ABSTRACT
Retinoids have significant clinical activity in several human cancers, yet the factors determining retinoid sensitivity in cancer cells are still unclear. Retinoid-induced expression of retinoic acid receptor (RAR) beta(2) is a necessary component of the retinoid anticancer signal in cancer cells. We have previously identified the Estrogen-responsive B Box Protein (EBBP), a member of the Tripartite Motif (TRIM) protein family, as a novel RARbeta2 transcriptional regulator in the retinoid signal. Here we examined the mechanism of the EBBP effect on the retinoid anticancer signal. We assessed retinoid-responsive RARbeta2 transcription in retinoid-resistant breast and lung cancer cells in the presence of chromatin modifying agents. A histone deacetylase (HDAC) inhibitor alone, or in combination with retinoid, was more effective than a demethylating agent in restoring RARbeta2 transcription in resistant cells. Overexpression of EBBP alone markedly increased histone acetylation. The effect of EBBP on retinoid-responsive transcription appeared to be limited to genes with the retinoic acid response element (betaRARE) regulatory sequence, such as CYP26A1. EBBP inhibited cell growth by effects on cyclin D1 and Phospho-Rb, and, reduced cell viability in retinoid-resistant cancer cells. The viability of non-cancer cells was unaffected by EBBP overexpression. Taken together our data suggests that EBBP acts to de-repress transcription of RARbeta2 and CYP26A1, by modifying histone acetylation in retinoid-resistant cancer cells, and, is an important target for drug discovery in retinoid-resistant cancers.
Subject(s)
DNA-Binding Proteins/physiology , Histones/metabolism , Neoplasms/drug therapy , Transcription Factors/physiology , Tretinoin/therapeutic use , Acetylation , Apoptosis , Cell Line, Tumor , Cell Survival , Cyclin D1/metabolism , Cytochrome P-450 Enzyme System/physiology , Drug Resistance, Neoplasm , Humans , Neoplasms/metabolism , Phosphorylation , Receptors, Retinoic Acid/genetics , Receptors, Retinoic Acid/physiology , Retinoblastoma Protein/metabolism , Retinoic Acid 4-Hydroxylase , Tripartite Motif Proteins , Ubiquitin-Protein LigasesABSTRACT
Retinoic acid (RA) induces growth arrest, cell death, and differentiation in many human cancer cells in vitro and has entered routine clinical use for the treatment of several human cancer types. One mechanism by which cancer cells evade retinoid-induced effects is through repression of retinoic acid receptor beta (RARbeta) gene transcription. The RA response element beta (betaRARE) is the essential DNA sequence required for retinoid-induced RARbeta transcription. Here we show that the estrogen-responsive B box protein (EBBP), a member of the RING-B box-coiled-coil protein family, is a betaRARE-binding protein. EBBP undergoes serine threonine phosphorylation and enhanced protein stability after RA treatment. Following RA treatment, we also observed increased nuclear EBBP levels in aggregates with the promyelocytic leukemia protein at promyelocytic leukemia nuclear bodies. EBBP enhanced RA-responsive RARbeta transcription in RA-sensitive and -resistant cancer cells, which were resistant to both a histone deacetylase inhibitor and a demethylating agent. EBBP-specific small interfering RNA reduced basal and RA-induced RARbeta expression. EBBP increased betaRARE-transactivating function through its coiled-coil domain. Taken together, our work suggests that EBBP may have a pivotal role in the retinoid anti-cancer signal.
Subject(s)
Antineoplastic Agents/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Signal Transduction/physiology , Transcription Factors/chemistry , Transcription Factors/metabolism , Tretinoin/metabolism , Amino Acid Sequence , Antineoplastic Agents/pharmacology , Breast Neoplasms , Cell Division/physiology , Cell Line, Tumor , DNA-Binding Proteins/genetics , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/physiology , Humans , Lung Neoplasms , Molecular Sequence Data , Neoplasm Proteins/metabolism , Neuroblastoma , Nuclear Proteins/metabolism , Promyelocytic Leukemia Protein , Protein Structure, Tertiary , RNA, Small Interfering , Receptors, Retinoic Acid/metabolism , Transcription Factors/genetics , Transcription, Genetic , Tretinoin/pharmacology , Tripartite Motif Proteins , Tumor Suppressor Proteins/metabolism , Ubiquitin-Protein LigasesABSTRACT
N-myc has emerged as a member of a transcriptional regulatory network which impinges directly on the machinery of cell growth and proliferation. Critical during neural crest embryogenesis, N-myc is rapidly down-regulated as tissues become terminally differentiated and growth-arrested. The involvement of N-myc in these fundamental cellular processes necessitates an intricate strategy for its regulation, which is still being elucidated. Deregulated N-myc over-expression has clear transforming ability in vitro and in vivo. The transcriptional target genes responsible for this activity are beginning to be unravelled.