L’hyperplasie bilatérale macronodulaire des surrénales (HBMS) conduit au développement de nodules corticosurrénaliens bilatéraux entraînant un syndrome de Cushing diagnostiqué souvent au cours de la cinquième décennie. L’étiologie de cette maladie n’était que partiellement connue mais le caractère bilatéral de l’atteinte surrénalienne et l’observation de cas familiaux suggéraient, au début de ce travail, une origine génétique. L’analyse de l’ADN tumoral et leucocytaire d’une série de 33 patients opérés a permis de mettre en évidence chez 25% des patients une perte d’hétérozygotie neutre en nombres de copies (LOH) de tout le bras court du chromosome 16 (16p) au niveau du tissu surrénalien. Un séquençage complet du génome de 5 patients (ADN germinal et tumoral) a permis de mettre en évidence une mutation du gène ARMC5 localisé en 16p pour 4 patients. Le séquençage direct des parties codantes d’ARMC5 à partir de l’ADN somatique et germinal de l’ensemble des patients opérés de la cohorte a montré qu’au total, 55% des patients avaient une mutation d’ARMC5. L’inactivation du gène se fait selon la théorie de Knudson (un événement germinal inactivant le premier allèle associé à un autre événement somatique inactivant le second allèle) ce qui laisse supposer qu’ARMC5 est un gène suppresseur de tumeur. L’analyse de la cohorte d’HBMS de nos collaborateurs américains au National Institute Health (laboratoire de CA. Stratakis, Bethesda, USA) a permis de confirmer que les mutations d’ARMC5 étaient un événement fréquent. Des variants de ce gène sont aussi associés à l’hypertension à rénine basse chez les patients noirs-américains. Afin de déterminer des corrélations génotype-phénotype, notre cohorte initiale a été élargie pour constituer une série consécutive de 98 cas index de patients présentant des formes légères à sévères de la maladie, opérées ou non. Vingt-quatre patients (25%) présentaient une altération d’ARMC5. Par ailleurs, 31 nodules surrénaliens de 19 patients ont été analysés en somatique. Le second événement était une mutation dans 68% des cas et une LOH du locus pour les 32% restant. Chez un même patient, le second événement était différent dans chaque nodule présenté. Les patients mutés avaient un syndrome de Cushing plus sévère cliniquement et biologiquement par rapport aux patients non mutés. La taille de leurs surrénales étaient plus grandes avec un plus grand nombre de nodules. Les patients mutés étaient aussi plus jeunes au diagnostic et plus souvent hypertendus. Ces patients étaient ainsi plus souvent opérés. La fonction de la protéine ARMC5 n’était pas connue lors de son identification comme gène de l’HBMS. In vitro, la surexpression du gène sauvage induit l’apoptose. La surexpression des mutants faux-sens et du mutant p.F700del retrouvés chez les patients entraîne moins d’apoptose qu’ARMC5 sauvage. La protéine ARMC5 contient des domaines Armadillo et BTB, connus pour être impliqués dans l’interaction protéine-protéine. En physiologie, l’ACTH stimule la production d’AMPc et la voie de la protéine kinase A (PKA) est impliquée dans différentes pathologies corticosurrénaliennes. Nous avons pu montrer qu’ARMC5 interagissait avec la sous-unité catalytique alpha de la PKA. L’invalidation d’ARMC5 conduit in vitro à une diminution de l’expression d’enzymes de la stéroïdogénèse, de la production de cortisol et une diminution de l’activité PKA. Ainsi, l’hypothèse pour expliquer les HBMS liées à une inactivation d’ARMC5 est que la perte d’apoptose conduit à une hyperplasie nodulaire du tissu corticosurrénalien et que, même si la production de cortisol est diminuée à l’échelle unicellulaire, l’effet de masse global conduit au total à un hypercortisolisme. Nos travaux ont donc identifié et caractérisé un premier gène causal, ARMC5, fréquemment impliqué dans l’HBMS et associé à des formes plus sévères de la maladie. Cette découverte ouvre des perspectives pour le diagnostic familial et la prise en charge des patients. (...) / Primary bilateral macronodular adrenal hyperplasia (PBMAH) is a rare cause of adrenal Cushing’s syndrome and bilateral adrenal tumors. We suspected a genetic origin of the disease on the basis of the report of some familial cases and the involvement of both adrenal glands. The aim of this study was to find a genetic cause of non syndromic PBMAH. To look at chromosomal abnormalities, we use single-nucleotide polymorphism (SNP) arrays and microsatellite markers analysis in a first series of 33 patients all operated for PBMAH. We realize whole genome sequencing of 5 patients (blood and tumor DNAs matched). Then we genotyped by Sanger sequencing the gene Armadillo Repeat Containing 5 (ARMC5) in this first series and 66 additional patients. Clinical data were collected to establish genotype-phenotype correlation. In addition, the cohort of patients of our collaborators at the National Institute Health (Dr. Stratakis, Bethesda, USA) was studied. The effects of ARMC5 inactivation and overexpression and the partners of the protein were sought in cell-culture models. The most frequent somatic alteration was a loss of heterozygosity at 16p observed in tumors of 25% of the patients. The gene ARMC5, located at 16p11.2, was the most frequently mutated by whole genome sequencing: a mutation was found in 4/5 patients. 55% of the patients of the first cohort (33 patients treated by adrenalectomy for PBMAH) had ARMC5 alteration. One patient presented with germline microdeletion of the locus identified by SNP array. Every patient had two events: either a mutation or a deletion at the germline level, either a second mutation or a LOH at the somatic level. We showed that the two events were present on different alleles suggesting that ARMC5 is a tumor suppressor gene. In addition, we showed for several patients that the second hit was different in each adrenal nodules of a same patient. This first cohort included only operated patients with serious forms of the disease. The study of the American cohort and the analysis of the total cohort of our lab including non-operated patients and milder forms showed an alteration of ARMC5 in about 25% of the patients. Genotype-phenotype correlation showed that ARMC5 defects are associated with younger age at the diagnosis, higher hypercortisolism, bigger adrenals and higher number of nodules. In addition, a mutation of ARMC5 was shown in a patient with a PBMAH secreting both aldosterone and cortisol. Analysis of a series of patient affected by primary hyperaldosteronism suggested that ARMC5 may be associated with hypertension especially in African-American subjet. Overexpression of ARMC5 leads in vitro to cell apoptosis. We showed that this apoptosis was reduced when transfecting vector harboring missense mutations or single amino-acid deletion found in our cohort. Invalidation of ARMC5 leads to a decreased steroidogenic enzymes expression, cortisol production and reduced protein kinase A (PKA) activity. We showed that ARMC5 interacts with the calaytic subunit alpha of the PKA dissociated from the cAMP-bound regulatory subunits. More than one quarter of sporadic PBMAH patients present a pathogenic germline ARMC5 defect and these index cases present a more severe disease. Systematic genotyping of ARMC5 may help for early diagnosis of PBMAH, familial counseling, and patients’ management. ARMC5 appears to be a new regulator of PKA and might represent a new target for the development of pharmacological agents controlling PKA function and cortisol production.

