Novel Candidate Biomarkers in Melanomas Figure 6. Epigenetic Drugs Modulated the Expression of Genes Recognized by Affecting Melanocyte Biology and Response to Therapy. Expression of genes recognized by affecting (A-B) melanocyte biology, and (C-E) response to cancer therapy was deregulated in a panel of five patient-derived metastatic melanoma cell lines in comparison to FM305 primary melanocytes, as assessed by RT-qPCR approach. (A-E) Epigenetic compounds 5AzaCdR and TSA modulated the expression of genes NDRG2, VDR, NSD1, CTCF and SRC in normal melanocytes and metastatic melanoma cells. Relative gene expression and Statistics were calculated as described in figure 5. 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.g006 Interestingly, deregulation on the expression of epigenetic machinery components such as CTCF and the NSD1 histone methyltransferase-coding genes was noted in human metastases (Figure 6C-6D, Figure 7). An epigenetic signature was also found deregulated in 4C11+ mouse metastatic melanoma in relation to 4C112 non-metastatic melanoma cells (Figure 2E). Together, these results could suggest the involvement of epigenetic abnormalities in melanoma progression. However, the regulatory mechanisms have yet to be determined. To illustrate some potential contribution of our findings in the context of melanoma progression, the interaction between the pair of genes CTCF/ SERPINE1 (Figure 7B) might impair P53-mediated senescence in melanomas even in the absence of direct genetic or epigenetic lesions. It is justified since SERPINE1 gene is a critical downstream target of P53 gene in the induction of replicative senescence [43]. Additionally, the P53 gene promoter was reported to be regulated by the CTCF transcription factor in transformed cell lines [44]. In fact, evidence is emerging that epigenetic changes might play a significant role in tumor cell plasticity and the metastatic phenotype of melanoma cells [10]. Notably, FM305 human primary melanocytes were able to alter their morphology (Figure 5A) and gene expression (Figure 5C- 5E, Figure 6) in response to 5AzaCdR plus TSA exposure. To our knowledge, this is the first report suggesting an epigenetic flexibility in normal primary melanocytes in response to alterations in their environment promoted by the exposure to epigenetic modifier drugs. If this ‘epigenetic flexibility’ can translate in plasticity and changes on cellular behavior will need to be carefully evaluated in the future. Finally, accumulating evidences implicate the molecular cou- pling of differentiation program patterns and melanoma stem cells in the establishment of drug tolerance and response to melanoma therapy. In addition, resistance to apoptosis and the chromatin state of genes act as hallmarks of drug resistance [1,45–48]. To identify mechanisms underlying successful melanoma therapy, some points need to be carefully discussed: i. This study was performed by using a mouse model of melanoma progression in which sequential detachment/re-adhesion cycles of non-tumori- genic melan-a melanocyte lineage caused cells to adopt 3D cluster morphology, a progressive anoikis-resistance phenotype [15,49] and the acquisition of malignant phenotype and a metastatic behavior of 4C11+ melanoma cell line as well (Figure 1); ii. Melanoma stem cell markers are dynamically regulated in each transition associated with melan-a tumorigenic conversion (Fig- ure S3). Noteworthy, Cheli and coworkers (2011) reported that the inhibition of microphthalmia-associated transcription factor (MITF), the master regulator of melanocyte lineage commitment and differentiation, increased the tumorigenic potential of human melanoma cells whereas up-regulated stem cell markers and enhanced the number of melanoma-initiating cells in human melanoma spheroids. Thus, authors concluded that MITF acts as the key molecular switch between human melanoma initiating cells and their differentiated progeny [48]. Melan-a, 4C, 4C112 and 4C11+ cell lines vary in their differentiation program patterns (Figure S3; Camila Ferreira de Souza, manuscript in prepara- tion). Despite this fact, it is so premature to conclude that the spheroid cells found after the anchorage blockage of 4C pre- malignant melanocytes are cancer stem cells. Further studies need to be carefully conducted to address this issue. Such characteriza- tion might carry important clinical implications as cancer stem cells show increased resistance to chemotherapy; iii. The chromatin modifying enzymes such as increased JARID1A and/ or increased histone deacetylases (HDACs) can reversibly mediate the acquisition of a drug tolerant state in melanomas [46]. Importantly, we previously reported that many histone marks differ among melan-a, 4C, 4C112 and 4C11+ cell lines, which could be associated with aberrant gene expression [11] (and data not shown). The notion that epigenetics is associated with widespread gene expression changes in cancer is not new. Some studies have pointed out that transcriptional factors, epigenetic complexes and pluripotency markers cooperate to modulate the expression of XIST gene. In addition, the presence of histone acetyltransferases (HATs) are related to the expression of VDR gene [50–53]. However, the relationship between them at a given stage of disease appears to be complex and certainly merits a careful exploration. Curiously, Do and coworkers (2008) showed an enhancement in Dnmt3a RNA levels after TSA treatment in female neurosphere cells suggesting that Dnmt3a gene expression is controlled by histone modification. Importantly, depletion of Dnmt3a mRNA was sufficient to inhibit the de novo methylation of the Xist region after TSA treatment, although Xist RNA level was slightly reduced. Together, these findings indicate that Dnmt3a has a direct effect on Xist methylation acting as a negative regulator of Xist gene as well [51]. We previously showed that the expression of Dnmt1 and Dnmt3a at protein level was detected in melan-a melanocytes. Besides that, the up-regulation of Dnmt3a protein was only found for 4C11+ metastatic melanoma cell line [11]. Interestingly, a slight decrease in the relative intensity of Xist gene expression was noted when TSA was only given to melan-a melanocytes and 4C11+ metastatic cells (Figure 4A). Thus, it is reasonable to hypothesize that no one molecular mechanism accounts for the properties of DNA methyltransferase and histone deacetylase inhibitors, even at a specific gene locus. It could explain, for example, others apparent opposite results, such as: i) The treatment of the 4C pre-malignant melanocytes and 4C112 non-metastatic melanoma cell line with 5AzaCdR plus TSA resulted in the highest up-regulation of Xist expression whereas a more robust up-regulation of Xist gene expression in 4C11+ metastatic melanoma cell line was observed when 5AzaCdR alone was given (Figure 4A); ii) The treatment of melan-a melanocytes as well as 4C112 non-metastatic and 4C11+ metastatic melanoma cell lines with 5AzaCdR in combination to TSA resulted in the highest up-regulation of Hspb1 gene expression whereas no significant change was observed at pre-malignant stage (4C melanocyte lineage) except a slight increase when 5AzaCdR was done (Figure 4B); iii) VDR gene expression was up-regulated in FM305 primary melanocytes after combined 5AzaCdR and TSA treatment whereas a slight decrease on VDR gene expression was observed when the combinatorial treatment protocol was given to Mel-2 and Mel-3 patient-derived metastatic melanoma cells PLOS ONE | www.plosone.org 13 September 2012 | Volume 7 | Issue 9 | e44800