Gene expression analyses in human tissues show that CEBPD expression is downregulated in breast cancer and is part of a 70-gene expression signature that predicts longer patient survival. These observations and studies in cell lines had suggested that C/EBPdelta functions as a tumor suppressor. Our recent contributions to elucidate the tumor suppressor-like functions of C/EBPdelta in mammary epithelial cells include the finding that C/EBPdelta inhibits the transformation of breast epithelial cell lines by Src kinase(s) (Sarkar et al., 2012). This study provides new mechanistic insight regarding breast tumor cell transformation by Src tyrosine kinase(s) and evidence that the C/EBPdelta transcription factor functions as a tumor suppressor in breast epithelial cell. First, we identified a novel pathway of Src kinase signaling which contributes to activation of the SIAH2 E3 ligase and targets the transcription factor C/EBPdelta for degradation. The Src family of tyrosine kinases is both upstream and downstream of many tumor promoting signaling pathways, and this study provides new molecular insights into its mechanisms of action and those of Src-kinase inhibitors, which are in clinical trials for breast cancer. Second, while SIAH2 has been linked to the oncogenic Ras/MAPK and hypoxia pathway, clinical studies on its role in breast cancer have been contradictory. Our study places SIAH2 downstream of Src and identifies C/EBPdelta as a relevant target of SIAH2 with tumor suppressor activity. Third, we identified a novel mechanism for regulation of C/EBPdelta at the level of protein stability and how this mechanism is biologically relevant for induction of cyclin D1 expression and inactivation of the tumor suppressor retinoblastoma protein (Rb) by Src kinase signaling. In summary this study provides insights about the role of C/EBPdelta in human breast epithelial cells and reveals a new molecular pathway which links Src kinases, the SIAH2 E3 ligase, and the C/EBPdelta transcription factor, providing a mechanism by which Src can inactivate tumor suppressor proteins. Previously, we had obtained the first in vivo evidence of C/EBPdelta's tumor-suppressing role, by crossing Cebpd null mice with transgenic MMTV-Neu mice, a model for stochastic mammary tumorigenesis (Balamurugan et al., 2010). Unexpectedly, this approach revealed an additional role of C/EBPdelta in promoting metastatic tumor progression. We had discovered that C/EBPdelta augments cell survival under hypoxic by suppressing expression of FBXW7alpha (Balamurugan et al., 2010). FBXW7alpha is a bona fide tumor suppressor for several epithelial cancers because it targets many oncoproteins for degradation. We subsequently investigated this pathway in the context of inflammation, which - like hypoxia - is known to promote cancer progression. Our new findings provide important mechanistic insights into the innate inflammatory signaling pathway through our investigations of C/EBPdelta/FBXW7alpha signaling in primary mouse and human monocytes/macrophages and monocyte/macrophage cell lines. First, C/EBPdelta had previously been reported as an amplifier of inflammatory signaling that is essential to clear gram-negative (LPS) infection in mice through functions downstream of the TLR4 receptor (Litvak et al., 2009, Nat Immunol). We have identified the LPS receptor subunit TLR4 as a target gene of C/EBPdelta, which in fact places C/EBPdelta upstream of LPS signaling. This is only the second report characterizing transcriptional regulation of the TLR4 gene. In addition, these data call for a re-evaluation of the precise role of C/EBPdelta in inflammatory signaling downstream of TLR4. Second, we show that C/EBPdelta mediates macrophage activation in part through activation of the HIF-1 pathway, suggesting that the pathway we previously characterized in tumor cells has its physiological origin in inflammation-associated signaling pathways. This data sheds new light on the intersection of inflammation and cancer cell signaling. Third, we identify a negative feedback loop between C/EBPdelta and FBXW7alpha, whereby FBXW7alpha targets C/EBPdelta for degradation. Perhaps most importantly, this reveals FBXW7alpha as a potent attenuator of pro-inflammatory gene expression. We provide the first report of FBXW7alpha expression and function in mature immune effector cells, which opens up new avenues for investigations into FBXW7alpha functions in macrophages, inflammation, and hematopoietic malignancies with potentials for discovery of novel therapeutics. Our finding that C/EBPdelta represses FBXW7alpha in tumor cells and macrophages suggested alternative roles for C/EBPdelta in tumor development, both within mammary epithelial cells and in the tumor microenvironment (Balamurugan et al., 2010, 2013). In addition, we discovered two degradation pathways that downregulate C/EBPdelta protein levels: the pro-oncogenic SIAH2 ubiquitin ligase pathway and a pathway requiring the tumor suppressor FBXW7 ubiquitin ligases (Sarkar et al., 2012, Balamurugan et al., 2013). These findings corroborate a dual nature of C/EBPdelta's functions as both a tumor promoter and tumor suppressor. We anticipate that future investigations into the molecular mechanisms of C/EBPdelta's dual functions will provide insights into the complexity and diversity of cellular processes that lead to breast cancer development.
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