Abstract Mutations in the currently known breast cancer susceptibility genes account for only 25–30% of all familial cases. Novel susceptibility genes can be identified by several methods, including candidate gene re-sequencing and genome-wide microarrays. We have applied microarrays for the detection of a new genomic variation class, copy number variants (CNVs), which potentially could disrupt genes in multiple pathways related to breast cancer susceptibility. The aim of the current study was to evaluate the role of the RNF8, UBC13, MMS2 and RAD51C DNA damage response genes in breast cancer susceptibility as well as to study if rare CNVs are associated with the predisposition to this disease. The analysis of 123 familial breast cancer cases revealed altogether nine different changes in the RNF8 and UBC13 candidate genes. However, none of the observed alterations were considered pathogenic. No alterations were observed in MMS2. The obtained results suggest that breast cancer predisposing alterations in RNF8, UBC13 and MMS2 are rare, or even absent. The RAD51C mutation screening of 147 familial breast cancer cases and 232 unselected ovarian cancer cases revealed two deleterious mutations: c.-13_14del27 was observed in a breast cancer case with familial history of ovarian cancer and c.774delT in an ovarian cancer case. Both mutations were absent in the control cohort. The results of the study support the hypothesis that rare variants of RAD51C predispose predominantly to ovarian cancer. A genome-wide scan of CNVs was performed for 103 familial breast cancer cases and 128 controls. The biological networks of the genes disrupted by CNVs were different between the two groups. In familial breast cancer cases, the observed mutations disrupted genes, which were significantly overrepresented in cellular functions related to maintenance of genomic integrity (P=0.0211). Biological network analysis showed that the disrupted genes were closely related to estrogen signaling and TP53-centered tumor suppressor network, and this result was confirmed by the analysis of an independent young breast cancer cohort of 75 cases. These results suggest that rare CNVs represent an alternative source of genetic variation contributing to hereditary risk for breast cancer. / Tiivistelmä Tunnetut rintasyöpäalttiusgeenien mutaatiot selittävät vain 25–30 prosenttia kaikista perinnöllisistä rintasyöpätapauksista. Uusia alttiusgeenejä voidaan tunnistaa useilla eri menetelmillä, kuten kandidaattigeenien mutaatiokartoituksella ja genomin-laajuisilla mikrosirutekniikoilla. Tässä tutkimuksessa sovelsimme mikrosirutekniikkaa uuden geneettisen variaatioluokan, kopiolukuvariaation (CNV), tutkimiseen. CNV:t voivat vaurioittaa lukuisia rintasyöpäalttiuteen liittyviä biokemiallisia reittejä. Tämän tutkimuksen tarkoitus oli arvioida RNF8-, UBC13-, MMS2- ja RAD51C -DNA- vauriovastegeenien sekä harvinaisten CNV:iden yhteyttä rintasyöpä-alttiuteen. 123 familiaalisen rintasyöpätapauksen analyysissä löytyi yhteensä yhdeksän muutosta RNF8- ja UBC13-geeneistä, joista yksikään ei osoittautunut patogeeniseksi. MMS2-geenissä ei havaittu muutoksia. Tulosten perusteella rintasyövälle altistavat muutokset RNF8-, UBC13- ja MMS2- geeneissä ovat joko erittäin harvinaisia tai niitä ei esiinny lainkaan. RAD51C-geenin mutaatiokartoitus 147 familiaalisesta rintasyöpätapauksesta sekä 232 valikoimattomasta munasarjasyöpätapauksesta paljasti kaksi haitallista mutaatiota. c.-13_14del27 havaittiin rintasyöpäpotilaalla, jonka suvussa esiintyi munasarjasyöpää, ja c.774delT todettiin munasarjasyöpäpotilaalta. Kumpaakaan mutaatiota ei havaittu verrokkiaineistossa. Tulokset vahvistavat hypoteesia RAD51C-geenin harvinaisten varianttien yhteydestä pääasiassa munasarjasyöpäriskiin. CNV:iden genomin-laajuinen skannaaminen suoritettiin 103 familiaaliselle rintasyöpätapaukselle ja 128 verrokille. CNV:iden häiritsemien geenien muodostamat biologiset verkostot olivat erilaiset näiden kahden ryhmän välillä. Familiaalisilla rintasyöpätapauksilla havaitut CNV:t vaikuttivat geeneihin, jotka olivat voimakkaasti korostuneita genomin eheyttä ylläpitävissä tehtävissä (P=0.0211). Biologisten verkostojen analyysi paljasti, että CNV:iden vahingoittamat geenit liittyivät läheisesti estrogeenisignalointiin sekä TP53-tuumorisupressoriverkostoon, ja tämä tulos vahvistettiin analysoimalla riippumatonta nuorista rintasyöpäpotilaista koostuvaa kohorttia (N=75). Tutkimuksen tulosten mukaan harvinaiset CNV:t ovat vaihtoehtoinen geneettisen variaation lähde perinnölliseen rintasyöpäalttiuteen.

L’ubiquitination est une modification post-traductionnelle des protéines qui consiste à attacher, d’une manière covalente, le groupement ubiquitine sur un résidu lysine de la protéine cible. Cette modification peut avoir un impact considérable sur la fonction, la localisation et la stabilité de ces cibles. Une fois établie par des enzymes appelées E3 ligases, l’ubiquitination peut être enlevée par des enzymes spécifiques appelées déubiquitinases, modulant ainsi les effets causés par cette modification. BAP1 (BRCA1-Associated Protein 1) est une déubiquitinase de la famille des UCH (Ubiquitin C-terminal Hydrolases) qui a été initialement identifiée comme partenaire du suppresseur de tumeurs BRCA1 (BReast Cancer Associated gene 1). De nombreux groupes de recherche, incluant le nôtre, ont montré que BAP1 est associée avec d’autres cofacteurs formant un large complexe multiprotéique. Ce dernier est impliqué dans plusieurs processus cellulaires comme la transcription des gènes, la régulation de la chromatine, la coordination du cycle cellulaire et la réponse aux dommages à l’ADN. La cible majeure de BAP1 est l’histone H2A ubiquitinée sur la lysine 119, une marque d’histone qui a été souvent associée avec une conformation répressive de la chromatine. Quels sont les mécanismes régulant le complexe BAP1 lui permettant d’exécuter ces fonctions biologiques? Cela implique-t-il des modifications post-traductionnelles touchant les partenaires de BAP1 ? Ces questions restent encore sans réponse définitive. Ainsi, les objectifs de cette thèse sont de caractériser le mécanisme et la fonction du complexe BAP1 en étudiant les modifications post-traductionnelles de ses partenaires. Pour répondre à ces questions nous avons étudié les modifications post-traductionnelles touchant BAP1 et ses cofacteurs mutuellement exclusifs ASXL1 et ASXL2 (Additional Sex Comb-like 1,2). Nous avons démontré qu’ASXL1 et ASXL2 sont monoubiquitinés uniquement lorsqu’ils sont associés à BAP1. Sachant que les complexes BAP1/ASXLs sont conservés au cours de l’évolution, nous avons aussi démontré que la monoubiquitination des ASXLs est conservée chez la Drosophile. En utilisant des méthodes de déplétion de protéines par siARN et CRISPR/Cas9 ainsi que des mutants de perte de fonction de BAP1 et ASXL2, nous avons identifié les enzymes responsables de la monoubiquitination des ASXLs ainsi que leur effet sur l’activité catalytique de BAP1. D’autre part, nous avons étudié le développement chez la Drosophile ainsi que le cycle cellulaire des cellules humaines pour identifier la fonction biologique de la monoubiquitination de ASXL2. Nos résultats démontrent que la monoubiquitination d’ASXL2 sur la lysine 370 en présence de BAP1 est une modification post-traductionnelle conservée et catalysée directement par la famille UBE2Es des enzymes de conjugaison de l’ubiquitine (UBE2E1,2,3 chez les mammifères et UbcD2 chez la Drosophile). Cette monoubiquitination stimule l’activité catalytique de BAP1 chez les mammifères et de son orthologue Calypso chez la Drosophile envers H2Aub. Le blocage de la monoubiquitination des ASXLs par des mutations ciblant la lysine K370 induit une inhibition de l’activité de BAP1, ce qui cause une dérégulation du cycle cellulaire chez les cellules mammifères et une transformation homéotique haltère-aile chez la Drosophile. De plus, il nous a été possible de constater l’importance de cette monoubiquitination dans le cancer en démontrant la forte corrélation d’expression de BAP1/ASXL2 et les UBE2Es au niveau du mésotheliome, un cancer connu pour la dérégulation de BAP1. Nos résultats indiquent l’importance des modifications post-traductionnelles, dont la monoubiquitination, dans la régulation de la fonction et la stabilité du complexe BAP1. De plus, nous décrivons un nouveau mécanisme d’activation d’une deubiquitinase par la monoubiquitination de son cofacteur. D’autres études seront nécessaires afin de comprendre le lien entre l’activation de BAP1/ASXL2 par monoubiquitination et la fonction suppresseur de tumeurs de BAP1 via la deubiquitination d’H2Aub. D’autre part, nous avons fait l’observation que la déplétion de la deubiquitinase associée à la particule régulatrice du protéasome, PSMD14, induit non seulement une réduction drastique d’H2Aub dans la cellule, mais aussi une mort cellulaire rapide. Ceci nous a poussé initialement à investiguer l’implication de l’activité catalytique du protéasome dans la régulation d’H2Aub en lien avec la mort cellulaire. Malgré le fait que nous n’ayons pas trouvé un lien direct entre PSMD14 et la deubiquitination d’H2Aub, nous avons identifié plusieurs candidats (DUBs et E2s) impliqués dans l’induction de la mort cellulaire tout en surmontant une résistance acquise contre des inhibiteurs ciblant l’activité catalytique du protéasome. Ces candidats pourraient représenter des cibles intéressantes pour développer des inhibiteurs spécifiques afin de contrecarrer la résistance aux inhibiteurs du protéasome. / Ubiquitination is a post-translational modification of proteins that involves covalently attaching the ubiquitin moiety to the lysine residues of the target protein. This modification has been reported to have a significant impact on the function, localization and stability of these targets. Once established by enzymes called E3 ligases, ubiquitination can be removed by specific enzymes called deubiquitinases, thus modulating the effects caused by this modification. BAP1 (or BRCA1-Associated Protein1) is a deubiquitinase, from the UCH (Ubiquitin C-terminal Hydrolases) family, that was originally identified as a partner of the BRCA1 (BReast Cancer Associated gene 1) tumor suppressor. We and other research groups have shown that BAP1 is associated with other co-factors