Novel Candidate Biomarkers in Melanomas Figure 3. Demethylating Agent 5-aza-29-deoxycytidine Differentially Modulated Gene Expression at Specific Stages of Melanoma Progression. (A) The 4C pre-malignant melanocytes, 4C112 non-metastatic and 4C11+ metastatic melanoma cell lines displayed the same percent of global genomic DNA demethylation upon 5AzaCdR treatment. (B-D) Genome-wide association studies of melan-a, 4C, 4C112 and 4C11+ cell lines untreated and previously exposed to 5AzaCdR revealed re-expressed genes at a given transition associated with melan-a malignant transformation. Heatmaps were generated by unsupervised hierarchical clustering using the Pearson correlation as metric distance and average-linkage as algorithm as well. The saturation of either color (scale from green to red) reflects the magnitude of the difference on gene expression level. ma: immortalized, but non-tumorigenic, melan-a melanocyte lineage; 4C: pre-malignant melanocyte lineage; 4C112: non-metastatic melanoma cell line, and 4C11+: metastatic melanoma cell line. 5AzaCdR: 5-aza-29-deoxycytidine. doi:10.1371/journal.pone.0044800.g003 candidate markers implicated in malignant transformation of melan-a melanocytes. Demethylating Agent 5AzaCdR Combined with the HDAC Inhibitor TSA Modulate Cell Morphology and Gene Expression in Human Primary Melanocytes To better follow up the steps of melanoma progression, we first examined the effect of TSA, 5AzaCdR, and 5AzaCdR plus TSA treatments on morphology of FM305 human primary melano- cytes. The primary melanocytes exhibited the highest change on cell morphology when treated with 5AzaCdR in combination to TSA (Figure 5A). Based on this, we next investigated whether the differentially expressed genes validated as candidate markers for melan-a malignant transformation could respond to epigenetic compounds in human specimens. To do this, HSPB1, SERPINE1 and FBLIM1 genes were further validated in two patient-derived right supraclavicular lymph node metastases (Mel-2 and Mel-3 cell lines) previously exposed to 10 mM 5AzaCdR for 48 hours followed by 40 nM TSA for 16 hours. Interestingly, we observed the same profile of drug responsiveness for FM305 human primary melanocytes (Figure 5C-5E) and melan-a mouse melanocyte Figure 4. Candidate Markers for melan-a Malignant Transformation. (A) Xist, (B) Hspb1, (C) Serpine1 and (D) Fblim1 genes were assessed for relative quantification (RQ) in mouse cell lines across the melanoma progression spectrum (melan-a, 4C, 4C112 and 4C11+) non-treated (NT) or previously treated with 40 nM TSA for 16 hours, 10 mM 5AzaCdR for 48 hours, and 10 mM 5AzaCdR for 48 hours plus 40 nM TSA for 16 hours. 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. ma: immortalized, but non-tumorigenic, melan-a melanocyte lineage; 4C: pre-malignant melanocyte lineage; 4C112: non-metastatic melanoma cell line, and 4C11+: metastatic melanoma cell line. TSA: Trichostatin A; 5AzaCdR: 5-aza-29-deoxycytidine. doi:10.1371/journal.pone.0044800.g004 PLOS ONE | www.plosone.org 8 September 2012 | Volume 7 | Issue 9 | e44800