HDAC inhibitors induce NOX5 expression in human uveal melanoma cells in a Brd4-Sp1/Sp3-dependent manner

Epigenetic reprogramming, particularly histone deacetylation (HDAC), and reactive oxygen species (ROS) play key roles in melanoma progression and the development of drug resistance. While various histone deacetylase inhibitors (HDACi) are already in clinical use for hematological cancers, recent studies indicate their potential effectiveness in melanoma models as well. Given the increasing interest in enhancing the therapeutic efficacy of HDACi, we investigated the molecular mechanisms underlying their association with ROS formation in melanomas. Uveal melanoma (UM), a rare and aggressive eye cancer, has been identified in the TCGA dataset as expressing the highest levels of the superoxide-producing enzyme NADPH oxidase 5 (NOX5) among all human cancers. Our study demonstrates that HDACi treatment increases NOX5 expression in UM cell lines. This upregulation is accompanied by elevated extracellular ROS, which is suppressed when NOX5 is silenced. The use of actinomycin D and cycloheximide significantly reduced HDACi-induced NOX5 expression, suggesting regulation at both transcriptional and translational levels. HDACi treatment also led to accumulation of acetylated histones H3 and H4. Since histone acetylation (particularly H4K5/8/12Ac) recruits BRD4 to facilitate transcription, we evaluated the effect of BRD4 knockdown or exposure to BRD4 inhibitors on NOX5 levels in UM cells. Both approaches reduced NOX5 expression induced by HDACi. Furthermore, dual silencing of the transcription factors Sp1 and Sp3 markedly attenuated both basal and HDACi-induced expression of NOX5. ChIP assays revealed that HDACi treatment increases chromatin-associated acetylated histone H3, phosphorylated RNA polymerase II (p-Ser2), BRD4, Sp1, and Sp3 at the NOX5 promoter region. Collectively, these findings suggest that HDACi-mediated upregulation of NOX5 in UM cells occurs, at least in part, via transcriptional activation of the NOX5 promoter. This process is driven by HDACi-induced histone hyperacetylation, which enhances the recruitment of BRD4, Sp1, and Sp3 to the promoter region, thereby facilitating RNA polymerase II-dependent transcription. Given that HDAC inhibition elevates both NOX5 expression and ROS production, co-targeting HDACs and NOX5 may offer a novel and effective therapeutic strategy in the treatment of UM.