Cancers arise when cells escape intrinsic controls on proliferation due to activation of oncogenes, together with loss/inactivation of tumor suppressors that normally promote apoptosis or senescence of pre-malignant cells. Proto-oncogenes encoding Ras GTPases are frequently activated in cancer cells and the p53 and RB tumor suppressor pathways are disrupted in nearly all tumors. Elucidating the components of these oncogenic and anti-oncogenic pathways is essential for understanding how cancers develop and thus to identify unique vulnerabilities of tumor cells that can be exploited therapeutically. Our laboratory is interested in the C/EBP (CCAAT/enhancer binding protein) family of bZIP transcription factors, especially their roles in regulating cell proliferation and tumorigenesis. Our research focuses on the function and regulation of C/EBPbeta as a downstream effector of Ras. Our studies have also implicated C/EBPb in oncogene-induced senescence (OIS) in primary mouse fibroblasts, where it acts together with RB:E2F complexes to inhibit proliferation of cells expressing oncogenic Ras. C/EBPb may therefore possess both pro- and anti-tumorigenic properties. We seek to understand these opposing functions in more mechanistic detail and to illuminate their contributions to cancer development or tumor suppression in vivo. Role of C/EBPb in tumorigenesis: Work from our laboratory and others revealed a critical role for C/EBPb in the development of certain cancers. For example, C/EBPb-deficient mice are completely resistant to skin tumors induced by carcinogens that cause Ras mutations. C/EBPb is also essential for Myc/Raf-induced transformation of monocyte/macrophages and is required for growth factor-independent suppression of apoptosis (self-sufficiency), a hallmark of leukemias and other tumor cells. We identified the IGF-1 gene as a transcriptional target of C/EBPb in transformed macrophages and demonstrated that the survival defect of C/EBPb-deficient cells is caused by impaired autocrine IGF-1 signaling. To determine whether C/EBPb affects other kinds of cancers we are investigating the consequences of C/EBPb deficiency on ENU-induced tumorigenesis in mice. Thus far the results indicate that C/EBPb KO animals develop fewer lymphomas than WT mice and show reduced incidence/malignancy of several other cancers. To extend these findings we are crossing the C/EBPb KO mice to a latent activatable K-ras strain that develops lung tumors at high frequency. Preliminary results show that animals lacking C/EBPb develop fewer and less malignant tumors. Finally, we are examining the expression and function of C/EBPb in human tumor cell lines to extend our observations to human cancers. C/EBPb regulates systemic insulin/IGF-1 and the tumor environment: To further investigate regulation of IGF-1 by C/EBPb, we examined IGF-1 levels and related physiological phenotypes of C/EBPb KO mice. The mutant animals display decreased fat content and bone density and contain reduced circulating levels of IGF-1, insulin and leptin. Transplanted colon cancer cells showed diminished tumorigenicity in C/EBPb null mice compared to WT animals, indicating that C/EBPb contributes to a more favorable tumor environment. This effect may be due to the lower levels of tumor-promoting growth factors and hormones in C/EBPb-deficient mice. Our findings indicate that C/EBPb promotes tumor development through both cell-autonomous and non-cell-autonomous mechanisms. Role of C/EBPb in cellular senescence: In many primary (non-immortalized) cells, elevated levels of activated Ras or other oncogenes induce senescence, a stable form of cell cycle arrest that requires induction of the ARF-p53 and p16Ink4a-RB tumor suppressor pathways. Recent work indicates that oncogene-induced senescence (OIS) serves as a bonafide tumor surveillance mechanism in vivo. We found that C/EBPb null MEFs fail to undergo Ras-mediated cell cycle arrest, demonstrating a requirement for C/EBPb in RasV12-induced cellular senescence. In addition, C/EBPb over-expression in normal MEFs or human diploid fibroblasts induces cell cycle arrest and a senescent cellular morphology. In contrast, expression of C/EBPb in