Clustered DNA methylation changes in polycomb target genes in early-stage liver cancer Yao-Li Chen a,b , Chih-Jan Ko a,c , Ping-Yi Lin a , Wan-Ling Chuang a , Chia-Chen Hsu d , Pei-Yi Chu e , Mei-Yu Pai d , Chun-Chun Chang d , Ming-Han Kuo d , Yi-Ru Chu d , Chun-Hsin Tung d , Tim H.-M. Huang f , Yu-Wei Leu d,⇑ , Shu-Huei Hsiao d,⇑ a Transplant Medicine & Surgery Research Centre, Changhua Christian Hospital, Changhua, Taiwan b School of Medicine, Chung Shan Medical University, Taichung, Taiwan c Department of General Surgery, Changhua Christian Hospital, Changhua, Taiwan d Human Epigenomics Center, Department of Life Science, Institute of Molecular Biology and Institute of Biomedical Science, National Chung Cheng University, Chiayi, Taiwan e Department of Pathology, St. Martin De Porres Hospital, Chiayi, Taiwan f Cancer Therapy and Research Center, Department of Molecular Medicine and Institute of Biotechnology, School of Medicine, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA article info Article history: Received 16 July 2012 Available online 25 July 2012 Keywords: DNA methylation Epigenetic Liver cancer Chromatin immunoprecipitation abstract Polycomb-group proteins mark specific chromatin conformations in embryonic and somatic stem cells that are critical for maintenance of their ‘‘stemness’’. These proteins also mark altered chromatin modi- fications identified in various cancers. In normal differentiated cells or advanced cancerous cells, these polycomb-associated loci are frequently associated with increased DNA methylation. It has thus been hypothesized that changes in DNA methylation status within polycomb-associated loci may dictate cell fate and that abnormal methylation within these loci may be associated with tumor development. To assess this, we examined the methylation states of four polycomb target loci – Trip10, Casp8AP2, ENSA, and ZNF484 – in liver cancer. These four targets were selected because their methylation levels are increased during mesenchymal stem cell-to-liver differentiation. We found that these four loci were hypomethylated in most early-stage liver cancer specimens. For comparison, two non-polycomb tumor suppressor genes, HIC1 and RassF1A, were also examined. Whereas the methylation level of HIC1 did not differ significantly between normal and tumor samples, RassF1A was significantly hypermethylated in liver tumor samples. Unsupervised clustering analysis classified the methylation changes within poly- comb and non-polycomb targets to be independent, indicating independent epigenetic evolution. Thus, pre-deposited polycomb marks within somatic stem cells may contribute to the determination of meth- ylation changes during hepatic tumorigenesis. Ó 2012 Elsevier Inc. All rights reserved. 1. Introduction Numerous aberrant genetic and epigenetic mutations/modifica- tions act in cooperation to create tumor variations among individuals and between different cancers. This complexity has also confounded the search for effective diagnostic biomarkers and therapeutic targets [1–3]. Instead of a function-dependent classification, statistical analyses followed by arbitrary cut-offs are often used to identify profiled loci where the expressional/ epigenetic differences could significantly classify cancer diseases pathologically [4]. The effectiveness of using the most frequently found loci as can- cer biomarkers was addressed by Wood et al. [1] Their study dem- onstrated that a series of minor (less frequent) expressional differences/mutations can be more relevant to tumorigenesis and have a better chance of predicting the development of cancer in a tissue-specific manner. Thus, better tracking of tumor evolution may require other ways to identify target loci. Epigenetic changes within polycomb group-governed loci have been reported to be sufficient to change cell fate [5–7]. Similar polycomb-governed loci have been identified in embryonic stem cells (ES) [8], somatic stem cell-like mesenchymal stem cells (MSCs) [9], and cancer stem cell-like cells [8]. 0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bbrc.2012.07.084 Abbreviations: MSC, mesenchymal stem cell; 5-Aza, 5-aza-2 0 -deoxycytidine; H3K27me3, histone 3 lysine 27 trimethylation. ⇑ Corresponding authors. Address: Department of Life Science, Institute of Molecular Biology and Institute of Biomedical Science, National Chung Cheng University, Chiayi 621, Taiwan. Fax: +886 5 272 2871. E-mail addresses: bioywl@ccu.edu.tw (Y.-W. Leu), bioshh@ccu.edu.tw (S.-H. Hsiao). Biochemical and Biophysical Research Communications 425 (2012) 290–296 Contents lists available at SciVerse ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc