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

O carcinoma epidermoide do esôfago continua sendo a principal neoplasia maligna esofágica na população brasileira. Os objetivos desta investigação foram: avaliar a imuno-expressão de um grupo de proteínas relacionadas à via intrínseca da apoptose (bax, APAF-1 e citocromo c) e da proteína p53 em um grupo de carcinomas epidermoides do esôfago; confrontar estes resultados com a atividade proliferativa medida pela imuno-expressão do antígeno Ki67 e com a atividade apoptótica medida pela imuno-expressão da caspase 3 clivada; e confrontá-los com parâmetros implicados no estadiamento do carcinoma epidermoide do esôfago (invasão local ou pT, estado dos linfonodos regionais ou pN, grau de diferenciação do tumor primário e local do tumor primário no esôfago) e com o tamanho do tumor primário. De um grupo inicial de 91 carcinomas esofágicos consecutivos, 66 carcinomas epidermoides do esôfago foram revistos, alocados em micromatrizes teciduais e submetidos à técnica de imuno-peroxidase com anticorpos primários anti: bax, APAF-1, citocromo c, p53, Ki67 e caspase 3 clivada. Suas imuno-expressões foram semiquantificada de 0 a 5+, exceto caspase 3 clivada que foi contada em 1000 células. Apresentaram amostras válidas um conjunto de 63 carcinomas epidermoides do esôfago. A mediana de imuno-expressão destas 6 proteínas foi: 2+, 5+, 5+, 5+, 3+ e 26, respectivamente. Houve correlação positiva entre a imunoexpressão do antígeno Ki67 e a de caspase 3 clivada (coeficiente rho de Spearman =0,373, p=0,003). Houve associação entre a imunoexpressão de APAF-1 e o grau de diferenciação, com valores maiores de APAF-1 para os carcinomas epidermoides do esôfago bem diferenciados (mediana de 5+ para tumores bem diferenciados, contra mediana de 2+ para tumores pouco diferenciados, p<0,001, teste de Kruskal-Wallis). Houve associação entre o tamanho do tumor primário e o nível de invasão local do tumor primário, com tamanhos maiores quanto maior o nível de invasão local dos carcinomas epidermoides do esôfago (mediana de 32,5 mm para os tumores pT1 e mediana de 50,0 mm para os tumores pT3 ou pT4, p=0,027, teste de Kruskal-Wallis). Não houve associação entre as demais variáveis. Embora atividade proliferativa e atividade apoptótica caminhem juntas nos carcinomas epidermoides do esôfago, no estágio invasivo do principal tipo histológico de carcinoma esofágico da população brasileira, não são mais os fatores ligados à via intrínseca da apoptose que influenciam a sua progressão. Além disso, se a imunoexpressão aumentada da proteína APAF-1 estimula a diferenciação nos carcinomas epidermoides esofágicos, não o faz através de estímulo da atividade apoptótica pura e simplesmente / Squamous cell carcinoma of the esophagus remains as the major malignant esophageal neoplasm in the Brazilian population. The objectives of this study were: to assess the immunoexpression of a group of proteins related to the intrinsic pathway of apoptosis (bax, APAF-1 and cytochrome c) and to p53 protein in squamous cell carcinoma of the esophagus; to confront these results with proliferative activity measured by the immunoexpression of Ki67 and with apoptotic activity measured by the immunoexpression of cleaved caspase 3; and to confront them with parameters involved in the staging of squamous cell carcinoma of the esophagus(local invasion or pT, lymph node status or pN, grade of differentiation of primary tumor and site of primary tumor in the esophagus) and with size of primary tumor. From a starting group of 91 consecutive carcinomas of the esophagus, 66 squamous cell carcinomas of the esophagus were selected, revised, placed in tissue microarrays blocks, and submitted to immunoperoxidase technique with primary antibodies to: bax, APAF-1, cytochrome c, p53, Ki67 and cleaved caspase 3. The immunoexpression was semiquantified in a scale from 0 to 5+, except for cleaved caspase 3, whicht was counted in 1000 cells. Sixty three squamous cell carcinomas of the esophagus displayed valid cores for analysis. The median immunoexpression of these 6 proteins were: 2+, 5+, 5+, 5+, 3+ and 26, respectively. A positive correlation was found between Ki67 antigen and cleaved caspase 3 immunoexpression (Spearmans rho coefficient =0.373, p=0.003). There was association between the immunoexpression of APAF-1 and the grade of differentiation, with higher values of APAF-1 for well differentiated squamous cell carcinomas of the esophagus (median of 5+ for well differentiated tumors and median of 2+ for poorly differentiated tumors, p<0.001, Kruskal-Wallis test). The size of primary tumor was statistically associated to the degree of local invasion of primary tumor, with higher size associated to deeper local invasion (median of 32.5 mm for pT1 tumors and median of 50.0 mm for pT3 or pT4 tumors, p=0.027, Kruskal-Wallis test). There was no association among the other variables. Although proliferative activity and apoptotic activity go together in squamous cell carcinomas of the esophagus, the factors involved in the intrinsic pathway of apoptosis does not differ significantly according to the histological parameters in the invasive phase of the development of squamous cell carcinoma of esophagus. Moreover, , if increased immunoexpression of APAF-1 stimulates differentiation of squamous cell carcinomas of the esophagus, it does not work through direct higher apoptotic activity

Abstract Lymphomas are a heterogeneous group of malignancies, which usually have a good prognosis and high cure rates. Lymphomas are sensitive to chemotherapy and radiotherapy, and many patients can be cured even after a relapse, resulting in a need for effective follow-up. However, the cost-benefit ratio of radiological imaging in predicting the forthcoming relapses is poor. Consequently, there is a need for biological prognostic and predictive markers to distinguish patients at the highest risk of relapse at the time of diagnosis or during follow-up. Despite rapid progress in lymphoma treatments, some patients still die from lymphoma. Thus, more data on the basic biological features of lymphomas are also needed. Gelatinases (MMP-2 and MMP-9) and their tissue inhibitors (TIMP-1 and TIMP-2) have been found to play a role in the progression of solid tumours. TP53 is a tumour suppressor gene, the mutations and protein over-expression of which have been demonstrated to be associated with survival in most cancer types. There is also some evidence that these proteins could have prognostic significance in lymphomas as well. In the present study, the tissue expression, plasma concentrations and clinical value of gelatinases and their tissue inhibitors were evaluated in lymphomas. 249 primary tissue samples from patients with Hodgkin, follicular, or diffuse large B-cell lymphoma were analysed for expression of gelatinases and/or their inhibitors using immunohistochemistry. In follicular lymphoma, p53 protein expression was also investigated. The plasma samples of 126 lymphoma patients and a control group of 44 healthy volunteers were collected and studied by ELISA. TIMP-1 expression correlated with bulky tumour and nodular sclerosis subtype of Hodgkin lymphoma. In follicular lymphoma, p53 over-expression was an independent adverse prognostic factor for survival and a predictor of histological transformation. Plasma MMP-2-TIMP-2 complex appeared to be a potential follow-up marker predicting the risk of relapse in lymphoma patients. Plasma levels of the MMP-2-TIMP-2 complex, proMMP-2, TIMP-2 and proMMP-2/TIMP-2 ratio were at abnormal levels both in patients with newly diagnosed lymphoma and those in remission compared to healthy controls. The clinical significance of these markers needs further studies.

La spleen tyrosine kinase (Syk) est une protéine kinase cytoplasmique qui intervient dans la signalisation immunitaire. Notre équipe a montré pour la première fois que Syk est exprimée aussi dans les cellules épithéliales mammaires et que son expression est perdue au cours de l’acquisition d’un phénotype invasif/métastatique. Syk agit comme un suppresseur de tumeurs et de métastases dans des modèles de xénogreffes de cancer du sein. Ces observations ont été étayées par des études cliniques qui montrent que la perte d’expression de Syk correspond à un risque accru de développement de métastases (facteur de mauvais pronostic) dans le cancer du sein et d’autres carcinomes. Par une approche de phospho-protéomique quantitative (SILAC), nous avons pu identifier de nouveaux substrats potentiels de Syk dans les cellules de cancer du sein. De façon intéressante, ressortent de nombreuses protéines impliquées dans l'adhésion intercellulaire (E-cadhérine/caténines) et la polarisation épithéliale (eg ZO3, occludine, claudine-3). Ces protéines, qui se localisent aux jonctions d'adhésion et d'occlusion, sont connues comme composants plate-formes de signalisation et exercent souvent une fonction de suppresseur de tumeur.Dans ce travail de thèse je me suis focalisé principalement sur :(i) le rôle de l’activité kinase de Syk dans la régulation du complexe E-cadhérine/caténines et(ii) les conséquences de l’invalidation conditionnelle de Syk dans la glande mammaire murine (développement mammaire et tumorigenèse).Par une approche de phosphorylation in vitro, nous avons montré que la E-cadhérine et différentes caténines sont des substrats directs de Syk. Les résidus tyrosines phosphorylés dans ces protéines ont été identifiés par spectrométrie de masse et les anticorps phospho-spécifiques correspondants ont été générés. En immunofluorescence, Syk endogène colocalise avec la E-Cadhérine au niveau des jonctions adhérentes et la surexpression de Syk stimule la phosphorylation de la E cadhérine et différentes caténines au niveau des jonctions intercellulaires. Des expériences d’immunoprécipitation montrent que les protéines E-cadhérine et caténines phosphorylées restent associées dans un complexe au niveau des jonctions adhérentes. L’extinction de Syk par shRNA dans une lignée de cancer de sein inhibe