No 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.

The epigenetic changes associated with melanoma progression to advanced and metastatic stages are still poorly understood. To shed light on the CpG methylation dynamics during melanoma development, we analyzed the methylome profiles of a four-stage cell line model of melanoma progression: non-tumorigenic melanocytes (melan-a), premalignant melanocytes (4C), non-metastatic melanoma cells (4C11−), and metastatic melanoma cells (4C11+). We identified 540 hypo- and 37 hypermethylated gene promoters that together characterized a malignancy signature, and 646 hypo- and 520 hypermethylated promoters that distinguished a metastasis signature. Differentially methylated genes from these signatures were correlated with overall survival using TCGA-SKCM methylation data. Moreover, multivariate Cox analyses with LASSO regularization identified panels of 33 and 31 CpGs, respectively, from the malignancy and metastasis signatures that predicted poor survival. We found a concordant relationship between DNA methylation and transcriptional levels for genes from the malignancy (Pyroxd2 and Ptgfrn) and metastasis (Arnt2, Igfbp4 and Ptprf) signatures, which were both also correlated with melanoma prognosis. Altogether, this study reveals novel CpGs methylation markers associated with malignancy and metastasis that collectively could improve the survival prediction of melanoma patients.

Recently, the non-coding RNAs (ncRNAs) have been classified in different categories, and its importance in regulating different cellular processes has been unravelled. The long non-coding RNAs (lncRNAs) can interact with DNA, other RNAs and proteins, including epigenetic modifiers. Some lncRNAs are related to genomic imprinting and are associated with chromatin-modifying complexes that can regulate gene transcription. It is well established that cancer cells have different epigenetic alterations and some of these modifications are associated with lncRNAs. Studies of cancer-associated lncRNAs have defined its function in the process of tumorigenesis, its impact on cell proliferation, cellular signalling, angiogenesis and metastasis. Therefore, having a better knowledge of their role might contribute to a better understanding of the diseases. In this chapter, we will discuss about lncRNA classification and functions, epigenetic marks and how they can guide transcription. Nevertheless, we will discuss how these mechanisms can interact and guide gene expression, as well as recently findings of dysregulation of lncRNAs in cancer.

Metastatic melanomas are extremely aggressive tumors, accounting for 70% of skin cancer deaths, which incidence is increasing globally. Despite the advances in their treatment, metastatic melanomas are still associated with a poor prognosis, with a median survival of 6 to 12 months. The study of the molecular alterations affecting key pathways during melanoma progression may contribute to a better understanding of the biology underlying the aggressiveness of the disease and lead to the identification of biomarkers for prognosis and novel targets for therapy. In addition to mutations, epigenetic alterations are important contributors to malignant transformation and tumor progression. Aberrant DNA methylation is an epigenetic hallmark of cancer, known to play important roles in melanoma development. Although different genes were described as presenting methylation alterations in their promoters in melanomas, the dynamics of these alterations along melanocyte malignant transformation and melanoma progression is still unclear. Our laboratory developed a linear model of melanoma progression in which different pre-malignant and melanoma cell lines were established after subjecting murine melanocytes to a sustained stressful condition (adhesion impediment). In this study, we used the spontaneously immortalized melanocyte lineage (melan-a); pre-malignant melanocytes (4C), obtained after subjecting melan-a cells to 4 cycles of adhesion impediment; a non-metastatic tumorigenic cell line (4C11-), obtained after a limiting dilution of spheroids formed by 4C adhesion impediment; and a metastatic cell line (4C11+), resulting from the spontaneously loss of p53 by 4C11- cells. The aim of this work was to identify genes that have their expression altered by promoter and gene body methylation that are related to the malignant transformation of melanocytes and metastasis. Through the integrative analysis of methylome (ERRBS) with transcriptome (RNAseq) data, it was possible to identify the dynamic changes in DNA methylation in promoters and gene bodies, and their relation with gene expression in both early and late stages of melanoma progression. Among those genes are Foxd1, Lrrk2, Adcy1 and Adcy3, which had their expression validated by qPCR, and their epigenetic regulation validated by the treatment of the cells with epigenetic drugs (5-aza-2'-deoxycytidine and Trichostatin A). The gene expression and promoter methylation profiles found in the murine model of melanoma progression were correlated with clinical data from melanoma patients available in cancers cohorts, revealing potential novel prognostic markers. Supported by FAPESP, CNPQ and CAPES Citation Format: Débora Papaiz, Flávia E. Rius, Diogo Pessoa, Eduardo M. Reis, Christopher E. Mason, Gangning Liang, Miriam G. Jasiulionis. Deregulated genes by abnormal promoter methylation in early and late stages in a linear model of melanoma progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 140.