forming a multi-protein complex involved in several cellular processes such as gene transcription, chromatin regulation, cell cycle regulation and DNA damage response. The major target of BAP1 is ubiquitinated histone H2A, a histone mark that has been frequently associated with a repressive chromatin conformation. What are the mechanisms regulating the BAP1 complex allowing it to perform its biological functions? Does this involve post-translational modifications affecting BAP1 partners? These questions are still incompletely answered. Thus, the objectives of our studies are to characterize the mechanism and the function of the BAP1 complex by studying the post-translational modifications that could affect its obligate partners including ASXLs. To address these questions, we studied the post-translational modifications affecting BAP1 and its two mutually exclusive co-factors ASXL1 and ASXL2 (Additional Sex Comb-like 1,2). We demonstrated that ASXL1 and ASXL2 are mono-ubiquitinated only when associated with BAP1. Taking into account that the BAP1/ASXLs complexes are highly conserved during evolution, we also demonstrated that the mono-ubiquitination of ASXLs is important for Drosophila development. Using RNAi and CRISPR/Cas9 gene depletion methods and loss-of-function mutants of BAP1 and ASXL2, we identified the precise site of ASXLs ubiquitination, the enzymes responsible for establishing this mono-ubiquitination as well as its effect on catalytic activity of BAP1. On the other hand, we investigated Drosophila development as well as human cell cycle progression to identify the biological function of ASXLs mono-ubiquitination. Our results indicate that the mono-ubiquitination of ASXL2 on lysine 370 in the presence of BAP1 is a conserved post-translational modification catalyzed directly by the UBE2E family of ubiquitin-conjugating enzymes (UBE2E1, 2, 3 in mammals and UbcD2 in Drosophila). This mono-ubiquitination event stimulates the catalytic activity of BAP1 in mammals and its Drosophila ortholog Calypso towards H2Aub in vivo and in vitro. Blocking the mono-ubiquitination of ASXLs, by mutations targeting lysine K370, induces an inhibition of BAP1 catalytic activity causing a deregulation of human cell cycle progression and a haltere-to-wing homeotic transformation in Drosophila. In addition, we were able to assess the importance of ASXLs mono-ubiquitination in cancer using the mesothelioma tumor model, demonstrating a strong correlation between the expression of BAP1/ASXL2 and UBE2Es. Our results indicate the importance of post-translational modifications, including mono-ubiquitination, in the regulation of the function and stability of the BAP1 complex. Moreover, we describe a novel mechanism of activation of a deubiquitinase by the mono-ubiquitination of its co-factor. Further studies will be needed to shed more light on the link between BAP1/ASXLs activation by mono-ubiquitination and the tumor suppressor function of BAP1 via H2Aub deubiquitination. On the other hand, we have noticed that the depletion of PSMD14, a deubiquitinase associated with the proteasome regulatory particle, induces not only a drastic reduction of H2Aub in the cell, but also rapid cell death. This prompted us initially to investigate the involvement of the catalytic activity of the proteasome in the regulation of H2Aub in connection with cell death. Although we did not find a direct link between PSMD14 and H2Aub deubiquitination, we identified several candidates (DUBs and E2s) involved in the induction of cell death while overcoming acquired resistance against proteasome catalytic inhibitors. These candidates may represent attractive targets for developing specific inhibitors to counteract resistance to proteasome inhibitors.

Indiana University-Purdue University Indianapolis (IUPUI) / While breast cancer is the second most commonly diagnosed cancer worldwide, its causes and natural history are not well defined. The female mammary organ is unique in that it does not reach full maturity until the lactation cycle following pregnancy. This cycle entails extensive growth and reorganization of the primitive epithelial ductal network. Following lactation, these same epithelial cells undergo an equally extensive program of apoptosis and involution. The mammary gland's sensitivity to pro-growth and pro-apoptotic signals may partly explain its proclivity to develop cancers. For epithelial cells to become transformed they must lose intracellular organization known as polarity as differentiated epithelial tissues are refractory to aberrant growth. One essential component of epithelial to mesenchymal transition is the intrinsic capacity of cells to repurpose polarity constituents to promote growth. Recently, a novel mechanism of organ size control has been shown to repurpose the apical junctional associated protein Yap into the nucleus where it functions as a transcriptional coactivator promoting growth and dedifferentiation. The focus of my work has been on a family of adaptor proteins termed Amots that have been shown to scaffold Yap and inhibit growth signaling. Specifically, I have shown that the 80KDa form of Amot, termed Amot80, acts as a dominant negative to the other Amot proteins to promote cell growth while reducing cell differentiation. Amot80 was found to promote the prolonged activation of MAPK signaling. Further, Amot80 expression was also found to enhance the transcriptional activity of Yap. This effect likely underlies the ability of Amot80 to drive disorganized overgrowth of MCF10A cells grown in Matrigel̈™. Overall, these data suggest a mechanism whereby the balance of Amot proteins controls the equilibrium between growth and differentiation within mammary epithelial tissues.

