Le carcinome à cellules rénales claires métastatique (CCC) demeure résistant aux thérapies actuelles. Les travaux précédents ont montré des similitudes mécanistiques entre tumorigenèse et néphrogenèse. Parmi les cibles de la voie oncogénique Sonic Hedgehog-Gli, le facteur développemental Lim1 a été identifié comme un nouvel oncogène dans le CCC. Les études préliminaires suggèrent que Lim1 aurait un rôle dans l’invasion cellulaire. Aucun inhibiteur de Lim1 n’est disponible; l’ARN interférence reste l’outil le plus efficace et le plus spécifique pour l’extinction des gènes. Dans le but de développer un nouvel outil d’inhibition de Lim1 applicable en clinique, basée sur des siRNAs véhiculés par des systèmes nanométriques, une nouvelle génération de véhicules de siRNAs a été étudiée. Il s’agit de polymères diacétyléniques photo-polymérisables sous forme de nanofibres (PDA-Nf). Ces travaux démontrent le rôle invasif de Lim1 dans la dissémination métastatique et identifient les PDA-Nf comme un nouvel outil de délivrance de siRNAs très prometteur en clinique. L’avantage du ciblage de Lim1 avec cette approche, est l’absence de son expression à l’âge adulte. / Clear cell renal cell carcinoma (CCC) remains resistant to current therapies, despite the development of anti-angiogenic and the new immunotherapeutic approaches. Previous work of the laboratory has shown mechanistic similarities between tumorigenesis and nephrogenesis. Among the targets of the Sonic Hedgehog-Gli pathway which is found oncogenic in CCC, the developmental factor Lim1 has been identified as a new oncogene in CCC. Preliminary studies suggest that Lim1 would have a role in cell invasion. No Lim1 inhibitors are available; RNA interference remains the most effective and specific tool for gene silencing. In order to develop a new clinically applicable inhibition tool for Lim1, based on siRNAs carried by nanoparticles, a new generation of siRNA vehicles has been investigated. These are photo-polymerizable diacetylenic polymers in the form of nanofibers (PDA-Nf). These works demonstrate the invasive role of Lim1 in metastatic dissemination and identifies PDA-Nf as a new promising siRNA delivery tool in clinical practice. The advantage of targeting Lim1 with this nano-carrier approach is that Lim1 is not expressed in adulthood.

Resumo: Os dados de seqüência genômicas humanas depositados em bancos públicos têm fornecido uma oportunidade sem precedentes para pesquisadores decifrarem a funcionalidade do genoma humano. Essas informações são extremamente valiosas na prevenção, diagnóstico e tratamento do câncer. O Cancer Genome Anatomy Project (CGAP), o Gene Expression Omnibus (GEO), o Genome Data Mining (GDM) e o Babelomics funtional analysis of genome-scale experiments representam quatro importantes ferramentas para o estudo da genética funcional. O objetivo do presente trabalho foi o de explorar bancos de dados públicos para selecionar e validar a expressão de genes relacionados com o desenvolvimento e a progressão de carcinoma renal de células claras (CRcc). Utilizando ferramentas de bioinformática foi analisada a expressão gênica diferencial entre duas bibliotecas de SAGE elaboradas a partir de tecidos renais normais e de CRcc. Os resultados obtidos demonstraram alterações na expressão de genes participantes de importantes vias metabólicas. Baseando-se nessas análises, os genes CDK7, CLDN1, CSTB, EPC2 e ZNF706 foram selecionados e a expressão gênica diferencial desses foi avaliada por PCR em tempo real em 35 amostras de pacientes portadores de CRcc em diferentes estágios de progressão da doença e 1 amostra metastática. Paralelamente, utilizando-se a metodologia de RaSH foram elaboradas bibliotecas subtrativas entre o tecido neoplásico e o tecidos normal, sendo selecionados os genes MATR3 e PITPNB. O ZNF706 apresentou super expressão em relação aos tecidos normais, nas amostras tumorais analisadas independentemente do grau de evolução tumoral. O mesmo comportamento foi apresentado pelo gene CSTB, sendo que os maiores níveis de expressão foram observados nas amostras de estágio III de progressão tumoral. O gene CDK7 apresentou níveis de expressão dependente do grau de evolução da... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The data of human genomic sequences available in public database have provided an unpredictable opportunity for researchers to decipher the utility of human genome. This information is extremely valuable in prevention, diagnosis and cancer treatment. The Cancer Genome Anatomy Project (CGAP), the Gene expression Omnibus (GEO), the Genome Data Mining (GDM) and the Babelomics Functional Analysis of Genome-Scale Experiments do represent four important tools for functional genetics studies. This work aimed to explore public database to select and validate expression of genes related to development and progression of Clear Cell Renal Cell Carcinoma (ccRCC). The differential gene expression of two SAGE libraries from normal renal tissues and ccRCC was analyzed using bioinformatics tools. The results showed altered gene expression pattern in genes that participate in important metabolic pathways. Based on these analysis, the genes CDK7, CLDN1, CSTB, EPC2 and ZNF706 were selected and their differential gene expression was evaluated by Real Time PCR in 35 tissue samples from patients with ccRCC in different stages of disease progression and one metastatic sample. Simultaneously through RaSH methodology were elaborated subtractive libraries between normal and neoplasic tissues so the genes MATR3 and PITPNB were selected. The ZNF706 presented over expression in tumor tissues independent of tumor progression grade. The same behavior was presented by CSTB where the higher expression levels were observed in samples from stage IV of tumor progression. The CDK7 gene presented expression levels dependent of disease evolution grade where the lowers levels were in stages I, II, III and presented over expression in samples from stage IV and retroperitoneal metastasis. This result shows a clear relationship between CDK7 expression and the aggressiveness of ccRCC and suggests that this gene... (Complete abstract click electronic access below) / Orientador: Paulo Peitl Junior / Coorientador: Paula Rahal / Banca: Raphael Bessa Parmagiani / Banca: Sônia Maria Oliani / Mestre

