{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T21:42:22Z","timestamp":1788298942921,"version":"build-2803163510"},"reference-count":48,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2026,4,27]],"date-time":"2026-04-27T00:00:00Z","timestamp":1777248000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000057","name":"National Institute of General Medical Sciences","doi-asserted-by":"publisher","award":["P20-GM109005"],"award-info":[{"award-number":["P20-GM109005"]}],"id":[{"id":"10.13039\/100000057","id-type":"DOI","asserted-by":"publisher"}]},{"award":["P20-GM109005"],"award-info":[{"award-number":["P20-GM109005"]}],"id":[{"id":"https:\/\/ror.org\/04q48ey07","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/100008231","name":"Arkansas Bioscience Institute","doi-asserted-by":"crossref","id":[{"id":"10.13039\/100008231","id-type":"DOI","asserted-by":"crossref"}]},{"id":[{"id":"https:\/\/ror.org\/04ptmx344","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Winthrop P. Rockefeller Cancer Institute"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Hepatocellular carcinoma (HCC) is a highly aggressive malignancy with poor prognosis and limited therapeutic options. Although epigenetic dysregulation is a hallmark of HCC, rational combinatorial targeting strategies remain incompletely defined. Here, we identify cooperative oncogenic functions of the chromatin modifiers enhancer of zeste homolog 2 (EZH2) and lysine-specific demethylase 1 (LSD1) in HCC. Analysis of the TCGA-LIHC cohort revealed that co-elevated EZH2 and LSD1 expressions are significantly associated with reduced overall survival. Gene set enrichment analysis demonstrated enrichment of Sonic Hedgehog (SHH) signaling and stress-responsive transcriptional programs in tumors with high EZH2\/LSD1 expression. Functionally, dual pharmacological inhibition of EZH2 (GSK126) and LSD1 (SP2509) suppressed HCC cell proliferation, induced G1-phase arrest, and enhanced apoptosis, as evidenced by increased caspase-3\/7 activity and decreased pro-caspase levels. Dual inhibition also impaired migration, invasion, tumor sphere formation, and stemness-associated gene expression. Mechanistically, co-targeting disrupted SHH signaling through the suppression of GLI1 expression. Chromatin immunoprecipitation revealed reduced EZH2, LSD1, and STAT3 occupancy at the GLI1 promoter following dual inhibition, leading to the repression of GLI1 and its downstream targets. Collectively, these findings demonstrate that EZH2 and LSD1 cooperatively sustain GLI1-dependent SHH signaling in HCC, and that dual epigenetic inhibition represents a mechanistically defined therapeutic strategy.<\/jats:p>","DOI":"10.3390\/ijms27093886","type":"journal-article","created":{"date-parts":[[2026,4,28]],"date-time":"2026-04-28T11:33:37Z","timestamp":1777376017000},"page":"3886","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Dual Targeting of EZH2 and LSD1 Suppresses Hepatocellular Carcinoma via Disruption of Sonic Hedgehog Signaling"],"prefix":"10.3390","volume":"27","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9263-5606","authenticated-orcid":false,"given":"HongDuck","family":"Yun","sequence":"first","affiliation":[{"name":"Department of Pharmacology and Toxicology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR 72225, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0008-3847-0602","authenticated-orcid":false,"given":"Ponmari","family":"Guruvaiya","sequence":"additional","affiliation":[{"name":"Department of Pharmacology and Toxicology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR 72225, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Olena","family":"Levurdiak","sequence":"additional","affiliation":[{"name":"Department of Pharmacology and Toxicology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR 72225, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9115-6283","authenticated-orcid":false,"given":"Alexei G.","family":"Basnakian","sequence":"additional","affiliation":[{"name":"Department of Pharmacology and Toxicology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR 72225, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2249-5203","authenticated-orcid":false,"given":"Marjan","family":"Boerma","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR 72225, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2115-3060","authenticated-orcid":false,"given":"Stephen","family":"Safe","sequence":"additional","affiliation":[{"name":"Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, TX 77843, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7240-2768","authenticated-orcid":false,"given":"KyoungHyun","family":"Kim","sequence":"additional","affiliation":[{"name":"Department of Pharmacology and Toxicology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR 72225, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,4,27]]},"reference":[{"key":"ref_1","first-page":"209","article-title":"Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries","volume":"71","author":"Sung","year":"2021","journal-title":"CA Cancer J. Clin."