Mitochondrial protein homeostasis is crucial for cellular function and integrity. It is ensured by many specific mitochondrial proteases with possible chaperone functions located across the various mitochondrial subcompartments. In the first part, we have focused on characterization of functional overlap and cooperativity of proteolytic subunits AFG3L2 and YME1L of the mitochondrial inner membrane complexes m- and i-AAA in HEK293 cells. The double AFG3L2/YME1L knockdown cells showed severe alteration in OPA1 protein processing, marked elevation in OMA1 protease and severe reduction in SPG7. Our results reveal cooperative and partly redundant involvement of AFG3L2 and YME1L in the maintenance of mitochondrial protein homeostasis and further emphasize their importance for mitochondrial and cellular function and integrity. The aim of the second part was to characterize the cellular function of LACE1 (lactation elevated 1) in mitochondrial protein homeostasis. LACE1 protein is a human homologue of yeast Afg1 (ATPase family gene 1) ATPase. We show that LACE1 is a mitochondrial integral membrane protein that exists as a part of three complexes of approximately 140, 400 and 500 kDa. We demonstrate that LACE1 mediates degradation of nuclear-encoded complex IV subunits COX4, COX5A and COX6A. Using affinity...

Le suppresseur de tumeurs BAP1 est la déubiquitinase la plus fréquemment mutée dans le cancer humain. Ce dernier est impliqué dans la régulation des gènes cibles des facteurs de transcription E2Fs, qui sont des régulateurs centraux de la prolifération cellulaire en contrôlant, les points de contrôle du cycle cellulaire, la mitose, la réparation de l'ADN et l'apoptose. Dans le complexe BAP1, nous notons plusieurs protéines liant la chromatine, telles que les facteurs de transcription FOXKs (FOXK1 et FOXK2), qui sont connues pour recruter BAP1 sur ses gènes cibles. En effet, le complexe BAP1 est recruté à la chromatine pour assurer sa fonction de déubiquitination de la marque d’histone répressive H2AK119ub, et ainsi, réguler l'expression des gènes. Dans cette étude, nous avons cherché à étudier l'axe BAP1-FOXKs dans la régulation des fonctions cellulaires associées à ce complexe. Fait intéressant, FOXK1, mais pas FOXK2, est connue pour avoir des propriétés oncogéniques, car des niveaux d'expression plus élevés de FOXK1 ont été observés dans une variété de cancers et sont corrélés avec la progression tumorale, l'invasion et les métastases. Ce fait soulève de nombreuses questions sur la nature de la régulation entre ces protéines dont la question suivante : s’agit-il d’une relation de coopération ou antagonisme, entre le suppresseur de tumeurs BAP1 et l’oncogène FOXK1 ? De façon intéressante, nous avons découvert que les protéines FOXK1 et FOXK2 forment deux complexes mutuellement exclusifs avec le complexe BAP1, ce qui suggère qu'elles ont des fonctions moléculaires et cellulaires distinctes à travers ce complexe. Nos études phénotypiques en utilisant des fibroblastes primaires humains montrent que FOXK1 et FOXK2 régulent différemment la prolifération cellulaire, la sénescence cellulaire et la transformation oncogénique. En effet, FOXK1, mais pas FOXK2, favorise la prolifération cellulaire via l’activation de l'expression d'E2F1 et de ses gènes cibles. En conséquence, FOXK1 retarde la sénescence cellulaire et favorise une réplication prolongée des fibroblastes primaires. De plus, la surexpression de FOXK1 favorise la transformation oncogénique des fibroblastes primaires transduits par les oncoprotéines E1A et RAS V12G en augmentant la pénétrance et en diminuant le temps de latence de formation des tumeurs. Ces résultats confirment que ces facteurs disposent de fonctions de signalisation cellulaire distinctes et suggèrent qu'ils sont régulés de manière différentielle au niveau moléculaire. Afin d’investiguer davantage le mécanisme moléculaire qui pourrait distinguer entre les fonctions cellulaires de FOXK1 et FOXK2, nous avons cherché à étudier les modifications post-traductionnelles de ces facteurs. Fait intéressant, nos données de spectrométrie de masse ont révélé que seul FOXK1, mais pas FOXK2, est modifié par O-GlcNAcylation, une modification post-traductionnelle catalysée par l'enzyme OGT. Nos essaies in-vitro montrent que cette modification régule la fonction de FOXK1 dans la prolifération cellulaire car le mutant de la O-GlcNAcylation réduit l'impact prolifératif de FOXK1 sur des cellules. Nos essaies in-vivo, en utilisant un modèle de xénogreffe de cellules cancéreuses humaines chez la souris, confirment que la O-GlcNAcylation de FOXK1 favorise la croissance tumorale. Plus intéressant, le mutant de la O-GlcNAcylation de FOXK1 affecte la transformation oncogénique des fibroblastes primaires transduits par E1A et RAS V12G, en augmentant le temps de latence tumorale et diminuant la taille finale de la tumeur. Au niveau de la chromatine, la O-GlcNAcylation de FOXK1 favorise le recrutement de BAP1 sur les gènes cibles des facteurs E2Fs afin deubiquitiner H2AK119Ub et permettre leur expression. En conclusion, la O-GlcNAcylation est un mécanisme clé qui régule la fonction de FOXK1 dans la prolifération cellulaire, et qui contribue à son activité oncogénique. Dans une autre perspective de cette étude, et afin d'investiguer l'importance de l'axe de signalisation BAP1-FOXKs dans la fonction de suppression tumorale de BAP1, nous avons étudié le rôle fonctionnel de l’interaction BAP1-FOXKs dans un modèle murin. Nous avons généré un modèle de souris en utilisant le système d'édition de gènes CRISPR/Cas9 pour muter la thréonine 492 du gène bap1, en alanine et ainsi perturber l'interaction entre les FOXKs et BAP1 chez la souris. Des descendants hétérozygotes