O carcinoma renal de células claras é o tumor renal maligno mais frequentemente diagnosticado nos adultos. Uma série de defeitos genéticos tem sido observada no tecido tumoral renal e tais achados podem estar envolvidos na gênese ou progressão desses tumores. Alterações metabólicas e genéticas da mitocôndria são fatores que contribuem para muitas doenças humanas, incluindo o câncer. Objetivo: Estabelecer método para extração mitocondrial sem contaminação pelo DNA nuclear; verificar se existe contaminação por inserções nucleares de origem mitocondrial (NUMTs) no sequenciamento do genoma mitocondrial no carcinoma renal de células claras. Métodos: Para o estudo foram selecionados quatro pacientes portadores de carcinoma renal de células claras. Após a cirurgia obtivemos de cada paciente um fragmento de tumor e um fragmento de parênquima renal sem comprometimento neoplásico, as amostras de cada paciente foram extraídas de forma que ao final pudéssemos obter quatro amostras de DNA, sendo duas com isolamento da mitocôndria e duas sem o isolamento da mitocôndria. As amostras obtidas foram submetidas às seguintes análises genéticas: Sequenciamento completo do mtDNA; Reação em cadeia da polimerase para avaliação da contaminação do mtDNA obtido com isolamento da organela por DNA nuclear; avaliação do número de cópias do mtDNA (depleção) e patogenicidade das mutações. Resultados: Com os resultados obtidos neste estudo podemos afirmar que é possível a realização da extração do DNA mitocondrial sem a contaminação do genoma nuclear; e que o DNA mitocondrial extraído de maneira clássica do DNA total não apresentou contaminação por inserções nucleares de origem mitocondrial (NUMTs). / Clear cell renal carcinoma is the most frequently diagnosed malignant renal tumor in adults. A number of genetic defects have been observed in renal tumor tissue and such findings may be involved in the genesis or progression of these tumors. Metabolic and genetic changes in mitochondria are contributing factors to many human diseases, including cancer. Objective: To establish a method for mitochondrial extraction without nuclear DNA contamination; check for contamination by nuclear inserts of mitochondrial origin (NUMTs) in the sequencing of the mitochondrial genome in clear cell renal carcinoma. Methods: Four patients with clear cell renal carcinoma were selected for the study. After surgery, we obtained from each patient a tumor fragment and a renal parenchyma fragment without neoplastic involvement, the samples from each patient were extracted so that in the end we could obtain four DNA samples, two with mitochondrial isolation and two without the mitochondria isolation of mitochondria. The obtained samples were submitted to the following genetic analyzes: Complete sequencing of mtDNA; Polymerase chain reaction to evaluate the contamination of mtDNA obtained with organelle isolation by nuclear DNA; evaluation of mtDNA copy number (depletion) and pathogenicity of mutations. Results: With the results obtained in this study we can affirm that it is possible to perform mitochondrial DNA extraction without contamination of the nuclear genome; and that the mitochondrial DNA extracted from the classical DNA of the total DNA was not contaminated by nuclear inserts of mitochondrial origin (NUMTs).

Les carcinomes rénaux représentent environ 3% des cancers chez l’adulte. Les plus fréquents parmi ces tumeurs sont les carcinomes rénaux à cellules claires (CRCC) (70% des cas). Dans une première partie, nous avons analysé le caryotype de 89 patients ayant subi une néphrectomie pour CRCC et avons corrélé les déséquilibres chromosomiques avec les principaux facteurs histo-pronostiques et cliniques de ces tumeurs. Cette étude nous a permis de confirmer l’impact diagnostique et/ou pronostique d’anomalies chromosomiques. Certaines étaient déja connues dans la littérature comme la perte du bras court d’un chromosome 3 à impact diagnostique ou la perte d’un chromosome 9 ou de son bras court associée à un pronostic défavorable. Nous avons ensuite, dans une seconde partie, sélectionné selon des critères cliniques et histologiques, deux lignées cellulaires R-180 et R-305 établies à partir de prélèvements chirurgicaux de CRCC de patients dont l’évolution clinique était défavorable pour le patient R-180 (survie de 1 an) et favorable pour le patient R-305 (survie de 7 ans). Nous avons analysé les profils cytogénétiques des deux lignées cellulaires et recherché des marqueurs d’intérêt. Nous avons mis en évidence une amplification du gène pard3 dans la lignée R-180 correspondant au patient qui est décédé 1 an après le diagnostic. Cette amplification a été associée à la surexpression de la protéine correspondante PAR-3 et à des modifications de l’organisation du cytosquelette. La diminution de l’expression de PAR-3 par transfection de siRNA dans les cellules R-180 a permis la restauration de l’organisation du cytosquelette et la réduction des capacités de migration cellulaire par rapport aux cellules non transfectées. Ce résultat suggère un rôle de PAR-3 dans la migration cellulaire des cellules R-180. Afin de valider la pertinence de ce nouveau biomarqueur dans le CRCC, nous avons étudié 101 tumeurs par immunohistochimie. Nous avons montré une corrélation significative entre la surexpression de PAR-3 dans la tumeur primitive des patients et une diminution de la survie globale et de la survie sans progression indépendamment d’autres facteurs pronostiques importants comme les métastases. De plus la surexpression de PAR-3 a été significativement associée aux facteurs histopathologiques et cliniques de mauvais pronostic : grades nucléaires de Fuhrman III ou IV, nécrose tumorale, composante sarcomatoide, atteinte surrénale, invasion de la graisse rénale ou hilaire, composante éosinophile, statut non-inactivé du gène VHL, grade tumoral plus élevé, envahissement ganglionnaire ou métastatique, et score ECOG (Eastern Cooperative Oncology Group) péjoratif. L’ensemble de nos résultats suggèrent que la surexpression de PAR-3 est associée à un risque significatif de progression