Das Klasse II Tumorsuppressor-Gen H-REV107-1, ist in normalen Geweben ubiquitär exprimiert, zeigt jedoch häufig Expressionsverluste, vorzugsweise in Tumoren des Brustgewebes, des Ovars und der Lunge. Das H-REV107-1 Protein wirkt in vitro und in vivo als Wachstumssuppressor. Um den Mechanismus der H-REV107-1 bedingten Wachstumshemmung zu verstehen, haben wir mit Hilfe des LexA-basierten Hefe-2-Hybrid Systems interagierende Proteine identifiziert. Diese Suche wurde mit einem H-REV107-1 Deletionskonstrukt durchgeführt, dem die Membran-bindende Domäne fehlte. Diese Analyse lieferte eine Vielzahl von potentiellen Interaktionspartnern, darunter der Retinsäure Rezeptor RARG, das Calcium-bindende Proteine S100A6, der Translations-Elongationsfaktor ETF und das weitgehend unbekannte Protein p14.5 Die Bindungen des H-REV107-1 Proteins an die beiden potentiellen Kandidaten, den Transkriptionsfactor PC4 und die regulatorische Untereinheit der Protein Phosphatase 2A (PR65), wurden genauer untersucht. Wir haben dabei einen Proteinkomplex aus H-REV107-1, PC4 und STAT1 (Signal Transducer and Activator of Transcription 1) identifiziert, der vermutlich eine Rolle in der IFNgamma - abhängigen Wachstumshemmung in Ovarialkarzinom Zellen spielt. Da sich die Expression des H-REV107-1 Gens durch IFNgamma über den Transkriptionsfaktor IRF-1 stimulieren läßt, und in verschiedenen Zelllinien sowohl zur Hemmung des Wachstums, als auch zur Apoptose führt, vermuteten wir verschiedene Mechanismen der Wachstumshemmung durch den IFNgamma-Signalweg und H-REV107-1. Weitere Analysen der H-REV107-1 - vermittelten Apoptose zeigten, daß die Interaktion zwischen H-REV107-1 und PR65 eine wichtige Rolle in diesem Prozeß spielt. Um die Proteindomäne zu identifizieren, welche für die direkte Wechselwirkung von H-REV107-1 mit PR65 verantwortlich ist, wurden H-REV107-1 Mutanten generiert und mittels Co-Immunpräzipitation getestet. Die Prolin-reiche Sequenz am N-Terminus des H-REV107-1 Proteins konnte als verantwortliche Domäne für die Interaktion bestimmt werden. Die funktionelle Analyse dieser Interaktion zeigte die Hemmung der Protein Phosphatase 2A (PP2A) Aktivität in Ovarialkarzinom Zellen durch H-REV107-1. Der Einsatz der Mutanten im Phosphatase-Aktivitätstest zeigte, daß die selbe Domäne, die die Interaktion vermittelt, auch für die Hemmung der Phosphatase 2A verantwortlich ist. Diese Fakten deuteten auf eine wichtige Rolle der Phosphatase 2A in Ovarialkarzinom Zellen hin, weil sowohl die Verwendung des PP2A Inhibitors (Okadainsäure), als auch die Transfektion der Zellen mit einem H-REV107-1 - Expressionsplasmid zur Apoptose führten. Damit konnten wir zeigen, daß PP2A für das Überleben der Ovarialkarzinomzellen notwendig ist, und die Reaktivierung des H-REV107-1 Proteins durch IFNgamma zur Hemmung der Phosphatase und damit zur Apoptose führt. / The H-REV107-1 class II tumor suppressor gene is ubiquitously expressed in normal tissues and down-regulated in human breast, ovarian and lung tumours. H-REV107-1 has the capacity to suppress growth of tumour cells in vitro and in vivo. To understand the mechanism of H-REV107-1 mediated growth suppression I performed a search for H-REV107-1 interacting proteins using a LexA-based yeast two-hybrid system. I screened a human kidney cDNA library with a truncated form of the H-REV107-1 as a bait. This analysis revealed numerous potential interaction partners. Among them the retinoic acid receptor gamma (RARG), the calcium-binding protein S100A6, the translation termination factor ETF1, and the potential translational inhibitor protein P14.5. The interaction of H-REV107-1 with the transcriptional co-activator PC4 and with the regulatory subunit A of protein phosphatase 2A (PR65) was analysed in detail. H-REV107-1 binds ectopically expressed and endogenous PC4. In addition, a multiprotein complex between H-REV107-1, PC4 and the signal transducer and activator of transcription 1 (STAT1) was demonstrated. This complex is likely to be involved in IFNgamma-mediated growth suppression of ovarian carcinoma cells. Endogenous H-REV107-1 can be induced after application of IFNgamma through the IRF-1 transcription factor. This up-regulation of H-REV107-1 leads either to growth suppression via a G1 arrest or to apoptosis depending on the cell line, suggesting different mechanisms of IFNgamma-, and H-REV107-1- mediated growth suppression. Further investigation of the mechanism of H-REV107-1-dependent apoptosis revealed an important role of the interaction between H-REV107-1 and the PR65 protein. The use of several H-REV107-1 mutant proteins generated after disruption of the highly conserved domains identified the proline-rich N-terminal domain responsible for the interaction with PR65 in Co-immunoprecipitation studies. Functional investigation of the H-REV107-1 - PR65 interaction demonstrated that wild-type H-REV107-1 is able to inhibit PP2A activity, however a mutant protein lacking the N-terminal domain was devoid of this function. We sought to identify the functional relevance of the PP2A activity in ovarian carcinoma cells with normally have suppressed the H-REV107-1 gene. Treatment of OVCAR-3 cells with the PP2A inhibitor Okadaic acid and transient transfection of the cells with wild-type H-REV107-1 resulted in the activation of caspase-9, suggesting a role for PP2A in the survival of ovarian carcinoma cells. We suggest, that the down-regulation of H-REV107-1 in ovarian carcinomas is a prerequisite for the PP2A-dependent activation of yet unknown signalling pathways mediating tumour cell survival. Reactivation of H-REV107-1 gene expression via IFNgamma leads to the inhibition of PP2A activity and tumour cell death.