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"6","DOI":"10.1038\/s41572-020-00240-3","article-title":"Hepatocellular carcinoma","volume":"7","author":"Llovet","year":"2021","journal-title":"Nat. Rev. Dis. Primers"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1301","DOI":"10.1016\/S0140-6736(18)30010-2","article-title":"Hepatocellular carcinoma","volume":"391","author":"Forner","year":"2018","journal-title":"Lancet"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"926","DOI":"10.1097\/HEP.0000000000000237","article-title":"Mechanisms of drug resistance in HCC","volume":"79","author":"Ladd","year":"2024","journal-title":"Hepatology"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"917","DOI":"10.1007\/s11912-022-01247-7","article-title":"The Treatment Landscape of Advanced Hepatocellular Carcinoma","volume":"24","author":"Wong","year":"2022","journal-title":"Curr. Oncol. Rep."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1894","DOI":"10.1056\/NEJMoa1915745","article-title":"Atezolizumab plus Bevacizumab in Unresectable Hepatocellular Carcinoma","volume":"382","author":"Finn","year":"2020","journal-title":"N. Engl. J. Med."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"78","DOI":"10.1016\/j.jhep.2019.03.007","article-title":"A comprehensive study of epigenetic alterations in hepatocellular carcinoma identifies potential therapeutic targets","volume":"71","author":"Bayo","year":"2019","journal-title":"J. Hepatol."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Czauderna, C., Poplawski, A., O\u2019Rourke, C.J., Castven, D., P\u00e9rez-Aguilar, B., Becker, D., Heilmann-Heimbach, S., Odenthal, M., Amer, W., and Schmiel, M. (2021). Epigenetic modifications precede molecular alterations and drive human hepatocarcinogenesis. JCI Insight, 6.","DOI":"10.1172\/jci.insight.146196"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"2893","DOI":"10.1101\/gad.1035902","article-title":"Histone methyltransferase activity associated with a human multiprotein complex containing the Enhancer of Zeste protein","volume":"16","author":"Kuzmichev","year":"2002","journal-title":"Genes Dev."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"622","DOI":"10.1002\/hep.25679","article-title":"Enhancer of zeste homolog 2 epigenetically silences multiple tumor suppressor microRNAs to promote liver cancer metastasis","volume":"56","author":"Au","year":"2012","journal-title":"Hepatology"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1754","DOI":"10.1038\/sj.bjc.6602531","article-title":"Clinicopathological significance of EZH2 mRNA expression in patients with hepatocellular carcinoma","volume":"92","author":"Sudo","year":"2005","journal-title":"Br. J. Cancer"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"52","DOI":"10.1186\/s40164-023-00405-2","article-title":"EZH2 in hepatocellular carcinoma: Progression, immunity, and potential targeting therapies","volume":"12","author":"Wang","year":"2023","journal-title":"Exp. Hematol. Oncol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"941","DOI":"10.1016\/j.cell.2004.12.012","article-title":"Histone demethylation mediated by the nuclear amine oxidase homolog LSD1","volume":"119","author":"Shi","year":"2004","journal-title":"Cell"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"3188","DOI":"10.1038\/onc.2015.129","article-title":"Lysine-specific demethylase 1 promotes the stemness and chemoresistance of Lgr5(+) liver cancer initiating cells by suppressing negative regulators of \u03b2-catenin signaling","volume":"34","author":"Lei","year":"2015","journal-title":"Oncogene"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"938","DOI":"10.1158\/0008-5472.CAN-17-1236","article-title":"LSD1 Stimulates Cancer-Associated Fibroblasts to Drive Notch3-Dependent Self-Renewal of Liver Cancer Stem-like Cells","volume":"15","author":"Liu","year":"2018","journal-title":"Cancer Res."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1445","DOI":"10.1158\/0008-5472.CAN-14-1560","article-title":"Lysine Demethylase LSD1 Coordinates Glycolytic and Mitochondrial Metabolism in Hepatocellular Carcinoma Cells","volume":"75","author":"Sakamoto","year":"2015","journal-title":"Cancer Res."