Orientador: Mariângela Esther Alencar Marques / Resumo: O uso da imuno-histoquímica pode aumentar a acurácia na detecção de melanócitos neoplásicos na derme. Com o objetivo de pesquisar microinvasão, cento e nove casos previamente diagnosticados como lentigo maligno (LM) foram resgatados dos arquivos do Departamento de Patologia da Faculdade de Medicina de Botucatu, da Universidade Estadual Paulista (FMB/UNESP) no período de 01/01/2002 a 01/01/2014. As lâminas histológicas de todos os casos foram revisadas pelos autores para confirmação do diagnóstico e seleção do bloco mais representativo para realização de estudo imuno-histoquímico com Melan-A e MITF. Em 25 casos (22,9%) foi observada invasão focal da derme por melanócitos neoplásicos claramente imunomarcados pelo Melan-A. Nos locais onde se evidenciou invasão foi realizada a medida da espessura de Breslow que variou de 0,1 a 0,45 mm. A coloração imuno-histoquímica com MITF evidenciou positividade focal na derme, porém a identificação das células coradas não permitiu a mesma segurança da coloração pelo Melan-A. Todas as lâminas de imuno-histoquímica foram contracoradas pelo Giemsa para diferenciar positividade para o anticorpo testado e melanina. O presente estudo permitiu identificar microinvasão dérmica em 22,9% dos casos de lentigo maligno, mostrando a possibilidade de estadiamento e tratamento inadequado quando utilizada a técnica de rotina. Os achados são um alerta para os patologistas e clínicos, especialmente em lesões de grandes dimensões e associadas com infiltrado linf... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The use of immunohistochemistry can enhance the accuracy in detecting neoplastic melanocytes in the dermis. In order to search for microinvasion, one hundred and nine cases previously diagnosed as lentigo maligna (LM) were retrieved from the archives of the Department of Pathology, Faculty of Medicine of Botucatu, Universidade Estadual Paulista (FMB / UNESP) in the period of 01/01 / 2002 to 01/01/2014. The histological slides of all cases were reviewed by the authors to confirm the diagnosis and selection of the most representative block for performing immunohistochemical study with Melan-A, and MITF. In 25 cases (22.9%) was observed focal dermal invasion by neoplastic melanocytes clearly immunostained for Melan-A. In these cases the Breslow thickness ranged between 0.1 and 0.45mm. Immunohistochemical staining showed MITF focal positivity in the dermis, but did not allow the same certainty of Melan-A staining. In order to distinguish melanin in macrophage cytoplasm from brown-staining melanocytes, the slides were counterstained by Giemsa. This study identified dermal microinvasion in 22.9% cases of lentigo maligna, showing the possibility of inadequate staging and treatment when using the routine technique. The findings are a warning for pathologists and clinicians, especially in large lesions and associated with lymphocytic infiltrate that obscures their limits. / Doutor

The p300 transcriptional coactivator has been implicated in the development of a large number of malignancies; however, the precise mechanism of p300-associated tumorigenesis remains unclear. Here, we demonstrate the functional impact of p300 in human melanomas using both genetic and chemical approach. Depletion of p300 in human melanoma cells was associated with cellular growth arrest and senescence. Microarray analysis identified the Microphthalmia-associated transcription factor (MITF), a critical lineage-specific transcription factor in melanocytes and melanomas, as a major downstream target of p300 in human melanoma. Ectopic expression of MITF in p300-depleted melanoma cells allowed rescue of the p300-silencing phenotype, suggesting a critical regulatory axis involving p300 and MITF. Chromatin immunoprecipitation studies revealed direct regulation of MITF transcription through p300 acetylation of proximal regulatory domains. Critically, we identified that Forkhead Box M1 (FOXM1), a potent pro-proliferation transcription factor, is a target of the p300-MITF signaling axis. Further evaluation of p300 regulation of melanoma cell growth was performed using a highly selective p300/CBP HAT inhibitor, 228-1. Inhibition of p300/CBP histone acetyltransferase (HAT) activity was found to significantly inhibit proliferation of multiple melanoma lines in an MITF-dependent fashion. Together, these data support the role of p300 as a promising therapeutic target in human melanoma and suggest particular therapeutic efficacy of small molecule inhibitors of p300 HAT activity in tumors expressing high levels of MITF. / 2018-12-14T00:00:00Z