cells lacking all three RB family members (pRb, p107 and p130) or expressing dominant negative E2F1 stimulates proliferation. These findings indicate that RB:E2F complexes act in concert with C/EBPb to regulate growth of fibroblasts and possibly other cells. Consistent with this idea, C/EBPb binds to and represses E2F-regulated genes such as c-Myc and cyclin A2. Whereas oncogenic Ras provokes premature senescence in primary fibroblasts, it transforms immortalized cells such as NIH 3T3 fibroblasts, which carry a deletion that removes the p16, p15 and p19Arf tumor suppressors. We found that Ras increases C/EBPb levels in MEFs but silences C/EBPb expression in NIH 3T3 cells. This difference was traced to p19Arf, since re-expression of Arf but not p16 or p53 restored C/EBPb levels in Ras-expressing NIH 3T3 cells. Ras also decreased C/EBPb expression in p16/p19Arf null MEFs. Forced expression of C/EBPb blocked Ras-induced transformation and proliferation, showing that C/EBPb is capable of inducing senescence in NIH 3T3 cells. These observations indicate that C/EBPb is a p53-independent target of the Arf tumor suppressor pathway in fibroblasts, and Ras-induced down-regulation of C/EBPb may be an important event in bypassing OIS and allowing tumor progression in some cell contexts. Post-translational regulation of C/EBPb activity: Because C/EBPb mediates cellular responses to oncogenic Ras, we are investigating post-translational regulation of C/EBPb activity by Ras signaling. We have found that the DNA-binding activity of C/EBPb is auto-inhibited and becomes activated in cells expressing oncogenic Ras or stimulated with growth factors. C/EBPb auto-inhibition requires three regions in the N-terminal half of the protein, including the transactivation domain, which are predicted to adopt secondary structure. Mutagenesis studies support the idea that these auto-inhibitory sequences participate in folding interactions that reciprocally inhibit C/EBPb DNA-binding and transactivation functions. A parallel objective is to characterize Ras-induced post-translational modifications (PTMs) on C/EBPb that mediate signal-dependent de-repression. We identified a RSK kinase site in the leucine zipper that serves as an important regulator of C/EBPb DNA-binding activity and also modulates dimerization specificity. We are presently investigating how other phosphorylation sites and different classes of modifications such as lysine acetylation and Lys/Arg methylation control C/EBPb DNA-binding, dimerization, and transactivation function. Ultimately we seek to determine how specific PTMs affect the biological and oncogenic properties of C/EBPb. Involvement of C/EBPgamma in cell growth regulation through heterodimerization with C/EBPb: C/EBPg is a ubiquitously-expressed protein that forms heterodimers with other C/EBP family members, including C/EBPb. We are investigating its role in cell growth regulation by analyzing C/EBPg-deficient mice and cells derived from these animals. C/EBPg-deficient MEFs proliferate very poorly in culture and express reduced levels of pro-mitotic r [summary truncated at 7800 characters]

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National Cancer Institute (NCI)
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Staiger, Jennifer; Lueben, Mary J; Berrigan, David et al. (2009) C/EBPbeta regulates body composition, energy balance-related hormones and tumor growth. Carcinogenesis 30:832-40
Spooner, Chauncey J; Sebastian, Thomas; Shuman, Jon D et al. (2007) C/EBPbeta serine 64, a phosphoacceptor site, has a critical role in LPS-induced IL-6 and MCP-1 transcription. Cytokine 37:119-27
Loomis, Kari D; Zhu, Songyun; Yoon, Kyungsil et al. (2007) Genetic ablation of CCAAT/enhancer binding protein alpha in epidermis reveals its role in suppression of epithelial tumorigenesis. Cancer Res 67:6768-76
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Suh, Hyung Chan; Gooya, John; Renn, Katie et al. (2006) C/EBPalpha determines hematopoietic cell fate in multipotential progenitor cells by inhibiting erythroid differentiation and inducing myeloid differentiation. Blood 107:4308-16
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