de Bap1T492A/+ ont été croisés pour générer des individus homozygotes et étudier leur phénotype. Fait intéressant, nos résultats montrent que les souris homozygotes Bap1T492A/T492A meurent au stade embryonnaire. De plus, les quelques souris homozygotes Bap1T492A/T492A viables échappant à la barrière de létalité (qui représentent seulement 4.7 % au lieu de 25%) sont remarquablement plus petites et certaines d'entre elles ont présenté des anomalies de développement. De plus, lors de l'analyse du système immunitaire des souris adultes homozygotes Bap1T492A/T492A, nous avons constaté une réduction importante dans les proportions des cellules NKT qui sont des combattants du système immunitaire pouvant éliminer les cellules cancéreuses. Ensemble, ces données montrent que l'axe BAP1-FOXKs est important pour le développement et fournissent de nouvelles informations sur la manière dont les événements de signalisation entre le suppresseur de tumeurs BAP1 et les facteurs FOXKs doivent être étroitement contrôlés pour maintenir une homéostasie cellulaire normale et prévenir le cancer. / The tumor suppressor BAP1 is one of the most frequently mutated deubiquitinase in human cancer. This latter is involved in the regulation of the E2F target genes, which are central regulators of cellular proliferation by controlling, cell cycle checkpoints, mitosis, DNA repair, and apoptosis. Importantly, in the BAP1 complex, we note several chromatin-binding proteins, such as FOXKs transcription factors, which are known to recruit BAP1 to its target genes. Indeed, the BAP1 complex is recruited to chromatin to ensure its deubiquitinating function of the repressive histone mark H2AK119ub and thus regulating gene expression. In this study, we sought to investigate the BAP1-FOXKs axis in regulating associated cellular functions. Interestingly, FOXK1 but not FOXK2, is known to have oncogenic properties as higher expression levels of FOXK1 has been observed in a variety of cancers and is correlated with tumor progression, invasion, and metastasis. These results raise many questions about the nature of the regulation between these proteins, including whether there is a cooperative or antagonistic relationship between the tumor suppressor BAP1 and the oncogene FOXK1? Interestingly, we found that FOXK1 and FOXK2 proteins are mutually exclusive for their interactions with the BAP1 complex, which suggests that they have distinct molecular and cellular functions within this complex. Our functional studies using human primary fibroblasts show that FOXK1 and FOXK2 regulate differentially cell proliferation, cellular senescence, and oncogenic transformation. Indeed, FOXK1, but not FOXK2, promotes cellular proliferation through the activation of the expression of E2F1 and its target genes. As a consequence, FOXK1 delays cellular senescence and promotes prolonged primary cell replication. In addition, overexpression of FOXK1 promotes oncogenic transformation of primary fibroblasts transduced by E1A and RAS V12G oncogenes, by increasing tumor penetrance and decreasing latency of tumor formation. These results confirm that these factors have distinct cellular signalling functions and suggest that they are regulated differentially on the molecular level. To further investigate the molecular mechanism, which could distinguish FOXK1 and FOXK2 cellular functions, we sought to study posttranslational modifications of these factors. Interestingly, our mass spectrometry data revealed that only FOXK1, but not FOXK2, is modified by O-GlcNAcylation, a protein posttranslational modification catalyzed by the enzyme OGT. Our in vitro data shows that this modification regulates FOXK1 function in cellular proliferation as the expression of the O-GlcNAc mutant reduces the proliferative potential of FOXK1. Our in-vivo data, using human cancer cell xenografts on mice, confirms that FOXK1 O-GlcNAcylation promotes tumor growth. More interestingly, FOXK1 O-GlcNAcylation mutants affect the oncogenic transformation of primary fibroblasts expressing E1A and RAS V12G by increasing tumor latency and decreasing tumor size. At the chromatin level, O-GlcNAcylation of FOXK1 promotes the recruitment of BAP1 to target genes of E2Fs factors in order to deubiquitinate H2AK119Ub and allow their expression. In conclusion, O-GlcNAcylation is a key mechanism that regulates the function of FOXK1 in cell proliferation, which contributes to its oncogenic activity. In another perspective of this study, and in order to investigate the importance of BAP1- FOXKs signaling axis in the tumor suppression function of BAP1, we sought to study the functional role of BAP1-FOXKs interaction in a murine model. We generated a mouse model using the CRISPR/Cas9 gene-editing system by mutating BAP1-threonine 492 into alanine and thus disrupting the interaction between FOXKs and BAP1. Heterozygous offspring of Bap1T492A/+ were crossed to generate homozygous litters to study their phenotypes. Interestingly, our results show that homozygous Bap1T492A/T492A mice are embryonic lethal. Moreover, a few homozygous Bap1T492A/T492A mice can escape the embryonic lethality (representing only 4.7% instead of 25%), are remarkably smaller, and some of them showed developmental abnormalities. Moreover, when analyzing the immune system of adult homozygous Bap1T492A/T492A mice, we found a significant reduction of NKT cells proportions, which could be central fighters against cancer cell development. Altogether, these data show that BAP1-FOXKs axis is important for development and provide novel insights into how signaling events between the tumor suppressor BAP1 and FOXKs should be tightly controlled to maintain normal cellular homeostasis and prevent cancer.