A expressão do gene RECK é ubíqua em tecidos normais e não detectável nas linhagens celulares tumorais testadas e em fibroblastos transformados por diversos oncogenes. Inicialmente isolado como um gene indutor da reversão fenotípica tumoral→normal em fibroblastos transformados por v-Ki-ras, o gene RECK codifica uma glicoproteína de membrana que suprime a invasão tumoral e metástase através da regulação da metaloprotease de matriz-9. Para entender os mecanismos de inibição da expressão do gene RECK, mediada por oncogenes, isolou-se e caracterizou-se a região 5\'- flanqueadora do gene RECK de camundongo (mRECK). Ensaios de atividade promotora utilizando mutantes de deleção da região 5\' - flanqueadora e o gene repórter luciferase, revelaram que a sequência de 52 pb, imediatamente \"upstream\" ao gene, possui uma atividade promotora que é suprimida pelo produto do oncogene Ha-ras (V12). Esta sequência contém dois sítios de ligação a Sp1 (SplA e SplB), um sítio de ligação a cEBPb e um CAAT box. EMSAs e ensaios de atividade promotora, utilizando mutantes pontuais nestes sítios, revelaram que as proteínas Spl e Sp3 se ligam a ambos os sítios Spl, e que a resposta a Ras é mediada apenas pelo sítio SplB. Análise por Southern blot utilizando uma enzima de restrição sensível à metilação e por Northern blot de mRNA extraído de células tratadas com um agente desmetilante, sugeriram que o mecanismo de metilação de DNA não está envolvido na regulação transcricional do gene RECK. O envolvimento de RECK em diversos sistemas de proliferação, invasão e reversão celular foi analisado através de Northern blot. Os resultados sugerem que a expressão de RECK é regulada por soro na linhagem A3l de fibroblastos normais de camundongo. / The RECK gene is ubiquitously expressed in normal human tissues, but is downregulated both in tumor cell lines and in oncogenically transformed fibroblasts. Initially isolated as a tumor→normal phenotypic reversioninducing gene in v-ki-ras-transformed fibroblast, RECK encodes a membrane-anchored glycoprotein that suppresses tumor invasion and metastasis by regulating the matrix metalloproteinase-9. In order to understand the mechanism of oncogene-mediated suppression of RECK gene expression, we have isolated and characterized the 5\'-flanking region of the mouse RECK gene (mRECK). Deletion mutants constructs of this 5\' -flanking region with the luciferase reporter gene, revealed that the 52 base pairs upstream displays promoter activity which is suppressed by the Ha-ras (V12) oncogene. This region contains two Spl-binding motifs (SplA and SplB), one cEBPb-binding motif, and one CAAT box. Gel shift analysis and reporter gene assays, in combination with site-directed point mutations in these elements, revealed that both Spl sites associate with Spl as well as Sp3 proteins, although Ras- responsiveness seems to be mediated only by the downstream Spl site (SplB). Southern blot analysis using a methylation-sensitive restriction enzyme and Northern blot analysis with mRNA extracted from cells treated with a demethylating agent, indicated that the mechanism of DNA methylation is not involved in the regulation of RECK gene transcription. The role of RECK in several systems of cell proliferation, invasion and reversion, analysed by Northern blot, suggested that RECK expression is cell cycle regulated by serum in normal A3l mouse fibroblast cell line.

O carcinoma de célula renal (CCR) subtipo célula clara é o câncer mais letal e prevalente do sistema urinário. A transformação maligna no CCR está possivelmente associada à mudanças no perfil de expressão de oncogenes e genes supressores de tumor, e acredita-se que estas alterações sejam críticas para o desenvolvimento do fenótipo maligno. Para identificar novos genes e vias moleculares associadas à transformação maligna no CCR célula clara, foram analisados perfis de expressão gênica de amostras pareadas de tumor e tecido não tumoral adjacente de 6 pacientes. Foi utilizada uma plataforma de microarrays de cDNA contendo 2.292 sondas mapeando éxons de genes codificadores e 822 sondas de RNAs não-codificadores mapeando em regiões intrônicas. A transcrição intrônica foi detectada em todos os tecidos normais e neoplásicos. Utilizando uma combinação de dois testes estatísticos e uma validação por leave-one-out, foi selecionado um subconjunto de 64 transcritos com expressão significativamente alterada em CCR célula clara em relação ao tecido não tumoral adjacente, estando a maior parte (86%) com expressão diminuída em CCR. Entre os transcritos com expressão diminuída, 49 mapearam em regiões não-traduzidas ou éxons de genes codificadores e 6 mapearam em regiões intrônicas de genes codificadores conhecidos. Os níveis de expressão diminuída de SIN3B, TRIP3, SYNJ2BP e NDE1 (p < 0,02), e de transcritos intrônicos derivados dos loci de SND1 e ACTN4 (p < 0,05), foram confirmados em CCR célula clara por Real-time RT-PCR. Um subconjunto de 25 transcritos se mostrou alterado em 6 amostras adicionais de CCR não célula clara, indicando alterações transcricionais comuns em CCR independentemente do subtipo histológico ou do estado de diferenciação do tumor. Além disso, foi analisado o perfil de metilação dos genes com expressão diminuída em tumor SIN3B, TRIP3, SYNJ2BP e GPX3. Nossos resultados indicam um novo conjunto de candidatos a gene supressor de tumor, que 8 podem desempenhar um papel importante na transformação maligna de células renais normais. / The clear cell subtype of renal cell carcinoma (RCC) is the most lethal and prevalent cancer of the urinary system. The carcinogenesis in RCC is thought to be associated with changes in the expression of several genes, and this alteration in gene expression is believed to be critical to the development of the malignant phenotype. To investigate new genes and molecular pathways associated with malignant transformation in clear cell RCC, gene expression profiles of matched samples of tumor and adjacent non-neoplastic tissue obtained from 6 patients were analysed. A custom-built cDNA microarray platform was used, comprising 2,292 probes that map to exons of genes and 822 probes for noncoding RNAs mapping to intronic regions. Intronic transcription was detected in all normal and neoplastic renal tissues. A subset of 64 transcripts with levels significantly deregulated in clear cell RCC relative to the matched non-tumor tissue, mostly (86%) downregulated in CCR, was

