Oncogene (2013) 32, 307 --317 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc ORIGINAL ARTICLE Zinc-finger protein 331, a novel putative tumor suppressor, suppresses growth and invasiveness of gastric cancer JYu1,4, QY Liang1,4, J Wang1,4, Y Cheng2, S Wang1, TCW Poon1,MYYGo1,QTao2, Z Chang3 and JJY Sung1 Zinc-finger protein 331 (ZNF331), a Kruppel-associated box zinc-finger protein gene, was identified as a putative tumor suppressor in our previous study. However, the role of ZNF331 in tumorigenesis remains elusive. We aimed to clarify its epigenetic regulation and biological functions in gastric cancer. ZNF331 was silenced or downregulated in 71% (12/17) gastric cancer cell lines. A significant downregulation was also detected in paired gastric tumors compared with adjacent non-cancer tissues. In contrast, ZNF331 was readily expressed in various normal adult tissues. The downregulation of ZNF331 was closely linked to the promoter hypermethylation as evidenced by methylation-specific PCR, bisulfite genomic sequencing and reexpression by demethylation agent treatment. DNA sequencing showed no genetic mutation/deletion of ZNF331 in gastric cancer cell lines. Ectopic expression of ZNF331 in the silenced cancer cell lines MKN28 and HCT116 significantly reduced colony formation and cell viability, induced cell cycle arrests and repressed cell migration and invasive ability. Concordantly, knockdown of ZNF331 increased cell viability and colony formation ability of gastric cancer cell line MKN45. Two-dimensional gel electrophoresis and mass spectrometry-based comparative proteomic approach were applied to analyze the molecular basis of the biological functions of ZNF331. In all, 10 downstream targets of ZNF331 were identified to be associated with regulation of cell growth and metastasis. The tumor-suppressive effect of ZNF331 is mediated at least by downregulation of genes involved in cell growth promotion (DSTN, EIF5A, GARS, DDX5, STAM, UQCRFS1 and SET) and migration/invasion (DSTN and ACTR3), and upregulation of genome-stability gene (SSBP1) and cellular senescence gene (PNPT1). A novel target of ZNF331 (DSTN) was functionally validated. Overexpression of DSTN in BGC-823 cells increased colony formation and migration ability. In conclusion, our results suggest that ZNF331 possesses important functions for the suppression of gastric carcinogenesis as a novel functional tumor-suppressor gene. Oncogene (2013) 32, 307--317; doi:10.1038/onc.2012.54; published online 27 February 2012 Keywords: Zinc-finger protein 331 (ZNF331); gastric cancer; tumor suppressor; methylation; invasion; 2-D gel electrophoresis INTRODUCTION associated box-A box and a zinc-finger domain with 12 Cys2His2 Gastric cancer is one of the leading causes of cancer-related death zinc fingers and locates on chromosome 19q13.4,5 The role of in China. Although the molecular mechanisms of gastric carcino- ZNF331 in tumor development remains largely unknown. The genesis remain unclear, epigenetic silencing of tumor-related functions of zinc-finger proteins are extraordinarily diverse, genes by promoter hypermethylation has recently emerged as an including DNA recognition, RNA packaging, transcriptional activa- important mechanism of tumorigenesis. The promoter hyper- tion, regulation of apoptosis, and so on. Structural studies of zinc- methylation profile differs in each cancer type and within each finger proteins have shed new insights into their extraordinary gene, providing tumor type- and gene-specific hypermethylation diversity of structure and function.6 Proteins containing the profiles that may involve in the corresponding molecular mechan- classical Cys2His2 zinc finger are among the most abundant in ism of tumorigenesis. The identification of a novel gene targeted eukaryotic genomes. Many of these proteins function by by promoter hypermethylation may provide insights into the recognition of specific DNA sequences. Because all the Kruppel- mechanisms for the inactivation of the tumor-suppressive path- associated box zinc-finger genes so far have been shown to act ways and is important for the identification of tumor markers in only as transcriptional repressors,7--9 ZNF331 is considered to be gastric cancer.1,2 Recently, using suppression subtraction hybridi- a transcriptional repressor.10 The engineered chimeric fusion zation,3 we have identified zinc-finger protein 331 (ZNF331)asa proteins of Kruppel-associated box domains combined with candidate tumor-suppressor gene. We aimed to clarify the different DNA-binding domains of oncogenic transcription factors epigenetic regulation and biological functions of ZNF331 in gastric have been shown to target their respective oncogenes and cancer in this study. specifically