Retinoids induce growth arrest, differentiation, and cell death in many cancer cell types. One factor determining the sensitivity or resistance to the retinoid anticancer signal is the transcriptional response of retinoid-regulated target genes in cancer cells. We used cDNA microarray to identify 31 retinoid-regulated target genes shared by two retinoid-sensitive neuroblastoma cell lines, and then sought to determine the relevance of the target gene responses to the retinoid anticancer signal. The pattern of retinoid responsiveness for six of 13 target genes (RARb2, CYP26A1, CRBP1, RGS16, DUSP6, EGR1) correlated with phenotypic retinoid sensitivity, across a panel of retinoid-sensitive or -resistant lung and breast cancer cell lines. Retinoid treatment of MYCN transgenic mice bearing neuroblastoma altered the expression of five of nine target genes examined (RARb2, CYP26A1, CRBP1, DUSP6, PLAT) in neuroblastoma tumour tissue in vivo. In retinoid-sensitive neuroblastoma, lung and breast cancer cell lines, direct inhibition of retinoid-induced RARb2 expression blocked induction of only one of eight retinoid target genes (CYP26A1). DNA demethylation, histone acetylation, and exogenous overexpression of RARb2 partially restored retinoid-responsive CYP26A1 expression in RA-resistant MDA-MB-231 breast, but not SK-MES-1 lung, cancer cells. Combined, rather than individual, inhibition of DUSP6 and RGS16 was required to block retinoid-induced growth inhibition in neuroblastoma cells, through phosphorylation of extracellularsignal-regulated kinase. In conclusion, sensitivity to the retinoid anticancer signal is determined in part by the transcriptional response of key retinoid-regulated target genes, such as RARb2, DUSP6, and RGS16.
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  (RAR) gene transcription. The RA response element  (RARE) is the essential DNA sequence required for retinoid-induced RAR 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 RARE-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 RAR 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 RAR expression. EBBP increased RARE-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.Over the past decade, basic and clinical research has demonstrated a therapeutic role for retinoids, in particular RA 2 in human cancer (1-3). Molecular events at the point of transcriptional regulation appear to be critical in determining cellular responses to retinoids. Upon entering the nucleus by simple diffusion, the retinoid ligand binds to retinoic acid receptors (RARs) or retinoid X receptors (each with ␣, , and ␥ subtypes). The ligand-receptor complex initiates the retinoid signal by changes that occur at consensus DNA sequences or RA response elements (RAREs) in the regulatory 5Ј-untranslated sequence of so-called "target" genes.RAR is a retinoid target with a well defined RARE, which is necessary but not sufficient for the RA anti-cancer signal (4). Repression of basal or RA-induced RAR transcription is a common event in a wide range of human tumor types, and numerous studies indicate that in specific circumstances RAR acts as a tumor suppressor gene (2, 5-8).A frequent mechanism of RAR repression is DNA methylation-induced silencing (9). Additionally, histone deacetylation is also associated with retinoid resistance, in the presence and absence of RAR2 hypermethylation, indicating that multiple mechanisms appear to contribute to RAR repression (10).Intrinsic and acquired retinoid resistance has limited the clinical activity of retinoid-based therapy and chemoprevention. RA treatment in acute promyelocytic leukemia has been particularly valuable in illuminating mechanisms of retinoid resistance. Most acute promyelocytic leukemia cases present with the reciprocal t(15,17) translocation, resulting in the fusion product PML-RAR␣ (11). Dominant negative effects of the PML-RAR␣ fusion protein have been associated ...
Increased retinoic acid receptor b (RARb 2 ) gene expression is a hallmark of cancer cell responsiveness to retinoid anticancer effects. Moreover, low basal or induced RARb 2 expression is a common feature of many human cancers, suggesting that RARb 2 may act as a tumour suppressor gene in the absence of supplemented retinoid. We have previously shown that low RARb 2 expression is a feature of advanced neuroblastoma. Here, we demonstrate that the ABC domain of the RARb 2 protein alone was sufficient for the growth inhibitory effects of RARb 2 on neuroblastoma cells. ATP7A, the copper efflux pump, is a retinoid-responsive gene, was upregulated by ectopic overexpression of RARb 2 . The ectopic overexpression of the RARb 2 ABC domain was sufficient to induce ATP7A expression, whereas, RARb 2 siRNA blocked the induction of ATP7A expression in retinoid-treated neuroblastoma cells. Forced downregulation of ATP7A reduced copper efflux and increased viability of retinoid-treated neuroblastoma cells. Copper supplementation enhanced cell growth and reduced retinoid-responsiveness, whereas copper chelation reduced the viability and proliferative capacity. Taken together, our data demonstrates ATP7A expression is regulated by retinoic acid receptor b and it has effects on intracellular copper levels, revealing a link between the anticancer action of retinoids and copper metabolism.
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