O complexo da esclerose tuberosa (TSC) é um transtorno genético, sistêmico, com expressividade variável e herança autossômica dominante. Clinicamente manifesta-se devido ao desenvolvimento de hamartomas e hamártias em diferentes tecidos, principalmente no cérebro, rins, coração, pele e pulmões, podendo causar disfunção do órgão. Mutação em um de dois genes supressores tumorais, TSC1 ou TSC2, são responsáveis pelo TSC. Os genes TSC1 e TSC2 codificam para hamartina e tuberina, respectivamente. Ambas as proteínas interagem fisicamente formando um complexo citosólico que inibe mTOR (mammalian target of rapamycin). Testes moleculares para TSC1 e TSC2 são úteis para auxiliar no diagnóstico de casos clínicos difíceis, em aconselhamento genético e estudos de associação genótipo-fenotípica, além de permitirem a caracterização molecular de mecanismos patogenéticos da formação dos hamartomas e análises funcionais de seus produtos gênicos. Apesar de o diagnóstico de TSC ser basicamente clínico, a partir da revisão de seus critérios em 2012 por um grupo de especialistas, o achado de uma mutação em TSC1 ou TSC2 passou a ser considerado suficiente para o diagnóstico definitivo da doença. O estudo apresentado aqui é parte de um projeto em andamento para estabelecer condições ao desenvolvimento de análise de mutações causadoras de TSC nos genes TSC1 e TSC2. Nosso objetivo neste trabalho foi avaliar por sequenciamento de Sanger o DNA genômico de 28 pacientes brasileiros com diagnóstico definitivo de TSC, procedentes dos estados de São Paulo ou Paraná, tendo como alvo a sequência codificadora do gene TSC1, parte de seus segmentos intrônicos, o promotor basal, bem como o segmento imediatamente a 5\' deste. Sete pacientes (25%) apresentaram mutações de sentido trocado (nonsense) ou com deslocamento da leitura à tradução (frameshift) no gene TSC1. Entre 31 outras variantes de DNA encontradas, 23 são polimorfismos conhecidos e oito apresentaram frequência inferior a 1%, como verificado in silico entre mais de mil sequências de genomas humanos. Entre essas oito variantes de DNA novas ou raras, quatro foram detectadas em pacientes para os quais uma mutação patogênica havia sido identificada e, por isso, foram reclassificadas como polimorfismos. Duas e uma variantes de DNA do mesmo paciente flanqueavam um sítio de ligação em potencial para um fator de transcrição específico, a 5\' do promotor basal de TSC1. Por fim, uma nova variante de DNA na região não codificadora do éxon 2 do gene TSC1 foi predita com potencial para alterar um elemento candidato a acentuador de splicing. Em resumo, como observado em estudos anteriores, descrevemos aqui 25% dos pacientes com TSC apresentando mutações patogênicas na sequência codificadora do gene TSC1. Nossos dados mostraram quatro novas variantes de DNA em regiões potencialmente reguladoras da expressão do gene TSC1, que podem revelar-se como mutações patogênicas e, portanto, necessitam ser testadas experimentalmente / Tuberous sclerosis complex (TSC) is a multisystem disorder, with variable expression and autosomal dominant inheritance. Clinically it is due to hamartia and hamartoma development in different tissues, notably in the brain, kidneys, heart, skin and lungs, causing organ dysfunction. Mutations in either tumor suppressor gene, TSC1 or TSC2, are responsible for TSC. TSC1 and TSC2 genes code for hamartin and tuberin, respectively. Both proteins physically interact forming a cytosolic complex that inhibits the mammalian target of rapamycin (mTOR). TSC1 and TSC2 molecular testing has been useful in diagnosing clinically challenging cases, in genetic counseling and genotype/phenotype association studies, besides evaluation of the molecular basis of hamartoma formation and functional analyses of both gene products. Although TSC diagnosis is basically clinical, since the 2012 specialist panel review the finding of a TSC1 or TSC2 mutation has been considered sufficient for the definite diagnosis of the disease. The results presented here are part of an ongoing project to establish conditions for TSC1 and TSC2 mutation studies. Our first aim is to evaluate by Sanger sequencing TSC1 coding sequence, and an average of 132 base pairs of intronic regions next to exon boundaries from TSC patients, in addition to the gene core promoter. We present preliminary results of a sample of 28 patients with definite TSC diagnosis, from São Paulo and Paraná states. Seven patients (25%) displayed TSC1 nonsense or frameshift mutations. Among 31 other DNA variants identified, 23 were known polymorphisms, and eight had frequencies below 1% as verified in silico among more than a thousand human genomes. Out of eight novel or rare DNA variants, four were detected in patients for whom a pathogenic mutation had been found. Two and one additional DNA point variants from the same patient flanked a putative transcription factor binding site. Finally, a novel DNA variant residing in the TSC1 noncoding exon 2 was predicted to change the sequence potential to behave as a splicing enhancer. In summary, similar to previous studies, we describe 25% of TSC patients with mutations in the TSC1 coding sequence. Differently from other reports, our data disclose four novel DNA variants in TSC1 potentially regulatory regions that are likely to unravel novel pathogenic mutations, and thus need to be experimentally tested

As células tumorais prosperam como consequência da capacidade de resistir aos mecanismos de morte celular e de evasão da vigilância imunológica. Nós propomos que, em cânceres que possuem o supressor de tumor p53 selvagem, a remediação de ambas dessas defesas pode ser promovida pela transferência genica combinada de vetores adenovirais portadores dos transgenes de p19Arf (proteína supressora de tumor, parceira funcional de p53) e de interferon-beta (IFNbeta, citocina imunomoduladora). De fato, em resultados anteriores, notamos que a transdução combinada (p19Arf/IFNbeta), mas não os tratamentos individuais, em células de melanoma murino B16F10 resulta em aumento massivo de morte celular. Porém a capacidade destas células em processo de morte de desencadear imunidade antitumoral não foi analisada. Nesta tese e em estudos complementares, buscamos investigar os mecanismos moleculares de morte celular envolvidos na resposta imune estimulada por p19Arf/IFNbeta e explorar sua