partiellement la ré-agrégation intercellulaire (2D/3D) et augmente l’invasion et la migration cellulaires et la croissance en 3D dans le Matrigel. Inversement, la surexpression de Syk inhibe la migration et l’invasion et favorise l'adhérence intercellulaire. Syk semble par la phosphorylation du complexe E-cadhérine/caténines consolider leurs interactions renforçant ainsi les jonctions intercellulaires et l’intégrité de l’épithélium, ce qui pourrait révéler un mécanisme majeur responsable de son activité anti-invasive. Leurs mécanismes moléculaires ont été explorés.Ces modèles cellulaires in vitro ont ensuite été étendus vers un modèle intégré murin. L'invalidation homozygote du gène SYK étant létale, nous avons développé un modèle d’invalidation conditionnelle de Syk dans la glande mammaire (Syk-flox:WAP-Cre). Ce modèle nous a permis tout d’abord d’étudier le rôle de Syk dans le développement et la physiologie de la glande mammaire au cours de la lactation et de l’involution, les glandes Syk-négatives montrant des défauts de développement. Il permet à plus long terme également d’évaluer l'implication de Syk dans la formation et la progression du cancer du sein chez des souris cKO Syk, après croisement ou non avec des souris transgéniques exprimant l’oncogène MMTV-Neu/Her2.Déterminer si Syk est un bona fide suppresseur de tumeurs est crucial car un inhibiteur de Syk est en cours d’étude clinique pour le traitement de l’arthrite rhumatoïde. L’identification des voies de signalisation gouvernées par Syk pourrait ultérieurement déboucher sur le développement de nouvelles thérapies ciblant ces protéines et bloquant l'évolution cancéreuse. / The spleen tyrosine kinase (Syk) is a cytoplasmic protein kinase involved in immune-response signaling. Our team showed for the first time that Syk is also expressed in mammary epithelial cells and that its expression is lost during acquisition of an invasive/metastatic phenotype. Syk acts as a tumor and metastasis suppressor in breast cancer xenograft models. Clinical studies corroborated that loss of Syk expression is correlated with a decreased survival and an increased risk of metastasis development (poor prognosis) in breast cancer and other carcinomas. Using a quantitative phospho-proteomic SILAC approach in breast cancer cells, our group identified new potential Syk substrates. Interestingly, many proteins are involved in intercellular adhesion (E-cadherin/catenin) and epithelial polarization (eg ZO3, occludin, claudin-3). These proteins are localized at the adherens and tight junctions and are known as signaling platforms and components often presenting a tumor suppressor function.In this thesis I mainly focused on:(i) the role of the Syk kinase activity in the regulation of the E-cadherin/catenin complex and(ii) the consequences of the conditional Syk knockout in the mouse mammary gland on breast development and tumorigenesis.Using in vitro kinase assays, we demonstrated that E-cadherin (E-Cdh) and different catenins are direct Syk substrates. The phosphorylated tyrosine residues were identified by mass spectrometry and corresponding phospho-specific antibodies were generated. By immunofluorescence, we observed that endogenous Syk and E-Cdh colocalize at adherens junctions (AJ) and that Syk overexpression stimulates Syk-dependent phosphorylation of E-cadherin and different catenins at AJ. Immunoprecipitation experiments indicate phosphorylated E-cadherin and catenin proteins are associated in a complex. Using functional tests, Syk knockdown by shRNA in breast cancer cells partially inhibited intercellular re-aggregation (2D/3D) and increased cell invasion, migration and 3D-growth in Matrigel. Conversely, Syk overexpression inhibited migration and invasion and promoted intercellular adhesion. Thus, Syk seems to strengthen the intercellular junctions and the integrity of the epithelium via the phosphorylation of the E-cadherin/catenin complex of which its molecular mechanisms were explored. This could be a major mechanism responsible for its anti-invasive activity.These in vitro observations were subsequently extended to an integrated mouse model. As the homozygous SYK gene knockout is lethal; we developed a conditional Syk deletion model in the murine mammary gland (Syk-flox:WAP-Cre).This model allowed us to study the role of Syk in the development and physiology of the mammary gland during lactation and involution, the Syk-negative glands showing developmental defects. On a long-term basis, it also allows to assess the involvement of Syk in the formation and progression of breast cancer in aging cKO Syk mice, bred or not with transgenic mice expressing the MMTV-Neu / Her2 oncogene.Whether Syk is a bona fide tumor suppressor is a crucial issue as Syk inhibitors are being evaluated in clinical studies for the treatment of rheumatoid arthritis. Identification of the signaling pathways governed by Syk could lead to the development of new therapies targeting these proteins and blocking tumor development and progression.

