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 and its role 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. 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 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). C/EBPbeta was previously shown to be phosphorylated by activated ERK kinase, and we identified a RSK kinase site in the leucine zipper that serves as an important regulator of C/EBPbeta DNA-binding and homodimerization. We have also mapped a CK2 phosphorylation site that is required for Ras-induced DNA binding. An important finding from our lab was the discovery 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. Hence, C/EBPbeta is uncoupled from Ras signaling and fails to undergo phosphorylation and activation by ERK and CK2. 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 anticipate that UPA-like mechanisms may regulate many proteins to coordinate cellular responses to Ras signaling. We are currently investigating this possibility by studying the regulation of other pro-oncogenic and anti-oncogenic transcription factors by their 3'UTR sequences. In cells expressing oncogenic Ras, p-ERK and CK2 are present in structures that we call """"""""perinuclear signaling complexes"""""""" (PSCs). PSCs are associated with endosomes and require the MAPK scaffold, KSR1 (kinase suppressor of Ras 1). Our research shows that in addition to its known ability to facilitate Raf-MEK-ERK signal transmission, KSR1 plays a key role in establishing the perinuclear localization of Ras effector kinases. We found that PSCs also form in response to growth factor signals, but with delayed kinetics (4-6 hr after GF stimulation). We propose that oncogenic Ras signaling mirrors this late phase of GF signaling in which effector kinases become localized to a perinuclear compartment, where they access key substrates such as C/EBPbeta in a UPA-regulated manner. We have observed similar localized signaling complexes in several kinds of tumor cells, and we suggest that PSCs are a common or universal feature of the oncogenic signaling landscape in cancer cells. In the future, PSC components may prove to be effective targets for cancer therapies. Their presence might also be used as a biomarker to monitor the efficacy of anti-cancer drugs targeting the Ras pathway. In a separate project we are investigating the regulatory and biological functions of the small C/EBP family member, C/EBPgamma. C/EBPgamma is a dimeric partner of C/EBPbeta, and one of its roles is to modulate the activity of C/EBPbeta through heterodimerization. Cebpg knockout MEFs display severe proliferative defects, increased senescence, and elevated expression of senescence-associated secretory phenotype (SASP) genes, effects that are at least partly due to increased levels of C/EBPbeta homodimers. Cebpg KO cells also exhibit oxidative stress that was traced to defective synthesis of the cellular anti-oxidant, glutathione. The growth defects in these cells were reversed by addition of the anti-oxidant, N-acetyl cysteine (NAC). Many adverse conditions, including oxidative and ER stresses, induce the bZIP transcription factor ATF4, which serves as a """"""""master"""""""" regulator of many cellular stress responses. We found that ATF4:C/EBPgamma heterodimeric complexes are induced in stressed cells and bind to genomic C/EBP:ATF response elements (CAREs), which regulate numerous stress-induced genes. Our studies have identified C/EBPgamma as a novel and essential C/EBP partner of ATF4. Cebpg knockout mice die shortly after birth due to defective lung inflation and respiratory failure. These defects could be substantially reversed by in utero administration of NAC to alleviate oxidative stress. C/EBPgamma also has an important role in cancer, and gene expression analysis suggests correlations between elevated CEBPG mRNA levels and increased malignancy in several human cancers. Furthermore, depletion of C/EBPgamma led to senescence and oxidative stress in human lung and breast tumor cell lines. Our data suggest that cancer cells rely on C/EBPgamma:ATF4 and/or C/EBPgamma:ATF2 heterodimers to mitigate stress arising from increased ROS, hypoxia, and nutrient deprivation.
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