et de mortalité dans le CRCC. Sa mise en évidence par immunohistochimie en routine hospitalière pourrait être utile pour identifier les patients à haut risque de progression, même en l’absence des paramètres pronostiques habituels. Des études complémentaires sont en cours pour intégrer ce biomarqueur dans les nomogrammes ainsi que pour évaluer l’impact de cette dérégulation dans la résistance des CRCC aux thérapies ciblées. / Kidney cancers represent about 3% of all adults’ malignancies. The most common form of kidney cancer is renal carcinoma of which 70 % of cases are defined as clear cell Renal Cell Carcinoma (ccRCC). We undertook a systematic review of all ccRCCs with a total of 89 patients who underwent nephrectomy surgery. We assessed the karyotype profile of all patients that we correlate with an immunohistochemical features and tumor symptoms. This study demonstrates a high impact of chromosomal abnormalities on patients’ diagnosis and prognosis. Some of these abnormalities have been submitted in other publications as the loss of the chromosome 3 p-arm which has a diagnosis impact, and the loss of the chromosome 9 or it s p-arm that have a poor prognosis impact. We selected two cell lines (R-180 and R-305) derived from ccRCC surgical specimens of a patient with unfavorable clinical course (R-180 cells) and a patient with favorable prognosis (R-305 cells) to identify genetic and molecular features that may explain the survival difference of the two patients. The cytogenetic analysis of these cell lines revealed that the pard3 gene was amplified only in the R-180 cell line that was derived from an aggressive ccRCC. The pard3 gene amplification was associated with overexpression of the encoded protein and altered cytoskeleton organization. PAR-3 knockdown in R-180 cell restored the cytoskeleton organisation and reduced cell migration in comparison to non-transfected cells. These results suggest PAR-3 role in R-180 migration cells line. With a view to corroborate the relevance of this new biomarker PAR-3 in ccRCC, we have studied 101 tumors using immunohistochemical methods. We proved a significant correlation between PAR-3 overexpression in the primitive tumor and, the decreasing of overall and free progression survival independently of other risk factors as metastasis. We also fund that the overexpression of PAR-3 is associated with an unfavorable clinical and immunohistochemical prognosis factors such as: stage III -IV in fuhrman system grading ,tumor necrosis, sarcomatoide component, supra renal metastasis, cancer spreading (surrounding fat and hilar), eosinophil component , none inactivate VHL gene, high tumor stage, lymph nodes spread, metastasis and ECOG scale. Our results reveal that the PAR-3 overexpression is associated with significant risk of ccRCCs mortality and spreading tumor. Immunohistochemical screening may be usefulness to identify patient’s high spreading risk whether the lack of the habitual prognosis parameters. Other studies are in progress to integrate this biomarker in nomograms and also to evaluate the impact on ccRCC’s resistance to targeted therapy.

HYAL-1 (hyaluronidase-1) appartient à la famille des hyaluronidases connues pour leur rôle dans la dégradation de l’acide hyaluronique. L’expression de HYAL-1 est élevée dans de nombreux type de cancers, notamment dans le cancer de la prostate, de la vessie, des reins et du sein où il est impliqué dans la croissance tumorale et les métastases. Récemment notre laboratoire a aussi démontré une expression élevée de HYAL-1 dans le cancer épithélial de l’ovaire (CEO) de type mucineux et à cellules claires, expression qui est inversement corrélée à celle du récepteur de l’oestrogène alpha (REα). Cependant, malgré le fait que le rôle de HYAL-1 dans le cancer soit bien établit, le mécanisme de sa régulation reste encore inconnu. Le REα est un facteur de transcription qui suite à sa liaison avec son ligand va réguler l’expression de plusieurs gènes. Le REα ainsi stimulé par l’hormone va activer la transcription de ces gènes cibles mais il est connu maintenant qu’une grande partie des gènes régulés par le REα sont en réalité réprimés par ce récepteur. Dans ce travail nous proposons d’étudier le mécanisme de la régulation du gène HYAL-1 par le REα dans le CEO à cellules claires et dans le cancer du sein. L’expression ectopique du REα dans la lignée TOV21G (RE-) de même que le traitement de la lignée MCF-7 (RE+) avec de l’oestrogène a induit une diminution du niveau d’expression de l’ARN m de HYAL-1. Ces résultats nous ont permis de confirmer que HYAL-1 est un gène cible du REα. Il est aussi connu que le REα peut exercer son action par différents mécanismes d’action, entre autres en interagissant avec une séquence d’ADN appelée élément de réponse à l’oestrogène (ERE), retrouvé sur le promoteur des gènes cibles ou bien indirectement par des interactions protéine-protéine en se liant à d’autres facteur de transcription tels que Sp1. Après avoir identifiés de telles séquences sur le promoteur proximal de HYAL-1, (1 ERE proximal à -900 pb, 3 distaux à -32350 pb, 48430, -50130 pb du site d’initiation de la transcription) en plus des 2 Sp1 connus (-60 et – 1020pb), nous avons démontrés par immunoprécipitation de la chromatine que le REα est recruté sur le promoteur de HYAL-1 au niveau de l’ERE proximal -900 pb et du distal -32350 pb de même que sur le site Sp1 -1020 pb. De plus, l’activité biologique de l’ERE -900 pb et du ii Sp1-1020pb à été confirmée par des essais de gènes rapporteurs à la luciférase. Avec son rôle connu dans la tumorigenèse, l’identification de HYAL-1 comme gène cible du REα pourrait être une avenue intéressante pour le traitement des cancers hormono-indépendants. / HYAL-1 (hyaluronidase-1) belongs to the hyaluronidase family of enzymes that degrade hyaluronic acid. HYAL-1 expression is elevated in many types of cancers including prostate, bladder, liver and breast cancer where it is involved in tumor growth and metastasis. In accordance to these observations, our group has also demonstrated high expression of HYAL-1 in clear cell and mucinous epithelial ovarian cancer (EOC) subtypes which was inversely correlated to that of estrogen