Cancer development involves aberrant control of cellular proliferation, resulting from activation of oncogenes and inactivation of tumor suppressors. The latter provide an intrinsic barrier to de-regulated growth and cancer by promoting cell death or inducing 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 nearly universally disabled. Loss of tumor suppressor pathways renders cells susceptible to transformation by Ras and other oncogenes by disrupting cell death or senescence responses. 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 roles in cell proliferation and tumorigenesis. Our research focuses primarily on C/EBPbeta as a downstream target of Ras signaling. Studies using Cebpbeta 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/EBPbeta 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/EBPbeta acts to arrest cellular proliferation through a pathway requiring RB:E2F. Thus, C/EBPbeta 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/EBPbeta 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/EBPbeta activity: C/EBPbeta is an intrinsically repressed (auto-inhibited) protein whose activity can be stimulated by oncogenic Ras or growth factor signaling through the Raf-MEK-ERK cascade. C/EBPbeta is auto-inhibited by 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/EBPbeta 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/EBPbeta DNA-binding and homodimerization. We also showed that the activated, homodimeric form of C/EBPbeta is 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. Pro-oncogenic role of C/EBPbeta: Previous studies showed that C/EBPbeta-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/EBPbeta 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/EBPbeta KO mice than in WT animals, probably due to decreased circulating levels of the growth factor IGF-1 in mutant mice. Thus, C/EBPbeta exerts both cell-autonomous and non-cell-autonomous effects on tumorigenesis. Regulation of C/EBPbeta activity by heterodimerization with C/EBPgamma: C/EBPgamma is a ubiquitously-expressed C/EBP protein that heterodimerizes with C/EBPbeta and other C/EBP family members. However, its biological functions are not well understood. We found that C/EBPgamma-deficient MEFs have reduced proliferative potential and display increased replicative senescence in vitro. This defect is partly due to the ability of C/EBPgamma to form heterodimers with C/EBPbeta and suppress its cytostatic activity. C/EBPgamma 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 and are activated by C/EBPbeta homodimers. Furthermore, by analyzing gene expression databases for several human cancers we found that increased CEBPG levels are frequently linked to poorer clinical outcomes, including lung tumors. Depletion of CEBPG in a human lung tumor cell line caused impaired cell growth and increased senescence. Thus, C/EBPgamma positively regulates cell proliferation/survival and may have a critical role in promoting the rapid growth of many tumor cells. We also found that Cebpg-/- MEFs exhibit elevated levels of reactive oxygen species (ROS) and increased apoptosis. This phenotype is due to reduced levels of the cellular antioxidant, glutathione, due to defective expression of genes regulating glutathione biosynthesis. Notably, we observed that C/EBPgamma forms heterodimers with the stress-induced bZIP protein, ATF4. ATF4 is a central regulator of the integrated stress response, which regulates cellular adaptation to several types of stress including oxidative stress, amino acid deprivation, and ER stress. Many stress-induced genes contain binding sites for C/EBP:ATF heterodimers (CARE elements), and it has been widely believed that CARE sites bind C/EBPbeta:ATF4 heterodimers. Our work demonstrates that C/EBPgamma is in fact the critical partner of ATF4 in stressed cells. This regulatory role of C/EBPgamma may also be important for its pro-oncogenic activity. A novel function for the Cebpb 3'UTR in regulating C/EBPbeta protein activity: We recently discovered that the Cebpb 3'untranslated region (3'UTR) inhibits Ras-induced post-translational activation of the C/EBPbeta protein, thereby suppressing its pro-senescence and cytostatic activities in tumor cells. The 3'UTR blocks activation of C/EBPbeta DNA-binding and transcriptional activities that are otherwise induced by oncogenic Ras. The 3'UTR also prevented C/EBPbeta-driven expression of SASP genes, while promoting expression of genes linked to cancers and TGFbeta signaling. The 3'UTR inhibitory effect was mapped to a region bearing A/U rich elements (AREs) and also required the ARE-binding protein, HuR. Notably, these components excluded Cebpb transcripts from a perinuclear region of the cytoplasm where the C/EBPbeta kinases p-ERK1/2 and CK2 reside in Ras-transformed cells. These findings indicate that the intracellular site of C/EBPbeta translation is critical for Ras-induced activation via effector kinases such as p-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/EBPbeta proceeds and OIS can be implemented to suppress tumorigenesis. We 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. 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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