Yes / Topoisomerase inhibitors are in common use as chemotherapeutic agents although they can display reduced efficacy in chemotherapy-resistant tumours, which have inactivated DNA damage response (DDR) genes, such as ATM and TP53. Here, we characterise the cellular response to the dual-acting agent, Alchemix (ALX), which is a modified anthraquinone that functions as a topoisomerase inhibitor as well as an alkylating agent. We show that ALX induces a robust DDR at nano-molar concentrations and this is mediated primarily through ATR- and DNA-PK- but not ATM-dependent pathways, despite DNA double strand breaks being generated after prolonged exposure to the drug. Interestingly, exposure of epithelial tumour cell lines to ALX in vitro resulted in potent activation of the G2/M checkpoint, which after a prolonged arrest, was bypassed allowing cells to progress into mitosis where they ultimately died by mitotic catastrophe. We also observed effective killing of lymphoid tumour cell lines in vitro following exposure to ALX, although, in contrast, this tended to occur via activation of a p53-independent apoptotic pathway. Lastly, we validate the effectiveness of ALX as a chemotherapeutic agent in vivo by demonstrating its ability to cause a significant reduction in tumour cell growth, irrespective of TP53 status, using a mouse leukaemia xenograft model. Taken together, these data demonstrate that ALX, through its dual action as an alkylating agent and topoisomerase inhibitor, represents a novel anti-cancer agent that could be potentially used clinically to treat refractory or relapsed tumours, particularly those harbouring mutations in DDR genes.

Tumor-induced immunosuppression can occur by multiple mechanisms, each posing a significant obstacle to immunotherapy. Evidence presented in this dissertation suggests that aberrant cytokine signaling, as a result of altered metabolism of Chronic Lymphocytic Leukemia (CLL) cells, confers a selective advantage for tumor survival and growth. Cells from CLL patients with aggressive disease (as indicated by high-risk cytogenetics) were found to exhibit prolongation in Interferon (IFN)-induced STAT3 phosphorylation, and increased levels of reactive oxygen species (ROS) in these cells reflected these signaling processes. Changes in the relative balance of phospho-STAT3 and phospho-STAT1 levels, in response to combinations of IL-2 + Toll-like receptor (TLR)-7 agonist + phorbol esters, as well as IFN, were associated with the immunosuppressive and immunogenic states of CLL cells. In addition, immunosuppressive leukemic cells were found to express high levels of proteins with O-linked N-acetylglucosamine (O-GlcNAc) modifications, due to increased metabolic activity through the Hexosamine Biosynthetic Pathway (HBP), which caused impaired intracellular signaling responses and affected disease progression. A conclusion of the studies presented here is that the intrinsic immunosuppressive properties of leukemic cells may be overcome by agents such as Resveratrol that target metabolic pathways of these cells.

Tumor-induced immunosuppression can occur by multiple mechanisms, each posing a significant obstacle to immunotherapy. Evidence presented in this dissertation suggests that aberrant cytokine signaling, as a result of altered metabolism of Chronic Lymphocytic Leukemia (CLL) cells, confers a selective advantage for tumor survival and growth. Cells from CLL patients with aggressive disease (as indicated by high-risk cytogenetics) were found to exhibit prolongation in Interferon (IFN)-induced STAT3 phosphorylation, and increased levels of reactive oxygen species (ROS) in these cells reflected these signaling processes. Changes in the relative balance of phospho-STAT3 and phospho-STAT1 levels, in response to combinations of IL-2 + Toll-like receptor (TLR)-7 agonist + phorbol esters, as well as IFN, were associated with the immunosuppressive and immunogenic states of CLL cells. In addition, immunosuppressive leukemic cells were found to express high levels of proteins with O-linked N-acetylglucosamine (O-GlcNAc) modifications, due to increased metabolic activity through the Hexosamine Biosynthetic Pathway (HBP), which caused impaired intracellular signaling responses and affected disease progression. A conclusion of the studies presented here is that the intrinsic immunosuppressive properties of leukemic cells may be overcome by agents such as Resveratrol that target metabolic pathways of these cells.

