5331

Over the past decade basic and clinical research has demonstrated a therapeutic role for retinoids, in particular, retinoic acid (RA) in human cancer. Molecular events at the point of transcriptional regulation appear to be critical in determining cellular responses to retinoids. The ligand-receptor complex initiates the retinoid signal by changes that occur at consensus DNA sequences or RA response elements (RAREs) in the regulatory 5’ untranslated sequence of target genes. One mechanism by which cancer cells evade retinoid induced effects is through repression of retinoic acid receptor β (RARβ) gene transcription. The RA response element beta (βRARE) is the essential DNA sequence required for retinoid-induced RARβ transcription. To identify novel regulators of retinoid signaling, we developed a technique for directly isolating βRARE-associated proteins, to better understand the dynamic changes in protein-protein and protein-DNA complexes at this focal point of the RA anti-cancer signal. Using this approach, we have identified the Estrogen-responsive B Box Protein (EBBP) as a βRARE-associated protein. EBBP is a poorly characterized member of the RING-B box-coiled-coil (RBCC)/Tripartite Motif (TRIM) protein family. In this report, we provide the first evidence that EBBP is a novel co-regulator of RARβ and, play a significant role in the retinoid signaling pathway. Our data indicate that RA treatment of retinoid-sensitive cells results in EBBP phosphorylation, and, elevated EBBP nuclear expression in aggregates with promyelocytic leukemia (PML) at PML nuclear bodies. Over-expression of EBBP increases the level of exogenous and endogenous βRARE transactivation. Transfection of EBBP siRNA significantly decreased RARβ gene expression in neuroblastoma cells. Over-expression of EBBP induces growth inhibition in RA-sensitive neuroblastoma, as well as RA-resistant lung, breast and acute promyelocytic leukemia cancer cells. Most importantly, EBBP restores RA-responsive RARβ transcription in RA-resistant lung and breast cancer cells, which were resistant to both a histone deacetylase inhibitor and demethylating agent. In addition, deletion \. mutant analysis localized the βRARE transactivating function to the coiled-coil domain of EBBP, a region thought to be responsible for protein-protein interactions.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]