aplicação como imunoterapia do câncer. Inicialmente, em modelo de vacinação profilática, revelamos que o tratamento combinado em células B16F10 promove a expressão de IL-15, ULBP1, dos receptores de morte FAS/APO1 e KILLER/DR5, assim como uma resposta de células natural killer que rejeitam estas células tratadas quando inoculadas em camundongos imunocompetentes singênicos. Após desafio tumoral no flanco oposto, a progressão desses tumores foi fortemente reduzida devido ao engajamento de linfócitos T CD4+ e CD8+, que apresentaram produção aumentada das citocinas IFN-? e TNF-alfa e medeiam proteção antitumoral de longo prazo. Em seguida, explorando um contexto de imunização diferente, a transferência de gênica in situ foi realizada em carcinoma heterotópico de pulmão e exibiu proteção significativa contra um desafio tumoral secundário, apenas quando o tumor primário foi tratado com p19Arf/IFNbeta. Análise de transcriptoma destes tumores indicou uma assinatura quimiotáxica de neutrófilos e linfócitos T CD8+ através das quimiocinas CCL3, CXCL3 e da IL-1beta. Em apoio destas observações, análises mecanicistas in vitro revelaram que células tratadas com p19Arf/IFNbeta ativam programas apoptóticos de p53 e antivirais de IFNbeta, enquanto sucumbem a um processo de morte por necroptose que também libera moléculas de morte celular imunogênica (MCI), calreticulina, ATP e HMGB1. No entanto, procurando potencializar ainda mais o benefício terapêutico dos nossos vetores, exploramos sua associação com o quimioterápico imunogênico doxorrubicina (Dox), que também é indutor de MCI. E nesta associação, percebemos que a Dox aumenta não apenas os níveis de morte celular, mas também a imunogenicidade das células tratadas, proporcionando em um modelo de vacina terapêutica, um controle tumoral superior em camundongos que já portavam antes da vacinação tumores B16F10 ou MCA205. Além disso, a associação in situ destas terapias restaurou a eficácia de uma dose sub-terapêutica de Dox, que em contraste com sua dose terapêutica, não prejudica a função cardíaca. Finalmente, também exploramos a associação com o bloqueio dos pontos de controle imunológicos PD-1 ou CTLA-4, que no modelo de vacina terapêutica, sua associação induziu maior rejeição completa de tumores B16F10. Em conclusão, aqui apresentamos evidências sobre a capacidade da combinação p19Arf/IFNbeta de induzir morte celular e estimulação imunológica. E ressaltamos seu potencial como uma estratégia de imunoterapia do câncer / Cancer cells thrive as a consequence of resisting cell death mechanisms and escaping from immune surveillance. We propose that, in cancers that harbor the wild-type tumor suppressor p53, remediation of both of these defenses can be achieved by harnessing the adenoviral vector mediated gene transfer of p19Arf (tumor suppressor protein, p53 functional partner) together with interferon-beta (IFNbeta, immunomodulatory cytokine). Indeed, in our initial observations, it was noticed that combined-transduction (p19Arf/IFNbeta), but not the individual treatments, of B16F10 mouse melanoma cells results in massive cell death levels. Yet, the capability of these dying cells to unleash antitumor immunity was not investigated. Here in this thesis and in complementary studies, we sought to investigate the molecular mechanisms of cell death involved in the p19Arf/IFNbeta immune stimulation and explore its potential as a mediator of cancer immunotherapy. First, in a prophylactic B16F10 vaccine model, we revealed that the dual treatment led to the up-regulation of IL-15, ULBP1, FAS/APO1 and KILLER/DR5 death receptors, plus a natural killer cell response that completely rejects treated cells when inoculated in syngeneic immunocompetent mice. Whereas, upon a contralateral tumor challenge, progression was strongly reduced by engaging both CD4+ and CD8+ T cells, which displayed augmented production of IFN-? and TNF-alpha cytokines and provided long term antitumor protection. Next, exploring different immunization context, in situ gene transfer in a heterotopic lung carcinoma exhibited significant protection against a secondary tumor challenge only when the primary tumor was treated with p19Arf/IFNbeta. Transcriptome analysis of these treated tumors indicated a chemotaxic signature of neutrophils and CD8+ T cells with the involvement of CCL3, CXCL3 chemokines and IL-1beta. Moreover, in support of this evidence, mechanistic in vitro studies revealed that p19Arf/IFNbeta treated cells reactivate p53 apoptotic and IFNbeta antiviral programs, while succumbing to a necroptosis cell death processes that also releases immunogenic cell death (ICD) molecules, calreticulin, ATP and HMGB1. Yet, aiming to potentiate therapeutic benefit of our vectors, we explored their association with doxorubicin (Dox) immunogenic chemotherapy, which is also an inducer of ICD. And in this setting, this association with Dox enhances not only cell death levels but also immunogenicity of treated cells, providing superior tumor control in a therapeutic vaccine model, where mice were already bearing B16F10 tumors or MCA205 sarcomas before vaccination. Moreover, associated use of these therapies in situ rescued efficacy of a sub-therapeutic dose of Dox, which in contrast to its therapeutic dose, does not impair cardiac function. Finally, we also evaluated the association with PD-1 or CTLA-4 checkpoint blockade immunotherapy, which in the therapeutic vaccine model induced full tumor rejection in a greater number of mice. In sum, here we provide compelling evidence for the ability of the p19Arf/IFNbeta combined gene transfer to promote cell death and immunogenic stimuli and underscored its potential to be applied as a cancer immunotherapy strategy

