Transcriptional signatures underlying dynamic phenotypic switching and novel disease biomarkers in a linear cellular model of melanoma progression

Abstract

Despite advances in therapeutics, the progression of melanoma to metastasis still confers a poor outcome to patients. Nevertheless, there is a scarcity of biological models to understand cellular and molecular changes taking place along disease progression. Here, we characterized the transcriptome profiles of a multi-stage murine model of melanoma progression comprising a nontumorigenic melanocyte lineage (melan-a), premalignant melanocytes (4C), nonmetastatic (4C11-) and metastasis-prone (4C11+) melanoma cells. Clustering analyses have grouped the 4 cell lines according to their differentiated (melan-a and 4C11+) or undifferentiated/“mesenchymal-like” (4C and 4C11-) morphologies, suggesting dynamic gene expression patterns associated with the transition between these phenotypes. The cell plasticity observed in the murine melanoma progression model was corroborated by molecular markers described during stepwise human melanoma differentiation, as the differentiated cell lines in our model exhibit upregulation of transitory and melanocytic markers, whereas “mesenchymal-like” cells show increased expression of undifferentiated and neural crest-like markers. Sets of differentially expressed genes (DEGs) were detected at each transition step of tumor progression, and transcriptional signatures related to malignancy, metastasis and epithelial-to-mesenchymal transition were identified. Finally, DEGs were mapped to their human orthologs and evaluated in uni- and multivariate survival analyses using gene expression and clinical data of 703 drug-naïve primary melanoma patients, revealing several independent candidate prognostic markers. Altogether, these results provide novel insights into the molecular mechanisms underlying the phenotypic switch taking place during melanoma progression, reveal potential drug targets and prognostic biomarkers, and corroborate the translational relevance of this unique sequential model of melanoma progression.

Genes regulated by DNA methylation are involved in distinct phenotypes during melanoma progression and are prognostic factors for patients

“…As mentioned before, our melanoma progression cellular model was established through continuous stress instead of genetic manipulation, resulting in epigenetic alterations both very early and late in tumor progression as previously described [ 12 , 13 ]. In a recent study, Preston‐Alp and colleagues showed that UV radiation alters DNA methylation both in primary human melanocytes and in murine melan‐a melanocytes in CpG sites found to be prognostic of overall survival of melanoma patients [ 29 ].…”

“…Studies have demonstrated a negative correlation between gene expression and promoter DNA methylation, but a positive correlation of gene expression with gene body DNA methylation [ 3 , 30 ]. Therefore, we analyzed previous data from RNA‐seq (transcriptome) [ 13 ] and ERRBS (methylome) of the four cell lines and integrated these data to unravel how methylation in different regions of the gene may affect gene expression. Our approach was to select genes that showed, in each comparison expression, changes positively correlated with alterations in gene body DNA methylation, as well as genes that showed a negative correlation between changes in expression and promoter DNA methylation (Fig.…”

“…Differently, only slight changes were observed in the number of genes downregulated by gene body DNA hypomethylation after comparing melan‐a to 4C ( n = 84), 4C11− ( n = 83), or 4C11+ ( n = 55) or genes upregulated by gene body DNA hypermethylation ( n = 64 for melan‐a vs. 4C, n = 44 for melan‐a vs. 4C11−, and n = 32 for melan‐a vs. 4C11+), suggesting that changes in gene body methylation are less prevalent during initial melanoma progression. When comparing gene regulation between 4C and 4C11− cells, we identified a small number of genes with changes in promoter or gene body methylation, most likely due to their very similar phenotypic characteristics [ 13 ]. When comparing cells corresponding to initial stages of melanoma progression (4C and 4C11−) to late‐stage 4C11+ cells, we observed more pronounced gene expression changes that are associated with gene body or promoter DNA methylation aberrancies (Fig.…”

“…After limiting dilution of 4C spheroids formed by another deadhesion, it was established the cell line 4C11−, an undifferentiated/mesenchymal and slow‐growth melanoma cell line capable of developing tumors in vivo . After the spontaneous loss of p53, 4C11− gave origin to the differentiated/pigmented and highly proliferative 4C11+ cell line, capable of forming fast‐growing tumors and developing lung metastasis in vivo [ 11 , 12 , 13 , 14 ]. Alterations in the global level of 5mC content and histone modifications, as well as in the expression of several epigenetic machinery components were previously described in this model [ 14 , 15 , 16 , 17 , 18 ].…”

Comprehensive Gene Expression Analysis to Identify Differences and Similarities between Sex- and Stage-Stratified Melanoma Samples

“…This goes in line with the well-reported positive correlation of melanoma heterogeneity with stage progression. Consequently, increased genetic instability in the tumor causes transcriptomic and proteomic diversity, which in turn allows microenvironment-driven or cell-intrinsic phenotype-switching, i.e., reversible switching between different phenotypes of proliferative and invasive potentials [ 30 , 31 ].…”