Development of cancer involves aberrant control of cellular proliferation due to activation of oncogenes as well as inactivation of tumor suppressors, which protect against unregulated growth by inducing apoptosis or growth arrest (senescence) of 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 exploited for therapeutic purposes. 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 C/EBPbeta and its role as a downstream effector of oncogenic Ras signaling. Tumor studies using Cebpb null mice as well as analysis of human and rodent tumor cells has revealed that C/EBPb has pro-oncogenic functions. However, in RasV12-expressing fibroblasts (MEFs) C/EBPb is also required for oncogene-induced senescence (OIS), an intrinsic tumor suppression mechanism in vitro and in vivo. In the context of OIS C/EBPb acts to arrest cellular proliferation by a pathway requiring RB:E2F. C/EBPb thus possesses both pro- and anti-tumorigenic activities. Because it plays an important role in cellular responses to Ras, we wish to elucidate the mechanisms by which C/EBPb expression and activity are controlled by oncogenic Ras signaling and understand the basis for C/EBPbs dual activities in suppressing or promoting cancer. Post-translational regulation of C/EBPb activity: C/EBPb is an intrinsically repressed protein whose activity can be stimulated by oncogenic Ras or growth factor signaling through a pathway critically dependent on MEK-ERK signaling. 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 core sequesters the basic region and transactivation domain, inhibiting both DNA binding and transactivation. C/EBPb activation by Ras signaling involves several inducible post-translational modifications (PTMs). We previously identified a RSK kinase site in the leucine zipper 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, possibly due to decreased circulating levels of IGF-1 in the 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 senescent-like growth arrest 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. Regulation of C/EBPb activity by heterodimerization with C/EBPgamma: C/EBPg is a ubiquitously expressed protein that preferentially heterodimerizes with C/EBPb and other C/EBP family members. However, its biological functions are not well understood. We found that C/EBPg-deficient MEFs display reduced proliferative potential in vitro. This defect is in part due to the ability of C/EBPg to suppress the growth-inhibitory effects of C/EBPb by forming heterodimers. C/EBPg knockout cells also display elevated levels of proinflammatory cytokines such as IL-6, which are involved in senescence induction and may account for the decreased proliferative potential of C/EBPg-deficient cells. Thus, C/EBPg is a key modulator of C/EBPb cytostatic activity. Role of the Cebpb 3'UTR in regulating C/EBPb protein activity: We recently made the unexpected discovery that the Cebpb 3'UTR inhibits the Ras-induced post-translational of C/EBPb, thus quelling its pro-senescence and cytostatic activities specifically in tumor cells. The 3'UTR suppresses the activation of C/EBPb DNA-binding and transcriptional potential in response to oncogenic Ras signaling. The 3UTR blocked the induction of senescence-associated C/EBPb target genes, while promoting expression of genes linked to cancers and TGFb signaling. The 3'UTR inhibitory effect was mapped to an A/U rich element (ARE)-containing region and requires the ARE-binding protein, HuR. These components also excluded Cebpb mRNA from a perinuclear cytoplasmic region that contains activated ERK1/2, indicating that the site of C/EBPb translation controls de-repression by Ras signaling via effector kinases such as ERK. Notably, 3UTR inhibition and Cebpb mRNA compartmentalization were absent in primary fibroblasts, allowing Ras-induced C/EBPb activation and OIS to proceed. In summary, our findings reveal a novel mechanism, termed 3'UTR regulation of protein activity or UPA, whereby non-coding mRNA sequences selectively regulate C/EBPb activity and suppress its anti-oncogenic functions. The UPA system may represent an attractive target for novel therapeutic interventions designed to reactivate tumor suppression programs in cancer cells.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010328-12
Application #
8348976
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
12
Fiscal Year
2011
Total Cost
$1,089,789
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Salotti, Jacqueline; Sakchaisri, Krisada; Tourtellotte, Warren G et al. (2015) An Arf-Egr-C/EBPβ pathway linked to ras-induced senescence and cancer. Mol Cell Biol 35:866-83
Bhaumik, Prasenjit; Davis, Jamaine; Tropea, Joseph E et al. (2014) Structural insights into interactions of C/EBP transcriptional activators with the Taz2 domain of p300. Acta Crystallogr D Biol Crystallogr 70:1914-21
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Yan, Chunguang; Tang, Huifang; Gao, Hongwei et al. (2013) Authors' reply. Am J Pathol 182:1459-60
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Basu, Sandip K; Malik, Radek; Huggins, Christopher J et al. (2011) 3'UTR elements inhibit Ras-induced C/EBPýý post-translational activation and senescence in tumour cells. EMBO J 30:3714-28

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