Cancers arise when cells escape normal controls on proliferation and survival through activation of oncogenes as well as loss or inactivation of tumor suppressors that normally induce apoptosis or growth arrest (senescence) in 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 to identify unique vulnerabilities of tumor cells that can be exploited for therapeutic advantage. Our laboratory studies the C/EBP (CCAAT/enhancer binding protein) family of bZIP transcription factors, with particular emphasis on their involvement in cell proliferation and tumorigenesis. Our current research focuses on the functions and regulation of C/EBPbeta as a downstream effector of Ras in tumor development. Work from our laboratory has also implicated C/EBPbeta in oncogene-induced senescence (OIS) in primary mouse fibroblasts, where it acts to restrain proliferation of cells expressing activated Ras. C/EBPbeta 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 in vivo. Post-translational regulation of C/EBPbeta activity: The DNA-binding activity of C/EBPbeta is intrinsically repressed and becomes activated in cells expressing oncogenic Ras or in response to growth factors. C/EBPbeta auto-inhibition requires at least three regions in the N-terminal half of the protein which are predicted to form secondary structure and are probably involved in folding interactions that mediate auto-repression. We have investigated Ras-induced sites of phosphorylation on C/EBPbeta and identified a RSK kinase site in the leucine zipper as an important regulator of C/EBPbeta DNA-binding activity and dimerization. We are continuing to investigate how phosphorylation and other modifications such as lysine acetylation and methylation control C/EBPbeta DNA-binding, dimerization, and transactivation and such modifications affect its biological and oncogenic properties. C/EBPbeta and tumorigenesis: Work from our laboratory and others demonstrates a critical role for C/EBPbeta in the development of certain cancers. C/EBPbeta-deficient mice were shown to be completely resistant to skin tumors induced by carcinogens that cause Ras mutations. C/EBPbeta is also essential for Myc/Raf-induced transformation of monocyte/macrophages and is required for these cells to evade apoptosis in the absence of extrinsic growth factors, a hallmark of leukemias and other tumor cells. We identified the IGF-I gene as a transcriptional target of C/EBPbeta in transformed macrophages and demonstrated that the survival defect of C/EBPbeta null cells stems from impaired autocrine signaling by IGF-I. To determine whether C/EBPbeta affects other kinds of tumors, we are investigating the effect of C/EBPbeta deficiency on carcinogen-induced cancers. Preliminary results indicate that C/EBPbeta KO mice exposed to ENU are impaired in lymphomagenesis and show reduced incidence or malignancy of several other cancers. To extend these findings we are currently intercrossing C/EBPbeta KO mice with transgenic strains that develop tumors in specific tissues. We are also examining the expression and function of C/EBPbeta in human tumor cell lines to extend our findings to human malignancies. C/EBPbeta regulates systemic insulin/IGF-I and the tumor environment: We found that C/EBPbeta KO mice display decreased fat content and bone density and have reduced circulating levels of IGF-I, insulin and leptin. Transplanted colon cancer cells showed diminished tumorigenicity in C/EBPbeta null mice compared to wild type animals, indicating that C/EBPbeta 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/EBPbeta-deficient mice. Along with previous work from our laboratory, these studies indicate that C/EBPbeta promotes tumor development through both cell-autonomous and non-cell-autonomous mechanisms. Role of C/EBPbeta 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 demonstrated a requirement for C/EBPbeta in RasV12-induced cellular senescence, as C/EBPbeta null MEFs fail to undergo Ras-mediated cell cycle arrest. In addition, C/EBPbeta over-expression in normal MEFs or human diploid fibroblasts induces a senescent-like cell cycle arrest. In contrast, expression of C/EBPbeta in cells lacking all three RB family members (pRb, p107 and p130) or expressing dominant negative E2F1 accelerates their proliferation. These findings indicate that RB:E2F complexes determine the growth-regulatory effects of C/EBPbeta in fibroblasts and possibly in other cells. Consistent with this idea, we found that C/EBPbeta binds to and represses several E2F-regulated genes. Current studies include determining whether C/EBPbeta contributes to OIS in vivo, elucidating where and how C/EBPbeta functions within the p53 and RB checkpoint pathways, and investigating the mechanism by which C/EBPbeta confers opposing effects on cellular proliferation in different contexts. Involvement of C/EBPgamma in cell growth regulation through heterodimerization with C/EBPbeta: C/EBPgamma is a ubiquitously expressed protein that forms heterodimers with other C/EBP family members, including C/EBPbeta. We are investigating its role in cell growth regulation by analyzing C/EBPgamma-deficient mice and cells and studying mechanisms that control its heterodimerization with C/EBPbeta. Our results indicate that C/EBPgamma positively regulates cell proliferation in part by opposing the growth-inhibitory effects of C/EBPbeta. Current work focuses on the mechanism by which C/EBPgamma regulates cell proliferation and its potential role in tumorigenesis
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