Cancer involves aberrant control of cellular proliferation due to activation of oncogenes and inactivation of tumor suppressors. Tumor suppressors 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, since such mutations disrupt apoptotic or senescence responses to oncogenic stress. 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. Analysis of Cebpb null mice and 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 mouse fibroblasts (MEFs), C/EBPb is also required for oncogene-induced senescence (OIS). In senescing cells, C/EBPb arrests 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 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 the DNA-binding and transactivation functions of C/EBPb. C/EBPb becomes activated by RAS signaling via several inducible post-translational modifications (PTMs). C/EBPb was previously shown to be phosphorylated by ERK kinase. We identified a RSK kinase site in the leucine zipper that regulates C/EBPb DNA binding and homodimerization, and recently mapped a CK2 phosphorylation site 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 the DNA-binding and transcriptional activities of C/EBPb, which are otherwise induced by oncogenic RAS. The 3'UTR inhibitory effect was mapped to a region bearing G/U rich elements (GREs), and 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 of the protein 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 addition, we are identifying proteins (in addition to HuR) that regulate Cebpb mRNA trafficking and are required for UPA regulation. In cells expressing oncogenic RAS or BRAF, p-ERK and CK2 become re-localized to nuclear-proximal structures we call perinuclear signaling complexes or PSCs. PSCs are associated with endosomes and require the MAPK scaffold KSR1 (kinase suppressor of Ras 1) for their formation. KSR1 also undergoes RAS-induced targeting to PSCs and colocalizes with p-ERK and CK2. Thus, besides its known ability to facilitate RAF-MEK-ERK signaling, KSR1 plays a key role in regulating subcellular localization of RAS effector kinases. We found that PSCs are also induced by serum growth factors in normal cells with delayed kinetics (4-6 hr after GF stimulation). We propose that mutant RAS constitutively activates this late phase of GF signaling, where effector kinases become localized to a perinuclear compartment and access key substrates that drive cell proliferation. We have detected PSCS in several kinds of human tumor cells and in KRAS-induced mouse lung tumors, suggesting that this localized complexes are a ubiquitous feature of the cancer signaling landscape. Our current studies are aimed at elucidating the molecular basis for RAS-induced formation of PSCs, focusing on the role of signaling adaptor proteins associated with specific classes of perinuclear endosomes. In the future, PSC components may prove to be effective targets for cancer therapy, as well as providing novel 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.