ZHX2 Restricts Hepatocellular Carcinoma by Suppressing Stem Cell-Like Traits Through KDM2A-Mediated H3K36 Demethylation

Abstract

Background: Liver cancer stem cells (CSCs) are critical determinants of cancer relapse and therapeutic resistance, but the mechanisms underlying the maintenance of CSCs are poorly understood. We aimed to explore the role of tumor repressor Zinc-fingers and homeoboxes (ZHX2) in liver CSCs. Methods: CD133+ or EPCAM+ stem-like liver cancer cells were sorted from tumor tissues of HCC patients and cell lines by flow cytometry. Also, sorafenib-resistant cells, tumor-sphere forming cells and side population (SP) cells were respectively cultured and isolated as hepatic CSCs. The tumor-initiating and chemoresistance properties of ZHX2-overexpressing and ZHX2-knockdown cells were analyzed in vivo and in vitro. Microarray, luciferase reporter assay, chromatin immunoprecipitation (ChIP) and ChIP-on-chip analyses were performed to explore ZHX2 target genes. The expression of ZHX2 and its target gene were determined by quantitative RT-PCR, western blot, immunofluorescence and immunohistochemical staining in hepatoma cells and tumor and adjacent tissues from HCC patients. Results: The expression of ZHX2 was reduced in liver CSCs from different origins. ZHX2 deficiency led to enhanced liver tumor progression and expansion of CSC populations in vitro and in vivo. Re-expression of ZHX2 restricted capabilities of CSCs in supporting tumor initiation, self-renew and sorafenib-resistance. Mechanically, ZHX2 suppressed liver CSCs via inhibiting KDM2A-mediated demethylation of histone H3 lysine 36 (H3K36) at the promoter regions of stemness-associated transcription factors, such as NANOG, SOX4 and OCT4. Moreover, patients with lower expression of ZHX2 and higher expression of KDM2A in tumor tissues showed significant poor survival. Conclusion: ZHX2 counteracts stem cell traits through transcriptionally repressing KDM2A in HCC. Our data will aid in a better understanding of molecular mechanisms underlying HCC relapse and drug resistance. Funding Statement: This work was supported by grants from the National Natural Science Fund for Outstanding Youth Fund (81425012), the National Key Research and Development Program (No.2016YFE0127000), the National Science Foundation of China (No. 81830017, 81370527, 81702647), Taishan Scholarship (No.tspd20181201), Shandong Provincial Key Innovation project (No.2018FYJH0503) and Collaborative Innovation Center of Technology and Equipment for Biological Diagnosis and Therapy in Universities of Shandong. Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: Informed consent was obtained from all patients before the study was initiated with approval of the Shandong University Medical Ethics Committee in accordance with the Declaration of Helsinki. All animal procedures were performed in according to protocols approved by the Shandong University Animal Care Committee and conducted with an animal ethical approval.