Cutaneous melanoma is associated with significant morbidity and mortality representing the most significant cutaneous malignancy. As it is known that early diagnosis and treatment are the most efficient approaches to cure cutaneous melanoma, an improved understanding of the molecular pathogenesis of melanoma and exploration of more reliable molecular biomarkers are particularly essential. Two different types of molecular biomarker for melanoma have been investigated in this thesis. microRNAs (miRNAs) are single-stranded RNA molecules of 20-23 nucleotides in length that are found in both animal and plant cells. miRNAs are involved in the RNA interference (RNAi) machinery to regulate gene expression posttranscriptionally. miRNAs have important roles in cancer: by controlling the expression level of their target genes they can affect cell signalling pathways and have been shown to have both prognostic and therapeutic potential. Importantly for melanoma research, reproducible miRNA expression profiles from formalin-fixed paraffin-embedded (FFPE) tissues can be obtained that are comparable to those from fresh-frozen samples. The aims of the miRNA project were: first, to identify a melanoma-specific miRNA expression profile; secondly, to investigate roles of some of the melanoma-specific miRNAs identified in melanomagenesis. Using miRNA microarray on FFPE samples, I obtained a melanoma-specific miRNA expression profile. 9 of these differentially expressed miRNAs between benign naevi and melanomas (7 downregulated, 2 upregulated in malignancies) were verified by qRT-PCR and the functions of four of these miRNAs were studied. Ectopic overexpression of miR- 200c and miR-205 in A375 melanoma cells inhibited colony forming ability in methylcellulose, an in vitro surrogate assay for tumourigenicity. Moreover, elevation of miR-200c resulted in increased expression levels of E-cadherin through negative regulation of the zinc finger E-box-binding homeobox 2 (ZEB2) gene. Ectopic overexpression of miR-211 in A375 melanoma cells repressed both colony formation in methylcellulose and migratory ability in matrigel, an in vitro surrogate assay for invasiveness. These findings indicate that miR-200c, miR-205 and miR-211 act as tumour suppressors in melanomagenesis. The second biomarker investigated, mutated BRAF, has been seen in 50-70% of spontaneous cutaneous melanoma. The commonest mutation in melanoma is a glutamic acid for valine substitution at position 600 (V600E). Oncogenic BRAF controls many aspects of melanoma cell biology. The aim of this part of the work was: firstly, to study BRAF V600E mutation status in our melanoma tissue microarray (TMA) panel; secondly, to correlate this mutation to various clinicopathological features and evaluate its prognostic value through statistical analyses. BRAF V600E mutations were seen in 20% of the primary and 69% of the metastatic melanomas, respectively. More BRAF V600E mutations were seen in males relative to females. The mutation was also related to cell pigmentation, but not to age, ulceration or solar elastosis. Melanoma patients with the BRAF V600E mutation relapse earlier than patients without this mutation. However, no significant association between the BRAF V600E mutation and overall survival and melanoma specific survival was found.

O carbaril (metilcarbamato de naftila), um inseticida de amplo espectro, foi recentemente associado ao desenvolvimento de melanoma cutâneo em estudo epidemiológico de coorte com trabalhadores agrícolas norte-americanos, expostos também à radiação solar, o principal fator etiológico para o desenvolvimento de tumores cutâneos. Apesar de abrangente e bem planejado, aquele estudo epidemiológico não é suficiente para caracterizar a contribuição direta do inseticida e da radiação solar na melanomagênese. Diversos estudos têm explorado o efeito sinérgico de determinadas substâncias químicas à radiação UV, potencializando seus efeitos deletérios sobre a pele, e possivelmente contribuindo para o desenvolvimento de tumores. A hipótese deste trabalho é de que a exposição ao carbaril associada à radiação solar possa estimular a transformação de melanócitos. Esse estudo visou caracterizar melanócitos humanos após exposição individual ou combinada ao carbaril (100uM) e à radiação solar (375 mJ/ cm2). Em ensaio de microarray, o carbaril, mas não a radiação solar, induziu uma importante resposta a estresse oxidativo, evidenciada pelo aumento da expressão de genes antioxidantes, como o Hemeoxigenase-1 (HMOX1), e pela diminuição da expressão do gene MiTF, regulador da atividade melanocítica; os resultados foram confirmados por qRT-PCR. Além disso, tanto o carbaril quanto a radiação solar induziram respostas que sugerem dano ao DNA e alteração de ciclo celular. A expressão dos genes nestas categorias, como p21 e BRCA1/2, foi notavelmente mais intensa no grupo de tratamento combinado e de fato, ensaios por citometria de fluxo demonstraram parada de ciclo celular na fase S, redução do número de células em apoptose e indução mais rápida de lesões do tipo CPD neste grupo experimental. Nossos dados sugerem que o carbaril é genotóxico para melanócitos humanos, especialmente quando associado à radiação solar / Carbaryl (1-naphthyl-methylcarbamate), a broad spectrum insecticide, has recently been associated with the development