Yes / Hypothalamic tanycytes in median eminence (ME) are emerging as a crucial cell population that regulates endocrine output, energy balance and the diffusion of blood-born molecules. Tanycytes have recently been considered as potential somatic stem cells in the adult mammalian brain, but their regenerative and tumorigenic capacities are largely unknown. Here we found that Rax+ tanycytes in ME of mice are largely quiescent but quickly enter the cell cycle upon neural injury for self-renewal and regeneration. Mechanistically, Igf1r signaling in tanycytes is required for tissue repair under injury conditions. Furthermore, Braf oncogenic activation is sufficient to transform Rax+ tanycytes into actively dividing tumor cells that eventually develop into a papillary craniopharyngioma-like tumor. Together, these findings uncover the regenerative and tumorigenic potential of tanycytes. Our study offers insights into the properties of tanycytes, which may help to manipulate tanycyte biology for regulating hypothalamic function and investigate the pathogenesis of clinically relevant tumors.

Inflammation has been found to play a role in the development of many different tumors. However, a tumor's ability to evade immune cell recognition can be integral to its progression as well. The following works explore this complicated role with a focus on histiocytic sarcoma (HS) and breast cancer. Chapter 1 opens with a broad overview of inflammation in tumorigenesis while Chapter 2 focuses on a review and discussion of current HS literature. Our investigations into the role of inflammation specifically in HS are initiated in Chapter 3 where we explore the role of the regulatory NLR, NLRX1, in the development of HS in mice. NLRX1 is an intracellular patter recognition receptor that functions to regulate pro-inflammatory cell pathways. Our studies reveal that in carcinogen-induced HS in mice, NLRX1 acts as a tumor suppressor. Moreover, when NLRX1 is lost, tumors that develop are associated with increases in expression of genes in NF-κB and AKT pathways. Though uncommon, HS is a clinically relevant tumor in dogs. In Chapter 4, we further investigate the role of the pathways identified in Chapter 3 in canine patients. Not only were these pathways increased, but our results also revealed previously unreported differences in tumors diagnosed as HS versus those diagnosed as hemophagocytic HS. To improve the use of canine HS both as an experimental and translational model, we sought to create a murine xenograft model. In Chapter 5, we discuss the development of our model and the results of pilot studies using targeted drug therapy. The focus of Chapters 3-5 is to further explore the role of inflammation in the development of HS. However, as aforementioned, the role of inflammation in tumorigenesis is quite complicated. In Chapter 6, we aim to address the concept that the lack of inflammation through immune evasion, can also be important in tumors. Breast cancer in humans is traditionally recognized as being highly immunosuppressive. In this final chapter, we investigate the use of an attenuated strain of bacteria to treat these tumors by way of shifting the immunosuppressive tumor microenvironment to a more pro-inflammatory state. / Ph. D.

In the presented study, we demonstrate that the alternative splicing of caspase 9 was dysregulated in a large percentage of NSCLC tumors and cell lines. These findings led to the hypothesis that survival pathways activated by oncogenic mutation regulated this mechanism. Indeed, the oncogenic PI3-Kinase/Akt pathway was demonstrated to regulate the alternative splicing of caspase 9. Further mechanistic studies demonstrate that multiple Akt isoforms can regulate the alternative splicing of caspase 9 in NSCLC. Akt was additionally shown to mediate the exclusion of the exon 3,4,5,6 cassette of caspase 9 via the phospho-state of the RNA trans-factor, SRp30a. Mutagenesis studies identified serine 199, serine 201, serine 227, and serine 234 as critical residues regulating the alternative splicing of caspase 9, as well as playing a role in the anchorage-independent growth of A549 cells. Since dysregulation of this splicing mechanism correlated with NSCLC tumors/cell lines and constitutively active Akt, oncogenic factors for NSCLC known to activate the PI3-Kinase/Akt pathway were examined in HBEC-3KT cells. In contrast to k-ras V12 expression, the overexpression/mutation of EGFR affected the alternative splicing of caspase 9 in a pro-oncogenic manner, dramatically lowering the caspase 9a/9b mRNA ratio. Stable downregulation of caspase 9b by shRNA blocked the ability of E746-A750 del EGFR expressing HBEC-3KTs to induce anchorage-independent growth, suggesting a role for caspase 9b as a cooperative oncogenic factor. These findings were further corroborated by the ability of caspase 9b expression to completely block the inhibition of clonogenic colony formation by erlotinib. Therefore, this study demonstrates that oncogenic factors activating the PI3-Kinase/Akt pathway regulate the alternative splicing of caspase 9, to produce caspase 9b, via a coordinated mechanism involving the phosphorylation of SRp30a. In additional studies, we demonstrate that the PI3-Kinase/PKCι pathway, a pathway important for cancer cell survival and transformation of lung epithelial cells, regulates the alternative splicing of Bcl-x pre-mRNA via modulation of SAP155 expression to produce an anti-apoptotic phenotype in NSCLC. Therefore, these studies link oncogenic mechanisms in NSCLC to the therapeutically relevant and distal target mechanisms of caspase 9 and Bcl-x pre-mRNA splicing.

