Epigenetic Reprogramming in Oral Epithelial Dysplasia using Sodium Valproate: an in vitro study parallel to the SAVER trial

Abstract

Background Current management of oral epithelial dysplasia (OED) focuses on preventing malignant transformation of the index lesion without addressing the risk of cancer development at other oral sites. Sodium valproate (SV) was identified as a potential chemopreventive agent for head and neck cancer owing to the dose- and time-dependent protective effect seen in a large retrospective cohort study. Objective Determine the effect of SV on cell proliferation, histone deacetylase (HDAC) activity, and protein expression in dysplasia cell lines in monolayer and develop 3D organotypic models to assess the effect of SV on invasion. Methods • Dysplasia cell lines (D20, DOK, and D35), normal oral keratinocytes (FNB6), and oral cancer fibroblasts (LIV9 and LIV22) cultured in monolayer. • MTT assays, HDAC-glo assay, Western blotting, and LINE-1 pyrosequencing. • 3D collagen models (cancer fibroblasts and dysplastic epithelial cells) exposed to 1 mM SV for 7 days before processing. Changes in invasion pattern and depth are assessed on hematoxylin and eosin–stained slides. Results Exposure to 1 mM SV for 48 hours reduces cell proliferation and HDAC activity and results in increased expression of acetylated histones vs control. SV has no effect on global DNA methylation. 3D models have been developed and are being optimized for gene expression studies. Conclusions This in vitro study runs parallel to the SAVER trial, a multicenter double-blind randomized controlled trial of SV vs placebo in 110 patients with high-risk OED. We have confirmed the mechanism of action of SV as an HDAC inhibitor with higher expression of acetylated histones when dysplasia cell lines are exposed to SV at clinically relevant concentrations.