receptor alpha (ERα). However, despite the fact that the role of HYAL-1 in cancer is well established, the mechanism of its regulation is still unknown. ERα is a transcriptional factor that regulates target-gene expression following ligand binding. Upon hormone stimulation, activated ERα will upregulate transcription of many target genes. However, it has been recently well documented that a large number of ERα responsive genes are in fact repressed. In this work we propose to study the mechanism by which ERα regulates HYAL-1 expression in clear cell EOC subtype as well as in breast cancer. The ectopic expression of ERα in TOV21G cell line (ERα -) and estrogen treatment of MCF-7 cells (ERα +) decreased HYAL-1 mRNA expression and allowed us to confirm that HYAL-1 is an ERα target gene. It is also known that ERα may exert its action through different mechanisms of action including interacting with a DNA sequence called estrogen response element (ERE) found in the promoter of target genes or indirectly by protein-protein interactions by binding to other transcription factor such as Sp1. Having identified such sequences in the proximal promoter of HYAL-1, (one proximal ERE -900 bp, 3 distals at -32350, -48430, -50130 bp from the start site of transcription) in addition to the two known Sp1 (-60 and -1020pb), we have demonstrated by chromatin immunoprecipitation that ERα is recruited at the HYAL-1 promoter at the ERE sites -900 pb and -32350 pb as well as at the Sp1 site -1020. Furthermore, the biological activity of the proximal ERE -900 and Sp1 -1020 sites were further confirmed by luciferase reporter gene assay. Given its known role in tumorigenesis, identification of HYAL-1 as an ERα target may provide an interesting approach for the treatment of hormono-independent cancer.

O carcinoma de célula renal (RCC) subtipo célula clara é o câncer mais letal e prevalente do sistema urinário. O diagnóstico deste tipo de câncer frequentemente é tardio em conseqüência da falta de sintomas perceptíveis aos pacientes. Um dos objetivos deste trabalho é a identificação de novos marcadores moleculares para diagnóstico precoce, o que ajudaria a diminuir a mortalidade em função de complicações resultantes do avanço da doença. Outro objetivo é a identificação de um conjunto de marcadores moleculares de prognóstico, de modo à prever com acurácia a evolução clínica da doença e, por conseqüência, o tempo de sobrevida do paciente. As modificações transcricionais associadas à carcinogênese e à progressão do câncer de rim ainda não foram completamente elucidadas. Além dos oncogenes e genes supressores de tumor, RNAs não-codificadores (ncRNAs) recentemente foram apontados como importantes reguladores da expressão gênica em humanos, e podem ter um papel importante na transformação maligna do câncer de rim. Para analisar a expressão gênica de ncRNAs e de genes codificadores para proteína foram utilizados dois microarranjos desenvolvidos por nosso grupo, enriquecidos em sondas para ncRNAs. Uma das plataformas possui 4 mil sondas de cDNA, das quais 822 sondas são para ncRNAs mapeando em regiões intrônicas. Outra possui 44 mil elementos e combina sondas de oligonucleotídeos (60-mer) intrônicas e exônicas de um mesmo locus genômico. Análises estatísticas foram feitas com a ferramenta Significance Analysis of Microarrays (q &#8804; 0,05) combinadas ou com a técnica de \"patient leave-one-out\" (genes com presença em 8 100% dos subconjuntos), ou alternativamente com o teste discriminante de Golub (p &#8804; 0,01 ou p < 0,05). Com a plataforma de 4 mil sondas foram estudadas 30 amostras de tecido renal de 18 pacientes com RCC subtipo célula clara. Um conjunto de 36 ncRNAs foi identificado como diferencialmente expresso entre amostras tumorais e não-tumorais. Uma assinatura adicional de 265 genes codificadores de proteínas foi identificada, indicando possíveis novos marcadores moleculares. Uma análise estatística supervisionada com dados de 16 pacientes identificou uma assinatura de ncRNAs correlacionada com sobrevida de 5 anos, formada por 27 ncRNAs com significativa expressão alterada em pacientes livres da doença em comparação com pacientes que morreram em função da doença. Uma assinatura adicional de 64 genes codificadores de proteínas também foi identificada como significativamente correlacionada com o acompanhamento clínico dos pacientes. Com a plataforma de 44 mil sondas foram analisados 17 pacientes, com amostras pareadas de tecido renal tumoral e não-tumoral agrupadas em 8 pools, sendo 4 de amostras tumorais e 4 de não-tumorais. Um conjunto de 66 ncRNAs parcialmente intrônicos antisenso e outro de 52 ncRNAs totalmente intrônicos antisenso foram identificados como diferencialmente expressos. Identificamos um subconjunto de 28 ncRNAs totalmente intrônicos antisenso e senso cuja expressão do gene codificador de proteína do mesmo locus estava simultaneamente alterada. Estes dados apontam para possíveis redes de regulação da expressão gênica dos ncRNAs em câncer. A extensa lista de ncRNAs e de genes codificadores para proteína identificados neste estudo podem ser promissores marcadores moleculares de carcinoma renal subtipo célula clara. / Renal cell carcinoma (RCC) is the most common malignancy of the adult kidney, and the clear cell subtype is the most prevalent and lethal cancer of the urinary system. Late diagnosis for this type of cancer is frequent, usually as a consequence of the lack of symptoms. One of the objectives of the present work is the identification of new molecular markers for the early diagnosis, which would help decrease mortality that develops as a function of disease progression. Another objective is the identification of a set of prognosis molecular markers, so as to accurately predict the clinical outcome of the disease, and consequently, patient survival. Transcriptional changes associated to carcinogenesis and to kidney cancer progression have not been entirely elucidated. Besides oncogenes and tumor suppressor genes, non-coding RNAs (ncRNAs) have been recently indicated as important regulators of gene expression in humans, and could have an important role in the malignant transformation in renal cancer. In order to measure ncRNA and protein-coding gene expression we have used two microarray platforms developed by our group, which are enriched in ncRNA probes. One of the platforms has 4 thousand cDNA probes, of which 822 are for ncRNAs that map to intronic regions. Another has 44 thousand elements and combines 60-mer oligonucleotide probes for intronic and exonic regions from the same genomic locus. Statistical analyses have been performed with the Significance Analysis of Microarrays tool (q &#8804; 0.05) combined with a patient leave-one-out approach (genes present in 100% of the sub-sets), or alternatively with Golubs discriminant test (p &#8804; 0.01 or p < 0.05). 