Aproximadamente 80% dos casos com Neoplasia endócrina múltipla tipo 1 (NEM1) possuem mutações germinativas no gene supressor de tumor MEN1, que os predispõem a tumores nas glândulas paratireóides, pâncreas endócrino e hipófise, além de outros tumores não endócrinos. A tumorigênese dos mais de 20 diferentes tipos de neoplasias já descritas na NEM1 ocorre pela presença da mutação germinativa MEN1 associadas a um segundo evento mutacional nas células desses tecidos, levando à perda de heterozigose (LOH) do locus do gene MEN1 (11q13) e à inativação da proteína supressora de tumor codificada por esse gene, a proteína MENIN. Recentemente, mutações germinativas em outros genes foram descritas em casos com NEM1 sem mutações no gene MEN1. Esses novos genes (CDKN1A, CDKN1B, CDNK2B e CDKN2C) codificam proteínas envolvidas no controle do ciclo celular (p21, p27, p15 e p18), chamadas proteínas inibidoras de quinases dependentes de ciclinas. Outro gene, chamado AIP, que codifica uma proteína chaperona de mesmo nome, também foi recentemente descrito associado à NEM1. Esses trabalhos descreveram o papel desses novos genes na NEM1, em nível germinativo, entretanto não esclareceu se esses novos genes estão inativados nos tumores de pacientes com NEM1 com mutação MEN1. O presente estudo investigou, pela primeira vez, o status somático do gene p27Kip1 em pacientes com mutação MEN1 e identificou quatro possíveis perda de heterozigose (LOH) em tumores de paratireóides e pâncreas, sugerindo que além de 11q13LOH, os tumores NEM1 podem sofrer raras perdas adicionais do gene supressor tumoral p27Kip1. Essas são as primeiras evidências na literatura de um processo de tumorigênese multi-step na NEM1, envolvendo três eventos genéticos: 1- Mutação germinativa MEN1; 2- 11q13-LOH; 3- Perda somática do gene supressor de tumor p27Kip1/CDKN1B / Approximately 80% of cases with multiple endocrine neoplasia type 1 (MEN1) harbor a germline mutation in the tumor suppressor gene MEN1, which predisposes these patients to tumors comprehending the parathyroid and pituitary glands, endocrine pancreas and others non-endocrine tumors. The tumorigenesis of the more than 20 different types of tumors already described in the MEN1 syndrome occurs due to a MEN1 germline mutation associated with a second mutational event in the cells of these tissues, leading to loss of heterozygosity (LOH) of the MEN1 gene locus (11q13) and therefore inactivation of tumor suppressor protein encoded by this gene, MENIN protein. Recently, germline mutations in other genes have been described in cases with MEN1 without any detectable mutations in the MEN1 gene. These novel genes (CDKN1A, CDKN1B, CDNK2B and CDKN2C) encode proteins involved in the control of the cell cycle (p21, p27, p15 and p18), called cyclin dependent kinases inhibitors. Another gene, called AIP, which encodes a chaperon protein with the same name, was recently described associated with MEN1 phenotypes. These data described a role for these novel genes in the germline level, however whether they are inactivated in tumors of patients with MEN1 mutation is so far not clarified. The present study investigated for the first time, the somatic status of p27KIP1 gene mutation in patients with MEN1 and identified four possible loss of heterozygosity (LOH) in tumors of the parathyroid and pancreas, suggesting that in addition 11q13-LOH, MEN1 tumors may suffer rare loss additional tumor suppressor gene p27KIP1. These are the first evidence in the literature of a process of tumorigenesis in MEN1 multi-step, involving three genetic events: 1-MEN1germlinemutation; 2-11q13LOH; 3-Loss of somatic tumor suppressor gene p27Kip1/CDKN1B

Cancer is the leading cause of death in Hong Kong (21,300 new cases and 11,500 deaths in 2003), with nasopharyngeal carcinoma (NPC), esophageal cancer (ESCC), and colorectal cancer (CRC) among the common ones. For these tumors, most patients present with advanced stage disease and poor treatment outcome, with an urge of early detection. Epigenetic inactivation of tumor suppressor genes (TSG) by CpG methylation represents an important mechanism of tumorigenesis, in addition to genetic abnormalities. Tumor-specific methylation can also be used as biomarkers for the identification of novel TSGs and for cancer early diagnosis and prognosis prediction. / Finally, for the purpose of development of epigenetic biomarker for cancer molecular diagnosis, I screened gene methylation in the serum samples. Aberrant methylation of PCDH10 and DLC1 was detected in serum samples (2/14 (14%) and 4/14 (29%) respectively) from tumor patients but not in normal controls. It suggests that screening for PCDH10 and DLC1 methylation in sera could be a tumor-specific and non-invasive epigenetic biomarker for molecular diagnosis and prognostics. (Abstract shortened by UMI.) / In the second approach, 1-Mb array-based comparative genomic hybridization (aCGH) was carried out to detect DNA copy number aberrations, which contain potential TSG loci, in a panel of NPC and ESCC cell lines. Frequent deletions include: 1p36.3, 3p14-11, 4p16-15, 5p13-q12, 6p21-12, 8p22-cent, 9p, 9q22-31, 10p, 13q12, 14q32, 16q23-24, 17q11.2, 18q in NPC, and 1p21, 4q21, 7p21, 7q35, 8p22-23, 8q11, 10p11, 11q22, 13q31, 14q32, 18q11-23 in ESCC. Several deletions (3p14-11 and 16q23) were further investigated in detail in this study. More than 12 genes were identified to be frequently silenced by methylation in tumors, including FHIT (3p14), WNT5A (3p14), ADAMTS9 (3p14), FEZF2 (3p14), ROBO (3p12), CADM2 (3p12), EPHA3 (3p11), RAB (11q22), ADAMTS18 (16q23), and TUSC8 (16q23), while homozygous deletion of these genes was infrequently detected. Aberrant methylation of these genes was also frequently detected in primary tumors in a tumor-specific manner. The tumor suppressor functions of TUSC8, WNT5A, CADM2 and ROBO were further investigated and validated. Further experiment indicated that induction of tumor cell apoptosis may contribute to the tumor suppressor function of TUSC8. / Modified genomic methylation subtractive approaches using uracil-DNA glycosylase or combined with pharmacological demethylation were developed. GADD45G, PCDH10, ROR2, DLC1L1 were among a series of novel methylated targets identified by these approaches. Methylation-associated silencing of these genes was frequently detected in various types of tumor cell lines and primary tumors including NPC, ESCC and CRC, in a tumor-specific manner. Ectopic expression of these genes strongly suppressed tumor cell growth and colony formation of silenced tumor cells. Epigenetic inactivation of GADD45G is the major mechanism for the loss of its response to environmental stresses. Reintroduction of PCDH10 strongly suppressed tumor cell migration and invasion. Ectopic expression of DLC1L1 in silenced tumor cells resulted in a remarkable suppression of tumor cell clonogenicity, which depends on its GAP activity. Furthermore, DLC1L1, but not its inactivating mutants, inhibited Ras mediated oncogenic transformation. Thus, these identified genes are functional TSGs. / Ying Jianming. / "July 2007." / Adviser: Qian Tao. / Source: Dissertation Abstracts International, Volume: 69-01, Section: B, page: 0083. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 147-173). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

