irreversible, metastases will be genetically distinct from the primary tumor. In contrast of this, there is a hypothesis which suggests that metastases arise because of microenvironment-driven changes to a cell’s phenotype, leading to some cells acquiring a slow proliferating, stem cell-like phenotype with invasive potential. Given the appropriate signals, these cells will migrate to find a new niche where a different microenvironment will lead to either a resumption of proliferation and formation of a new metastasis or dormancy [5]. As the epigenetic modifications can be modulated by changes in the environment, leading to a reprogramming of gene expression patterns in mammals [6,7], it is plausible to hypothesize that there is a biological relationship between epigenetics and microenvironment-driven changes in gene expres- sion, which would lead to a phenotype-switching in melanomas. In fact, it has become clear that a dynamic reprogramming in epigenetic marks has been positively correlated with aberrant gene expression during melanocytic neoplasia, including our model of study [8–11]. Epigenetic information is propagated during somatic cell divisions, and is critical for preserving gene expression patterns and cellular identity. Epigenetic regulation of gene expression involves the interplay of DNA methylation, histone modifications, nucleosome occupancy, and microRNAs. DNA methylation patterns are tightly correlated with chromatin architecture. Active regions of the chromatin, which enable gene expression, are associated with hypomethylated DNA, whereas hypermethylated DNA is packaged in inactive chromatin. Histone deacetylation is a global mark of gene silencing. The interrelation between DNA methyltransferases (DNMTs) and histone deacetylases (HDACs) associated with the dynamic reversibility of epigenetic signatures in somatic cells, has been offered opportunities for testing drugs that can re-express critical silenced-genes in cancer [12–13]. The main purpose of our research was to identify novel markers in early and later stages of melan-a malignant transformation. The molecular markers deregulated earlier in melan-a malignant transformation could provide insights into the aetiology and pathogenesis of melanomas. In addition, they would be useful to predict a functional phenotype associated with metastasis. Apart from this, the molecular markers specifically deregulated at metastatic stage could confer selective clonal growth advantage. Here, we used the high-throughput Affymetrix microarray approach to conduct class-comparison experiments to identify significantly up- and down-regulated genes in each transition of malignant transforma- tion of melan-a mouse melanocyte lineage: from melan-a to 4C, 4C to 4C112, and 4C112 to 4C11+ cells. The 4C (pre-malignant melanocytes), 4C112 (non-metastatic melanoma) and 4C11+ (metastatic melanoma) cell lines were established after sequential detachment/re-adhesion cycles of melan-a melanocytes. Next, the class-comparison experiments were done in melan-a, 4C, 4C112 and 4C11+ cells previously exposed to the demethylating agent 5- aza-29-deoxycytidine (5AzaCdR). Genome-wide association stud- ies allowed us to identify candidate down-regulated genes whose expression could be modulated, directly or indirectly, by 5AzaCdR treatment at several stages of melanoma progression. A mining gene expression signature was found and validated in human specimens untreated and previously treated with 5AzaCdR in combination to the HDAC inhibitor Trichostatin A (TSA). Our data open new avenues for the detection of early melanocytic lesions with high risk for metastasis, meanwhile provide data which might be used in the future for the incorporation of new metastasis-related biomarkers into a model more closely related to translational research. Novel Candidate Biomarkers in Melanomas Results Tumorigenicity and Metastatic Capabilities Were Acquired by melan-a Melanocytes after Detachment/ Re-adhesion Cycles The metastatic capacity of a melanoma cell subpopulation almost always results in the successful of this neoplastic disease, as evaluated by growing mortality rates worldwide in patients with stage IV melanomas (American Joint Committee on Cancer) [2]. Based on this, we first investigated whether cell lines established from melan-a melanocytes after detachment/re-adhesion cycles showed tumorigenicity property. As previously described, melan-a and 4C melanocyte lineages were not able to grow as tumors when inoculated subcutaneously into C57Bl6 syngeneic mice, whereas 4C112 and 4C11+ cell lines showed tumorigenic potential [14,15]. Based on the in vivo growth profile, we characterized the 4C112 and 4C11+ cell lines as slow- and fast-growing melano- mas, respectively, as evaluated by differences in latency times for tumor appearance (P,0.01) (Figure 1A). We next evaluated whether 4C112 and 4C11+ melanoma cell lines presented metastatic capacity. Using C57Bl6 syngeneic mice, 4C11+ melanoma cell line was shown to be able to grow as macrometa- static pulmonary foci, as evaluated by tumor multiplicity (P,0.0001) (Figure 1B). Importantly, animals inoculated intra- venously with 4C11+ melanoma cell line (2.5x105 cells per mouse) displayed overall survival rates of 30 days (P,0.0001) (Figure 1C). Karyotype Evolution The karyotypic instability can act as an indicator of cancer aggressiveness [16]. As such, we next analyzed karyotypic aberrations that accompanied the tumorigenic conversion of melan-a melanocyte lineage. It is worth mentioning that melan-a is a cell line that was first described as having a normal diploid karyotype [14]. In addition, melan-a cell line displayed no structural chromosomal abnormalities [17]. In this article, evaluation of chromosomal abnormalities of well-established melanoma cell lines was performed using spectral karyotyping (SKY). Twenty metaphases per cell line examined were analyzed. In general, the results of our chromosome analyses combined with the findings previously reported by Silva et al [17] revealed specific non-random structural abnormalities in chromosomes 6, 8, 11, 14, 15, and X in pre-malignant melanocytes and early melanomas (4C and 4C112 cell lines, respectively). Importantly, here we are showing that the tumorigenic phenotype was marked by the appearance of a unique translocation of T[7;X], which was seen in 6 of 20 metaphases examined. Besides that, the 4C11+ metastatic melanoma cell line displayed a complex pattern of numerical and structural aberrations that may contain potential pathological consequences. Such metastasis-related aberrations could readily be distinguished by imbalances in the number of chromosomes, Robertsonian fusions involving the chromosomes 8 and 12, and less centromere fragments than 4C112 non-metastatic melanoma cell line as well (P,0.0001) (Table 1, Figure S1). Gene Expression Profiles of melan-a Melanocytes and melan-a-Derived Cell Lines Revealed Pathways Commonly Perturbed in Pre-Malignant Melanocytes and Metastatic Melanomas As the first step in identifying up- and down-regulated genes associated with melan-a malignant transformation and melanoma progression, we identified significantly differentially expressed genes with mRNA extracted from melan-a, 4C, 4C11–– and 4C11+ cell monolayers. The high-throughput Affymetrix micro- PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e44800