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  • Periodic Report Summary 1 - EPIMIRMEL (Crosstalk between microRNAs and epigenetics in melanocyte differentiation: A new approach to unravel the complexity of melanoma progression and metastasis.)

Periodic Report Summary 1 - EPIMIRMEL (Crosstalk between microRNAs and epigenetics in melanocyte differentiation: A new approach to unravel the complexity of melanoma progression and metastasis.)

Melanoma is the most aggressive form of skin cancer and one of the most invasive tumor types. Melanoma incidence keeps increasing worldwide and is expected to double every 20 years in the absence of lifestyle changes. Despite remarkable advances that have led to recent FDA approval of new compounds, outcomes for metastatic melanoma patients remain poor. Unfortunately, relapse with treatment-refractory disease (MAPK inhibitors) and adverse side effects and eventual emergence of resistance (immunotherapy) are common occurrences (Lo et al., 2014; Macdonald et al., 2015; Dranoff, 2013). The lack of treatments with durable responses may be due, at least in part, to an incomplete understanding of the molecular mechanisms that regulate tumor initiation and/or progression to metastasis. Therefore, there is an urgent need to find new strategies to expand our knowledge of melanomagenesis.
Strong evidence supports the notion that melanoma cells behave like stem cells (reviewed in Zabierowski et al., 2008): they frequently exhibit characteristics of neural crest or embryonic stem cells (Hendrix et al., 2003), retain their morphologic and biological plasticity despite repeated cloning (Kath et al., 1991), often express developmental genes, and they can differentiate into a wide range of cell lineages, including neural, mesenchymal, and endothelial cells. Complementing these findings, gene expression profiling of melanoma has shown that tumor progression and increased invasive behavior correlate with loss of differentiation features (Ryu et al., 2007) and expression of neural crest markers (Uong et al., 2010; White et al., 2011).
We reasoned that studying a model of differentiation of melanocytes from neural crest stem cells could be useful to identify the mechanisms that modulate melanoma stem-like properties, probably among the most significant contributing features to the tumor aggressiveness. The plasticity and reversibility of the epigenetic mechanisms make them ideal orchestrators of these dynamic processes. Then, we hypothesize that epigenetically controlled miRNAs could be involved in normal differentiation and in tumorigenesis when altered. Thus, aberrant epigenetic modifications could be responsible for reactivation (or retention) of stem-like properties in melanoma cells.
Our initial strategy to study the epigenetic changes occurring during differentiation was to develop a melanocyte differentiation model. We tried different protocols, but they failed to render melanocytes. As alternative strategy, we use stem cell lines, neural crest cell line and normal melanocytes to generate the DNA methylation profiles to perform the analyses proposed in our project. As planned, we identified and validated differentially methylated regions comprising not only miRNAs but also protein-coding genes. We integrated those results with DNA methylation profiles from human melanoma patients to identified the best candidates with a potential role during melanoma progression and metastasis. Finally, we perform functional validation both in vitro and in vivo, using melanoma cell lines and mouse metastasis models, respectively, as planned in our project.
Comparing the DNA methylation profiles from stem cells, neural crest (NC) and melanocytes, we identified miR-24-1, miR-944 and miR-125b1 as miRNAs epigenetically regulated during differentiation. miR-24-1 and miR-944 are hypermethylated in stem and NC cell lines, but DNA methylation is lost in melanocytes. An opposite pattern was found for miR-125b1, hypomethylated in stem cells and hypermethylated in some melanocytes. Next, we analyzed DNA methylation profiles from 71 primary and 267 metastasis melanoma samples available in The Cancer Genome Atlas (TCGA) aimed to examined the possible role in melanoma metastasis. Intriguingly, for those three miRNAs, metastatic melanoma samples recapitulate the methylation profile that we found in stem cell lines.
Interestingly, miR-24-1 is located in a cluster that also includes miR-23b and miR-27b, and expression of miR-27b was also significantly decreased in metastatic samples. miR-24-1 and other members of the cluster have been recently described as tumor suppressors in bladder (Inoguchi, S. et al., 2014) and prostate cancer (Goto et al., 2014: Ishteiwy et al., 2012). However, to our knowledge, epigenetic regulation and functional role of miR-24-1 in melanoma progression have not been previously reported. Regarding miR-944, recent studies have shown that this miRNA is upregulated in cervical cancer and promotes cell proliferation, migration and invasion in human cervical cancer cells (Xie et al., 2015; Witten et al., 2010), but again epigenetic control and role in melanoma remain unknown. In contrast, a role of miR-125b in melanoma (Glud et al., 2010; Kappelmann et al., 2013) and CpG hypermethylation-associated gene silencing in melanoma cell lines (Kim et al., 2014) have been previously described. However, the link between the role of those miRNAs in stemness-differentiation and melanoma progression has not been previously reported for any of them. Additional analyses are planned for the next period.