Poon Cho Sun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 83-91). / Abstract also in Chinese. / ACKNOWLEDGEMENT --- p.v / ABSTRACT --- p.vi / LIST OF ABBREVIATIONS --- p.x / Chapter Chapter 1 --- Introduction --- p.1 / Chapter Chapter 2 --- Literature review --- p.5 / Chapter 2.1 --- Epidemiology and aetiology of cervical cancer --- p.5 / Chapter 2.1.1 --- Incidence and mortality --- p.5 / Chapter 2.1.2 --- Aetiology --- p.6 / Chapter 2.1.2.1 --- Oral contraceptive pills and cervical cancer --- p.7 / Chapter 2.1.2.2 --- Human papilloma virus (HPV) and cervical cancer --- p.7 / Chapter 2.1.2.3 --- Immunity and cervical cancer --- p.8 / Chapter 2.1.2.4 --- Socio-economic differences and cervical cancer --- p.9 / Chapter 2.1.2.5 --- Smoking and cervical cancer --- p.9 / Chapter 2.1.2.6 --- Male role and cervical cancer --- p.9 / Chapter 2.1.2.7 --- Nutrition and cervical cancer --- p.10 / Chapter 2.2 --- Oncogenes and tumour suppressor genes --- p.10 / Chapter 2.2.1 --- Oncogene --- p.10 / Chapter 2.2.2 --- Tumour suppressor gene --- p.13 / Chapter 2.2.3 --- Alterations of oncogene in cervical cancer --- p.16 / Chapter 2.2.4 --- Alterations of tumour suppressor genes in cervical cancer --- p.18 / Chapter 2.3 --- Alterations of chromosome 1 in cervical cancer --- p.19 / Chapter 2.3.1 --- Cytogenetic tudy --- p.19 / Chapter 2.3.2 --- Molecular genetic study --- p.21 / Chapter 2.4 --- Loss of heterozygosity (LOH) --- p.21 / Chapter Chapter 3 --- Materials and methods --- p.24 / Chapter 3.1 --- Materials --- p.24 / Chapter 3.1.1 --- Patients --- p.24 / Chapter 3.1.2 --- Specimens --- p.24 / Chapter 3.1.2.1 --- Blood samples --- p.24 / Chapter 3.1.2.2 --- Tumour tissue specimens --- p.24 / Chapter 3.1.3 --- Chemicals and reagents --- p.25 / Chapter 3.1.3.1 --- Chemicals --- p.25 / Chapter 3.1.3.2 --- Reagents --- p.27 / Chapter 3.1.3.3 --- Markers --- p.29 / Chapter 3.1.4 --- Major equipment --- p.33 / Chapter 3.2 --- Methodology --- p.33 / Chapter 3.2.1 --- DNA extraction --- p.33 / Chapter 3.2.2 --- DNA amplification --- p.35 / Chapter 3.2.2.1 --- Validation of PCR primers and optimisation of PCR condition --- p.35 / Chapter 3.2.2.2 --- End labelling of the primer by (γ-32p)ATP --- p.35 / Chapter 3.2.2.3 --- PCR for LOH detection --- p.36 / Chapter 3.2.2.4 --- Electrophoresis --- p.37 / Chapter 3.2.2.5 --- Gel dry and radioautography --- p.38 / Chapter 3.2.2.6 --- PCR analysis of the D1S80 and D1S76 loci --- p.39 / Chapter 3.3 --- Determination of Loss of heterozygosity (LOH) --- p.39 / Chapter 3.4 --- Statistical analysis --- p.40 / Chapter Chapter 4 --- Results --- p.41 / Chapter 4.1 --- LOH analysis in cervical cancer --- p.41 / Chapter 4.2 --- LOH and age in cervical cancer --- p.60 / Chapter 4.3 --- LOH and pathological grade in cervical cancer --- p.62 / Chapter 4.4 --- LOH and clinical stage in cervical cancer --- p.64 / Chapter 4.5 --- LOH and clinical status in cervical cancer --- p.66 / Chapter Chapter 5 --- Discussion --- p.68 / Chapter 5.1 --- Microsatellite markers --- p.69 / Chapter 5.2 --- PCR condition --- p.70 / Chapter 5.3 --- LOH in cervical cancer --- p.72 / Chapter 5.4 --- Correlation of LOH with clinico-pathologic characteristics of cervical cancer --- p.76 / Chapter 5.4.1 --- LOH and age --- p.78 / Chapter 5.4.2 --- LOH and clinical stage --- p.78 / Chapter 5.4.3 --- LOH and pathologic grade --- p.79 / Chapter 5.4.4 --- LOH and clinical status --- p.79 / Chapter Chapter 6 --- Conclusion --- p.80 / Chapter Chapter 7 --- References --- p.83

As maiores taxas de incidência de câncer de mama ocorrem em mulheres idosas, que apresentam tumores com expressão de receptores de estrógeno e/ou progesterona, de baixo estadiamento e menor taxa de proliferação, se comparado com as jovens. Um dos fatores de predisposição ao câncer de mama é mutação germinativa nos genes BRCA1 ou BRCA2, que podem compreender entre 5-10% das pacientes diagnosticadas. A grande maioria dos casos são ditos esporádicos, em que não há como estabelecer um único fator determinante. Dentre o escopo de possíveis causas estão as mutações somáticas, acumuladas no tecido mamário ao longo da vida. A identificação destas mutações permite melhor compreensão da carcinogênese e possibilita a criação de tratamentos cada vez mais personalizados. O gene PIK3CA, por exemplo, já está determinado como driver (responsáveis pela obtenção de vantagem seletiva de um determinado clone) para câncer de mama. As mutações patogênicas que ocorrem neste gene levam a ativação da via de Akt/mTOR, entre outras, que mantém o ciclo celular ativo. Um gene que vem sendo estudado recentemente é o PRKD1, cujas funções parecem estar ligadas à manutenção do fenótipo epitelial das células do tecido mamário. Assim, o objetivo desse trabalho identificar mutações germinativas nos genes BRCA1 e BRCA2, analisando também o histórico familiar para câncer de mama/ovário/próstata, e mutações somáticas no gene PRKD1 em pacientes pós menopausadas,. Foram incluídas quarenta e nove pacientes diagnosticadas com carcinoma ductal invasivo em idade superior a 54 anos, que preenchessem critérios da NCCN (National Comprehensive Cancer Network) para Síndrome de Câncer de Mama e/ou Ovário Hereditário e tinham disponível um fragmento tumoral emblocado em parafina coletado na ausência de tratamento neo adjuvante. A extração de DNA foi realizada a partir do sangue periférico para sequenciamento de BRCA1 e BRCA2, realizado através da plataforma Ion Torrent(TM) ou pelo método de Sanger. Os resultados obtidos por Ion Torrent(TM) foram analisados, primeiramente, através da ferramenta online Ion Reporter e os de Sanger através do programa Mutation Surveyor v.3.20. Para a caractetização das variantes encontradas foram utilizados: os bancos de dados BIC, LOVD, LOVD-IARC, UMD e ClinVar além dos preditores in silico da conservação dos aminoácidos entre as espécies Polyphen-2, SIF, Provean e AlignGVGD e do preditor de efeito no splicing HSF e bancos de dados de frequência alélica ExAC, 1000 genomas e NHLBI GO Exome Sequencing Project, seguindo os critérios da American College of Medical Genetics and Genomics em conjunto com a Association