Skin Pigmentation represents the major natural protection against the deleterious effects of Ultraviolet light and involves a crosstalk between keratinocytes and melanocytes. Pigment synthesis or melanogenesis is initiated by the binding of \(\alpha\)-Melanocyte Stimulating Hormone \((\alpha-MSH)\) to the Melanocortin 1 Receptor (MC1R), expressed by the melanocytes. α-MSH is generated by cleavage of pro-opiomelanocortin hormone (POMC), produced by both melanocytes and keratinocytes. Activation of MC1R leads to an increase in cAMP levels, causing the expression of the transcription factor MITF. MITF regulates the expression of the enzymes involved in melanogenesis as well as genes important for the survival and proliferation of melanocytes. Pigment synthesis, which occurs in specialized organelles called melanosomes, involves the regulation of different proteins as well as fine homeostatic tuning such as melanosomal pH regulation. The POMC derivative, \(\alpha-MSH\), begins the pigmentation pathway by activating the MC1R signaling pathway, and OCA2 regulates the end of this pathway by controlling tyrosinase activity. The OCA2 gene has been shown to be important in the control of intra-melanosomal pH to allow optimal conditions for the activity of Tyrosinase, the limiting enzyme of pigment (melanin) production. OCA2 polymorphisms have been linked to oculocutaneous albinism type 2 and to blue eye color, demonstrating the importance of this gene in fine pH regulation on pigment production. Polymorphisms in POMC have also been linked to red-haired/fair-skin color in humans. Despite the effort to dissect the mechanisms involved in the control of pigmentation, the transcriptional regulation of POMC and OCA2 are still not fully understood. In this study, we investigate the relevance of the cAMP/CREB pathway in the transcriptional regulation of these two proteins. Our data shows that both POMC and OCA2 expression increases after stimulation of the cAMP/CREB pathway. We demonstrate that MITF transcriptionally regulates OCA2: the cAMP/CREB pathway therefore induces OCA2 in a MITF-dependent manner. On the other hand, our data reveals that POMC may be regulated by cAMP in a MITF-independent fashion but consistent with the hypothesis of a positive feedback loop within the MC1R signaling pathway.

There is an increasing evidence that alterations in chromatin remodeling play an important role in tumorigenesis. The SWI/SNF chromatin remodeling complexes contribute to the regulation of gene expression by altering the local chromatin structure. Depending on the context, they can act as either transcriptional activators or repressors. All SWI/SNF subcomplexes contain one of two ATPase subunits, Brm (Brahma) or Brg1 (Brahma related gene 1), which provide the energy for remodeling. Malignant melanoma is an aggressive cancer and is known for its notorious resistance to conventional anticancer therapies. MITF (microphthalmia-associated transcription factor) plays an essential role in melanoma biology and is placed on the central crossroad in the regulation of melanocyte development, differentation, maintenance of lineage identity, and survival of both normal and malignant melanocytes. Our results show that the active SWI/SNF complex is strictly required for the expression of MITF. This complex is also required for expression of some transcriptional MITF targets. The survival of melanoma cells is absolutely dependent on functional SWI/SNF complex and all subunits of this complex are expressed at high levels in melanoma cell lines. Primarily, Brg1-containing subcomplexes are more important for MITF...

