Author Manuscript Published OnlineFirst on April 20, 2020; DOI: 10.1158/0008-5472.CAN-19-3226 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. ZNF143-mediated H3K9 trimethylation upregulates CDC6 by activating MDIG in hepatocellular carcinoma Lili Zhang1*, Qi Huo2*, Chao Ge1, Fangyu Zhao1, Qingqing Zhou1, Xiaoxia Chen3, Hua Tian1, Taoyang Chen4, Haiyang Xie5, Ying Cui6, Ming Yao1, Hong Li1#, Jinjun Li1# 1 State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200032, China 2 Department of Medical Oncology, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China. 3 Med-X Research Institute, Shanghai Jiao Tong University, Shanghai 200030, China 4 Qi Dong Liver Cancer Institute, Qi Dong 226200, China 5 Department of General Surgery, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310000, China 6 Cancer Institute of Guangxi, Nanning 530027, China * These authors contributed equally to this work. # Co-corresponding authors Running Title: ZNF143 promotes cell proliferation in HCC 1 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 20, 2020; DOI: 10.1158/0008-5472.CAN-19-3226 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. #Address for correspondence: Jinjun Li, Ph.D. or Hong Li, Ph.D. State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiaotong University School of Medicine Add: 25/Ln 2200 Xietu Road, Shanghai 200032, China E-mail: [email protected] or [email protected] Tel: +86-21-64432140 or +86-21-64067346 Conflict of interest statement: No potential conflicts of interest were disclosed. 2 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 20, 2020; DOI: 10.1158/0008-5472.CAN-19-3226 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Zinc finger protein 143 (ZNF143) belongs to the zinc finger protein family and possesses transcription factor activity by binding sequence-specific DNA. The exact biological role of ZNF143 in hepatocellular carcinoma (HCC) has not been investigated. Here we report that ZNF143 is overexpressed in HCC tissues and its overexpression correlates with poor prognosis. Gain- and loss-of-function experiments showed that ZNF143 promoted HCC cell proliferation, colony formation, and tumor growth in vitro and in vivo. ZNF143 accelerated HCC cell-cycle progression by activating cell division cycle 6 (CDC6). Mechanistically, ZNF143 promoted expression of CDC6 by directly activating transcription of histone demethylase mineral dust-induced gene (MDIG), which in turn reduced H3K9me3 enrichment in the CDC6 promoter region. Consistently, ZNF143 expression correlated significantly with MDIG and CDC6 expression in HCC. Collectively, we propose a model for a ZNF143-MDIG-CDC6 oncoprotein axis that provides novel insight into ZNF143 which may serve as a therapeutic target in HCC. 3 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 20, 2020; DOI: 10.1158/0008-5472.CAN-19-3226 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Introduction Hepatocellular carcinoma (HCC) is the most common cause of death worldwide, and the molecular mechanism of uncontrolled HCC progression remains unclear (1). Accumulating evidence has suggested that dysregulation of the cell cycle is one of the most important hallmarks of HCC (2). A selective CDK4/6 inhibitor showed encouraging results in preclinical models of HCC and might represent a novel therapeutic strategy for HCC treatment (3). Elucidation of the mechanisms underlying the pathogenesis and molecular biology of dysregulated cell cycle in HCC is fundamental for the development of effective therapeutic treatments. Zinc finger protein 143 (ZNF143) can be activated by IGF1 and is a transcription factor of the C2H2 family. This protein contains a transcription activation domain, 7 Kruppel-like C2H2 zinc finger motif domain, and a C-terminal domain (4,5). Some studies have shown that ZNF143 possesses potent oncogenic properties and can promote growth (6,7). In prostate cancer, ZNF143 is involved in cisplatin resistance by regulating the transcription of DNA repair genes (8). Additionally, ZNF143 is closely related to tumor malignancy via regulating cell motility and exerting tumor suppressive effects in breast cancer (9). Intriguingly, Haibara et al. found that two small molecules inhibit ZNF143 activity, suggesting an opportunity for cancer therapeutics (10). To date, the exact biological role of ZNF143 in HCC has not been investigated. 