NTTS 2017

Background: Metastatic melanoma cells exhibit an extraordinary phenotypic plasticity, not only in adapting to unfamiliar microenvironments, but also in surviving aggressive treatments and immune response. A major source of phenotypic variability is the alternative splicing of the messenger RNA (mRNA). This process is catalyzed by one of the most complex cellular machineries, the spliceosome, which is composed of ribonucleoproteins and polypeptides denominated spliceosome factors (SF). Changes in the expression level of SFs lead to a splicing reprogramming that has been linked to different types of cancer progression. We have recently demonstrated that the interplay between epigenomic and transcriptomic alterations of SFs generates melanoma cells with an enhanced predisposition for brain metastasis. Material and Methods: Using the Illumina Human 450K BeadChip technology, we generated genome-wide DNA methylation maps of 133 melanoma specimens, including melanocytes and nevi (n=9), primary melanomas (PRM; n=15), lymph node metastases (LNM; n=27), extracranial metastases (n=37), and brain metastases (MBM; n=45). This analysis included 15 patients with PRM or LNM paired with multiple (>2) distant organ metastases (DOM). To ensure highly representative DNA methylation patterns, all the specimens were microdissected. Additionally, DNA methylation and gene expression data for DOM (n=67) generated by the Cancer Genome Atlas (TCGA) were integrated to validate our findings. Results: This epigenomic profiling allowed us to evaluate changes in the DNA methylation levels of 6,370 genomic regions affecting 356 genes encoding splicing and mRNA editing factors. Overall, 58 genomic regions affecting 12 genes were differentially methylated during melanoma progression to metastasis (Absolute [PRM–DOM]>0.3; Wilcoxon test, FDR-p<0.01). In agreement with our previous studies, this analysis identified epigenetic alterations on ESRP1, ESRP2 and PTBP1 genes which, as we recently described, are involved in melanoma metastasis in an independent patient cohort. These regions were primarily located in gene body CpG island regions suggesting a positive influence on gene expression rates. Interestingly, liver metastases presented hypermethylation of 16 genomic regions overlapping the ADARB2 gene which regulates the splicing of pre-micro-RNAs. Conversely, bowel metastases showed hypomethylation of the ADARB2 gene. ESRP1 and ESRP2 genes were hypomethylated in spleen and liver metastases but hypermethylated in lung and brain metastases. Analysis of TCGA RNA-sequencing data showed changes in the splicing program of several targets of these SFs. Conclusions: Aberrant splicing has a significant impact on melanoma progression to metastasis. The characterization of epigenetic regulation of SF genes influenced by metastasis microenvironment opens up a new avenue for understanding the affinity of melanoma cells for specific metastatic niches.