11 With the 4-thousand probes platform we studied 30 samples from renal tissue of 18 RCC patients with clear cell subtype. A set of 36 ncRNAs has been identified as differentially expressed between tumor and non-tumor tissue. An additional signature of 265 protein-coding genes has been identified, indicating possible new molecular markers. A supervised statistical analysis with data from 16 patients has identified a ncRNA signature correlated to 5-year survival outcome, comprised of 27 ncRNAs with significantly altered expression in diseasefree patients compared to patients who died from cancer within the 5-year follow-up. An additional 64-gene signature of protein-coding genes has been identified as significantly correlated to clinical outcome. With the 44-thousand probes platform we have analyzed 17 patients, with paired tumor and non-tumor samples grouped into 8 pools, of which 4 were from tumor and 4 from nontumor samples. A set of 66 partially intronic antisense ncRNAs and another of 52 totally intronic antisense ncRNAs have been identified as differentially expressed between tumor and non-tumor tissue. A sub-set of 28 totally intronic antisense or sense ncRNAs were identified as having a simultaneous change in expression of the protein-coding gene from the same locus. Overall, the data point to a possible ncRNA regulatory network in cancer. The extensive lists of ncRNAs and of protein-coding genes identified in the present study can be seen as promising molecular markers of RCC from the clear-cell subtype.

Introdução: com o uso generalizado de métodos de imagem, aumentou-se a detecção de lesões renais como achados incidentais. Tais lesões podem ser tanto benignas, tais como os angiomiolipomas, e outras malignas, como os carcinomas de células renais, portanto torna-se importante um método não-invasivo com boa acurácia para sua distinção . A ultrassonografia é pouco específica para este fim. A tomografia computadorizada e a ressonância magnética são os métodos mais utilizados na caracterização de lesões incidentais renais. Na Ressonância Magnética, o uso de sequências convencionais ponderadas em T2W e T1W, antes e após a administração endovenosa de contraste paramagnético, mostrou-se pouco eficaz para este fim. Técnicas quantitativas associadas às imagens convencionais, tais como a oposição de fase e a restrição a difusão da água, vêm sendo estudadas, devido ao potencial para melhorar a caracterização não-invasiva, evitando nefrectomias parciais ou totais, e outras intervenções invasivas por lesões benignas. Objetivos: avaliar a acuidade diagnóstica de técnicas combinadas de ressonância magnética para diferenciação de angiomiolipomas pobres em gordura de lesões malignas do rim. Métodos: pacientes que obtiveram o diagnóstico histológico das lesões renais entre os anos de 2010 e 2014 e que realizaram exame pré-operatório. As lesões foram estudadas, colocando-se um ROI (region of interest, no inglês) na maior parte da lesão e córtex renal normal, evitando-se área de não-lesões, calculando a intensidade de sinal nas seqüências T1W in e out-phase, T2W, o Wash in, Wash out relativo e absoluto das lesões e o cálculo absoluto do sinal da lesão no ADC (coeficiente de difusão aparente, do inglês). Os resultados foram obtidos na forma de índices padronizados pelo córtex renal e baço, pelas fases pré e pós-contraste, e de forma absoluta pelo ADC. Os resultados foram confrontados com o diagnóstico final e feito associações estatísticas para observar a relevância. Resultados:Foram estudadas 85 lesões em 74 pacientes, sendo 40 do gênero masculino e 34 do feminino. O cálculo do teor de gordura se mostrou ineficaz para distinção entre CCRs e AMLpg; o índice de intensidade de sinal em T2W, lesão/córtex normal foi útil na diferenciação dos CCRs CC de AMLpg. Outro parâmetro importante foi a cálculo de wash out relativo que se mostrou mais acentuado no AMLpg que em todos os subtipos de CCRs estudados e da medida do ADC médio, que apresentou valores maiores nos casos de CCR CC, em comparação com os outros subtipos e com os AMLpg. Conclusão: As técnicas combinadas de RM, principalmente o índice de sinal T2W da lesão, Wash out relativo e IS do ADC, associados a dados epidemiológicos são viáveis, quando utilizados em conjunto, para a diferenciação de lesões malignas renais dos angiomiolipomas, podendo ter implicações na conduta terapêutica, com redução do número de nefrectomias por lesão benignas. / Introduction: With the widespread use of imaging methods, detection of incidental renal masses has steadily increased in recent years, and these may be either benign, such as angiomyolipoma, or malignant, such as renal cell carcinomas. Therefore, it is important to have a method that allows accurate characterization. Ultrassonography is not very specific for this purpose. CT and MRI are the methods used in the characterization of renal incidental lesions. In MRI, the use of conventional sequences, such as T1W and T2W before and after intravenous administration of paramagnetic contrast media, has proved ineffective for this purpose. Quantitative techniques associated with conventional images, such as chemical shift and diffusion weighted imaging (DWI), have played a key role in this differentiation, which aims to improve characterization, avoiding partial or total nephrectomy, and other invasive interventions for benign lesions. Objectives: to evaluate the diagnostic accuracy of combined techniques of MRI to differentiate fat-poor angiomyolipoma from renal cell carcinomas. Methods: Patients who had