Valproic Acid Inhibits Glioma Cell Proliferation by Inducing Neuronal‐Like Differentiation Partly Through HDAC1/REST Suppression

ABSTRACT Inducing differentiation of glioma cells, including glioma stem cells (GSCs), into neuron‐like cells represents a promising therapeutic strategy. Our previous drug repositioning study revealed that histone deacetylase (HDAC) inhibitors are potential inducers of glioma cell differentiation. In this study, the antiepileptic drug valproic acid (VPA), a known HDAC inhibitor with blood–brain barrier permeability, was investigated for its ability to induce neuronal differentiation in glioma. The results showed that VPA treatment induced pronounced neuronal‐like morphological changes and upregulated expression of neuronal markers in glioma cell lines U87 and LN229 and their derived GSCs, while concurrently inhibiting proliferation and attenuating stemness. Proteomic sequencing and bioinformatics analyses confirmed the enrichment of neuronal differentiation‐associated proteins and downregulation of cell cycle‐related proteins following VPA exposure. Mechanistically, VPA reduced the thermal stability of HDAC1 and RE‐1 silencing transcription factor (REST) and downregulated their protein levels. Pharmacological inhibition of HDAC1 or REST phenocopied VPA‐induced differentiation, whereas REST overexpression attenuated this effect. In vivo, VPA administration significantly delayed subcutaneous U87 xenograft tumor growth, prolonged survival in an orthotopic GSC xenograft model, and reduced HDAC1/REST expression while inducing neuronal marker expression in tumor tissues. Furthermore, Connectivity Map analysis identified mTOR inhibitors as potential synergistic agents, and the combination of VPA with rapamycin exhibited synergistic anti‐proliferative effects. Collectively, these findings demonstrate that VPA suppresses glioma growth by inducing neuronal‐like differentiation partly through HDAC1/REST inhibition, which uncovers a distinct differentiation‐based anti‐glioma mechanism of VPA, supporting its potential clinical utility and providing a rationale for combination strategies in glioma differentiation therapy.