O receptor aril de hidrocarbonetos (AhR) em melanomas: associação com mutações BRAF, com a progressão da doença e identificação de ligantes / The aryl hydrocarbon receptor (AhR) in melanomas: association with BRAF mutations, with disease progression and ligand identification

O melanoma é um tipo de câncer de pele geneticamente diverso, que surge diante das transformações em melanócitos. A mutação BRAFV600E está presente em mais de 90% de todas as mutações em BRAF, sendo assim ocorre em cerca de 50% dos casos registrados. As mutações em NRAS, ocupam o segundo lugar entre as mutações mais prevalentes, cerca de 20% dos casos. Informações sobre as assinaturas genéticas, permitiram o desenvolvimento de terapia alvo dirigida. O Vemurafenib, inibidor da quinase BRAFV600E, apresentou inicialmente resultados bastante satisfatórios, contudo existe registro de casos de recidiva e resistência. O receptor aril de hidrocarbonetos é expresso em vários componentes da pele, e assim está relacionado a homeostase e fisiopatologia da pele. Diante disso, a avaliação da expressão do receptor em um painel de linhagens mutadas para NRAS e BRAF, e BRAF resistentes, mostrou-se maior do que a encontrada em melanócitos. Também encontramos maior expressão de mRNA de AhR em linhagens de melanoma derivadas de sítio primário e metastático, mutadas para BRAFV600E, quando comparadas ao melanócito. Agregado a isto, a análise in silico no TCGA (The Cancer Genome Atlas) mostrou que há 18% de alteração genética em AhR, sendo em maior parte a alta regulação de mRNA. Também, a análise do banco público GSE12391, mostrou aumento de mRNA de AhR na fase de crescimento vertical do melanoma. Assim, concluímos que há maior expressão de mRNA e sua importância nas fases de desenvolvimento do melanoma, tanto nos processos iniciais quanto em processos de migração, invasão e metástase. Ainda, encontramos maior mRNA do receptor em linhagens resistentes ao Vemurafenib. Este resultado sustenta a hipótese de que AhR pode ser considerado um marcador de resistência em melanomas. O AhR, inicialmente no citoplasma, quando ativado pode atuar como fator de transcrição regulando vários genes que apresentam sequências definidas, participando de respostas carcinogênicas. Compostos halogenados e moléculas endógenas derivadas das vias de metabolização do triptofano são agonistas do receptor. Anteriormente, nosso grupo mostrou que linhagens de melanoma incubadas com triptamina e DMT exibiram menor clonogenicidade. Diante de uma literatura escassa sobre o papel do DMT no melanoma e com base nestes resultados, nosso objetivo foi avaliar o papel de AhR nesta interface DMT-melanoma. Para isto, nosso objetivo foi construir linhagem editada geneticamente para AhR através da ferramenta CRISPR-Cas9. Vários foram os esforços, sem sucesso, utilizados nas tentativas de comprovar a manutenção de células editadas na cultura. Atrelamos a este resultado a possibilidade de haver duas subpopulações editadas geneticamente pós CRISPR-Cas9, onde uma destas manteve o padrão de crescimento semelhante às células wild type. Devido a este crescimento diferencial, não obtivemos congruências nos ensaios e postulamos a perda do possível nocaute. A partir disso, realizamos ensaios de interactoma para avaliar a interação de DMT-AhR. Nosso resultado sugere a interação de DMT ao receptor sigma 1, e não ao receptor aril de hidrocarbonetos. Desta forma, o interactoma sustenta a hipótese de que DMT não é um ligante de AhR. Para certificar este resultado análises de docking associados a ensaios biológicos, avaliando o papel do receptor, devem ser realizados para averiguar a afinidade e seletividade de DMT como ligante do receptor na linhagem de melanoma

Melanoma is a genetically diverse type of skin cancer, which arises from changes in melanocytes. The BRAFV600E mutation is present in more than 90% of all BRAF mutations, so it occurs in about 50% of registered cases. Mutations in NRAS occupy the second place among the most prevalent mutations, about 20% of cases. Information on genetic signatures allowed the development of targeted therapy. vemurafenib, kinase inhibitor BRAFV600E, initially presented very satisfactory results, however there is a record of cases of relapse and resistance. The aryl hydrocarbon receptor is expressed in several components of the skin and is thus related to homeostasis and skin pathophysiology. Therefore, the evaluation of receptor expression in a panel of strains mutated to NRAS and BRAF, and resistant BRAF, proved to be greater than that found in melanocytes. We also found main expression of AhR mRNA in melanoma strains derived from primary and metastatic site, mutated to BRAFV600E, when compared to melanocyte. Added to this, the in silico analysis in TCGA (The Cancer Genome Atlas) showed that there is 18% of genetic alteration in AhR, being mostly the high regulation of mRNA. Also, an analysis by the public bank GSE12391, showed an increase in AhR mRNA in the vertical growth phase of melanoma. Thus, it is concluded that there is greater expression of mRNA and its importance in the stages of development of melanoma, both in recent processes and in the processes of migration, invasion and metastasis. In addition, we found higher receptor mRNA in strains resistant to vemurafenib. This result supports the hypothesis that AhR can be considered a marker of resistance in melanomas. AhR, initially in the cytoplasm, when activated can act as a transcription factor regulating several genes that have defined sequences, participating in carcinogenic responses. Along with this, we show that along the tumor progression, there is an increase in AhR in the radial growth phase of melanoma. Halogenated compounds and endogenous molecules derived from the tryptophan metabolism pathways are receptor agonists. Previously, our group showed that melanoma strains incubated with tryptamine and DMT exhibited less clonogenicity. In view of a scarce literature on the role of DMT in melanoma and based on these results, our objective was to evaluate the role of AhR in this DMT-melanoma interface. For this, our goal was to build genetically edited strain for AhR using the CRISPR-Cas9 tool. Several efforts were unsuccessful in attempts to prove the maintenance of cells edited in the culture. We linked to this result the possibility of having two subpopulations genetically edited after CRISPR-Cas9, where one of them maintained the growth pattern like wild type cells. Due to this differential growth, we did not obtain congruence in the tests and postulated the loss of the possible knockout. From that, we performed interactome assays to evaluate the DMT-AhR interaction. Our result suggests the interaction of DMT with the sigma 1 receptor, and not the aryl hydrocarbon receptor. Thus, the interactome supports the hypothesis that DMT is not an AhR ligand. To certify this result, docking analyses associated with biological assays, evaluating the role of the receptor, should be performed to ascertain the affinity and selectivity of DMT as a ligand of the receptor in the melanoma lineage