a histological diagnosis of renal lesions between 2010 and 2014 and underwent pre-operative exam. An ROI (region of interest)cwas placed in most of the lesion and normal renal cortex, avoiding area of non lesions, by calculating the signal intensity in all sequences, in and out phase T1W, T2W, and the wash in, wash out, relative and absolute, of the lesions and estimation of the ADC. The results were obtained in the form of standardized indices for renal cortex and spleen, the pre- and post-contrast phases, and absolute values for ADC. Results were confronted with the final diagnosis and statistical analysis to observe the relevancy. Results: the estimation of intracellular fat content was ineffective for characterization, while the T2W signal intensity index was used for differentiation between CCRs clear cells from fat-poor AML. Another important parameter was the \"wash out\", which was more prominent for AMLpg. ADC values was higher for CCR CC. Conclusion: We concluded that the combined techniques of MRI mainly T2W signal ratio, \"Wash out\" and ADC values, when used in association and correlated with epidemiological data may be feasible for the differentiation among fat-poor angiomyolipomas and renal malignancies, with important therapeutics implications, reducing unnecessary nephrectomies for benign lesions.

HYAL-1 (hyaluronidase-1) appartient à la famille des hyaluronidases connues pour leur rôle dans la dégradation de l’acide hyaluronique. L’expression de HYAL-1 est élevée dans de nombreux type de cancers, notamment dans le cancer de la prostate, de la vessie, des reins et du sein où il est impliqué dans la croissance tumorale et les métastases. Récemment notre laboratoire a aussi démontré une expression élevée de HYAL-1 dans le cancer épithélial de l’ovaire (CEO) de type mucineux et à cellules claires, expression qui est inversement corrélée à celle du récepteur de l’oestrogène alpha (REα). Cependant, malgré le fait que le rôle de HYAL-1 dans le cancer soit bien établit, le mécanisme de sa régulation reste encore inconnu. Le REα est un facteur de transcription qui suite à sa liaison avec son ligand va réguler l’expression de plusieurs gènes. Le REα ainsi stimulé par l’hormone va activer la transcription de ces gènes cibles mais il est connu maintenant qu’une grande partie des gènes régulés par le REα sont en réalité réprimés par ce récepteur. Dans ce travail nous proposons d’étudier le mécanisme de la régulation du gène HYAL-1 par le REα dans le CEO à cellules claires et dans le cancer du sein. L’expression ectopique du REα dans la lignée TOV21G (RE-) de même que le traitement de la lignée MCF-7 (RE+) avec de l’oestrogène a induit une diminution du niveau d’expression de l’ARN m de HYAL-1. Ces résultats nous ont permis de confirmer que HYAL-1 est un gène cible du REα. Il est aussi connu que le REα peut exercer son action par différents mécanismes d’action, entre autres en interagissant avec une séquence d’ADN appelée élément de réponse à l’oestrogène (ERE), retrouvé sur le promoteur des gènes cibles ou bien indirectement par des interactions protéine-protéine en se liant à d’autres facteur de transcription tels que Sp1. Après avoir identifiés de telles séquences sur le promoteur proximal de HYAL-1, (1 ERE proximal à -900 pb, 3 distaux à -32350 pb, 48430, -50130 pb du site d’initiation de la transcription) en plus des 2 Sp1 connus (-60 et – 1020pb), nous avons démontrés par immunoprécipitation de la chromatine que le REα est recruté sur le promoteur de HYAL-1 au niveau de l’ERE proximal -900 pb et du distal -32350 pb de même que sur le site Sp1 -1020 pb. De plus, l’activité biologique de l’ERE -900 pb et du ii Sp1-1020pb à été confirmée par des essais de gènes rapporteurs à la luciférase. Avec son rôle connu dans la tumorigenèse, l’identification de HYAL-1 comme gène cible du REα pourrait être une avenue intéressante pour le traitement des cancers hormono-indépendants. / HYAL-1 (hyaluronidase-1) belongs to the hyaluronidase family of enzymes that degrade hyaluronic acid. HYAL-1 expression is elevated in many types of cancers including prostate, bladder, liver and breast cancer where it is involved in tumor growth and metastasis. In accordance to these observations, our group has also demonstrated high expression of HYAL-1 in clear cell and mucinous epithelial ovarian cancer (EOC) subtypes which was inversely correlated to that of estrogen receptor alpha (ERα). However, despite the fact that the role of HYAL-1 in cancer is well established, the mechanism of its regulation is still unknown. ERα is a transcriptional factor that regulates target-gene expression following ligand binding. Upon hormone stimulation, activated ERα will upregulate transcription of many target genes. However, it has been recently well documented that a large number of ERα responsive genes are in fact repressed. In this work we propose to study the mechanism by which ERα regulates HYAL-1 expression in clear cell EOC subtype as well as in breast cancer. The ectopic expression of ERα in TOV21G cell line (ERα -) and estrogen treatment of MCF-7 cells (ERα +) decreased HYAL-1 mRNA expression and allowed us to confirm that HYAL-1 is an ERα target gene. It is also known that ERα may exert its action through different mechanisms of action including interacting with a DNA sequence called estrogen response element (ERE) found in the promoter of target genes or indirectly by protein-protein interactions by binding to other transcription factor such as Sp1. Having identified such sequences in the proximal promoter of HYAL-1, (one proximal ERE -900 bp, 3 distals at -32350, -48430, -50130 bp from the start site of transcription) in addition to the two known Sp1 (-60 and -1020pb), we have demonstrated by chromatin immunoprecipitation that ERα is recruited at the HYAL-1 promoter at the ERE sites -900 pb and -32350 pb as well as at the Sp1 site -1020. Furthermore, the biological activity of the proximal ERE -900 and Sp1 -1020 sites were further confirmed by luciferase reporter gene assay. Given its known role in tumorigenesis, identification of HYAL-1 as an ERα target may provide an interesting approach for the treatment of hormono-independent cancer.