for Molecular Pathology. Para caracterização de mutação somática do gene PRKD1 determinou-se duas regiões de maior importância para serem sequenciadas: Ser738/Ser742 e Ser910 que fosforilam o domínio quinase da proteína, ativando-o. Vinte e três amostras tumorais tiveram DNA extraído. Também foi realizada uma análise das informações sobre PRKD1 do banco de dados COSMIC (Catalogue of Somatic Mutations in Cancer) e a construção de curvas de sobrevida (Kaplan-Meier) da expressão de PRKD1 utilizando a ferramenta online KM Plotter. A idade mediana das pacientes foi de 62 anos ao diagnóstico e de 64 anos na época de inclusão no estudo. A maioria tinha tumores de grau histológico II (63,27%), estádio clinico II (20%) e do subtipo luminal B (53,06%). Trinta e duas relataram parentes de primeiro grau afetados com câncer de mama/ovário/ próstata. Trinta e oito pacientes tiveram sequenciamento completo de BRCA1 e BRCA2 por Ion Torrent(TM) e onze tiveram sequenciamento parcial de BRCA1 e BRCA2 por Sanger. Variantes patogênicas foram encontradas em quatro pacientes (BRCA1=2/BRCA2=2). Uma nova variante missense foi identificada em BRCA2: c.3371A > G (p.Q1124R). Para o sequenciamento de PRKD1 quinze foram sequenciadas para Ser910 e de oito foi possível analisar o resultado. Nenhuma variante patogênica foi encontrada. Os dados obtidos sobre PRKD1 no COSMIC foram: de 2773 amostras, em apenas 15 (0,54%) foram identificadas mutações em PRKD1, 46% (7/15) provém de mulheres com idade superior a 55 anos e subtipo molecular Luminal. PRKD1 apresenta maiores frequência de mutação em câncer de intestino grosso (4,22%) e pele (4,02%). As curvas de sobrevida construídas no KM Plotter demonstram a alta expressão do gene parece ter impacto positivo na sobrevida das pacientes. Apesar da baixa frequência de mutações no PRKD1 este gene, outros dados demonstram que parece ter um papel de gene supressor de tumor no câncer de mama, que deve ser inibido de através de outros mecanismos como metilaçao de DNA / The highest rates of breast cancer incidence occur in elderly women, who present estrogen and / or progesterone receptor tumors, with a low clinical staging and lower proliferation rate compared to the young women. One of the factors predisposing to breast cancer is germline mutation in the BRCA1 or BRCA2 genes, which may comprise between 5-10% of the diagnosed patients. The vast majority of cases are said to be sporadic, in which there is no way to establish a single determining factor. Among the scope of possible causes are somatic mutations, accumulated in the breast tissue throughout life. The identification of these mutations allows a better understanding of carcinogenesis and enables the creation of increasingly personalized treatments. The PIK3CA gene, for example, is already determined as a driver (responsible for the selective advantage of a particular clone) for breast cancer. The pathogenic mutations that occur in this gene lead to the activation of Akt / mTOR pathway, among others, which keeps the cell cycle active. One gene that has recently been studied is PRKD1, whose functions seem to be linked to the maintenance of the epithelial phenotype of the mammary tissue cells. Thus, the objective of this work was to identify germline mutations in BRCA1 and BRCA2 genes, also analyzing the family history for breast / ovarian / prostate cancer, and somatic mutations in the PRKD1 gene in postmenopausal patients. Forty-nine patients diagnosed with ipsilateral ductal carcinomas over the age of 54 years who completed NCCN (National Comprehensive Cancer Network) criteria for Breast Cancer and / or Hereditary Ovarian Syndrome and had a tumor paraffin embedded in paraffin collected in the absence of neo adjuvant treatment available. DNA extraction was performed from the peripheral blood for sequencing of BRCA1 and BRCA2, performed through the Ion Torrent (TM) platform or by the Sanger method. The results obtained by Ion Torrent (TM) were first analyzed through the online tool Ion Reporter and those by Sanger through the program Mutation Surveyor v.3.20. The BIC, LOVD, LOVD-IARC, UMD and ClinVar databases were used in addition to the in silico predictors of amino acid conservation among Polyphen-2, SIF, Provean and AlignGVGD species and the effect predictor in the HSF splicing and allelic frequency databases ExAC, 1000 genomes and the NHLBI GO Exome Sequencing Project, following the criteria of the American College of Medical Genetics and Genomics in conjunction with the Association for Molecular Pathology. In order to characterize the somatic mutation of the PRKD1 gene, we determined two regions of greater importance to be sequenced: Ser738 / Ser742 and Ser910 that phosphorylate the protein kinase domain, activating it. Twenty-three tumor samples had DNA extracted. An analysis of PRKD1 information from the COSMIC (Catalog of Somatic Mutations in Cancer) database and the construction of survival curves (Kaplan-Meier) for PRKD1 expression using the online KM Plotter tool was also performed. The median age of the patients was 62 years at diagnosis and 64 years at the time of inclusion in the study. Most of them had tumors of histological grade II (63.27%), clinical stage II (20%) and molecular subtype luminal B (53.06%). Thirty-two reported first-degree relatives affected with breast / ovarian / prostate cancer. Thirty-eight patients had BRCA1 and BRCA2 complete sequencing by Ion Torrent (TM) and eleven had BRCA1 and BRCA2 partial sequencing by Sanger. Pathogenic variants were found in four patients (BRCA1 = 2 / BRCA2 = 2). For PRKD1 sequencing, fifteen patients tumors were sequenced for Ser910 and in eight samples it was possible to analyze the result. No pathogenic variant was found. The data obtained on PRKD1 in COSMIC were: from 2773 samples, in only 15 (0.54%) mutations were identified in PRKD1, 46% (7/15) came from women aged over 55 years and had tumor molecular subtype Luminal. PRKD1 shows higher mutation frequency in cancer of the large intestine (4.22%) and skin (4.02%). The survival curves constructed in KM Plotter demonstrate the high expression of the gene seems to have a positive impact on the patients survival . Despite the low frequency of mutations in PRKD1 gene, other data demonstrate that it appears to play a role of tumor suppressor gene in breast cancer, which must be inhibited by other mechanisms such as DNA methylation