer binding protein (C/EBP) family of transcription factors in regulating cell growth and survival and demonstrates their involvement in 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 select target genes. Our laboratory uses knockout mice as well as cell lines derived from these animals to elucidate the functional roles of the various C/EBP family members in controlling cell proliferation and tumorigenesis. A major focus of our research is to elucidate the role of C/EBPbeta in cellular transformation induced by oncogenic Ras or its downstream effector kinases. C/EBPbeta is a widely expressed nuclear protein that is post-translationally activated by Ras signaling, suggesting that it could serve as a nuclear effector of oncogenic signals transmitted via the Ras pathway. In support of this idea, we previously showed that C/EBPbeta-deficient mice are completely resistant to the development of skin tumors induced by the carcinogen, DMBA (this project was a collaboration with Robert Smart, North Carolina State University, and Esta Sterneck, NCI-Frederick). Normal mice subjected to this protocol develop epidermal papillomas that carry mutations in the Ras proto-oncogene. Thus, C/EBPbeta is absolutely essential for skin tumorigenesis in an experimental animal model. We also found that C/EBPbeta null animals display significantly greater numbers of apoptotic cells in the epidermis after carcinogen treatment compared to wild type mice, suggesting that C/EBPbeta is involved in suppressing programmed cell death in pre-cancerous cells. To our knowledge, C/EBPbeta is the only mutation that completely blocks DMBA-induced skin tumor development. Since C/EBPbeta activity is regulated by Ras signaling, we hypothesize that C/EBPbeta is an essential component of the Ras-dependent tumorigenesis pathway in epidermal keratinocytes. We are currently investigating whether C/EBPbeta is essential for other cancers using a variety of mouse tumor models. If it has a widespread role in growth and survival of cancer cells, C/EBPbeta may be an promising target for the development of novel anti-tumorigenic agents. In another study, our laboratory demonstrated that C/EBPbeta function is required for neoplastic transformation of macrophages. In contrast to wild-type cells, C/EBPbeta-/- bone marrow-derived macrophages infected with a Myc/Raf-expressing retrovirus (J2) do not display growth factor independence. The ability to survive in the absence of exogenous growth factors (""""""""self sufficiency"""""""") is a hallmark of transformation in many leukemias and other cancers. We found that J2-transformed, C/EBPbeta nullizygous cells require macrophage-colony stimulating factor (M-CSF) or similar hematopoietic growth factors for survival, whereas wild type cells can evade apoptosis without a requirement for extrinsic factors. Several lines of evidence indicate that the survival defect in C/EBPbeta null cells results from impaired expression of IGF-I, a growth factor that has been shown to promote survival of many cancer cells but has not been implicated in myeloid leukemogenesis. Thus, our results demonstrate that C/EBPbeta-regulated production of IGF-I provides a critical autocrine prosurvival signal in a myeloid leukemic cell. Since C/EBPbeta is a critical regulator of the IGF-I gene in macrophages, we are interested in determining whether C/EBPbeta also may control serum IGF-I levels. Circulating levels of IGF-I are known to be a risk factor for the development and/or progression of many cancers. Serum IGF-I expression is modulated by environmental factors such as caloric intake. Therefore, we have undertaken studies to determine whether C/EBPbeta mediates dietary regulation of serum IGF-I levels and, subsequently, cancer risk. These experiments are currently in progress and are being performed in collaboration with the laboratories of Steve Hursting and Carl Barrett (CCR, NCI). The regulation of C/EBPbeta activity by Ras signaling is likely to underlie its role as an effector of Ras-induced transformation and tumorigenesis. Hence, our laboratory is interested in determining the mechanism by which C/EBPbeta is activated upon engagement of specific Ras effector pathways. In this regard, we and others have shown that the DNA-binding activity of C/EBPbeta is autorepressed. More recently, we observed that C/EBPbeta becomes de-repressed and is capable of binding to DNA in cells over-expressing oncogenic Ras or stimulated with growth factors. The sequences responsible for autorepression map to the N-terminal portion of the molecule and involve at least three distinct regions of predicted secondary structure. Studies to determine the structural basis for autorepression are currently in progress as a collaboration with Maria Miller and Alex Wlodawer (NCI-Frederick). We have also identified several sites of phosphorylation on C/EBPbeta that are induced by Ras signaling and we are currently investigating how these modifications affect C/EBPbeta activity. One novel phosphoacceptor site, Ser64, is a substrate for cyclin-dependent kinases (Cdk's) and becomes phosphorylated in a cell-cycle dependent manner. Mutation of Ser64 to alanine does not affect DNA binding but disrupts the ability of C/EBPbeta to enhance Ras-induced transformation of NIH 3T3 fibroblasts. Thus, Cdk-mediated phosphorylation of Ser64 during specific stages of the cell cycle appears to be necessary for unimpeded proliferation (or survival) of transformed fibroblasts.
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