A Síndrome de von Hippel-Lindau é uma doença hereditária multissistêmica, causada por mutações germinativas no gene VHL que predispõe o portador a manifestações benignas e malignas em diversos órgãos. Entre esses eventos, o carcinoma de células claras renais (CRC) é o de pior prognóstico, com uma penetração média de 25% e sendo a principal causa de morte nestes pacientes. Os CRCs são tumores agressivos, pouco responsivos à quimioterapia e imunoterapia, e muitas vezes são diagnosticados em estágios avançados. Podem estar associados a síndromes hereditárias como o VHL ou apresentar a forma esporádica. Caracteristicamente, o CRC é provocado pela inativação dos dois alelos do gene VHL. Nos casos associados ao VHL, um alelo do gene VHL sofre uma mutação germinativa e um segundo evento mutacional somático nas células do tumor. Por outro lado, na forma esporádica, o CRC é resultado de dois eventos somáticos adquiridos, que incluem uma combinação de metilação do promotor, mutações pontuais que afetam a sequência de leitura aberta (ORF) e rearranjos cromossômicos, principalmente perda de heterozigosidade (LOH). Embora os eventos somáticos nos CRCs esporádicos já tenham sido explorados em outros estudos, os mecanismos de inativação somáticos do gene VHL nos CRCs associados à síndrome ainda não foram bem descritos. Este estudo avaliou os eventos somáticos no gene VHL em CRCs retirados em procedimentos cirúrgicos de pacientes portadores da síndrome. Os eventos somáticos em vários tumores de um mesmo paciente foram comparados a fim de verificarmos se essas mutações são independentes e não clonais. Oito pacientes com amostras CRCs previamente armazenadas no BNT tiveram sua mutação germinativa no gene VHL caracterizada por sequenciamento ou MLPA. Todas as amostras foram submetidas a uma revisão da patologia e macrodissecadas sempre que necessário. Para a análise das manifestações somáticas do gene VHL, o DNA foi extraído de 30 CRCs conservados em RNA latter ou formaldeído (parafina). As amostras foram analisadas quanto à metilação da região promotora do gene pelo método MS-PCR e para mutações pontuais por sequenciamento. Fomos capazes de detectar a mutação somática em 25 dos 30 tumores, incluindo uma mutação pontual e dois tumores diferentes de um mesmo paciente, nenhuma microdeleção e 23 grandes deleções. Em contraste com a literatura, nenhum dos tumores apresentou metilação no promotor do VHL. Devido ao grande número de achados LOH e da resolução limitada da técnica de MLPA para avaliar a extensão dos rearranjos cromossômicos em 3p, não foi possível concluir a análise de clonalidade dos tumores. Um estudo exploratório para caracterizar ganhos e perdas genômicas utilizando a técnica CNV array está em andamento em nosso laboratório. / The von Hippel-Lindau syndrome (VHL) is a multissystemic hereditary disease, caused by germline mutations in the VHL gene that predisposes the carrier to benign and malignant manifestations in different organs. Among these events, the clear cell renal carcinoma (RCC) is the most fearful, with an average penetration of 25% being the leading cause of death in these patients. RCCs are aggressive tumors, poorly responsive to chemo- and immunotherapy that are often diagnosed in advanced stages. They can be associated with hereditary syndromes such as VHL or present in a sporadic form. Characteristically, RCCs carrier the inactivation of the two alleles of VHL gene. In cases associated with VHL, one allele of the VHL gene is mutated in the germline, and the second mutational event occurs in the somatic cells of the tumor. On the other hand, in the sporadic form, RCCs results of two acquired somatic events, which includes a combination of methylation of the promoter, point mutations affecting the ORF, and rearrangements mainly loss of heterozigosity (LOH). Although somatic events in sporadic RCC have been explored before by others, the mechanisms of somatic VHL gene inactivation in VHL-associated RCCs have been poorly characterized. This study evaluated the somatic mutational events in the VHL gene of RCCs removed from VHL patients in therapeutic surgical procedures. The somatic events in multiple tumors from the same patient were compared in order to analyze whether these mutations are independent and not clonal. Eight patients with RCCs samples previously stored at BNT had their germline VHL gene mutation characterized by sequencing or MLPA. All samples were submitted to a pathology review and macrodissected whenever necessary. For the analysis of somatic events of VHL gene, DNA from 30 RCCs were extracted from either RNA later or archival formalin-fixed, paraffin-embedded tissue sections. Samples were analyzed for VHL gene promoter methylation by MS-PCR, and for point mutation in the coding DNA by sequencing. We were able to detect the somatic mutation in 25 of the 30 tumors, including one point mutations in two different tumors of the same patient, no micro-deletions, and 23 large deletions. In contrast to the literature, none of the tumors have shown methylation on the VHL promoter. Because of the large number of LOH findings, and the limited resolution of MLPA to evaluate the extension of 3p chromosomal rearrangements, we could not conclude the analysis of tumor clonality. An exploratory study to characterize genomic gains and losses using CNV-array technique are ongoing in our laboratory.