Immunohistochemical analysis of BRAF V600E mutation in ameloblastomas.

OBJECTIVE: This study aimed to investigate the presence of BRAF V600E mutation in mandible ameloblastomas by correlating clinical and imaging data on the cases studied. METHODS: Eighty-four cases diagnosed as mandibular ameloblastoma were selected for analysis. The specimens were submitted to immunohistochemistry for detection of BRAF V600E mutated protein. Clinical-pathological data such as age, gender, tumour size, mandibular location, radiographic aspects, histological type and sub-type, and tumour status were collected. The clinical-pathological parameters were categorised and analysed according to BRAF V600E detection. RESULTS: Of the 84 patients, 78.6% (66 cases) demonstrated positivity for anti-BRAF V600E antibody, whereas 18 were negative (21.4%). The correlation between BRAF expression and variables showed statistical significances for mandibular location (P = 0.0353) and tumour size (P = 0.008), whereas no statistical significance was observed for gender, age, radiographic aspect, histological pattern, histological sub-type and tumour status. Multivariate logistic regression revealed a significant risk for BRAF positivity in tumours with posterior mandibular location (OR = 7.23, P = 0.0451) and size > 4 cm (OR = 7.29, P = 0.0150). CONCLUSION: BRAF V600E mutation is common in mandibular ameloblastomas, especially in cases of tumours larger than 4 cm and in the posterior region of the mandible. In addition, this mutation can occur regardless of histological type of the tumour, age, gender, radiographic aspect and tumour status. CLINICAL SIGNIFICANCE: The association between clinical-pathologic features and BRAF V600E mutation in ameloblastomas may provide directions for the treatment of this neoplasia. The use of BRAF inhibitors for targeted therapy could lead to an establishment of an alternative compared to the resective surgery.

Integrated data analysis reveals potential drivers and pathways disrupted by DNA methylation in papillary thyroid carcinomas.

BACKGROUND: Papillary thyroid carcinoma (PTC) is a common endocrine neoplasm with a recent increase in incidence in many countries. Although PTC has been explored by gene expression and DNA methylation studies, the regulatory mechanisms of the methylation on the gene expression was poorly clarified. In this study, DNA methylation profile (Illumina HumanMethylation 450K) of 41 PTC paired with non-neoplastic adjacent tissues (NT) was carried out to identify and contribute to the elucidation of the role of novel genic and intergenic regions beyond those described in the promoter and CpG islands (CGI). An integrative and cross-validation analysis were performed aiming to identify molecular drivers and pathways that are PTC-related. RESULTS: The comparisons between PTC and NT revealed 4995 methylated probes (88% hypomethylated in PTC) and 1446 differentially expressed transcripts cross-validated by the The Cancer Genome Atlas data. The majority of these probes was found in non-promoters regions, distant from CGI and enriched by enhancers. The integrative analysis between gene expression and DNA methylation revealed 185 and 38 genes (mainly in the promoter and body regions, respectively) with negative and positive correlation, respectively. Genes showing negative correlation underlined FGF and retinoic acid signaling as critical canonical pathways disrupted by DNA methylation in PTC. BRAF mutation was detected in 68% (28 of 41) of the tumors, which presented a higher level of demethylation (95% hypomethylated probes) compared with BRAF wild-type tumors. A similar integrative analysis uncovered 40 of 254 differentially expressed genes, which are potentially regulated by DNA methylation in BRAFV600E-positive tumors. The methylation and expression pattern of six selected genes (ERBB3, FGF1, FGFR2, GABRB2, HMGA2, and RDH5) were confirmed as altered by pyrosequencing and RT-qPCR. CONCLUSIONS: DNA methylation loss in non-promoter, poor CGI and enhancer-enriched regions was a significant event in PTC, especially in tumors harboring BRAFV600E. In addition to the promoter region, gene body and 3'UTR methylation have also the potential to influence the gene expression levels (both, repressing and inducing). The integrative analysis revealed genes potentially regulated by DNA methylation pointing out potential drivers and biomarkers related to PTC development.