of cutaneous melanoma in an epidemiological cohort study with U.S. farm workers also exposed to ultraviolet radiation, which is known to be the main etiologic factor for skin carcinogenesis. Although comprehensive and well designed, the epidemiological study is not sufficient to characterize the direct contribution of the insecticide and solar radiation in melanomagenesis. Several studies have explored the synergistic effect of certain chemicals with UV radiation, increasing its deleterious effects on the skin, possibly contributing to tumor development. We hypothesized that Carbaryl exposure associated with UV solar radiation may induce melanocyte transformation. This study aims to characterize human melanocytes after individual or combined exposure to Carbaryl (100uM) and solar radiation (375 mJ/ cm2). In a microarray analysis, Carbaryl, but not solar radiation, induced an important oxidative stress response, evidenced by the upregulation of antioxidant genes, such as Hemeoxygenase-1 (HMOX1), and downregulation of MiTF, the main regulator of melanocytic activity; results were confirmed by qRT-PCR. Moreover, both Carbaryl and solar UV induced a gene response that suggests DNA damage and cell cycle alteration. The expression of genes in these categories, such as p21 and BRCA1/2, was notably more intense in the combined treatment group in an additive manner and in fact, flow cytometry assays demonstrated cell cycle arrest in S phase, reduced apoptosis induction and faster induction of CPD lesions in this experimental group. Our data suggests that carbaryl is genotoxic to human melanocytes, especially when associated with solar radiation

O carbaril (metilcarbamato de naftila), um inseticida de amplo espectro, foi recentemente associado ao desenvolvimento de melanoma cutâneo em estudo epidemiológico de coorte com trabalhadores agrícolas norte-americanos, expostos também à radiação solar, o principal fator etiológico para o desenvolvimento de tumores cutâneos. Apesar de abrangente e bem planejado, aquele estudo epidemiológico não é suficiente para caracterizar a contribuição direta do inseticida e da radiação solar na melanomagênese. Diversos estudos têm explorado o efeito sinérgico de determinadas substâncias químicas à radiação UV, potencializando seus efeitos deletérios sobre a pele, e possivelmente contribuindo para o desenvolvimento de tumores. A hipótese deste trabalho é de que a exposição ao carbaril associada à radiação solar possa estimular a transformação de melanócitos. Esse estudo visou caracterizar melanócitos humanos após exposição individual ou combinada ao carbaril (100uM) e à radiação solar (375 mJ/ cm2). Em ensaio de microarray, o carbaril, mas não a radiação solar, induziu uma importante resposta a estresse oxidativo, evidenciada pelo aumento da expressão de genes antioxidantes, como o Hemeoxigenase-1 (HMOX1), e pela diminuição da expressão do gene MiTF, regulador da atividade melanocítica; os resultados foram confirmados por qRT-PCR. Além disso, tanto o carbaril quanto a radiação solar induziram respostas que sugerem dano ao DNA e alteração de ciclo celular. A expressão dos genes nestas categorias, como p21 e BRCA1/2, foi notavelmente mais intensa no grupo de tratamento combinado e de fato, ensaios por citometria de fluxo demonstraram parada de ciclo celular na fase S, redução do número de células em apoptose e indução mais rápida de lesões do tipo CPD neste grupo experimental. Nossos dados sugerem que o carbaril é genotóxico para melanócitos humanos, especialmente quando associado à radiação solar / Carbaryl (1-naphthyl-methylcarbamate), a broad spectrum insecticide, has recently been associated with the development of cutaneous melanoma in an epidemiological cohort study with U.S. farm workers also exposed to ultraviolet radiation, which is known to be the main etiologic factor for skin carcinogenesis. Although comprehensive and well designed, the epidemiological study is not sufficient to characterize the direct contribution of the insecticide and solar radiation in melanomagenesis. Several studies have explored the synergistic effect of certain chemicals with UV radiation, increasing its deleterious effects on the skin, possibly contributing to tumor development. We hypothesized that Carbaryl exposure associated with UV solar radiation may induce melanocyte transformation. This study aims to characterize human melanocytes after individual or combined exposure to Carbaryl (100uM) and solar radiation (375 mJ/ cm2). In a microarray analysis, Carbaryl, but not solar radiation, induced an important oxidative stress response, evidenced by the upregulation of antioxidant genes, such as Hemeoxygenase-1 (HMOX1), and downregulation of MiTF, the main regulator of melanocytic activity; results were confirmed by qRT-PCR. Moreover, both Carbaryl and solar UV induced a gene response that suggests DNA damage and cell cycle alteration. The expression of genes in these categories, such as p21 and BRCA1/2, was notably more intense in the combined treatment group in an additive manner and in fact, flow cytometry assays demonstrated cell cycle arrest in S phase, reduced apoptosis induction and faster induction of CPD lesions in this experimental group. Our data suggests that carbaryl is genotoxic to human melanocytes, especially when associated with solar radiation