Spleen tyrosine kinase (SYK) regulates mitogenic signaling, inflammatory responses and cell fate in a number of diverse cell types. KRAS is a proto-oncogene that controls cell growth and proliferation through several mitogenic pathways. In pancreatic cancer, KRAS is frequently mutated, resulting in constitutive activation in 90% of pancreatic cancer cell lines. We previously showed that SYK is highly expressed in a subset of KRAS-mutant pancreatic ductal adenocarcinoma (PDAC) cell lines. We demonstrated that SYK kinase inhibition with PRT062607 (SYKi) causes decreased cell proliferation of PDAC cell lines. Furthermore, combined SYKi and MEK inhibitor (MEKi) treatment promotes additive effects on suppression of PDAC cell proliferation and clonogenic growth. Mechanistically, SYK activates the mTORC1 kinase complex as shown by reduced phosphorylation of ribosomal S6 protein and its upstream kinase p70 S6 kinase (p70S6K) following SYKi treatment in PDAC cell lines. SYK-mediated mTORC1 activation occurs independently of MEK/ERK and PI3K/AKT effector signaling pathways. The mTORC1 complex suppresses lysosome biogenesis and macroautophagy (autophagy). Consequently, mTORC1 suppression via SYK inhibition or shRNA-mediated depletion causes accumulation of autolysosomes. These effects are mediated by the enhanced nuclear localization of MITF, a key transcriptional regulator of genes involved in lysosome biogenesis and autophagy pathway activation. In summary, SYK positively regulates mTORC1 activation in a subset of PDAC cell lines to suppress hyperactivation of autophagy. These findings open new avenues for further exploration of SYK as a critical regulator of the autophagy pathway in KRAS/mTORC1-dependent PDAC, and how this may be exploited for therapeutic benefit.

There is an increasing evidence that alterations in chromatin remodeling play an important role in tumorigenesis. The SWI/SNF chromatin remodeling complexes contribute to the regulation of gene expression by altering the local chromatin structure. Depending on the context, they can act as either transcriptional activators or repressors. All SWI/SNF subcomplexes contain one of two ATPase subunits, Brm (Brahma) or Brg1 (Brahma related gene 1), which provide the energy for remodeling. Malignant melanoma is an aggressive cancer and is known for its notorious resistance to conventional anticancer therapies. MITF (microphthalmia-associated transcription factor) plays an essential role in melanoma biology and is placed on the central crossroad in the regulation of melanocyte development, differentation, maintenance of lineage identity, and survival of both normal and malignant melanocytes. Our results show that the active SWI/SNF complex is strictly required for the expression of MITF. This complex is also required for expression of some transcriptional MITF targets. The survival of melanoma cells is absolutely dependent on functional SWI/SNF complex and all subunits of this complex are expressed at high levels in melanoma cell lines. Primarily, Brg1-containing subcomplexes are more important for MITF...

A síndrome de Waardenburg é uma síndrome geneticamente heterogênea, com uma taxa de penetrância muito alta e expressividade extremamente variável. O objetivo desse estudo foi a caracterização molecular de uma amostra brasileira de pacientes com SW, dando continuidade ao estudo clínico feito em Pardono (2005), por meio do estudo de 48 probandos classificados com a síndrome de Waardenburg tipo 1 ou 2. Foram estudados os genes PAX3, MITF, SOX10, SNAI2, EDN3 e EDNRB, por meio do sequenciamento pelo método de Sanger, e investigadas as microdeleções e microduplicações dos genes PAX3, MITF e SOX10 pela técnica de MLPA (Multiplex Ligationdependent Probe Amplification). Dentre os resultados obtidos, identificou-se 17 mutações potencialmente patogênicas (35,4% dos probandos). Dessas, seis são variações de número de cópias (12,5% dos probandos). Além disso, foi realizado um levantamento na base de dados LOVD (Leiden Open Variation Database), no qual constam 105 mutações não sinônimas exônicas consideradas causativas da SW. Diversos algoritmos foram utilizados para avaliar a possível patogenicidade dessas mutações, os quais levam em conta as frequências das mutações na base de dados do projeto 1000 genomas e 6500 exomas, anotam as previsões dadas pelos programas Polyphen2, MutationTaster, LRT e SIFT e verificam a conservação em mamíferos e primatas. Por meio dessa análise, verificou-se que em 19 mutações desse tipo (18%) faltam evidências de sua patogenicidade, colocando-se em dúvida a sua relação com a síndrome de Waardenburg / Waardenburg syndrome (WS) is a genetically heterogeneous syndrome, with a very high penetrance rate and highly variable expressivity. The focus of this study was the molecular characterization of a Brazilian sample of patients with WS (48 probands classified with Waardenburg syndrome type 1 or 2). The analysis of genes PAX3, MITF, SOX10, SNAI2, EDN3 and EDNRB were performed by the Sanger sequencing method. Microduplications and microdeletions in genes PAX3, MITF and SOX10 were investigated by MLPA technique (Multiplex Ligationdependent Probe Amplification). We detected 17 mutations considered as potentially pathogenic in 17 probands of the sample (35,4 % of probands). Among these, six are copy number variations (12,5% of probands). In addition, we performed a survey using the database of LOVD (Leiden Open Variation Database), which contains 105 non-synonymous exonic mutations considered causative of WS. Several algorithms were used to evaluate the possible pathogenicity of these mutations, taking into account the frequency of mutations in the database project in 1000 genomes and 6500 exomes, and using programs : Polyphen2, MutationTaster, LRT and SIFT. These algorithms also verify the conservation of the variations in mammals and primates. Through this analysis, lack of evidence was found for the pathogenicity of 19 non-synonymous mutations (18%) and association of these with Waardenburg syndrome is questioned