4 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 20, 2020; DOI: 10.1158/0008-5472.CAN-19-3226 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. An increasing body of evidence suggests that epigenetic changes such as DNA methylation and other post-translational modifications of chromatin play critical roles in HCC tumorigenesis (11,12). Of these epigenetic alterations, dynamic histone modifications play a pivotal role in the regulation of gene transcription (13). Histone methylation can be regulated by histone methyltransferase and demethylases. Mineral dust-induced gene (MDIG), also known as myc-induced nuclear antigen with a molecular weight of 53 kDa (MINA53), was first identified in alveolar macrophages obtained from coal miners (14), contains one JmjC domain and can demethylate H3K9me3 (15,16). Numerous studies have demonstrated that MDIG is overexpressed in malignancies and can promote cell migration, cell cycle transition and proliferation (14,17). Various studies have shown that functionally related genes involved in epigenetic reprogramming can be controlled by specific transcription factors (18,19). Elucidation of the epigenetic alterations induced by transcription factors will provide more comprehensive and precise insight into the regulatory mechanisms of transcription factors. In the current study, we identified cell division cycle 6 (CDC6) as a downstream target gene of ZNF143 during the regulation of cell cycle in HCC. ZNF143 promoted CDC6 expression by attenuating the enrichment of H3K9me3 in CDC6 promoter region through activation of MDIG. Our findings reveal the fundamental roles of ZNF143 in cell cycle control and identify the ZNF143-MDIG-CDC6 axis as a potential target for anticancer therapy. 5 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 20, 2020; DOI: 10.1158/0008-5472.CAN-19-3226 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Materials and Methods Human tissues Human primary HCC/matched adjacent noncancerous liver tissue specimens were obtained with informed consent from the Guangxi Cancer Institute (Nanning, China), Zhejiang University (Hangzhou, China) and the Qidong Liver Cancer Institute (Qidong, China). None of the patients received preoperative radiation or chemotherapy. This study was approved by the Research Ethics Committee of Renji Hospital, Shanghai Jiao Tong University School of Medicine and in accordance with Declaration of Helsinki. Written informed consent was received from participants prior to inclusion in the study. The Cancer Genome Atlas (TCGA) and HCCDB cohort The mRNA expression data were downloaded from TCGA database (https://tcga-data.nci.nih.gov/tcga/). HCCDB data were directly downloaded from the HCCDB dataset (http://lifeome.net/database/hccdb/home.html) (20). Immunohistochemistry (IHC) Tissue microarrays (TMAs) were constructed, and the diagnosis was confirmed by three pathologists. IHC was performed as previously described (21). Briefly, the sections were incubated overnight with ZNF143, MDIG and CDC6 at 4°C and then evaluated by two independent observers in a blinded manner. The antibodies used in this study were anti-ZNF143 (HPA003263, 1:10, Sigma-Aldrich), 6 Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 20, 2020; DOI: 10.1158/0008-5472.CAN-19-3226 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. anti-MDIG/MINA53 (PA5-31300, 1:25, Invitrogen), anti-CDC6 (HPA050114, 1:10, Sigma-Aldrich) and anti-Ki67 (GT209429, 1:50, GeneTech). Scores of staining intensity were: 0, negative; 1, weak; 2, moderate; 3, strong. Scores of positively stained cell proportion were: 0, no positive; 1, <10%; 2, 10%–35%; 3, 35%–75%; 4, >75%.The results were scored 0 to 4 by two independent investigators. A score of 0–2 was considered to represent low expression and a score of 3–4 was considered to represent high expression. Cell culture The human HCC cell lines MHCC-97L, MHCC-LM3, and MHCC-97H were kindly provided by the Liver Cancer Institute of Zhongshan