ATP7A is a novel target of retinoic acid receptor β2 in neuroblastoma cells

Abstract

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.

Neuroprotection by marine-derived compound, 11-dehydrosinulariolide, in an in vitro Parkinson’s model: a promising candidate for the treatment of Parkinson’s disease

“…Cell survival was determined after treatment with Alamar blue, a tetrazolium dye that is reduced by living cells to a fluorescent product. This assay is similar in principle to the 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide cell viability assay and has been previously validated as an accurate measure of the survival of SH-SY5Y cells (Bohlken et al 2009). Relative protection (percent) was calculated as 100×[(optical density (OD) of 6-OHDA/11-dehydrosinulariolide-treated cells−OD of 6-OHDA-treated cells) / (OD of control cells−OD of 6-OHDA-treated cells)] (Lee et al 2006).…”

Retinoic acid signaling and neuronal differentiation

“…RARb2 induces the expression of ATP7A, a Golgi-associated protein that removes copper from cells [204]. Knock down of RARb2 inhibits expression of ATP7A, reducing copper efflux.…”

“…Knock down of RARb2 inhibits expression of ATP7A, reducing copper efflux. Copper levels are apparently critical for the response of neuroblastoma cells to RA because copper supplementation induced proliferation, and copper chelation promoted differentiation [204]. Elongin A is an elongation factor that is essential for neuronal differentiation, in vivo [205].…”

Behind the Link between Copper and Angiogenesis: Established Mechanisms and an Overview on the Role of Vascular Copper Transport Systems

“…Conclusively, the possibility of hindering the spread of cancer by keeping vascular copper levels below the pathological threshold points to the ATP7A pump as a potential clinical target. Interestingly, among retinoids (vitamin A derivatives), known to inhibit the VEGF- and FGF-2-induced endothelial activation [158], retinoic acid receptor β is known to up-regulate the expression of ATP7A in BE(2)-C and SH-SY5Y human neuroblastoma cell models [159]. …”