lineage (Figure 4B-4D) in relation to the expression of genes HSPB1/Hspb1, SERPINE1/Serpine1, and FBLIM1/Fblim1. In spite of this, only the SERPINE1/Serpine1 gene showed the same treatment responsiveness when 5AzaCdR combined with TSA was given to 4C11+ mouse metastatic melanoma cell line as well as Mel-2 and Mel-3 human metastatic melanoma cells. The treatment conditions for all human cells were the same as those described for mouse cell lines, and cell viability was monitored by the Trypan Blue assays (data not shown). Furthermore, we investigated the differential expression of genes HSPB1, SERPINE1 and FBLIM1 in a panel of five patient-derived metastatic melanoma cell lines in comparison to FM305 primary melano- cytes. Based on the clinical staging criterion measured by the time from post treatment of primary tumors to re-occurrence of the diseases, we have distinguished two groups of metastases: M1 (Mel-2 and Mel-3: better prognosis), and M2 (Mel-11, Mel-14 and Mel-33: worst prognosis). RT-qPCR approach revealed a unifying profile of gene expression in which FBLIM1, HSPB1 and SERPINE1 up-regulation significantly distinguishes less aggressive metastases (Mel-2 and Mel-3) from those displaying more aggressiveness (Mel-11 and Mel-14) (Figure 5C-5E). Curiously, the expression profile exhibited for the genes HSPB1 and SERPINE1 in Mel-2 and Mel-3 human metastases was noted for mouse cell lines representing pre-malignant melanocytes and non- metastatic melanoma cells (4C and 4C112 cell lines, respectively) in comparison to melan-a melanocytes (Figure 4B-4C, Figure 5C-5D). Additionally, we investigated by RT-PCR the expression of XIST in two female patient-derived metastatic melanoma cell lines with high degree of aggressiveness (Mel-11 and Mel-33). The positive correlation on expression of Xist/XIST was seen for 4C11+ mouse metastatic melanoma cell line and Mel- 33 (inguinal lymph node metastasis), but not for Mel-11 (brain metastasis) (Figure 4A, Figure 5B). Treatment of Human Melanocytic Cells with Epigenetic Compounds Modulate the Expression of Genes Recognized by Affecting Melanocyte Biology and Response to Cancer Therapy As the first step in identifying genes implicated in melanocyte biology, whose expression were deregulated in the progression of melanomas, we performed a manual annotation of the 136 genes significantly down-regulated in the transition from 4C112 to 4C11+, and up-regulated in 4C11+ metastatic melanoma cells upon 5AzaCdR treatment. Two genes were selected for validation of gene expression (NDRG2 and VDR) (Figure 3D) [27–30]. Moreover, we were also interested in identifying genes potentially implicated in response to cancer therapy. Along this line, we selected the gene NSD1 (Figure 3D) [31] as well as two additional genes based on the literature searched for further validation (CTCF and SRC) [32,33]. Therefore, the expression pattern of human NDRG2, VDR, NSD1, CTCF and SRC genes in several human metastatic melanoma cell lines in comparison to primary melanocytes were analyzed at mRNA level by RT-qPCR methodology. The highest expression level of NDRG2 gene was found in the brain (Mel-11), axillary lymph node (Mel-14), and inguinal lymph node (Mel-33) metastases. On the other hand, NDRG2 mRNA was nearly undetectable in the two right supraclavicular lymph node metastases derivatives from the same patient before and after the re-occurrence of the disease (Mel-2 and Mel-3 cell lines, respectively). Interestingly, when 5AzaCdR treatment was given in combination to TSA, a significant up- regulation of NDRG2 expression was found in Mel-2 metastatic melanoma cells (Figure 6A). VDR gene expression was highly Novel Candidate Biomarkers in Melanomas positive in the metastases examined, except for Mel-33 cell line, which was established from inguinal lymph node metastasis. A slight decrease on VDR gene expression was observed when 5AzaCdR plus TSA treatment was given to Mel-2 and Mel-3 melanoma cells (Figure 6B). The significant up-regulation of human NSD1 gene was found in all metastases evaluated, except Mel-2, in comparison to primary melanocytes. The treatment based on 5AzaCdR plus TSA caused a slight decrease on NSD1 gene expression in Mel-2 melanoma cells (Figure 6C). An enhancement of CTCF gene expression was specifically found in the aggressive metastases examined (Mel-11, Mel-14 and Mel-33) in comparison to primary melanocytes. Mel-3 melanoma cells showed a reduced expression on CTCF gene upon 5AzaCdR combined with TSA treatment (Figure 6D). The SRC gene expression was higher in the more aggressive metastases (Mel-14 and Mel-33) when compared to those presenting less aggressive- ness (Mel-2 and Mel-3). Interestingly, the expression of SRC gene had undergone down- and up-regulation, respectively, when Mel- 2 and Mel-3 melanoma cells were treated with 5AzaCdR combined with TSA (Figure 6E). All genes evaluated had undergone up-regulation in FM305 primary melanocytes upon 5AzaCdR+TSA treatment (Figure 6A-6E). Hierarchical Clustering and Network Analyses Revealed Novel Gene Interactions in Melanoma Progression In an effort to elucidate the possible usefulness of the novel gene expression signature as a marker of melanoma progression, we performed an unsupervised hierarchical clustering (HCL) analysis by grouping the five human metastatic melanoma cell lines examined according to their patterns of gene expression (Pearson correlation metric distance; average linkage algorithm). To do this, mRNA levels of HSPB1, SERPINE1, FBLIM1, NDRG2, VDR, NSD1, CTCF and SRC were measured after RT-qPCR approach by using the 2-DDCq method, and plotted in a heatmap. Notably, unsupervised HCL of patient-derived metastatic melanoma cell lines was able to cluster samples into their theoretical cell type branches (Figure 7A). Finally, Relevance Networks were gener- ated allowing us to find, by an additional technique, some strong gene regulatory relationships as candidate markers in advanced melanomas. In such methodology, each pair of genes related by a correlation coefficient larger than a minimum threshold and smaller than a maximum threshold is connected by a line. Links colored in red represent genes that are positively correlated while links colored in blue represent genes negatively correlated. To illustrate some findings, the relationships between pairs of genes NDRG2/NSD1, VDR/NSD1, SRC/NSD1 as well as CTCF/ SERPINE1 were deregulated in four of five metastases examined. Additionally, interactions between pairs of genes HSPB1/SER- PINE1 as well as FBLIM1/SERPINE1 were found in three of five metastases evaluated. These results highlight the potential usefulness of the novel gene expression signature as a marker in melanoma progression (Figure 7B). Discussion Up to date, classical chemotherapy as well as discovered biomarkers have not been significant successful neither to diagnosis melanomas or to benefit patients, with rare exceptions, since only the gene CTLA4 and V600E mutational status of BRAF gene have been recognized as bona fide targets to treat metastatic disease [34,35]. In this study, we used a mouse model of melanoma progression in which the non-tumorigenic melan-a melanocyte lineage was submitted from anchorage-dependent to anchorage-independent PLOS ONE | www.plosone.org 9 September 2012 | Volume 7 | Issue 9 | e44800