Moreover, although our project was initially focused in miRNAs, one of the main hits identified in our analysis of differentially methylated CpGs in stem cells vs. melanocytes and primary vs. metastatic melanoma, was a nuclear receptor. We centered our effort in studying this candidate because previous studies have shown its essential role in development and differentiation during embryogenesis. More relevant considering our aims, it has been identified as an important transcriptional regulator of human neural crest. Thus, according to our results, it could be a developmental/stemness gene aberrantly reactivated during melanomagenesis. Epigenetic reactivation of this gene during metastasis could confer neural crest stem-like features that drive invasiveness, aggressiveness and metastatic potential of melanoma cells.
Unsupervised clustering analysis revealed a significant CpG hypermethylation in melanocytes in contrast to complete lack of methylation in stem and NC cell lines, associated to loss of expression in melanocytes. A significant CpG hypomethylation was also detected in metastasis vs. primary melanoma samples in the TCGA cohort (n=406) and validated in an independent cohort (n=76). Integration of CpG methylation and mRNA expression TCGA revealed a significant inverse correlation suggesting that DNA methylation regulates the expression in these tumors. Epigenetic regulation was then confirmed in MeWo methylated melanoma cell line, by restored expression upon treatment with DNA demethylating agent. Collectively, these data demonstrated that this gene is epigenetically regulated during melanocyte differentiation and melanoma progression. Interestingly, the reciprocal patterns of methylation and expression in NC stem cells/melanocytes versus primary/metastatic melanoma, support the concept of acquisition or reactivation of neural crest stem cell features by metastatic melanoma cells and suggest the possibility that its hypomethylation and expression contributes to such ‘reprogramming’ effect. Thus, we hypothesized that epigenetic activation could contribute to melanoma progression and metastasis. To test this hypothesis, we performed loss-of-function (LOF) and gain-of-function (GOF) in vitro and in vivo experiments. In melanoma cells carrying hypomethylation and expression, shRNA-mediated silencing had no effect on proliferation, but significantly suppressed the ability to form colonies in soft agar (a measure of anchorage-independent growth) and to form spheres from single cell suspensions in low attachment plates (a property of stem cells). Conversely, ectopic overexpression in melanoma cells harboring locus hypermethylation (MeWo) resulted in enhanced anchorage-independent growth and sphere formation.
In in vivo assays in mouse, gene silencing had no effect in tumor growth upon subcutaneous injection. However, we observed a significant reduction in the metastatic spread of sh-knockdown melanoma cells instilled by ultrasound-guided intra-cardiac injection. Conversely, the overexpression promoted melanoma metastasis in vivo. Overall, our results indicate a critical role of this factor in melanoma progression and metastasis, strongly supported by clinic-pathological data in melanoma patients and in vivo mouse models. We are now working in dissecting the underlying mechanism of its pro-metastatic behavior. With this aim, we performed and integrated RNAseq and ChIPseq experiments in our LOF and GOF cellular models. Overall, these studies will clarify how aberrant expression of this gene orchestrates transcriptional programs that promotes metastasis.
All together, our data highlight the relevance of epigenetic regulation in a potential ‘reprogramming’ of metastatic melanoma cells towards a stem-like epigenetic pattern. Characterization of epigenetically-mediated alterations in melanoma promises to be a good strategy to identify prognosis biomarkers, but also may provide new targets for therapeutic intervention. Nuclear receptors are among the top four families of drug targets and account for approximately 13% of all Food and Drug Administration (FDA)-approved drugs (Overington et el., 2006). The most well-known examples are hormone receptors, such as androgen and estrogen receptors that have been studied intensively, and represent established targets for a number of approved drugs in prostate and breast cancers (Dalal et al., 2014; Robinson-Rechavi et al., 2001). The relevance and impact of our finding is potentiated by the presence of distinctive ligand binding sites. Then, the nuclear receptor function could be potentially modulated by small molecules, which compete with endogenous ligands to act as either antagonists or agonists. Thus, this project not only will increase our scientific knowledge, but also could have significant clinical and socio-economic contribution in melanoma management.

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