Identification of a Src Tyrosine Kinase/SIAH2 E3 Ubiquitin Ligase Pathway That Regulates C/EBPδ Expression and Contributes to Transformation of Breast Tumor Cells

The transcription factor CCAAT/enhancer-binding protein delta (C/EBP delta, CEBPD) is a tumor suppressor that is downregulated during breast cancer progression but may also promote metastasis. Here, we have investigated the mechanism(s) regulating C/EBP delta expression and its role in human breast cancer cells. We describe a novel pathway by which the tyrosine kinase Src downregulates C/EBP delta through the SIAH2 E3 ubiquitin ligase. Src phosphorylates SIAH2 in vitro and leads to tyrosine phosphorylation and activation of SIAH2 in breast tumor cell lines. SIAH2 interacts with C/EBP delta, but not C/EBP beta, and promotes its polyubiquitination and proteasomal degradation. Src/SIAH2-mediated inhibition of C/EBP delta expression supports elevated cydin D1 levels, phosphorylation of retinoblastoma protein (Rb), motility, invasive properties, and survival of transformed cells. Pharmacological inhibition of Src family kinases by SKI-606 (bosutinib) induces C/EBP delta expression in an SIAH2-dependent manner, which is necessary for therapeutic responses to SKI-606 in vitro. Ectopic expression of degradation-resistant mutants of C/EBP delta, which do not interact with SIAH2 and/or cannot be polyubiquitinated, prevents full transformation of MCF-10A cells by activated Src (Src truncated at amino acid 531 [Src-531]) in vitro. These data reveal that C/EBP delta expression can be regulated at the protein level by oncogenic Src kinase signals through SIAH2, thus contributing to breast epithelial cell transformation.