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"6651","DOI":"10.3748\/wjg.v18.i45.6651","article-title":"Overexpression of lysine specific demethylase 1 predicts worse prognosis in primary hepatocellular carcinoma patients","volume":"18","author":"Zhao","year":"2012","journal-title":"World J. Gastroenterol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"757","DOI":"10.1007\/s00280-016-2990-1","article-title":"The novel EZH2 inhibitor, GSK126, suppresses cell migration and angiogenesis via down-regulating VEGF-A","volume":"77","author":"Chen","year":"2016","journal-title":"Cancer Chemother. Pharmacol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"2009","DOI":"10.1158\/0008-5472.CAN-18-2395","article-title":"EZH2 Inhibitor GSK126 Suppresses Antitumor Immunity by Driving Production of Myeloid-Derived Suppressor Cells","volume":"79","author":"Huang","year":"2019","journal-title":"Cancer Res."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1038\/s41392-019-0040-2","article-title":"Epigenetic drug library screening identified an LSD1 inhibitor to target UTX-deficient cells for differentiation therapy","volume":"4","author":"Wu","year":"2019","journal-title":"Signal Transduct. Target. Ther."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1902","DOI":"10.1158\/1535-7163.MCT-18-0373","article-title":"Therapeutic Targeting of KDM1A\/LSD1 in Ewing Sarcoma with SP-2509 Engages the Endoplasmic Reticulum Stress Response","volume":"17","author":"Pishas","year":"2018","journal-title":"Mol. Cancer Ther."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"2155","DOI":"10.1038\/leu.2014.119","article-title":"Highly effective combination of LSD1 (KDM1A) antagonist and pan-histone deacetylase inhibitor against human AML cells","volume":"28","author":"Fiskus","year":"2014","journal-title":"Leukemia"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"7252","DOI":"10.1158\/1078-0432.CCR-04-0713","article-title":"X-tile: A new bio-informatics tool for biomarker assessment and outcome-based cut-point optimization","volume":"10","author":"Camp","year":"2004","journal-title":"Clin. Cancer Res."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"158","DOI":"10.6004\/jadpro.2022.13.2.7","article-title":"Tazemetostat: EZH2 Inhibitor","volume":"13","author":"Straining","year":"2022","journal-title":"J. Adv. Pract. Oncol."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Noce, B., Di Bello, E., Fioravanti, R., and Mai, A. (2023). LSD1 inhibitors for cancer treatment: Focus on multi-target agents and compounds in clinical trials. Front. Pharmacol., 14.","DOI":"10.3389\/fphar.2023.1120911"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"587","DOI":"10.1016\/j.gpb.2022.01.004","article-title":"SynergyFinder Plus: Toward Better Interpretation and Annotation of Drug Combination Screening Datasets","volume":"20","author":"Zheng","year":"2022","journal-title":"Genom. Proteom. Bioinform."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Sang, N., Zhong, X., Gou, K., Liu, H., Xu, J., Zhou, Y., Zhou, X., Liu, Y., Chen, Z., and Zhou, Y. (2023). Pharmacological inhibition of LSD1 suppresses growth of hepatocellular carcinoma by inducing GADD45B. MedComm, 4.","DOI":"10.1002\/mco2.269"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"110068","DOI":"10.1016\/j.intimp.2023.110068","article-title":"Alteration of the tumor microenvironment by pharmacological inhibition of EZH2 in hepatocellular carcinoma","volume":"118","author":"Qiang","year":"2023","journal-title":"Int. Immunopharmacol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"37974","DOI":"10.18632\/oncotarget.16467","article-title":"Role of EZH2 in cancer stem cells: From biological insight to a therapeutic target","volume":"8","author":"Wen","year":"2017","journal-title":"Oncotarget"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"441","DOI":"10.1007\/s00441-023-03834-x","article-title":"lncRNA SNHG9 enhances liver cancer stem cell self-renewal and tumorigenicity by negatively regulating PTEN expression via recruiting EZH2","volume":"394","author":"Yang","year":"2023","journal-title":"Cell Tissue Res."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Leung, Y.K., Lee, S.G., Wang, J., Guruvaiah, P., Rusch, N.J., Ho, S.M., Park, C., and Kim, K. (2024). The Loss of an Orphan Nuclear Receptor NR2E3 Augments Wnt\/\u03b2-catenin Signaling via Epigenetic Dysregulation that Enhances Sp1-\u03b2 catenin-p300 Interactions in Hepatocellular Carcinoma. Adv. Sci., 11.","DOI":"10.1002\/advs.202308539"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"315","DOI":"10.1038\/s41392-023-01559-5","article-title":"Hedgehog signaling in tissue homeostasis, cancers, and targeted therapies","volume":"8","author":"Jing","year":"2023","journal-title":"Signal Transduct. Target. Ther."