INTRODUÇÃO: Os microRNAs (miRNAs) são uma classe de pequenas moléculas não codificadoras de proteínas que regulam a expressão gênica durante a etapa de tradução. Esta regulação é feita pelo pareamento de bases com o mRNA-alvo (RNA mensageiro), resultando na supressão da tradução ou na clivagem do mRNA. A depender se os miRNAs têm como alvo genes supressores de tumor ou oncogenes, eles podem atuar como supressores tumorais ou oncogenes. A imunoistoquímica triplo negativa, no câncer de mama, é, comumente, utilizada como substituto clínico para identificação dos tumores basaloides, que se caracterizam pela expressão de genes epiteliais basais, sendo associados a menores taxas de sobrevida livre de doença e sobrevida global. O câncer de mama triplo negativo faz com que seja necessária a descoberta de marcadores moleculares que possam servir de alvos terapêuticos ou, pelo menos, que sirvam como marcadores preditivos da resposta aos quimioterápicos. OBJETIVO: avaliar a expressão de microRNAs, por PCR em tempo real, no carcinoma mamário ductal invasivo (CDI) triplo negativo. MÉTODOS: Foram avaliados materiais em parafina de tumor de 31 pacientes com as seguintes características: carcinoma invasivo de mama, receptores de estrogênio e de progesterona negativos e HER 2 negativo, bem como tecido mamário histologicamente normal. Foram utilizados kit para extração de RNA de amostras fixadas e parafinadas - miRNeasy FFPE; kit para síntese de cDNA - miScript II RT; kit miScript SYBR Green PCR e miScript miRNA PCR Arrays para análise de 84 sequências de miRNA de câncer humano. Foram avaliados dados clínicos, como idade, paridade, amamentação, status menopausal; variáveis histológicas, como tamanho do tumor, status linfonodal, invasão linfática; características imunoistoquímicas, como expressão de Ki-67, EFGR e CK 5/6. O seguimento das pacientes buscou verificar a ocorrência e o tempo de aparecimento de recidiva loco regional, metástase à distância e óbito. Para análise estatística foi utilizado o software miScript miRNA PCR Array Data Analysis, que utiliza o método de quantificação relativa DeltaCt. RESULTADOS: A análise comparativa dos 31 casos de CDI triplo negativo com os 18 casos de parênquima mamário normal definiu microRNAs hiperexpressos, sendo eles: miR-96-5p (fold-regulation(FR) = 9,68, p = 0,000008), miR-21-5p (FR = 4,47, p = 0,00), miR-7-5p (FR = 5,8, p = 0,00137) , miR-182-5p (FR= 7,92, p = 0,000001), miR-210-3p (FR = 11,83, p = 0,000048), miR-18a-5p (FR = 9,51, p = 0,000034), miR-155-5p (FR= 4,40 , p = 0,00019) e miR-93-5p (FR= 4,15, p = 0,000023). Aponta, ainda, microRNAs com hipoexpressão, a saber: miR-204-5p (FR = -10,26, p = 0), miR-205-5p (FR= -4,07, p = 0,019822), miR-125b-5p (FR= -4,29, p=0) e let 7c-5p (FR= -4,91, p=0). CONCLUSÃO: a expressão de microRNAs no carcinoma ductal invasivo triplo negativo permite diferenciá-lo do tecido normal / INTRODUCTION: MicroRNAs (miRNAs) are a class of small non-coding protein molecules that regulate gene expression during the translation stage. This adjustment is made by base pairing with the mRNA (messenger RNA) target resulting in suppression of translation or cleavage of the mRNA. Depending on whether miRNAs target tumor suppressor genes or oncogenes, they can act as tumor suppressors or oncogenes. The triple negative immunohistochemistry in breast cancer is commonly used as a substitute for clinical identification of basaloid tumors, which are characterized by the expression of basal epithelial genes and are associated with lower rates of disease-free survival and overall survival. The triple negative breast cancer makes necessary the discovery of molecular markers that may serve as therapeutic targets or at least as predictive markers of response to chemotherapy. OBJECTIVE: evaluate the expression of microRNAs by RT-PCR in triple negative breast invasive ductal carcinoma (IDC). METHODS: Paraffin embedded tumor material from 31 patients with the following characteristics were evaluated: invasive breast carcinoma, negative estrogen and progesterone receptor, negative HER 2, and histologically normal breast tissue. Were used: Kit for RNA extraction from fixed and paraffin embedded samples - miRNeasy FFPE; cDNA synthesis kit - miScript II RT; miScript SYBR Green PCR Kit and miScript miRNA PCR Arrays for analysis of 84 miRNA sequences of human cancer. Clinical data such as age, parity, breastfeeding, menopausal status; histological variables such as tumor size, lymph node status, lymphatic invasion; immunohistochemical characteristics, such as expression of Ki-67, EFGR and CK 5/6 were evaluated. The follow-up of patients aimed to verify the occurrence and time of appearance of loco regional recurrence, distant metastasis and death. For statistical analysis the miScript miRNA PCR Array Data Analysis software, which uses the method of relative quantification DeltaCt, was used. RESULTS: A comparative analysis of 31 cases of triple negative IDC with 18 cases of normal breast parenchyma defined microRNAs overexpressed, as follows: miR-96-5p (fold-regulation (FR) = 9.68, p = 0.000008), miR -21-5p (FR = 4.47, p = 0.00), 5p, miR-7 (FR = 5.8, p = 0.00137), miR-182-5p (FR = 7.92, p = 0.000001), miR-210-3p (FR = 11.83, p = 0.000048), miR-18a-5p (FR = 9.51, p = 0.000034), miR-155-5p (FR = 4.40, p = 0.00019) and miR-93-5p (FR = 4.15, p = 0.000023). Furthermore, microRNAs with reduced expression, as follows: miR-204-5p (FR = -10.26, p = 0), miR-205-5p (FR = -4.07, p = 0.019822), miR -125b-5p (FR = -4.29, p = 0) and Let-7c 5p (FR = -4.91, p = 0). CONCLUSION: the expression of microRNAs in triple negative invasive ductal carcinoma allows to differentiate it from normal tissue

