The development of cancer involves aberrant control of cellular proliferation, owing mainly to the activation of oncogenes and inactivation of tumor suppressors. The latter proteins provide an intrinsic barrier to de-regulated growth and cancer by inducing apoptosis or permanent growth arrest (senescence) in pre-malignant cells. Ras proto-oncogenes are often mutationally activated in cancer cells, while the p53 or RB tumor suppressor pathways are almost universally disabled. Acquiring detailed knowledge of the various oncogenic and anti-oncogenic pathways is essential for understanding how cancers develop and to identify unique vulnerabilities of tumor cells that can be used to develop novel anti-cancer agents and strategies.Our laboratory studies the C/EBP (CCAAT/enhancer binding protein) family of transcription factors and their involvement in regulating cell proliferation and tumorigenesis. Our research focuses primarily on the role of C/EBPbeta as a downstream effector of Ras signaling. Studies using Cebpb null mice as well as analysis of human and rodent tumor cells have shown that C/EBPb has pro-oncogenic functions and is essential for the development of many cancers. However, in primary fibroblasts (MEFs) C/EBPb is also required for oncogene-induced senescence (OIS), an intrinsic tumor suppression mechanism that prevents neoplastic transformation in vitro and in vivo. In senescing cells, C/EBPb acts to arrest cellular proliferation through a pathway requiring RB:E2F. Thus, C/EBPb possesses both pro- and anti-tumorigenic activities. Because it plays an important role in cellular responses to Ras, we have undertaken studies to elucidate the mechanisms by which C/EBPb expression and its activity are controlled by oncogenic Ras signaling and to understand the molecular basis for its dual role in both suppressing and promoting cancer.Post-translational regulation of C/EBPb activity: C/EBPb is an intrinsically repressed (auto-inhibited) protein whose activity can be stimulated by oncogenic Ras or growth factor signaling through the Raf-MEK-ERK signal transduction pathway. C/EBPb auto-inhibition involves three short regions in the N-terminal half of the protein that, together with sequences at the C terminus, are predicted to fold into a hydrophobic core. The folded core sequesters the basic region and transactivation domain, inhibiting both DNA binding and transactivation. C/EBPb becomes activated by Ras signaling through several inducible post-translational modifications (PTMs). We previously identified a RSK kinase site in the leucine zipper that serves as an important regulator of C/EBPb DNA-binding and homodimerization. We also showed that the activated, homodimeric form of C/EBPb is specifically capable of associating with the transcriptional coactivator p300/CBP, and this interaction requires the three N-terminal auto-inhibitory elements. Thus, these regulatory sequences have bifunctional roles in C/EBPb regulation.Pro-oncogenic role of C/EBPb: Previous studies showed that C/EBPb-deficient mice are resistant to development of carcinogen-induced skin tumors. We have extended these findings by using other carcinogenesis protocols and mouse tumor models. For example, C/EBPb knockout mice treated with the carcinogen ENU exhibit impaired lymphomagenesis and reduced incidence/malignancy of a broad spectrum of other cancers compared to WT animals. C/EBPb null mice also develop fewer lung tumors than WT mice in a K-ras-induced carcinogenesis model. Furthermore, mouse colon carcinoma cells grow less efficiently in C/EBPb KO mice than in WT animals, probably due to decreased circulating levels of the growth factor IGF-1 in mutant mice. Thus, C/EBPb exerts both cell-autonomous and non-cell-autonomous effects on tumorigenesis.Tumor suppressor functions of C/EBPb: We have reported that C/EBPb is required for RasV12-induced cellular senescence in MEFs and showed that C/EBPb over-expression alone induces cell cycle arrest and senescence by a mechanism requiring RB:E2F. Cells lacking the tumor suppressor p19ARF bypass OIS and are transformed by Ras. We found that ARF, but not p53, is required to maintain C/EBPb levels in Ras-expressing fibroblasts, indicating that C/EBPb is part of an ARF-dependent tumor suppressor network. C/EBPb expression requires Egr family transcription factors and oncogenic Ras signaling decreases Egr levels in transformed fibroblasts, which at least partly explains down-regulation of C/EBPb in these cells. We are continuing to investigate how loss of ARF alters cellular responses to Ras and regulates Cebpb gene silencing. Our studies indicate that members of the Egr family of transcription factors are critical regulators of CEBPb gene transcription and their levels decrease upon expression of RasV12 in transformed fibroblasts. Regulation of C/EBPb activity by heterodimerization with C/EBPgamma: C/EBPg is a ubiquitously-expressed C/EBP protein that preferentially heterodimerizes with C/EBPb and other family members. However, its biological functions are not well understood. We discovered that C/EBPg-deficient MEFs have reduced proliferative potential and display increased replicative senescence in vitro. This defect is in part due to the ability of C/EBPg to form heterodimers with C/EBPb and suppress its cytostatic activity. C/EBPg knockout cells also display elevated levels of senescence-associated secretory phenotype (SASP) genes, which encode pro-inflammatory cytokines (e.g., IL-6, CXCL chemokines, etc.) that are involved in senescence induction. Furthermore, by analyzing gene expression databases we found that increased CEBPG levels are linked to poorer clinical outcomes in several human cancers, including lung tumors. Accordingly, depletion of CEBPG in a human lung tumor cell line caused impaired cell growth and increased senescence. Thus, C/EBPg is a key regulator of cell proliferation and survival and may have a critical role in promoting the rapid growth of many tumor cells.A novel function for the Cebpb 3'UTR in regulating C/EBPb protein activity: We recently made the unexpected discovery that the Cebpb 3'untranslated region (3'UTR) inhibits Ras-induced post-translational activation of C/EBPb, thereby suppressing its pro-senescence and cytostatic activities specifically in tumor cells. The 3'UTR blocks activation of C/EBPb DNA-binding and transcriptional activities that are otherwise induced by oncogenic Ras. The 3'UTR also prevented the C/EBPb-mediated induction of SASP genes, while promoting expression of genes linked to cancers and TGFbeta signaling. The 3'UTR inhibitory effect was mapped to an A/U rich element (ARE)-containing region and required the ARE-binding protein, HuR. Notably, these components excluded Cebpb mRNA from a perinuclear cytoplasmic region where the C/EBPb kinases p-ERK1/2 and CK2 reside in Ras-transformed cells. These findings indicate that the intracellular site of C/EBPb translation controls de-repression by Ras signaling via effector kinases such as ERK. Notably, 3'UTR inhibition and Cebpb mRNA compartmentalization were not observed in primary mouse and human fibroblasts. Consequently, in these cells Ras-induced activation of C/EBPb proceeds and OIS is implemented to suppress tumorigenesis. IWe are currently addressing whether other anti-oncogenic and oncogenic proteins are regulated by UPA-like mechanisms acting through 3'UTR sequences. We are also attempting to identify additional components of the 3'UTR regulatory pathway, as we anticipate that the UPA system will provide an attractive target for new therapeutic strategies aimed at reactivating intrinsic tumor suppression programs in cancer cells.
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