A síndrome de Waardenburg é uma síndrome geneticamente heterogênea, com uma taxa de penetrância muito alta e expressividade extremamente variável. O objetivo desse estudo foi a caracterização molecular de uma amostra brasileira de pacientes com SW, dando continuidade ao estudo clínico feito em Pardono (2005), por meio do estudo de 48 probandos classificados com a síndrome de Waardenburg tipo 1 ou 2. Foram estudados os genes PAX3, MITF, SOX10, SNAI2, EDN3 e EDNRB, por meio do sequenciamento pelo método de Sanger, e investigadas as microdeleções e microduplicações dos genes PAX3, MITF e SOX10 pela técnica de MLPA (Multiplex Ligationdependent Probe Amplification). Dentre os resultados obtidos, identificou-se 17 mutações potencialmente patogênicas (35,4% dos probandos). Dessas, seis são variações de número de cópias (12,5% dos probandos). Além disso, foi realizado um levantamento na base de dados LOVD (Leiden Open Variation Database), no qual constam 105 mutações não sinônimas exônicas consideradas causativas da SW. Diversos algoritmos foram utilizados para avaliar a possível patogenicidade dessas mutações, os quais levam em conta as frequências das mutações na base de dados do projeto 1000 genomas e 6500 exomas, anotam as previsões dadas pelos programas Polyphen2, MutationTaster, LRT e SIFT e verificam a conservação em mamíferos e primatas. Por meio dessa análise, verificou-se que em 19 mutações desse tipo (18%) faltam evidências de sua patogenicidade, colocando-se em dúvida a sua relação com a síndrome de Waardenburg / Waardenburg syndrome (WS) is a genetically heterogeneous syndrome, with a very high penetrance rate and highly variable expressivity. The focus of this study was the molecular characterization of a Brazilian sample of patients with WS (48 probands classified with Waardenburg syndrome type 1 or 2). The analysis of genes PAX3, MITF, SOX10, SNAI2, EDN3 and EDNRB were performed by the Sanger sequencing method. Microduplications and microdeletions in genes PAX3, MITF and SOX10 were investigated by MLPA technique (Multiplex Ligationdependent Probe Amplification). We detected 17 mutations considered as potentially pathogenic in 17 probands of the sample (35,4 % of probands). Among these, six are copy number variations (12,5% of probands). In addition, we performed a survey using the database of LOVD (Leiden Open Variation Database), which contains 105 non-synonymous exonic mutations considered causative of WS. Several algorithms were used to evaluate the possible pathogenicity of these mutations, taking into account the frequency of mutations in the database project in 1000 genomes and 6500 exomes, and using programs : Polyphen2, MutationTaster, LRT and SIFT. These algorithms also verify the conservation of the variations in mammals and primates. Through this analysis, lack of evidence was found for the pathogenicity of 19 non-synonymous mutations (18%) and association of these with Waardenburg syndrome is questioned