A Síndrome de von Hippel-Lindau é uma doença hereditária multissistêmica, causada por mutações germinativas no gene VHL que predispõe o portador a manifestações benignas e malignas em diversos órgãos. Entre esses eventos, o carcinoma de células claras renais (CRC) é o de pior prognóstico, com uma penetração média de 25% e sendo a principal causa de morte nestes pacientes. Os CRCs são tumores agressivos, pouco responsivos à quimioterapia e imunoterapia, e muitas vezes são diagnosticados em estágios avançados. Podem estar associados a síndromes hereditárias como o VHL ou apresentar a forma esporádica. Caracteristicamente, o CRC é provocado pela inativação dos dois alelos do gene VHL. Nos casos associados ao VHL, um alelo do gene VHL sofre uma mutação germinativa e um segundo evento mutacional somático nas células do tumor. Por outro lado, na forma esporádica, o CRC é resultado de dois eventos somáticos adquiridos, que incluem uma combinação de metilação do promotor, mutações pontuais que afetam a sequência de leitura aberta (ORF) e rearranjos cromossômicos, principalmente perda de heterozigosidade (LOH). Embora os eventos somáticos nos CRCs esporádicos já tenham sido explorados em outros estudos, os mecanismos de inativação somáticos do gene VHL nos CRCs associados à síndrome ainda não foram bem descritos. Este estudo avaliou os eventos somáticos no gene VHL em CRCs retirados em procedimentos cirúrgicos de pacientes portadores da síndrome. Os eventos somáticos em vários tumores de um mesmo paciente foram comparados a fim de verificarmos se essas mutações são independentes e não clonais. Oito pacientes com amostras CRCs previamente armazenadas no BNT tiveram sua mutação germinativa no gene VHL caracterizada por sequenciamento ou MLPA. Todas as amostras foram submetidas a uma revisão da patologia e macrodissecadas sempre que necessário. Para a análise das manifestações somáticas do gene VHL, o DNA foi extraído de 30 CRCs conservados em RNA latter ou formaldeído (parafina). As amostras foram analisadas quanto à metilação da região promotora do gene pelo método MS-PCR e para mutações pontuais por sequenciamento. Fomos capazes de detectar a mutação somática em 25 dos 30 tumores, incluindo uma mutação pontual e dois tumores diferentes de um mesmo paciente, nenhuma microdeleção e 23 grandes deleções. Em contraste com a literatura, nenhum dos tumores apresentou metilação no promotor do VHL. Devido ao grande número de achados LOH e da resolução limitada da técnica de MLPA para avaliar a extensão dos rearranjos cromossômicos em 3p, não foi possível concluir a análise de clonalidade dos tumores. Um estudo exploratório para caracterizar ganhos e perdas genômicas utilizando a técnica CNV array está em andamento em nosso laboratório. / The von Hippel-Lindau syndrome (VHL) is a multissystemic hereditary disease, caused by germline mutations in the VHL gene that predisposes the carrier to benign and malignant manifestations in different organs. Among these events, the clear cell renal carcinoma (RCC) is the most fearful, with an average penetration of 25% being the leading cause of death in these patients. RCCs are aggressive tumors, poorly responsive to chemo- and immunotherapy that are often diagnosed in advanced stages. They can be associated with hereditary syndromes such as VHL or present in a sporadic form. Characteristically, RCCs carrier the inactivation of the two alleles of VHL gene. In cases associated with VHL, one allele of the VHL gene is mutated in the germline, and the second mutational event occurs in the somatic cells of the tumor. On the other hand, in the sporadic form, RCCs results of two acquired somatic events, which includes a combination of methylation of the promoter, point mutations affecting the ORF, and rearrangements mainly loss of heterozigosity (LOH). Although somatic events in sporadic RCC have been explored before by others, the mechanisms of somatic VHL gene inactivation in VHL-associated RCCs have been poorly characterized. This study evaluated the somatic mutational events in the VHL gene of RCCs removed from VHL patients in therapeutic surgical procedures. The somatic events in multiple tumors from the same patient were compared in order to analyze whether these mutations are independent and not clonal. Eight patients with RCCs samples previously stored at BNT had their germline VHL gene mutation characterized by sequencing or MLPA. All samples were submitted to a pathology review and macrodissected whenever necessary. For the analysis of somatic events of VHL gene, DNA from 30 RCCs were extracted from either RNA later or archival formalin-fixed, paraffin-embedded tissue sections. Samples were analyzed for VHL gene promoter methylation by MS-PCR, and for point mutation in the coding DNA by sequencing. We were able to detect the somatic mutation in 25 of the 30 tumors, including one point mutations in two different tumors of the same patient, no micro-deletions, and 23 large deletions. In contrast to the literature, none of the tumors have shown methylation on the VHL promoter. Because of the large number of LOH findings, and the limited resolution of MLPA to evaluate the extension of 3p chromosomal rearrangements, we could not conclude the analysis of tumor clonality. An exploratory study to characterize genomic gains and losses using CNV-array technique are ongoing in our laboratory.