Les mutations du gène suppresseur de tumeur MEN1 sont connues depuis de nombreuses années, pour être à l'origine du syndrome des Néoplasies endocriniennes multiples de type 1 (Syndrome des NEM1). Ce syndrome constitue une maladie héréditaire associée à une perte d'hétérozygotie progressive du gène MEN1, affectant principalement les organes endocrines. Plus récemment, l'implication du gène MEN1 a émergé dans la tumorigénèse d'autres organes, et plus particulièrement dans dans le cancer du sein et le cancer de la prostate, où son rôle reste encore très controversé. Pour mieux déterminer le rôle joué par menin dans les cellules prostatiques (oncogène ou gène suppresseur), mon projet de thèse avait donc pour but de caractériser un nouveau modèle murin d'invalidation du gène Men1 spécifiquement dans les cellules luminales de la glande prostatique, le modèle Men1F/F Nkx3.1CreERT2+/-. Les examens anatomopathologiques réalisés sur ce nouveau modèle murin ont montré que la perte d'expression du gène Men1 conduisait à une accélération de la tumorigénèse dans la glande prostatique par rapport aux souris contrôles. D'autre part les analyses moléculaires issues de l'étude de notre nouveau modèle murin, ont montré que l'expression du récepteur aux androgènes (RA) était diminué dans les cellules déficientes pour le gène Men1 . Des analyses menées in vitro ont montré que la protéine menin, codée par le gène MEN1, joue le rôle de régulateur transcriptionnel du RA. De la même manière, mes travaux ont mis en évidence, que la protéine menin semble réguler l'expression du récepteur aux estrogènes alpha (RE?), en liant la région promotrice de ce dernier dans des lignées cellulaires du cancer du sein. De plus, les analyses cliniques ont révélé que l'expression réduite de menin corrélée avec la survenue du sous type luminal B du cancer du sein, connue pour exprimer faiblement le RE??Ainsi mes travaux de thèse ont permis de conforter notre hypothèse sur le rôle oncosuppressif du gène MEN1 dans le glande prostatique. D'autre part, nous avons mis en évidence l'implication de la protéine menin dans la régulation de l'expression des récepteurs nucléaires, dans le cancer du sein et de la prostate / For a long time, mutations of the MEN1 gene have been known to be responsible of the Multiple Endocrine Neoplasia type 1 (MEN1 syndrome), a hereditary disease affecting mainly endocrine organs. Recent advances highlighted the involvement of the MEN1 gene in the development of the breast cancer and prostate cancer. Nevertheless, the role played by the MEN1 gene in prostate cancer still remains unclear, described as on oncogene by some studies, or as a tumor suppressor by others. To further adress this issue, we generated a novel and inductible mouse model, Men1F/F-Nkx3.1Cre-/+, in which the Men1 gene can be specifically disrupted in luminal prostatic cells upon tamoxifen injection. Anatomopathologic examination of our model showed that the Men1 gene disruption accelerate the tumorigenesis in the prostatic gland compared to the control mice. Moreover, molecular analyses showed that the expression of androgen receptor (AR) decreased in Men1-deficient cells. In vitro study perfomed in prostate cancer cell lines showed that menin protein encoded by the Men1 gene is involved in the transcriptionnal regulation of AR.Similarly, my work showed that menin protein also involved in the transcriptionnal regulation of the estrogen receptor alpha (ER?) expression, through its binding on the promoter of the ER??gene. Moreover, clinical study revealed that decrease in menin expression correlates with the occurrence of luminal B subtype of breast cancer, in which ER??expression is reduced. Thus this thesis work, allowed to better characterized the oncosuppressive role of the Men1 gene in the prostatic gland. This work, also highlighted for the first time the involvement of menin protein in the regulation of nuclear receptor expression, in prostate and breast cancer

Les cancers du sein de sous-type luminal B sont associés à un mauvais pronostic. Afin de mieux comprendre la biologie de ce sous-type, nous avons étudié au sein de 188 tumeurs mammaires de différents sous-types, les anomalies du nombre de copies, les méthylations de l'ADN, les profils d'expression génique et les mutations somatiques dans 9 gènes sélectionnés. Un total respectif de 237 et 101 oncogènes et gènes suppresseurs de tumeurs (TSG) candidats présentaient une dérégulation de l'expression en relation avec leur CNA. 88% des TSG potentiels étaient localisés sur le bras chromosomique 6q. 101 oncogènes candidats ont été validés sur une série publique de 5765 cancers du sein, et l'expression de 67 gènes était associée à un mauvais pronostic au sein des tumeurs luminales. 