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

Abstract

The transcription factor CCAAT/enhancer-binding protein delta (C/EBPδ, 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/EBPδ expression and its role in human breast cancer cells. We describe a novel pathway by which the tyrosine kinase Src downregulates C/EBPδ 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/EBPδ, but not C/EBPβ, and promotes its polyubiquitination and proteasomal degradation. Src/SIAH2-mediated inhibition of C/EBPδ expression supports elevated cyclin 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/EBPδ 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/EBPδ, 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/EBPδ expression can be regulated at the protein level by oncogenic Src kinase signals through SIAH2, thus contributing to breast epithelial cell transformation.

ACKNOWLEDGMENTS

We thank Suzanne Specht for expert technical assistance. We are indebted to Ira Daar, Stanley Lipkowitz, and Allan Weissman (NCI) for generously providing reagents and advice. We thank Amy Tang (Mayo Clinic Cancer Center) for SIAH expression constructs, Rosalyn Irby (Penn State College of Medicine) for Src-531, Chris Albanese (Georgetown University) for the cyclin D1 promoter reporter, Lyuba Varticovski (NCI) for MDA-MB-231T cells, and Jaqueline Bromberg (Memorial Sloan-Kettering Cancer Center) for MDA-MB-468 cells. We thank Amit Chaudhary and Luana Scheffer for advice with confocal microscopy, Pralhada Rao Raghavendra for his advice with site-directed mutagenesis, Robert Leighty for help with statistical analysis, and Jiro Wada for preparation of the figures. We thank Ira Daar, Kuppusamy Balamurugan, Sara Courtneidge, and Allan Weissman for invaluable discussion and critical comments on the manuscript.

This research was supported by the Intramural Research Program of the NIH, National Cancer Institute and in part with federal funds under contract number HHSN261200800001E.

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