Inflammation and hypoxia are known to promote the metastatic progression of tumours. The CCAAT/enhancerbinding protein-d (C/EBPd, CEBPD) is an inflammatory response gene and candidate tumour suppressor, but its physiological role in tumourigenesis in vivo is unknown. Here, we demonstrate a tumour suppressor function of C/EBPd using transgenic mice overexpressing the Neu/Her2/ERBB2 proto-oncogene in the mammary gland. Unexpectedly, this study also revealed that C/EBPd is necessary for efficient tumour metastasis. We show that C/EBPd is induced by hypoxia in tumours in vivo and in breast tumour cells in vitro, and that C/EBPd-deficient cells exhibit reduced glycolytic metabolism and cell viability under hypoxia. C/EBPd supports CXCR4 expression. On the other hand, C/EBPd directly inhibits expression of the tumour suppressor F-box and WD repeat-domain containing 7 gene (FBXW7, FBW7, AGO, Cdc4), encoding an F-box protein that promotes degradation of the mammalian target of rapamycin (mTOR). Consequently, C/EBPd enhances mTOR/AKT/S6K1 signalling and augments translation and activity of hypoxia-inducible factor-1a (HIF-1a), which is necessary for hypoxia adaptation. This work provides new insight into the mechanisms by which metastasis-promoting signals are induced specifically under hypoxia.

Toll-like receptor 4 (TLR4) plays a pivotal role in innate immune responses, and the transcription factor CCAAT/enhancer binding protein delta (C/EBPδ, Cebpd) is a TLR4-induced gene. Here, we identify a positive feedback loop in which C/EBPδ activates Tlr4 gene expression in macrophages and tumour cells. In addition, we discovered a negative feedback loop whereby the tumour suppressor FBXW7α (FBW7, Cdc4), whose gene expression is inhibited by C/EBPδ, targets C/EBPδ for degradation when C/EBPδ is phosphorylated by GSK-3β. Consequently, FBXW7α suppresses Tlr4 expression and responses to the ligand lipopolysaccharide (LPS). FBXW7α depletion alone is sufficient to augment pro-inflammatory signalling in vivo. Moreover, as inflammatory pathways are known to modulate tumour biology, Cebpd null mammary tumours, which have reduced metastatic potential, show altered expression of inflammation-associated genes. Together, these findings reveal a role for C/EBPδ upstream of TLR4 signalling and uncover a function for FBXW7α as an attenuator of inflammatory signalling.

The transcription factor CCAAT/enhancer binding protein δ (C/EBPδ, CEBPD, NFIL-6β) has tumor suppressor function; however, the molecular mechanism(s) by which C/EBPδ exerts its effect are largely unknown. Here, we report that C/EBPδ induces expression of the Cdc27 (APC3) subunit of the anaphase promoting complex/cyclosome (APC/C), which results in the polyubiquitination and degradation of the prooncogenic cell cycle regulator cyclin D1, and also down-regulates cyclin B1, Skp2, and Plk-1. In C/EBPδ knockout mouse embryo fibroblasts (MEF) Cdc27 levels were reduced, whereas cyclin D1 levels were increased even in the presence of activated GSK-3β. Silencing of C/EBPδ, Cdc27, or the APC/C coactivator Cdh1 (FZR1) in MCF-10A breast epithelial cells increased cyclin D1 protein expression. Like C/EBPδ, and in contrast to cyclin D1, Cdc27 was down-regulated in several breast cancer cell lines, suggesting that Cdc27 itself may be a tumor suppressor. Cyclin D1 is a known substrate of polyubiquitination complex SKP1/CUL1/F-box (SCF), and our studies show that Cdc27 directs cyclin D1 to alternative degradation by APC/C. These findings shed light on the role and regulation of APC/C, which is critical for most cellular processes.