24 gènes présentaient une dérégulation de l'expression en relation avec un haut niveau de méthylation de l'ADN. FOXO3, PIK3CA et TP53 étaient les gènes les plus fréquemment mutés parmi les 9 testés. Dans une méta-analyse de séquençage de nouvelle génération regroupant 875 cancers du sein, les gènes les plus fréquemment mutés dans le sous-type luminal B étaient PIK3CA, TP53 et GATA3. Les nombreuses altérations moléculaires ciblaient des voies de signalisation communes, incluant 3 axes pouvant jouer un rôle majeur dans le sous-type luminal B : la voie TP53 et l'instabilité chromosomique, les voies de signalisation PI3K/AKT/MTOR/FOXO et MAPK/JNK, et les altérations des facteurs de transcription et épigénomiques. En conclusion, nous avons établi un répertoire de gènes candidats dans le sous-type luminal B qui pourraient être impliqués dans le développement et/ou l'hormonorésistance de ce sous-type. / Breast cancers (BCs) of the luminal B subtype have a poor prognosis. To better understand this subtype we studied in 188 BCs of various molecular subtypes, DNA copy number aberrations, DNA promoter methylation, gene expression profiles, and somatic mutations in nine selected genes. A total of 237 and 101 luminal B-specific candidate oncogenes and tumor suppressor genes (TSGs) presented a deregulated expression in relation with their CNAs. Interestingly, 88% of the potential TSGs are located within chromosome arm 6q. 101 candidate oncogenes were validated in a public series of 5,765 BCs and the overexpression of 67 was associated with poor survival in luminal tumors. 24 genes presented a deregulated expression in relation with a high DNA methylation level. FOXO3, PIK3CA and TP53 were the most frequent mutated genes among the nine tested. In a meta-analysis of next-generation sequencing data in 875 BCs, PIK3CA, TP53 and GATA3 were the most frequent mutated genes. Numerous molecular alterations targeted common signalling pathway, included 3 ways wich may play a major in the luminal B subtype: TP53 pathway and chromosomal instability, PI3K/AKT/MTOR/FOXO and MAPK/JNK pathway, and epigenomic and transcription factors alterations. In conclusion, we have reported a repertoire of luminal B candidate genes that may be involved in the development and/or hormone resistance of this subtype.

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

Einleitung: Der seit über 30 Jahren ausgiebig erforschte Transkriptionsfaktor p53 besitzt offenbar Funktionen, die über seine bekannte und gut untersuchte Aktivität als Tumorsuppressor hinausgehen. So scheint er auch an der Regulation der menschlichen Lebenserwartung - über die Vermittlung einer allgemeinen physischen Robustheit - sowie der weiblichen Fertilität beteiligt zu sein. Insbesondere Primaten zeichnen sich durch eine vergleichsweise lange Lebenserwartung und eine lange reproduktive Phase aus. Ob p53 hier eine Rolle spielen könnte, ist unbekannt. Unsere Arbeitsgruppe entdeckte vor einigen Jahren das humane endogene Retrovirus-K (HERV-K) Protein Np9, dessen Gen sich in mehreren Kopien nur bei Menschen, Schimpansen und Gorillas findet. Weitere Untersuchungen wiesen außerdem darauf hin, dass Np9 an den Tumorsuppressor p53 zu binden vermag. Ziele der Untersuchungen: Es stellte sich also die Frage, ob die Funktion von p53 durch die Bindung an Np9 moduliert werden kann. Eine derartige Modulation des multifunktionellen Transkriptionsfaktors wäre natürlich auf Hominiden beschränkt. In der vorliegenden Arbeit sollten einige Teilaspekte der Interaktion von p53 und Np9 näher untersucht werden. Material und Methoden: Für die Bindungskartierung von p53 und Np9 wurden GST-Pulldown-Analysen durchgeführt. Die GST-Protein-Plasmide wurden in E.coli BL21 transformiert und nach Induktion mit IPTG exprimiert. Sie dienten als „Fängerproteine“ und waren dank ihres Glutathion-S-Transferase-tags in der Lage an GST-Sepharose-Kügelchen zu binden. Der putative Interaktionspartner als „Beuteprotein“ wurde in vitro translatiert und in diesem Zuge auch mit 35S radioaktiv markiert. Dann wurde er mit den an die Beads gebundenen GST-Proteinen inkubiert und anschließend die Proben auf ein SDS-Gel aufgetragen und aufgetrennt. Das Gel wurde anschließend auf eine Membran übertragen und der Blot auf einen Radioaktivfilm aufgelegt, woraufhin die Protein-Protein-Bindungen anhand des radioaktiven Beuteproteins als Banden erkennbar waren. Abschließend wurde der Blot mit GST-Antikörper inkubiert, dann am Folgetag mit Anti-Mouse-Antikörper. Mittels ECL Substrat konnte nun die Bindung der GST-getaggten Proteine an die Sepharosebeads nachgewiesen werden. Für den Electrophoretic Mobility Shift Assay wurden verschiedene Versuchsansätze pipettiert, welchen nach einer Inkubationszeit das zuvor mit 32P radioaktiv