Novel Candidate Biomarkers in Melanomas Table 1. An overview of chromosomal aberrations during melanoma progression. Cell Line melan-a* 4C* 4C112 4C11+ Chromosome Number Centromere Fragments 54 51 51 70 21 31 31 5 Recurrent Structural Chromosomal Changes None T[8;14] {19}/T[14;8] {21}/T[15;X] {18}/F[8;6] {4}/Del[11] {18} T[8;14] {13}/T[14;8] {14}/ T[7;X] {6}/T[15;X] {11}/F[8;6] {19}/Del[11] {16} T[1;16] {19}/T[8;1] {20}/T[1;12] {10}/T[3;13] {19}/T[13;3] {15}/T[X;15] {15}/Rb[8.12] {14}/Rb[12.12] {30}/Del[1] {18}/Del[11] {16} The karyotype descriptions indicated by ’*’are derivatives from analyses previously performed by Silva et al [17]. The number of chromosomes and centromere fragments in the cell line panel are derivatives from the analyses of 20 metaphases per cell line examined. The braces indicate how often each rearrangement was seen in 20 methaphases. Del: deletion; F: fusion; Rb: Robertsonian fusion; T: translocation. doi:10.1371/journal.pone.0044800.t001 Triacylglycerol metabolism (Figure 2B-2C). Finally, we investigated whether the genes from different class-comparison experiments shared one or more cellular pathways. As a result, we found pathways commonly perturbed between the transitions from melan-a melanocytes to 4C pre-malignant melanocytes and 4C112 non-metastatic to 4C11+ metastatic melanoma cells. Therefore, we noted that genes from the pathways TGF-b, Notch, Insulin/IGF pathway-PKB, Insulin/IGF pathway MAPKK/MAPK, Cytoskeletal regulation by Rho GTPase, Cadherin signaling, and Alzheimer disease-presenilin signaling were over-represented in 4C pre-malignant melanocytes in relation to melan-a cells, and under-represented in 4C11+ metastatic melanoma cell line in relation to 4C112 non- metastatic melanoma cells. Furthermore, we also observed neuroendocrine pathways commonly down-regulated in the transition from melan-a melanocytes to 4C pre-malignant melanocytes, and up-regulated in the transition from 4C112 non-metastatic to 4C11+ metastatic melanoma cell lines, as follows: 5HT degradation, Adrenaline/NOR biosynthesis, Cortocotropin- releasing factor, Muscarinic acetylcholine receptor, Opioid and b-adrenergic receptor as well. Interestingly, the transition from 4C pre-malignant melanocytes to 4C112 non-metastatic melanoma cells was marked by the over-representation of genes from the Histamine synthesis-related pathway. Nonetheless, such pathway was over- represented in the transition from 4C112 non-metastatic mela- noma to 4C11+ metastatic melanoma cells. Therefore, we provide evidence that stress-driven changes in the environment can impair the neuroendocrine activity of the melanocytes in the initial stages associated with malignant transformation of melanocytes. Togeth- er, these results provide insights into the aetiology and pathogen- esis of malignant melanomas by suggesting that early melanocytic lesions would be useful to predict a functional phenotype associated with metastasis (Figure 2B-2D). Importantly, pertur- bations on the expression of genes associated with epigenetic machinery were identified as molecular markers of tumor progression in our melanoma model (Figure 2E) [11]. Demethylating Agent 5AzaCdR Differentially Modulated Gene Expression at Specific Stages of Melanoma Progression Previously, we reported that the differential expression of Dnmt1 and Dnmt3a at protein level were associated with melan-a malignant transformation. Additionally, we demonstrated that the treatment with 10 mM 5-aza-29-deoxycytidine (5AzaCdR) for 48 hours was able to inhibit the tumorigenicity capacity of the 4C11+ metastatic melanoma cell line in vivo [11]. Besides that, a growing body of data has shown that epigenetic modifier drugs can affect a number of tumor cell phenotypes, including the proliferation of melanoma cells [18,19]. As the first step in characterizing the global demethylation responses induced by DNA hypomethylating agents, melan-a, 4C, 4C112 and 4C11+ cell lines were treated with 10 mM 5AzaCdR for 48 hours. The establishment of this treatment condition for 5AzaCdR was justified since we aimed to maximize demethylation efficiency and to minimize drug toxicity as well. Cell toxicity and viability were monitored by using a standard methyl thiazol tetrazolium assay (MTT), as previously described [11]. Following treatment of cells, there was a substantial decrease of genomic DNA methylation, since all treated cells showed significant percent of demethylation when compared with untreated cells. The decrease in global DNA methylation was observed at the same extent in 4C pre-malignant melanocytes, 4C112 non-metastatic, and 4C11+ metastatic melanoma cells as well (Figure 3A). In order to identify candidate genes targeted, either directly or indirectly, by aberrant DNA methylation at specific stages of melanoma progression, a genome- wide screening was performed in melan-a, 4C, 4C112 and 4C11+ cell lines after their treatment with 5AzaCdR at the time and concentration indicated. The high-throughput Affymetrix micro- array approach was used as a platform screening, as described above. The experiments were carried out in two biological replicates. Class-comparison experiments were performed, and transcripts were selected as statistically significant by the pairwise two-class SAM analysis (FDR and Q-values,0.05) by using 500 random permutations as follows: I) gene expression profile of the parental melan-a melanocyte lineage versus its 5AzaCdR-treated counterpart cell: 250 up-regulated genes upon 5AzaCdR treat- ment; II) gene expression profile of 4C pre-malignant melanocyte lineage versus its 5AzaCdR-treated counterpart cell: 81 up- regulated genes upon 5AzaCdR treatment; III) gene expression profile of 4C112 non-metastatic melanoma cell line versus its 5AzaCdR-treated counterpart cell: 569 up-regulated genes upon 5AzaCdR treatment; IV) gene expression profile of 4C11+ metastatic melanoma cell line versus its 5AzaCdR-treated counterpart cell: 359 up-regulated genes upon 5AzaCdR treat- ment (Figure S2, Tables S1–S4). To identify candidate tumor suppressor genes whose expression could be modulated by epigenetic agents in distinct phases of melan-a malignant transformation, we performed genome-wide association studies for melan-a versus 4C, 4C versus 4C112, and 4C112 versus 4C11+ cell lines as follows: I) intersection between genes found significantly down-regulation in 4C pre-malignant melanocytes in comparison to melan-a melanocyte lineage, and our list of over- expressed genes upon 5AzaCdR treatment in 4C melanocytes; II) intersection between genes found significantly down-regulation in PLOS ONE | www.plosone.org 4 September 2012 | Volume 7 | Issue 9 | e44800