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1387","DOI":"10.1242\/dev.028167","article-title":"Sonic hedgehog signaling regulates reciprocal epithelial-mesenchymal interactions controlling palatal outgrowth","volume":"136","author":"Lan","year":"2009","journal-title":"Development"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"198","DOI":"10.1016\/S1476-5586(03)80052-0","article-title":"c-Myc enhances sonic hedgehog-induced medulloblastoma formation from nestin-expressing neural progenitors in mice","volume":"5","author":"Rao","year":"2003","journal-title":"Neoplasia"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"2961","DOI":"10.1182\/blood-2019-132054","article-title":"STAT3-induced Gli1 promotes the differentiation and maturation of myelofibrosis fibrocytes","volume":"134","author":"Manshouri","year":"2019","journal-title":"Blood"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"401","DOI":"10.18632\/oncotarget.27884","article-title":"STAT3 induces the expression of GLI1 in chronic lymphocytic leukemia cells","volume":"12","author":"Rozovski","year":"2021","journal-title":"Oncotarget"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"333","DOI":"10.1093\/bioinformatics\/18.2.333","article-title":"PROMO: Detection of known transcription regulatory elements using species-tailored searches","volume":"18","author":"Messeguer","year":"2002","journal-title":"Bioinformatics"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1465","DOI":"10.1126\/science.1227604","article-title":"EZH2 oncogenic activity in castration-resistant prostate cancer cells is Polycomb-independent","volume":"338","author":"Xu","year":"2012","journal-title":"Science"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"839","DOI":"10.1016\/j.ccr.2013.04.008","article-title":"Phosphorylation of EZH2 activates STAT3 signaling via STAT3 methylation and promotes tumorigenicity of glioblastoma stem-like cells","volume":"23","author":"Kim","year":"2013","journal-title":"Cancer Cell"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"3985","DOI":"10.1073\/pnas.1503152112","article-title":"STAT3-driven transcription depends upon the dimethylation of K49 by EZH2","volume":"112","author":"Dasgupta","year":"2015","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"436","DOI":"10.1038\/nature04020","article-title":"LSD1 demethylates repressive histone marks to promote androgen receptor-dependent transcription","volume":"437","author":"Metzger","year":"2005","journal-title":"Nature"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"505","DOI":"10.1016\/j.cell.2006.12.038","article-title":"Histone methylation-dependent mechanisms impose ligand dependency for gene activation by nuclear receptors","volume":"128","author":"Kwon","year":"2007","journal-title":"Cell"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"52","DOI":"10.1158\/0008-5472.CAN-24-0788","article-title":"LSD1 and CoREST2 potentiate STAT3 activity to promote enteroendocrine cell differentiation in mucinous colorectal cancer","volume":"85","author":"Ladaika","year":"2025","journal-title":"Cancer Res."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"1235","DOI":"10.1016\/j.chembiol.2006.09.018","article-title":"Stattic: A small-molecule inhibitor of STAT3 activation and dimerization","volume":"13","author":"Schust","year":"2006","journal-title":"Chem. Biol."},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"Zhou, X., Man, M., Cui, M., Zhou, X., Hu, Y., Liu, Q., and Deng, Y. (2024). Relationship between EZH2 expression and prognosis of patients with hepatocellular carcinoma using a pathomics predictive model. Heliyon, 10.","DOI":"10.1016\/j.heliyon.2024.e38562"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"362","DOI":"10.1111\/bph.16257","article-title":"Transcriptome-level discovery of survival-associated biomarkers and therapy targets in non-small-cell lung cancer","volume":"181","year":"2024","journal-title":"Br. J. Pharmacol."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"15545","DOI":"10.1073\/pnas.0506580102","article-title":"Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles","volume":"102","author":"Subramanian","year":"2005","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"1123","DOI":"10.1016\/j.chembiol.2006.11.001","article-title":"A new small-molecule Stat3 inhibitor","volume":"13","author":"McMurray","year":"2006","journal-title":"Chem. Biol."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/9\/3886\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,4,29]],"date-time":"2026-04-29T04:27:26Z","timestamp":1777436846000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/9\/3886"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,4,27]]},"references-count":48,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2026,5]]}},"alternative-id":["ijms27093886"],"URL":"https:\/\/doi.org\/10.3390\/ijms27093886","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,4,27]]}}}