markierte Oligonukleotid zugegeben wurde. Nach erneuter Inkubation wurden die Proben auf das nicht-denaturiende EMSA-Gel aufgetragen und elektrophoretisch aufgetrennt. Dabei wurden die Protein-Oligonukleotid-Verbindungen gemäß ihrer Ladung, Größe und Konformation getrennt. Die Gele wurden im Geltrockner getrocknet und direkt mit einer Verstärkerfolie auf den Radioaktivfilm in einer Radioaktivkassette aufgelegt. Ergebnisse: Zunächst war es notwendig, die Bindung der beiden Partner biochemisch zu kartieren. Dies geschah mittels der GST-Pulldown-Analyse, in der Fragmente der Proteine exprimiert, miteinander inkubiert und schließlich kopräzipitiert wurden. Es stellte sich heraus, dass p53 mit seinem C-Terminus an Np9 bindet. Np9 hingegen band mit seinen Aminosäureresten (aa) 1-64 (ohne den C-terminus mit den aa 65-74) an p53. Das Np9-Fragment 36-74 zeigte nur eine schwache Bindung an p53. Interessanterweise band das Np9-Fragment 36-64 stärker an p53 als Volllängen-Np9 (1-74), was auf eine die Interaktion hemmende Domäne im C-Terminus von Np9 hinweisen könnte. Um zu untersuchen, ob die Bindung von Np9 an den C-Terminus von p53 die p53-DNA-Interaktion beeinflusst, wurden Electrophoretic Mobility Shift Assays (EMSAs) durchgeführt. Es konnte gezeigt werden, dass Np9-zumindest in vitro-durch Bindung an die regulatorische Domäne von p53 und in Anwesenheit des p53-aktivierenden Antikörpers PAb421 in der Lage war, die spezifische Bindungsfähigkeit von p53 an DNA zu erhöhen und somit seine Funktion als Transkriptionsfaktor zu unterstützen. Schlussfolgerungen: Die Resultate weisen also erstmals darauf hin, dass das nukleäre HERV-K Protein Np9 spezifisch in Hominiden eine p53-abhängige Tumorsuppressoreigenschaft aufweisen könnte. Weitere Untersuchungen-insbesondere in vivo-sind nun notwendig. Dies könnte auch als Forschungsgrundlage zu endogenen Retrovirusproteinen beim Pferd dienen.:1 Einleitung und Zielsetzung der Arbeit 1 2 Literaturübersicht 2 2.1 Der Tumorsuppressor p53 2 2.2 Das Kernprotein Np9 7 2.2.1 Retroviren 7 2.2.2 Endogene Retroviren 8 2.2.3 Humane endogene Retroviren 8 2.2.4 HERV-K 10 2.2.5 Das nukleäre Protein Np9 10 3 Material und Methoden 15 3.1 Material 15 3.1.1 Chemikalien 15 3.1.2 Puffer und Lösungen 17 3.1.3 Antikörper 21 3.1.4 Enzyme 22 3.1.5 Reaktionskits 22 3.1.6 Bakterienstämme 23 3.1.7 Kulturmedien 23 3.1.8 Oligonukleotide für EMSA 23 3.1.9 Größenstandards 24 3.1.10 Plasmide 26 3.2 Methoden 28 3.2.1 Nukleinsäuretechniken 28 3.2.2 Protein-Methoden 31 3.2.3 Prokaryonten 40 4 Ergebnisse 42 4.1 Interaktion zwischen Np9 und dem Tumorsuppressorprotein p53 42 4.2 GST-Pulldown 43 4.2.1 Klonierung für die GST-Pulldown-Analysen 43 4.2.2 Induktion der Proteinexpression 48 4.2.3 GST-Pulldown-Experimente 53 4.3 EMSA (Electrophoretic Mobility Shift Assay) 57 4.3.1 Radioaktive Markierung der Sonden 58 4.3.2 EMSA-Experimente 58 5 Diskussion 63 6 Zusammenfassung 68 7 Summary 70 8 Literaturverzeichnis 72 Danksagung 86 Abbildungsverzeichnis 87 Tabellenverzeichnis 88 / Introduction: The transcription factor p53, extensively investigated for over 30 years, apparently has functions which exceeds his known and well examined activity as a tumor suppressor. It seems to be involved in the regulation of the human life expectancy – by providing a general physical robustness - as well as of the female fecundity. Primates too are characterized by a comparatively long life expectancy and long reproductive phases, yet the possible influence of p53 is unknown. Our research group has discovered some years ago the Np9 protein of human endogenous retrovirus K (HERV K), which is found in several copies only with humans, chimpanzees and gorillas. Other investigations by our group suggested that Np9 might be able to interact with the tumor suppressor p53. Objective of the investigations: To study whether the function of p53 can be modulated by the interaction with Np9. Such a modulation of the multifunctional transcription factor would of course be limited to hominids. In the present work some aspects of the interaction between p53 and Np9 were analysed. Materials and methods: For the mapping of the interaction of p53 and Np9, GST pulldown assays were carried out. The GST protein plasmids were transformed in E. coli BL21 and expressed after IPTG induction. They served as bait proteins and bound to GST sepharose beads because of their Glutathione S-transferase-tags. The putative interaction partner as a prey protein was translated in vitro and radioactively marked with 35S. After being incubated with the GST-proteins bound to the beads, the samples were transferred on a SDS gel and separated. The gel was transferred to a membrane and the blot was exposed to an X-ray film. Thus, the radioactively labelled prey