Le mélanome est un cancer de la peau très agressif. Microphthalmia-associated transcription factor (MITF) est un facteur de transcription clé contrôlant le développement de la lignée mélanocytaire, ainsi que la prolifération et l’invasion des cellules de mélanome. Pour mieux comprendre les fonctions de MITF, nous avons identifié ses cofacteurs impliques dans la régulation de la transcription. Nous avons montré que le complexe de remodelage de la chromatine NURF interagit avec MITF. Ma thèse a consisté à élucider le rôle de NURF dans le mélanome et les mélanocytes. La perte de BPTF, la principale sous-unité de ce complexe, induit un arrêt de la prolifération et une entrée en senescence des cellules de mélanome. Nous avons montré que BPTF et MITF coopèrent pour réguler l’expression de gènes impliqués dans la prolifération and invasion suggérant que BPTF et un cofacteur de MITF. De façon inattendue, l’inactivation de BPTF spécifiquement dans les mélanocytes entraine la perte progressive et totale de la pigmentation du pelage en raison de l’incapacité des cellules souches mélanocytaire à produire une descendance fonctionnelle. C’est la première fois que l’interaction fonctionnelle entre NURF et MITF est démontrée in vitro, complétée par des observations phénotypique uniques in vivo, contribuant à la compréhension de la biologie des mélanocytes et du mélanome. / Melanoma is a highly aggressive form of skin cancer. Microphthalmia-associated transcription factor (MITF) isa key regulator of development of the melanocyte lineage and proliferation and invasion of melanoma cells.To further elucidate the functions of MITF, we identified factors co-regulating transcription with MITF. We identified the NURF chromatin-remodeling complex as MITF interactor. My thesis aims to elucidate the role of NURF in melanoma and melanocytes. Loss of BPTF, the principal subunit of the complex, led to arrest of proliferation and entry into senescence of melanoma lines. We showed BPTF and MITF co-regulate genes involved in proliferation and invasion suggesting that BPTF acts as cofactor for MITF. Remarkably, the mouse model of melanocyte-specific BPTF ablation led to progressive and complete loss of coat pigmentation due to the inability of the melanocyte stem cells to produce functional progeny. This is the first report of NURF-MITF functional interaction in vitro, complemented with a unique in vivo phenotype, both adding to a general understanding of melanocyte and melanoma biology.

Le mélanome est un cancer très agressif en raison de sa capacité rapide à former des métastases et de développer une résistance aux traitements existants.
 MITF (Micropthalmia-associated Transcription Factor) est un facteur de transcription clé à toutes les étapes de développement du lignage mélanocytaire et dans la physiopathologie du mélanome. Afin de comprendre les mécanismes impliqués dans la régulation de l’activité et de la stabilité de MITF, nous avons identifié ses partenaires protéiques parmi lesquels figurent de nombreuses sous-unités des complexes de remodelage de la chromatine ATP-dépendant PBAF et NURF. Ce travail caractérise le rôle et l’étendue de la coopération entre BRG1/PBAF et plusieurs facteurs de transcription clés tels que MITF et SOX10 dans le fonctionnement des cellules de mélanome, qui recrutent activement de BRG1 à la chromatine et contribuent ainsi à la mise en place de la signature épigénétique caractéristique des cellules de mélanome prolifératives. Par ailleurs, l’utilisation de différents modèles murins a permis de révéler in vivo la contribution fonctionnelle distincte mais complémentaire de ces deux complexes de remodelage associé à MITF aux cours de trois stades majeurs du lignage mélanocytaire : le développement embryonnaire des mélanocytes, leur différentiation ainsi que lors de l’initiation et la progression du mélanome. Ce travail contribue ainsi à une meilleure compréhension du fonctionnement biologique des mélanocytes, du mélanome et du remodelage de la chromatine chez les eucaryotes. / Malignant melanoma is the most deadly form of skin cancer due to its quick metastatic spread and the development of resistance to available treatments.
MITF (Micropthalmia-associated Transcription Factor) is a transcription factor and master regulator of melanocyte lineage development and melanoma physiopathology. In order to investigate the mechanisms involved in the regulation of MITF activity and stability, we identified its numerous partners by tandem affinity purification coupled to mass spectrometry, which include several subunits of the PBAF and NURF ATP-dependant chromatin remodelling complexes. The present work characterizes the role and extent of cooperation between BRG1/PBAF and several key transcription factors including MITF and SOX10 in melanoma cell function, that actively recruit BRG1 to chromatin to establish the epigenetic landscape of proliferative melanoma cells. Furthermore, using different mouse models we revealed the distinct but complementary functional contribution of these two MITF-associated chromatin remodelers in vivo at three majors stages of melanocyte lineage development: embryonic development of melanocytes, their differentiation and during melanomagenesis. Thus, this work contributes to a better understanding of processes regulating the biological function of melanocytes, melanoma and more widely chromatin remodelling events in eukaryotes.