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

In addition to mutations, epigenetic alterations are important contributors to malignant transformation and tumor progression. The aim of this work was to identify epigenetic events in which promoter or gene body DNA methylation induces gene expression changes that drive melanocyte malignant transformation and metastasis. We previously developed a linear mouse model of melanoma progression consisting of spontaneously immortalized melanocytes, premalignant melanocytes, a nonmetastatic tumorigenic, and a metastatic cell line. Here, through the integrative analysis of methylome and transcriptome data, we identified the relationship between promoter and/or gene body DNA methylation alterations and gene expression in early, intermediate, and late stages of melanoma progression. We identified adenylate cyclase type 3 (Adcy3) and inositol polyphosphate 4-phosphatase type II (Inpp4b), which affect tumor growth and metastatic potential, respectively. Importantly, the gene expression and DNA methylation profiles found in this murine model of melanoma progression were correlated with available clinical data from large population-based primary melanoma cohorts, revealing potential prognostic markers.

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

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.

Identificação e anotação funcional de RNAs longos não codificadores associados à subtipos moleculares em tumores pancreáticos / Identification of long non coding RNAs associated to molecular subtypes in pancreatic tumors

O Adenocarcinoma Pancreático Ductal (Pancreatic Ductal Adenocarcinoma - PDAC) é a sé- tima causa de mortes por câncer no mundo, com uma taxa de sobrevida de apenas 6%. Embora alguns genes estejam recorrentemente mutados em grande parte dos tumores e sejam críticos para a oncogênese, a heterogeneidade das alterações moleculares tanto no tumor quanto em componentes do microambiente tumoral se reflete em diferentes características fenotípicas com comportamentos clínicos distintos e que têm sido associados a diferentes subtipos moleculares através da análise computacional de dados de alterações somáticas e transcricionais no PDAC. RNAs não codificadores longos (lncRNAs) têm sido reconhecidos como importantes reguladores da expressão gênica em doenças proliferativas mas sua associação com subtipos em PDAC e sua contribuição para o estabelecimento de diferentes fenótipos moleculares e clínicos da doença não foi explorada até o momento. Neste trabalho, foi implementada uma abordagem computacional com o objetivo de identificar e anotar funcionalmente lncRNAs associados a subtipos moleculares de PDAC. Inicialmente, a classificação não supervisionada por Fatoração Matricial Não Negativa (Non-Negative Matrix Factorization - NMF) de dados de expressão gê- nica global de amostras clínicas disponíveis publicamente (The Cancer Genome Atlas - TCGA) resultou na identificação de quatro subgrupos distintos de PDAC, que recapitulam os fenótipos Exócrino/Endócrino, Imunogênico, Escamoso e Progenitor descritos na literatura. Uma análise de expressão diferencial permitiu a identificação de assinaturas de expressão gênica características que incluem lncRNAs associados a cada subgrupo. Através da construção de redes de coexpressão de mRNAs e lncRNAs e a identificação de módulos da rede significativamente enriquecidos em genes que participam em vias moleculares conhecidas foi possível inferir possíveis funções biológicas à lncRNAs associados aos diferentes subtipos moleculares, tais como funções exócrinas/neuroendócrinas, imunogênicas, reparo de DNA/progressão do ciclo celular e progenitoras/morfogênicas. Entre ele, o subgrupo 3, enriquecido para fenótipo Escamoso e associado a hiper-expressão do supressor tumoral TP63, possui dois lncRNAS hiper-expressos neste subgrupo em relação aos outros subgrupos, sendo que o lncRNA antissenso FAM83A-AS1 tem a predição de interagir com as proteínas FGFR2, AXIN1, PTEN, BRAF, SMAD4, TGFBR2, TP53 e CDKN2A, que exercem funções importantes na transdução de sinal e supressão tumoral no câncer incluindo o de pâncreas. Entre os lncRNAs hipo-regulados no subgrupo 3 em relação ao outros subgrupos, alguns, como FLJ42875, LOC338651, C20orf56 e LOC38838 tem predição de interação com alta afinidade à proteína BRCA2, que está envolvida no reparo de DNA e participa de processos de resistência à quimioterápicos. As informações trazidas por este estudo permitem gerar hipóteses sobre a contribuição de lncRNAs para a definição de subtipos moleculares de PDAC e priorizar candidatos e experimentos para estudos funcionais de modo a contribuir para um melhor entendimento sobre os mecanismos de ação de lncRNAs na tumorigênese e agressividade do câncer de pâncreas

Pancreatic Ductal Adenocarcinoma (PDAC) is the seventh cause of worldwide cancer related deaths, with an overall survival rate of only 6%. Some genes might be recurrently mutated in a large number of tumors, and be critical for oncogenesis, molecular alteration heterogeneity both in the tumor as all as in the tumor microenvironment is reflected in diverse phenotypic features with distinct clinical outcomes, and this distinction in multiple molecular subtypes has been drawn through transcriptional and somatic alteration computational analysis within PDAC. Long Non Coding RNAs (lncRNAs) have been recognized as important gene expression regulators in proliferative diseases, but its association to molecular subtypes in PDAC and its contribution in the establishment of diverse molecular and clinical phenotypes hasnt been explored at length until the present. This work focused on the implementation of a computational approach with the objective of lncRNA identification and functional annotation associated to distinct molecular subtypes in PDAC. Initially, Non-negative Matrix Factorization (NMF), an unsupervised classification method, applied to global gene expression data from publicly available clinical samples (The Cancer Genome Atlas - TCGA) resulted in the identification of four distinct PDAC molecular subgroups reminiscent of Exocrine/Endocrine, Immunogenic, Squamous and Progenitor phenotypes. Differential expression analysis allowed a characteristic gene expression signature identification, including distinct molecular subtype associated lncRNAs. mRNA and lncRNA containing gene co-expression modules significantly enriched annotated pathways containing the molecular subtype associated lncRNAs allowed to designate possible molecular functions of the distinct molecular subtype associated lncRNAs, such as exocrine/neuroendocrine, immunogenic, DNA repair/cell cycle progression and progenitor/morphogenic functions. Subgroup 3, enriched with a Squamous phenotype and associated to TP63 over-expression contains two lncRNAs over-expressed compared to other subgroups; furthermore, the antisense lncRNA FAM83A-AS1 yielded a predicted lncRNA-protein interaction to FGFR2, AXIN1, PTEN, BRAF, SMAD4, TGFBR2, TP53 and CDKN2A, proteins that play important signal transduction and tumor suppressor roles in several cancer types, including pancreas. Among under-expressed lncRNAs in subgroup 3 compared to the other subgroups, some, such as FLJ42875, LOC338651, C20orf56 and LOC38838 yilded a high protein interaction prediction score with BRCA2, a protein involved in DNA repair and processes resuling in chemotherapy resistance. The information brought by this study allowed to generate hypothesis on lncRNA contribution to define PDAC molecular subtypes, helping prioritize candidates and experiments for functional studies, thus contributing to a better understanding on lncRNA mechanisms related to tumor progression and aggressiveness in pancreatic cancer