protein forms bands that identify the protein-protein interaction. Finally the blot was incubated with GST antibody, then on the following day with anti-mouse antibody. Using ECL-substrate it was now possible to demonstrate that the GST-tagged proteins bound to the sepharose beads. For the Electrophoretic Mobility Shift Assay different samples were prepared and, after an incubation time, the oligonucleotide radioactively marked with 32P was added. After additional incubation it was transferred on non-denaturating EMSA gel and separated by electrophoresis. Thus the protein oligonucleotide conjugates were separated according to charge, size and conformation. The gels were dried in the gel dryer, transferred to a membrane and placed against an X-ray film in a cassette. Results: Initially a biochemical mapping of the binding of the two partners had to be carried out. This was done by means of the GST pulldown assay, in which fragments of the proteins were extruded, incubated together and finally co-precipitated. It turned out that the C-terminus of p53 bound to Np9. However, Np9 bound to p53 with his amino acid residues (aa) 1-64 (lacking the C-terminal aa 65-74). The Np9 fragment 36-74 showed only a weak binding to p53. Interestingly the Np9 fragment 36-64 was binding stronger to p53 than a full length Np9 (1-74), which could point to a C-terminal domain in Np 9 inhibiting the interaction. In order to examine whether the binding of Np9 to the C-terminal of p53 affects the interaction of p53 with DNA, Electrophoretic Mobility Shift Assays (EMSAs) were carried out. It could be shown that Np9 was able to raise the specific binding ability of p53 with DNA and to support therefore its function as a transcription factor, by binding to the regulatory domain of p53 in presence of the activating p53 antibody PAB421. Conclusions: The results show for the first time that, specifically in hominids, the nuclear HERV-K protein Np9 could have a tumor suppressing quality that is dependent on p53. Further investigations, in particular in vivo, are necessary. This could be the starting point for research on equine endogenous retrovirusproteins in horses.:1 Einleitung und Zielsetzung der Arbeit 1 2 Literaturübersicht 2 2.1 Der Tumorsuppressor p53 2 2.2 Das Kernprotein Np9 7 2.2.1 Retroviren 7 2.2.2 Endogene Retroviren 8 2.2.3 Humane endogene Retroviren 8 2.2.4 HERV-K 10 2.2.5 Das nukleäre Protein Np9 10 3 Material und Methoden 15 3.1 Material 15 3.1.1 Chemikalien 15 3.1.2 Puffer und Lösungen 17 3.1.3 Antikörper 21 3.1.4 Enzyme 22 3.1.5 Reaktionskits 22 3.1.6 Bakterienstämme 23 3.1.7 Kulturmedien 23 3.1.8 Oligonukleotide für EMSA 23 3.1.9 Größenstandards 24 3.1.10 Plasmide 26 3.2 Methoden 28 3.2.1 Nukleinsäuretechniken 28 3.2.2 Protein-Methoden 31 3.2.3 Prokaryonten 40 4 Ergebnisse 42 4.1 Interaktion zwischen Np9 und dem Tumorsuppressorprotein p53 42 4.2 GST-Pulldown 43 4.2.1 Klonierung für die GST-Pulldown-Analysen 43 4.2.2 Induktion der Proteinexpression 48 4.2.3 GST-Pulldown-Experimente 53 4.3 EMSA (Electrophoretic Mobility Shift Assay) 57 4.3.1 Radioaktive Markierung der Sonden 58 4.3.2 EMSA-Experimente 58 5 Diskussion 63 6 Zusammenfassung 68 7 Summary 70 8 Literaturverzeichnis 72 Danksagung 86 Abbildungsverzeichnis 87 Tabellenverzeichnis 88

In response to telomere shortening, oxidative stress, DNA damage or aberrant activation of oncogenes, normal somatic cells exit the cell cycle and enter an irreversible growth arrest termed senescence. The limited proliferative capacity imposed by senescence on cells impedes the accumulation of mutations necessary for tumorigenesis and prevents proliferation of cells at risk of neoplastic transformation. Opposite to the tumor suppressor function, accumulation of senescent cells in adult organisms is thought to contribute to aging by depleting the renewal capacity of tissues and stem/progenitor cells, and by interfering with tissue homeostasis and functions. The Antagonistic Pleiotropy Theory of senescence proposes that senescence is beneficial early in life by acting as a tumor suppressor, but harmful late in life by contributing to aging. Recent studies have provided evidence strongly supporting the tumor suppressor function of senescence, however, direct evidence supporting the role of senescence in aging remains largely elusive. In this thesis, I describe studies to test the Antagonistic Pleiotropy Theory of senescence in tumorigenesis and aging. The approach that I have taken is to alter the senescence response in vivo by changing the expression of a senescence regulator in mice. The consequence of altered senescence response on tumorigenesis and stem cell self-renewal was investigated. The senescence regulator I studied is Smurf2, which has been shown previously