INTRODUÇÃO: Os microRNAs (miRNAs) são uma classe de pequenas moléculas não codificadoras de proteínas que regulam a expressão gênica durante a etapa de tradução. Esta regulação é feita pelo pareamento de bases com o mRNA-alvo (RNA mensageiro), resultando na supressão da tradução ou na clivagem do mRNA. A depender se os miRNAs têm como alvo genes supressores de tumor ou oncogenes, eles podem atuar como supressores tumorais ou oncogenes. A imunoistoquímica triplo negativa, no câncer de mama, é, comumente, utilizada como substituto clínico para identificação dos tumores basaloides, que se caracterizam pela expressão de genes epiteliais basais, sendo associados a menores taxas de sobrevida livre de doença e sobrevida global. O câncer de mama triplo negativo faz com que seja necessária a descoberta de marcadores moleculares que possam servir de alvos terapêuticos ou, pelo menos, que sirvam como marcadores preditivos da resposta aos quimioterápicos. OBJETIVO: avaliar a expressão de microRNAs, por PCR em tempo real, no carcinoma mamário ductal invasivo (CDI) triplo negativo. MÉTODOS: Foram avaliados materiais em parafina de tumor de 31 pacientes com as seguintes características: carcinoma invasivo de mama, receptores de estrogênio e de progesterona negativos e HER 2 negativo, bem como tecido mamário histologicamente normal. Foram utilizados kit para extração de RNA de amostras fixadas e parafinadas - miRNeasy FFPE; kit para síntese de cDNA - miScript II RT; kit miScript SYBR Green PCR e miScript miRNA PCR Arrays para análise de 84 sequências de miRNA de câncer humano. Foram avaliados dados clínicos, como idade, paridade, amamentação, status menopausal; variáveis histológicas, como tamanho do tumor, status linfonodal, invasão linfática; características imunoistoquímicas, como expressão de Ki-67, EFGR e CK 5/6. O seguimento das pacientes buscou verificar a ocorrência e o tempo de aparecimento de recidiva loco regional, metástase à distância e óbito. Para análise estatística foi utilizado o software miScript miRNA PCR Array Data Analysis, que utiliza o método de quantificação relativa DeltaCt. RESULTADOS: A análise comparativa dos 31 casos de CDI triplo negativo com os 18 casos de parênquima mamário normal definiu microRNAs hiperexpressos, sendo eles: miR-96-5p (fold-regulation(FR) = 9,68, p = 0,000008), miR-21-5p (FR = 4,47, p = 0,00), miR-7-5p (FR = 5,8, p = 0,00137) , miR-182-5p (FR= 7,92, p = 0,000001), miR-210-3p (FR = 11,83, p = 0,000048), miR-18a-5p (FR = 9,51, p = 0,000034), miR-155-5p (FR= 4,40 , p = 0,00019) e miR-93-5p (FR= 4,15, p = 0,000023). Aponta, ainda, microRNAs com hipoexpressão, a saber: miR-204-5p (FR = -10,26, p = 0), miR-205-5p (FR= -4,07, p = 0,019822), miR-125b-5p (FR= -4,29, p=0) e let 7c-5p (FR= -4,91, p=0). CONCLUSÃO: a expressão de microRNAs no carcinoma ductal invasivo triplo negativo permite diferenciá-lo do tecido normal / INTRODUCTION: MicroRNAs (miRNAs) are a class of small non-coding protein molecules that regulate gene expression during the translation stage. This adjustment is made by base pairing with the mRNA (messenger RNA) target resulting in suppression of translation or cleavage of the mRNA. Depending on whether miRNAs target tumor suppressor genes or oncogenes, they can act as tumor suppressors or oncogenes. The triple negative immunohistochemistry in breast cancer is commonly used as a substitute for clinical identification of basaloid tumors, which are characterized by the expression of basal epithelial genes and are associated with lower rates of disease-free survival and overall survival. The triple negative breast cancer makes necessary the discovery of molecular markers that may serve as therapeutic targets or at least as predictive markers of response to chemotherapy. OBJECTIVE: evaluate the expression of microRNAs by RT-PCR in triple negative breast invasive ductal carcinoma (IDC). METHODS: Paraffin embedded tumor material from 31 patients with the following characteristics were evaluated: invasive breast carcinoma, negative estrogen and progesterone receptor, negative HER 2, and histologically normal breast tissue. Were used: Kit for RNA extraction from fixed and paraffin embedded samples - miRNeasy FFPE; cDNA synthesis kit - miScript II RT; miScript SYBR Green PCR Kit and miScript miRNA PCR Arrays for analysis of 84 miRNA sequences of human cancer. Clinical data such as age, parity, breastfeeding, menopausal status; histological variables such as tumor size, lymph node status, lymphatic invasion; immunohistochemical characteristics, such as expression of Ki-67, EFGR and CK 5/6 were evaluated. The follow-up of patients aimed to verify the occurrence and time of appearance of loco regional recurrence, distant metastasis and death. For statistical analysis the miScript miRNA PCR Array Data Analysis software, which uses the method of relative quantification DeltaCt, was used. RESULTS: A comparative analysis of 31 cases of triple negative IDC with 18 cases of normal breast parenchyma defined microRNAs overexpressed, as follows: miR-96-5p (fold-regulation (FR) = 9.68, p = 0.000008), miR -21-5p (FR = 4.47, p = 0.00), 5p, miR-7 (FR = 5.8, p = 0.00137), miR-182-5p (FR = 7.92, p = 0.000001), miR-210-3p (FR = 11.83, p = 0.000048), miR-18a-5p (FR = 9.51, p = 0.000034), miR-155-5p (FR = 4.40, p = 0.00019) and miR-93-5p (FR = 4.15, p = 0.000023). Furthermore, microRNAs with reduced expression, as follows: miR-204-5p (FR = -10.26, p = 0), miR-205-5p (FR = -4.07, p = 0.019822), miR -125b-5p (FR = -4.29, p = 0) and Let-7c 5p (FR = -4.91, p = 0). CONCLUSION: the expression of microRNAs in triple negative invasive ductal carcinoma allows to differentiate it from normal tissue