Histone Deacetylase Inhibitor-Induced CDKN2B and CDKN2D Contribute to G2/M Cell Cycle Arrest Incurred by Oxidative Stress in Hepatocellular Carcinoma Cells via Forkhead Box M1 Suppression

Forkhead box protein M1 (FOXM1) is a pivotal regulator of G2/M cell cycle progression in many types of cancer. Previously, our study demonstrated that histone deacetylase inhibition (HDACi) sensitizes hepatocellular carcinoma cells (HCC) to oxidative stress through FOXM1 suppression. However, the mechanism underlying its suppression by HDACi still requires elucidation. We hypothesized that HDACi induce genes responsible for destabilizing and inactivating FOXM1. The transcriptome in the HepG2 was revealed by massive analysis of cDNA end (MACE). Expression of mRNA and proteins were analyzed by quantitative real-time PCR (qPCR) and western blot, respectively. Cell cycle was analyzed by fluorescence-activated cell sorting (FACS). Oxidative stress and HDACi suppressed CDK4/6 levels while enhancing CDK inhibitor 2B and 2D (CDKN2B and CDKN2D) expressions in HCC. Palbociclib, a specific inhibitor of CDK4/6, induced G2/M cell cycle arrest in HCC by down-regulating phosphorylation level of FOXM1, and its downstream target genes such as aurora kinase A (AURKA) and polo-like kinase 1 (PLK1). HDACi treatment increased the ubiquitination level of FOXM1 by suppressing ubiquitin-specific peptidase 21 (USP21), which deubiquitinates FOXM1. Inhibiting FOXM1 degradation with MG132 treatment affected neither palbociclib-induced G2/M cell cycle arrest nor expression of its target genes. Double knockdown of CDKN2B and CDKN2D reduced the oxidative stress and HDACi-induced G/2M cell cycle arrest. In conclusion, oxidative stress and HDACi synergistically cause G2/M cell cycle arrest via CDKN2 induction, which sequentially inhibits CDK4/6, FOXM1, and its downstream target genes AURKA, PLK1, and CCNB1 phosphorylation in HCC.