to activate senescence in culture. I hypothesized that the senescence response will be impaired by Smurf2 deficiency in vivo. Consequently, Smurf2-deficient mice will develop tumors at an increased frequency, but also gain enhanced self-renewal capacity of stem/progenitor cells with age. I generated a Smurf2-deficient mouse model, and found that Smurf2 deficiency attenuated p16 expression and impaired the senescence response in primary cells and tissues. Smurf2-deficient mice exhibited an increased susceptibility to spontaneous tumorigenesis, indicating that Smurf2 is a tumor suppressor. At the premalignant stage of tumorigenesis, a defective senescence response was documented in the Smurf2-deficient mice, providing a mechanistic link between impaired senescence response and increased tumorigenesis. The majority of tumors developed in Smurf2-deficent mice were B-cell lymphomas with an origin in germinal centers of the spleen and a phenotype resembling human diffuse large B-cell lymphoma (DLBCL). I discovered that Smurf2 mediated ubiquitination of YY1, a master regulator of germinal centers. Stabilization of YY1 in the absence of Smurf2 was responsible for increased cell proliferation and drove lymphomagenesis in Smurf2-deficient mice. Consistently, a significant decrease of Smurf2 expression was observed in human primary DLBCL samples, and more importantly, a low level of Smurf2 expression in DLBCL correlated with poor survival prognosis. Moreover, I found that hematopoietic stem cells (HSCs) in Smurf2-deficient mice had enhanced function compared to wild-type controls. This enhanced stem cell function was associated with increased cell proliferation and decreased p16 expression, suggesting that defective senescence response in Smurf2-deficient mice leads to increased self-renewal capacity of HSCs. My study, for the first time, offers direct genetic evidence of an important tumor suppressor function for Smurf2 as well as its function in contributing to stem cell aging. Collectively, these findings provide strong evidence supporting the Antagonistic Pleiotropy Theory of senescence in tumorigenesis and aging.

TNFα-induced programmed necrosis is a caspase-independent cell death program that is contingent upon the formation of a multiprotein complex termed the necrosome. The association of two of the components of the necrosome, receptor interacting protein 1 (RIP1) and RIP3, is a critical and signature molecular event during necrosis. Within this complex, both RIP1 and RIP3 are phosphorylated which are consequential for transmission of the pro-necrotic signal. Namely, it has been demonstrated that RIP3 phosphorylation is required for binding to downstream substrates. Nevertheless, the regulatory mechanisms governing necrosome activation remain unclear. Since necrosis is implicated in a variety of different diseases, understanding the biochemical signaling pathway can potentially yield future drug targets. I was interested in identifying other regulators of necrosis in hope of gaining a better understanding of the necrosis signaling pathway and regulators of the necrosome. To address this, I screened a cancer gene siRNA library in a cell line sensitive to necrosis. From this, I independently identified CYLD as a positive regulator of necrosis. Previous studies suggest that deubiquitination of RIP1 in the TNF receptor (TNFR)-1 signaling complex is a prerequisite for transition of RIP1 into the cytosol and assembly of the RIP1-RIP3 necrosome. The deubiquitinase cylindromatosis (CYLD) is presumed to promote programmed necrosis by facilitating RIP1 deubiquitination in this membrane receptor complex. Surprisingly, I found that TNFα could induce RIP1-dependent necrosis in CYLD-/- cells. I show that CYLD does not regulate RIP1 ubiquitination at the receptor complex. Strikingly, assembly of the RIP1-RIP3 necrosome was delayed, but not abolished in the absence of CYLD. In addition to the TNFR-1 complex, I found that RIP1 within the necrosome was also ubiquitinated. In the absence of CYLD, RIP1 ubiquitination in the NP-40 insoluble necrosome was greatly increased. Increased RIP1 ubiquitination correlated with impaired RIP1 and RIP3 phosphorylation, a signature of kinase activation. My results show that CYLD regulates RIP1 ubiquitination in the NP-40 insoluble necrosome, but not in the TNFR-1 signaling complex. Contrary to the current model, CYLD is not essential for necrosome assembly. Rather, it facilitates RIP1 and RIP3 activation within the necrosome and the corollary is enhancement of necrosome functionality and subsequent necrosis. My results therefore indicate that CYLD exerts its pro-necrotic function in the NP-40 insoluble necrosome, and illuminates the mechanism of necrosome activation.