Inhibition of histone demethylase KDM4 by ML324 induces apoptosis through the unfolded protein response and Bim upregulation in hepatocellular carcinoma cells

• Published in: Chemico-biological interactions 2022-02, Vol.353, p.109806-109806, Article 109806
• Main Authors: Kim, So Young, Hwang, Supyong, Lee, Bo Ra, Hong, Jung A., Sung, Young Hoon, Kim, Inki
• Format: Article
• Language: English
• Subjects: Antineoplastic Agents - chemistry; Antineoplastic Agents - pharmacology; Apoptosis - drug effects; Bcl-2-Like Protein 11 - genetics; Bcl-2-Like Protein 11 - metabolism; Bim; Carcinoma, Hepatocellular - pathology; Cell Line, Tumor; Cell Proliferation - drug effects; Death receptor 5; Hepatocellular carcinoma; Humans; Jumonji Domain-Containing Histone Demethylases - antagonists & inhibitors; Jumonji Domain-Containing Histone Demethylases - metabolism; Liver Neoplasms - pathology; Lysine demethylase 4; ML324; Oxyquinoline - chemistry; Oxyquinoline - pharmacology; Protein Binding; Receptors, TNF-Related Apoptosis-Inducing Ligand - antagonists & inhibitors; Receptors, TNF-Related Apoptosis-Inducing Ligand - genetics; Receptors, TNF-Related Apoptosis-Inducing Ligand - metabolism; RNA Interference; RNA, Small Interfering - metabolism; Transcription Factor CHOP - genetics; Transcription Factor CHOP - metabolism; Unfolded protein response; Unfolded Protein Response - drug effects; Up-Regulation - drug effects
• ISSN: 0009-2797; 1872-7786
• DOI: 10.1016/j.cbi.2022.109806

Hepatocellular carcinoma (HCC) is an extremely aggressive malignancy that ranks as the sixth-leading cause of cancer-associated death worldwide. Recently, various epigenetic mechanisms including gene methylation were reported to be potential next era HCC therapeutics and biomarkers. Although inhibition of epigenetic enzymes including histone lysine demethylase 4 (KDM4) enhanced cell death in HCC cells, the detailed mechanism of cell death machinery is poorly understood. In this study, we found that ML324, a small molecule KDM4-specific inhibitor, induced the death of HCC cells in a general cell culture system and 3D spheroid culture with increased cleavage of caspase-3. Mechanistically, we identified that unfolded protein responses (UPR) were involved in ML324-induced HCC cell death. Incubation of HCC cells with ML324 upregulated death receptor 5 (DR5) expression through the activation transcription factor 3 (ATF3)-C/EBP homologous protein (CHOP)-dependent pathway. Moreover, we identified BIM protein as a mediator of ML324-induced apoptosis using CRISPR/Cas9 knockout analysis. We showed that the loss of Bim suppressed ML324-induced apoptosis by flow cytometry analysis, colony formation assay, and caspase-3 activation assay. Interestingly, BIM protein expression by ML324 was regulated by ATF3, CHOP, and DR5 which are factors involved in UPR. Specifically, we confirmed the regulating roles of KDM4E in Bim and CHOP expression using a chromatin immune precipitation (ChIP) assay. Physical binding of KDM4E to Bim and CHOP promoters decreased the response to ML324. Our findings suggest that KDM4 inhibition is a potent anti-tumor therapeutic strategy for human HCC, and further studies of UPR-induced apoptosis and the associated epigenetic functional mechanisms may lead to the discovery of novel target for future cancer therapy. ●ML324 induced apoptotic cell death in HCC cells.●ML324 activates unfolded protein response which leads ATF3-CHOP dependent DR5 upregulation.●Bim protein is involved in ML324-induced cell death.●UPR pathway and Bim upregulation is associated in ML324-induced cell death.●KDM4E regulates ML324-induced apoptosis through binding to the Bim and CHOP promoter.