Cross-Platform Array Screening Identifies COL1A2 , THBS1 , TNFRSF10D and UCHL1 as Genes Frequently Silenced by Methylation in Melanoma Vanessa F. Bonazzi1*, Derek J. Nancarrow 1 , Mitchell S. Stark1, Ralf J. Moser2, Glen M. Boyle3, Lauren G. Aoude1, Christopher Schmidt4, Nicholas K. Hayward1 1 Oncogenomics Laboratory, Queensland Institute of Medical Research, Herston, Brisbane, Queensland, Australia, 2 SEQUENOM, Asia Pacific Office, Queensland Institute of Medical Research, Herston, Brisbane, Queensland, Australia, 3 Drug Discovery Group, Queensland Institute of Medical Research, Herston, Brisbane, Queensland, Australia, 4 Cancer Immunotherapy Group, Queensland Institute of Medical Research, Herston, Brisbane, Queensland, Australia Abstract Epigenetic regulation of tumor suppressor genes (TSGs) has been shown to play a central role in melanomagenesis. By integrating gene expression and methylation array analysis we identified novel candidate genes frequently methylated in melanoma. We validated the methylation status of the most promising genes using highly sensitive Sequenom Epityper assays in a large panel of melanoma cell lines and resected melanomas, and compared the findings with those from cultured melanocytes. We found transcript levels of UCHL1, COL1A2, THBS1 and TNFRSF10D were inversely correlated with promoter methylation. For THBS1 and UCHL1 the effect of this methylation on expression was confirmed at the protein level. Identification of these candidate TSGs and future research designed to understand how their silencing is related to melanoma development will increase our understanding of the etiology of this cancer and may provide tools for its early diagnosis. Citation: Bonazzi VF, Nancarrow DJ, Stark MS, Moser RJ, Boyle GM, et al. (2011) Cross-Platform Array Screening Identifies COL1A2, THBS1, TNFRSF10D and UCHL1 as Genes Frequently Silenced by Methylation in Melanoma. PLoS ONE 6(10): e26121. doi:10.1371/journal.pone.0026121 Editor: Ralf Krahe, University of Texas MD Anderson Cancer Center, United States of America Received April 11, 2011; Accepted September 20, 2011; Published October 20, 2011 Copyright: 2011 Bonazzi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was funded by Cure Cancer Australia, grant number 552466. NKH is a recipient of a Senior Principal Research Fellowship from the National Health and Medical Research Council of Australia, grant number 442919. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: vanessa.bonazzi@qimr.edu.au Introduction Aberrant epigenetic modifications are a feature of several human diseases, including cancer. While several forms of epigenetic modification are known to exist, so far DNA methylation is the only one shown to directly target DNA and to be frequently aberrant in many tumor types [1,2]. By far the most common form of DNA methylation occurs via covalent modification of cytosine bases which precede a guanine residue (CpG). The reaction is catalysed by DNA methyltransferases [3] and results in a methyl group being added to the 5th position of the pyrimidine ring of cytosine. In mammals the majority of CpG sequences are methylated, with the exception of CpG-rich stretches present within the 59 regulatory components of many genes, termed CpG islands [4]. There is strong evidence suggesting an inverse relationship between the presence of CpG island methylation and the level of target gene expression [5], although this suppression is not always evident. Both hypermethyla- tion of CpG islands located in the promoters of tumor suppressor genes (TSGs) and global hypomethylation seem to play an important role during cancer development. Often TSGs are not primarily inactivated through mutation or deletion, but rather through targeted CpG island methylation. Melanoma genomics studies have identified a large number of chromosomal loci that show repeated loss of heterozygosity (LOH), highlighting widespread chromosomal instability [6,7]. Additionally, aberrant promoter methylation may also occur and lead to inactivation of TSGs which play a role in progression to malignancy. During melanomagenesis, well-known TSGs, such as PTEN, CDKN2A/p16INK4A and RASSF1A often have expression reduced through CpG island methylation [8,9,10]. In the last decade, several studies have assessed genome-wide methylation using the DNA methyltransferase inhibitor 5-aza-2-deoxycytidine (5AzadC) and identified TSPY, HOXB13 and SYK as novel TSGs in melanoma [11,12,13]. In a previous study, we combined 5AzadC treatment with Trichostatin A (TSA), an inhibitor of class I and II histone deacetylase enzymes, and conducted a microarray-based analysis on a panel of melanoma cell lines identifying eight highly ‘reactivated’ genes (expression fold change .4), not previously known to be epigenetically silenced in melanoma [14]. For five of these genes there was no prior evidence of inactivation by promoter methylation in any other cancer type. Follow up of these genes was carried out in a larger panel of melanoma cell lines, in addition to fresh tumors and melanocyte cultures, using the highly sensitive Sequenom Epityper assay [15,16] and correlated with microarray based gene expression levels. Four genes: PPP1R3C (protein phosphatase 1, regulatory (inhibitor) subunit 3C), ENC1 (ectodermal-neural cortex 1), RARRES1 (retinoic acid receptor responder also known as tazarotene induced gene 1, TIG1) and TP53INP1 (tumor protein p53 inducible nuclear protein 1), had PLoS ONE | www.plosone.org 1 October 2011 | Volume 6 | Issue 10 | e26121