Cancer involves aberrant control of cellular proliferation due to activation of oncogenes and inactivation of tumor suppressors. The latter provide an intrinsic barrier to cell 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 targeted by novel anti-cancer 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 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 are studying the mechanisms by which C/EBPb expression and activity are controlled by oncogenic RAS signaling and the molecular basis for its dual role in both suppressing and promoting cancer. 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 cascade. C/EBPb 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 C/EBPb's DNA-binding and transactivation functions. C/EBPb becomes activated by RAS signaling through several inducible post-translational modifications (PTMs). C/EBPb was previously shown to be phosphorylated by activated ERK kinase, and we identified a RSK kinase site in the leucine zipper that regulates C/EBPb 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/EBPb protein, thereby suppressing its pro-senescence and cytostatic activities 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 C/EBPb-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 G/U rich elements (GREs) and also required the ARE/GRE-binding protein, HuR. These components act by directing Cebpb transcripts to the peripheral cytoplasm, excluding them from a perinuclear region where the C/EBPb kinases p-ERK1/2 and CK2 reside in RAS-transformed cells. In this location, newly-translated C/EBPb is uncoupled from RAS signaling and fails to undergo phosphorylation and activation by ERK and CK2. Thus, the intracellular site of C/EBPb translation is critical for RAS-induced activation via effector kinases such as p-ERK. Notably, 3'UTR inhibition and Cebpb mRNA compartmentalization are not observed in primary mouse and human fibroblasts. Consequently, RAS-induced activation of C/EBPb is permitted 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 whether the activities of other pro-oncogenic and anti-oncogenic transcription factors are controlled by 3'UTR sequences. In cells expressing oncogenic Ras, p-ERK and CK2 are found in structures we denote perinuclear signaling complexes or 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 signaling, KSR1 plays a key role in regulating perinuclear localization of RAS effector kinases. We found that PSCs are also induced by serum growth factors, but with delayed kinetics (4-6 hr after GF stimulation). We propose that oncogenic RAS signaling mirrors this late phase of the GF response in which effector kinases become localized to a perinuclear compartment where they access key substrates. In the case of C/EBPb and possibly other targets, phosphorylation occurs in a UPA-regulated manner. We have observed similar localized signaling complexes in several kinds of human tumor cells and in KRAS-induced mouse lung tumors, suggesting that PSCs are a ubiquitous feature of the signaling landscape in cancer cells. In the future, PSC components may prove to be effective targets for cancer therapies, as well as biomarkers to identify tumor cells in tissue samples. In addition, PSCs could be used to monitor tumor responses to anti-cancer drugs that inhibit the RAS-ERK pathway. In a separate project we are investigating the regulatory and biological functions of the small C/EBP family member, C/EBPgamma, which is a dimeric partner of C/EBPb. One of its roles is to modulate the activity of C/EBPb 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/EBPb homodimers. Cebpg KO cells also exhibit oxidative stress that was linked 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 redox imbalances and ER stress, induce the bZIP transcription factor ATF4, which serves as a master regulator of many cellular stress responses. We found that ATF4:C/EBPg heterodimeric complexes are induced in stressed cells and bind to genomic C/EBP:ATF response elements (CAREs), which regulate numerous stress response genes. These studies have identified C/EBPg 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/EBPg 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/EBPg led to senescence and oxidative stress in human lung and breast tumor cell lines. Cebpg-/- mice also develop significantly fewer malignant solid tumors than WT mice upon aging, consistent with the anti-oxidant functions of C/EBPg promoting cancer. In summary, our data indicate that cancer cells rely on C/EBPg:ATF heterodimers to regulate genes involved in mitigating stresses arising from increased ROS, hypoxia, and nutrient deprivation.
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