Epigenetic Gene Silencing in Human Melanoma Table 1. Summary of COL1A2, THBS1, TNFRSF10D and UCHL1 methylation in melanocytes, melanoma cell lines, fresh-frozen melanoma tumors and other cancer cell lines. % of methylation in melanoma Col1A2 THBS1 TNFRSF10D UCHL1 other cancer cell lines In melanocytes 2% 9% 5% 5% In MM cell lines 24% 31% 66% 42% MM/mel 10.48 3.43 13.03 8.10 In Fresh tumors 13% 15% 30% 21% FT/Mel 5.76 1.79 6.05 4.05 esophageal cancer 65% 2% 2% 82% glioma 5% 4% 33% 5% colon cancer 92% 43% 2% 90% Ratios to melanocytes are presented for the % of methylation in melanoma cell lines and melanocytes (MM/mel) and the fresh-frozen tumors (FT/mel). MM malignant melanoma. mel melanocytes. FT fresh-frozen tumors. doi:10.1371/journal.pone.0026121.t001 tumor samples respectively. In their systematic methylation profiling of several human cancer cell lines, Paz et al. reported reactivation of THBS1 expression following 5AzadC treatment in all 18 melanoma cell lines analysed [22]. Liu et al. [23] showed TNFRSF10D promoter methylation in 85% of their melanoma cell lines (17/20) and 80% of their fresh melanoma tumor samples (32/40). In their study of the molecular effects of low dose of 5AzadC (Decitabine) on 8 melanoma cell lines, Halaban et al. [24] presented evidence for changes in expression of COL1A2, TNFRSF10D, THBS1 and UCHL1. Here, we further document the link between these genes and melanoma by confirming the correlation between methylation and expression of these genes in a larger panel of melanoma cell lines. In our study, we assessed 45 melanoma cell lines, 30 fresh tumor samples and correlated methylation status and mRNA expression levels to observations made in pooled melanocytes. Furthermore, while the majority of the previous methylation studies were based on the analysis of gene re-expression post-5AzadC treatment but not on assesment of promoter methylation per se, we present here a study design which goes beyond 5AzadC treatment to include a precise CpG methylation profile via the Epityper assay, a sensitive and high- throughput method for DNA methylation analysis. Within the large collagen family, collagen type I is the most abundant, structural component of healthy connective tissue and consists of a heterotrimer of two a1 (COL1A1) and one a2 (COL1A2) chains. Cellular p53 negatively regulates COL1A2 through TGF-b signalling in normal dermal fibroblasts [25]. Evidence for COL1A2 aberrant promoter methylation has been described in different cancer cells such as breast cancer, medulloblastoma, hepatoma, colorectal cancer [26,27,28] and more recently in melanoma [12,21]. Sengupta et al. describe the possible advantages a decrease in collagen synthesis may confer on cancer cells, including faster cell growth and increased tumorigenic potential [28]. THBS1 (also known as TSP-1) encodes the glycoprotein thrombospondin, which is generally considered a tumor suppres- sor and mediates cell-to-cell and cell-to-matrix interactions important for platelet aggregation and angiogenesis [29]. Down-regulation of THBS1 by methylation has been described in several cancer types such as neuroblastoma [30,31], colorectal [32,33,34] and stomach cancers [35,36,37]. Promoter hyper- methylation of THBS1 was detected in brain metastases of solid tumors such as, melanoma, lung, ovarian and breast carcinomas [38] and more recently associated with bad prognosis in penile squamous cell carcinoma [39]. The protein encoded by TNFRSF10D (TRAIL4, DcR2) is a member of the TNF-receptor superfamily containing an extracel- lular TRAIL-binding domain and a truncated cytoplasmic death domain. This receptor does not induce apoptosis but has been shown to play an inhibitory role in TRAIL-induced apoptosis [40]. Like all the other genes encoding TRAIL receptors, TNFRSF10D expression is directly regulated by p53 and regulates cellular chemosensitivity [41]. TNFRSF10D promoter hypermethylation has been described as a mechanism of inactivating this gene in several cancer types [42,43,44]. The current study details UCHL1 (ubiquitin COOH-terminal esterase L1) inactivation by promoter methylation in melanoma. This gene encodes a peptidase activator of the ubiquitin-dependent protein degradation pathway. Originally identified in neurons and in cells of the diffuse neuroendocrine system, mutations in this gene have been associated with Parkinson disease [45,46]. UCHL1 has been attributed important roles in multiple cellular processes such as proliferation, cell cycle, apoptosis and intra-cellular signalling. Its role in tumorigenesis, TSG or oncogene, seems to be dependent on the tumor type [47,48]. UCHL1 methylation has been reported in multiple tumors [49], such as esophageal [50], gastric [36,51], renal [52], prostate [53], head and neck squamous [47], ovarian [54], hepatocellular and colorectal cancers [49,55]. Some studies even suggested the use of UCHL1 methylation as a biomarker for diagnosis and prognosis of certain tumors [50,55,56]. Li et al. recently showed in nasopharyngeal carcinoma cell lines that UCHL1 was a member of the p53/p14ARF/MDM2 complex [48]. Through its deubiquitinating activity, UCHL1 is involved in the stabilisation of p53 and p14ARF. In parallel, it also decreases the amount of MDM2 by promoting its degradation through ubiquitination. A reduction in UCHL1 expression has previously been associated with poor survival in melanoma [57]. Our study confirms that this is likely to occur via hypermethylation of the UCHL1 regulatory region. Interestingly, all four genes (COL1A2, THBS1, TNFRSF10D and UCHL1) we identify here as methylated in melanoma, encode components that fit within the p53 ontology pathway. Moreover, two of the genes we had identified in a previous methylation study are also associated with p53 function, either being induced by p53 (ENC1) or interacting with p53 (TP53INP1) [14]. In melanoma, direct inhibition of p53 by mutation is relatively infrequent (see [58] for review). Our finding of six candidate TSGs linked to p53 function that are subject to methylation in melanoma might indicate alternative mechanisms by which these cells abrogate p53 downstream signalling in this tumor type. Further functional validation of these methylated genes is necessary to confirm their importance in melanocytic neoplasia, their candidacy as potential TSGs, as well as their possible relationship to p53 status. PLoS ONE | www.plosone.org 5 October 2011 | Volume 6 | Issue 10 | e26121