Novel Candidate Biomarkers in Melanomas metastases was also determined by RT-qPCR. Relative gene expression was calculated according to 2-DDCq method, using cellular ACTB mRNA levels as endogenous reference control. Error bars in all cases represent SEM and P values were based on Students’ t test or One-Way ANOVA test followed by the post-hoc Tukey. *P,0.05, **P,0.01, ***P,0.001. FM305: human primary melanocytes from neonatal foreskin; Mel-2: right supraclavicular lymph node metastasis; Mel-3: counterpart of Mel-2 after the re-occurrence of the disease; Mel-11: brain metastasis; Mel-14: axillary lymph node metastasis, and Mel-33: inguinal lymph node metastasis. TSA: Trichostatin A; 5AzaCdR: 5-aza-29-deoxycytidine. doi:10.1371/journal.pone.0044800.g005 culture conditions [15]. We reported that melan-a melanocytes acquired features of aggressive melanoma cells after sequential detachment/re-adhesion cycles. This aggressive phenotype was characterized by a biological relationship between the complex karyotype with Robertsonian fusions and metastatic capacity [16] of 4C11+ melanoma cells in vivo (Figure 1B-1C, Table 1, Figure S1). Genome-wide screening of melan-a, 4C, 4C112 and 4C11+ cell lines revealed a large set of differentially up- and down- regulated genes in the consecutive transitions implicated in melan- a malignant transformation (from non-tumorigenic to metastatic stage) (Figure 2A). It is well known that mammalian development and cell type determination result from the tightly controlled regulatory mechanisms between signaling pathways and transcrip- tion factors in progenitor cells as well as environmental influences. Interestingly, we showed that the conversion from melan-a melanocytes to 4C pre-malignant melanocytes involved the down-regulation of neuroendocrine pathways [36,37], providing evidence that stress-driven changes in the environment can impair the neuroendocrine activity of the melanocytes in the initial stages of melan-a tumorigenic conversion. Additionally, we showed that the conversion from 4C pre-malignant melanocytes to 4C11+ metastatic melanoma cells involved, among others, the down- regulation on the expression of melanocyte stem cell-related signaling pathways, such as TGF-b [38] and Notch [39] (Figure 2B, 2D). This finding is in agreement with the data reported earlier that melan-a melanocytes and 4C11+ metastatic melanoma cells display an epithelial morphology, whereas 4C pre- malignant melanocytes and 4C112 non-metastatic melanoma cells exhibit a mesenchymal morphology with enhanced expres- sion of the gene Chd1, and the presence of the repressive histone mark H3K27 trimethylation plus the active mark H3K4 trimethylation [11]. Taken together, these findings would indicate a less differentiated chromatin state. In terms of human cancer biology, the stem cell chromatin state may enhance the likelihood for DNA hypermethylation on tumor suppressor-coding genes [40]. Based on the shared genetic background of the cells as well as commonly perturbed pathways over-represented in 4C pre- malignant melanocytes in relation to melan-a cells, and under- represented in 4C11+ metastatic melanoma in relation to 4C112 non-metastatic melanoma cells (Figure 2D), our rationale was that epigenetic reprogramming on gene expression occurring earlier in melan-a malignant transformation would be useful to predict a phenotype associated with metastasis. Using genome-wide expression arrays we identified patterns of gene expression following 5-aza-29-deoxycytidine (5AzaCdR) exposure. Genome-wide association studies between untreated cell lines and their 5AzaCdR-treated counterpart cells allowed us to identify candidate down-regulated genes whose expression could be modulated, directly or indirectly, by 5AzaCdR treatment at specific stages of melanoma progression (Figure 3B-3D). A gene expression signature was discovered and validated by RT- qPCR in melan-a, 4C, 4C112 and 4C11+ mouse cell lines (Figure 4A-4C). Moreover, candidate metastasis-related bio- markers were validated in early-passage cultures of patient-derived metastatic melanoma cell lines as well as primary melanocytes untreated and/or previously treated with 5AzaCdR in combina- tion to the HDAC inhibitor Trichostatin A (TSA) (Figure 5B-5E, Figure 6). A signature comprised by the genes Xist/XIST, Hspb1/HSPB1, Serpine1/SERPINE1, Fblim1/FBLIM1, CTCF, NDRG2, NSD1, SRC and VDR was selected for further validation by RT-qPCR approach. XIST (X inactive-specific transcript) is the master regulator of X-chromosome inactivation to equalize gene products between male and female mammals, by inactivating one of the X- chromosomes during the early embryonic development in females (Lyon hypothesis, 1962) [41]. Apart from this, DNA copy number changes of XIST gene was reported in colorectal carcinomas [22]. The positive correlation between HSPB1 (heat shock 27 KDa protein 1) and SERPINE1 (serpin peptidase inhibitor, clade E, member 1) overexpression induced reversal of the invasive and metastatic phenotype of melanoma cells in vitro [20,21]. The positive correlation between FBLIM1 (filamin binding LIM protein 1) and SRC (v-src sarcoma viral oncogene homolog [avian]) overexpression resulted in anoikis resistance contributing to tumorigenicity of established carcinoma cells [42]. Additionally, SRC activation was implicated with primary resistance to PLX4032 in patient-derived melanoma cells [33]. The protein encoded by the CTCF (CCCTC-binding factor) gene contributes to the organization of DNA into loops of transcriptionally repressive heterochromatin or into active euchromatin, which facilitates blocks or which connects distal enhancers and proximal promoters [12]. In terms of response to cancer therapy, the insulator protein CTCF mediated the down-regulation of telome- rase in response to sulforaphane exposure, a compound that has histone deacetylase inhibition activity, in human breast cancer cells [32]. NSD1 (nuclear receptor binding SET domain protein 1) gene silencing was associated with tamoxifen resistance in breast tumors [31]. The decrease on the expression of VDR (vitamin D receptor) gene was linked to progression of pigmented skin lesions as well as shorter patients’ melanoma overall survival [30]. Finally, down-regulation of the NDRG2 (NDRG family member 2) gene was reported to inhibit the metastatic potential of melanoma cells in vitro and in vivo [27,28]. Even though much has been discussed about the discrepancies between mouse and human mechanisms of carcinogenesis, network analysis showed, for example, the interaction between pairs of genes HSPB1/SERPINE1 as well as FBLIM1/SERPINE1 in some of the human metastases examined (Figure 7B). Moreover, up-regulation on the expression of Serpine1/SERPINE1 gene upon 5AzaCdR plus TSA treatment was found in 4C11+ mouse metastatic melanoma cells as well as Mel-2 and Mel-3 patient-derived metastases (Figure 4C, Figure 5D). In addition, melan-a mouse melanocyte lineage and FM305 human primary melanocytes shared the up-regulation on the expression of HSPB1, SERPINE1 and FBLIM1 genes when 5AzaCdR associated with TSA was given (Figure 4B-4D, Figure 5C-5E). Hierarchical clustering of human metastatic melanoma cells was able to group samples into their theoretical cell type branches (Figure 7A). Despite the fact that it could be a marker of molecular heterogeneity, network analysis pointed out gene interactions commonly perturbed among the metastases evaluated such as NDRG2/NSD1, VDR/NSD1, SRC/NSD1, CTCF/SER- PINE1, HSPB1/SERPINE1 and FBLIM1/SERPINE1 (Figure 7B). PLOS ONE | www.plosone.org 11 September 2012 | Volume 7 | Issue 9 | e44800