suppress malignant growth.11 The application of ZNF331, also known as ZNF361, ZNF463 and Rita, is a Kruppel- artificial zinc-finger protein transcription factors to repress the associated box zinc-finger protein gene consisting of a Kruppel- expression of cancer-related genes, such as human telomerase 1Institute of Digestive Disease and Department of Medicine and Therapeutics, Li Ka Shing Institute of Health Sciences, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, China; 2Cancer Epigenetics Laboratory, Department of Clinical Oncology, The Chinese University of Hong Kong, Hong Kong, China and 3State Key Laboratory of Biomembrane and Membrane Biotechnology, School of Medicine, National Engineering Laboratory for Anti-tumor Therapeutics, Tsinghua University, Beijing, China. 4These authors contributed equally to this work. Correspondence: Professor J Yu, Institute of Digestive Disease, Department of Medicine and Therapeutics, Li Ka Shing Institute of Health Sciences, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, NT, Hong Kong. E-mail: [email protected] or Professor JJY Sung, Institute of Digestive Disease, Department of Medicine and Therapeutics, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, NT, Hong Kong. E-mail: [email protected] Received 15 July 2011; revised 25 November 2011; accepted 10 January 2012; published online 27 February 2012 Zinc-finger protein 331 in gastric cancer JYuet al 308 reverse transcriptase, has presented a new promising strategy for silenced or downregulated in the tested gastric cancer cell lines inhibiting the growth of human cancer cells.12 These findings (12/17, 71%). Expression of variant 2 was detected only in 1 out demonstrate important tumor-suppressor functions of zinc-finger of the 17 cell lines (SNU719) by RT--PCR (Figure 1c). Real-time proteins and their promising application in cancer therapy. In this quantitative RT--PCR (qRT--PCR) indicated that expression of study, we investigated the gene-expression pattern of ZNF331, variant 2 in SNU719 was over 200 times higher than in the other molecular characterization of the promoter of ZNF331 and its cells. Dissimilarly, ZNF331 waslessfrequentlysilencedor tumor-suppressor function in gastric cancer. downregulated in colon, esophageal, liver and other human cancer cell lines (data not shown). These results implied that loss of ZNF331 expression, especially variant 2, may be mainly RESULTS involved in gastric carcinogenesis. Correlation of ZNF331 mRNA expression level with methylation Sequence analysis using both CpG island searcher (http:// status in gastric cancer cell lines cpgislands.usc.edu/) and CPGPLOT (http://www.cbib.u-bordeaux2. Three transcription variants of ZNF331 have been identified in fr/pise/cpgplot.html) revealed CpG islands around the transcrip- human tissues previously.4 The three variants are transcribed from tion start sites of both variants 1 and 2, indicating potential different loci and share the same open reading frame (ORF) epigenetic regulation of ZNF331 expression by DNA methylation. (Figure 1a), suggesting that they possess the same function and To evaluate the association of silencing or downregulation of might be transcriptionally regulated in a tissue-specific manner. ZNF331 variants 1 and 2 with methylation status, methylation- RT--PCR results indicated that variants 1 and 2 of ZNF331 were specific PCR was carried out. Heavy methylation of ZNF331 variant ubiquitously expressed in various normal human tissues, including 2 was observed in all silenced cell lines, whereas partial or full gastric tissue, whereas variant 3 was found only in the methylation of variant 1 was detected in cancer cells with reduced reproductive tissue, testis (Figure 1b). These results demonstrated or silenced expression (Figure 1d). These results indicated a that constitutive expression of ZNF331 in normal tissues mainly correlation between ZNF331 mRNA expression and its methylation involves variants 1 and 2 but not variant 3. So we further status in gastric cancer cell lines. analyzed the transcription of variants 1 and 2 in a series of cancer To further confirm that DNA methylation is indeed responsible cell lines. RT--PCR results indicated that variant 1 was frequently for the silencing or downregulation of ZNF331, six gastric cancer Figure 1. Transcriptional silencing/downregulation of ZNF331 is associated with DNA methylation. (a) Schematic for the three transcription variants of ZNF331, which are transcribed from different transcription start sites while containing the same ORF. (b) Ubiquitous mRNA expression of ZNF331 variants 1 and 2 in normal human adult tissues. (c) ZNF331 variant 1 and variant 2 expression were silenced or reduced in many gastric cancer cell lines. (d)