Inhibition of HDAC6 elicits anticancer effects on head and neck cancer cells through Sp1/SOD3/MKP1 signaling axis to downregulate ERK phosphorylation

Oxidative stress caused by reactive oxygen species (ROS) and superoxides is linked to various cancer-related biological events. Extracellular superoxide dismutase (SOD3), an antioxidant enzyme that removes superoxides, contributes to redox homeostasis and has the potential to regulate tumorigenesis. Histone deacetylase 6 (HDAC6), a major HDAC isoform responsible for mediating the deacetylation of non-histone protein substrates, also plays a role in cancer progression. In this study, we examined the potential effects of HDAC6 inhibition on SOD3 expression in head and neck cancer (HNC) cells and its impact on cell proliferation, which remains unaddressed. We found that functional inactivation of HDAC6, through the use of chemical inhibitors such as tubastatin A (TubA), gene knockdown, or overexpression of an inactive mutant, strongly upregulated protein and mRNA levels of SOD3 in HNC cell lines FaDu and Detroit562. Mechanistically, TubA induced acetylation of the transcription factor Sp1 at Lys703, which consequently enhanced its binding to the SOD3 proximal promoter region and increased SOD3 expression. An acetylation-defective Sp1 mutant (K703R) was much less effective in inducing SOD3 expression compared to wild-type Sp1. TubA reduced intracellular ROS and superoxide levels, and this antioxidative effect was attenuated in SOD3 knockdown cells. Similar to the changes in ROS levels, HDAC6 inhibition as well as SOD3 overexpression suppressed cell proliferation and the stimulatory phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), whereas SOD3 knockdown produced opposite effects in both resting and TubA-treated conditions. In addition, SOD3 overexpression prevented ROS-induced ERK1/2 phosphorylation and enhanced the protein stability of mitogen-activated protein kinase phosphatase 1 (MKP1), thereby counteracting ERK1/2 phosphorylation. We further showed that SOD3-mediated ERK1/2 dephosphorylation was moderated in MKP1 knockdown cells. Collectively, these results suggest that HDAC6 inhibition elicits anticancer effects on HNC cells by promoting Sp1 acetylation-dependent SOD3 upregulation, leading to MKP1 stabilization and subsequent ERK1/2 inactivation.