Mutações com ganho de função do proto-oncogene Ras se encontram entre umas das mais freqüentes modificações em cânceres humanos, além disso, tumores com esses caracterísitcas possuem, em geral, mau prognóstico. O objetivo inicial desta tese foi estudar novos mecanismos anti-proliferativos disparados por dois agentesmitogênicos, FGF2 (\"Fibroblast Growth Factor 2\") e PMA (\"Phorbol-12-Myristate-13-Acetate\", (um diéster de forbol), sobre células de camundongos transformadas por Ras e refratárias a apoptose. Para isso utilizamos duas linhagens celulares: uma linhagem naturalmente trtansformada por uma ampliação do gene K-Ras, que é derivada de um tumor de córtex adreno-cortical de camundongo e é denominada Y1, e uma sublinhagem derivada de Balb/c-3T3, transformada em laboratório com o oncogene H-RasV12 humano. A fim de se elucidar o mecanismo de ação de FGF2, foram selecionadas e caracterizadas múltiplas sublinhagens clonais resistentes a FGF2, derivadas das linhagens parentais Y1 e B61. Mostramos assim, que o FGF2 exerce um forte efeito negativo, de forma que os clones resistentes ao mesmo tendem a perder aos altos níveis de expressão da proteína Ras. Mostramos ainda que esses células passam a ser dependentes de FGF2 para crescer em cultura, perdem a capacidade de crescimento em suspensão e são menos tumorigênicas quando comparadas às células parentais. Em uma segunda etapa, caracterizamos o efeito citotóxico de PMA sobre células transformadas por Ras, e vimos que esse efeito é mais acentuado para células transformadas por K-Ras, mas é nulo sobre células imortalizadas não tumorigênicas. Mostramos ainda que esse efeito passa pela ativação da via de PKC. A inibição da proliferação por PMA se deve, ao menos parcialmente, à indução de senescência nessas células. De forma semelhante ao que foi para o estudo com FGF2, foram selecionados clones resistentes a PMA, derivados de Y1. Os clones obtidos se mostraram muito instáveis, pouco resistentes a PMA e dependentes de FGF2 para crescer. Todos os clones testados se mostram tumorigênicos, entretanto, apresentaram maior tempo de latência, estaticamente diferente da célula parental, Y1. Assim, neste trabalho, mostramos que duas substâncias, com caráter mitogênico e potencialmente oncogênico, são capazes de inibir seletivamente a proliferação de células transformadas por Ras, uma vez que elas não têm efeito sobre células não transformadas. Desvendar os mecanismos que causam a citotoxidade dessas substâncias deve trazer informações relevantes com possibilidades de impacto em terapia de tumores dependentes dos oncogenes Ras. / Amplification and gain of function mutations in ras proto-oncogenes are frequent genetic lesions in human cancers of bad prognostic. This thesis aimed to investigate novel anti-proliferative mechanisms induced by two mitogens, FGF2 (\"Fibroblast Growth Factor 2\") and PMA (\"Phorbol-12-Myristate-13-Acetate\", a phorbol diester), in murine cell lines transformed by ras and resistant to apoptosis. To this end, we took two different mouse malignant cell lines: Y1, a cell line derived from an adrenal tumor, naturally transformed by K-ras amplification and another one, 3T3-B61, obtained by transformation of Balb-3T3 fibroblasts with the H-rasV12 oncogene. To elucidate FGF2 mechanisms of action, we selected, isolated and characterized clonal sublines resistant to FGF2 from both Y1 and 3T3-B61 parental lines. FGF2-resistant clones are rare normal-like revertant sublines that no longer display Ras over expression, dependent on FGF2 for growth, do not grow in suspension cultures and exhibit low tumorigenicity in Nude mice. These results show that FGF2 exerts a strong selective pressure against ras-transformed cells, inducing senescence and irreversibly blocking proliferation. Differently from FGF2 , PMA citotoxic effect is completely dependent on PKC activity. In addition, PMA is highly toxic to K-Ras transformed Y1 cells, poorly toxic to H-Ras-transformed 3T3-B61 cells and not toxic to immortalized non tumorigenic cell lines. Attempts to select PMA-resistant cells fropm Y1 parental line have yielded very rare, highly clonal sublines, dependent on FGF2 for proliferation. In conclusion, two mitogens, FGF2 and PMA, can selectively inhibit Ras-driven proliferation, a phenomenon of great interest for biology and therapy of tumors dependent on ras oncogenes.

La télomérase est une ribonucléoprotéine, constituée d’un composant ARN (hTR) qui sert dematrice à l’addition des séquences télomériques aux extrémités des chromosomes et d’un composantprotéique catalytique à activité de transcriptase inverse (hTERT). La réactivation de la télomérasedans 90% des cancers compense le raccourcissement des télomères, permettant ainsil’immortalisation et la survie des cellules tumorales. Ce rôle canonique de la télomérase estaujourd’hui bien documenté. Cependant des travaux récents suggèrent que la télomérase pourraitavoir d'autres fonctions indépendantes de son rôle sur le maintien de la longueur des télomères dansla tumorigénèse et/ou la progression tumorale.Dans les neuroblastomes (NB), l’augmentation du niveau d’activité télomérase (AT) estassociée à un stade avancé de la maladie et à un mauvais pronostic. En effet, plusieurs études ontmontré que les neuroblastomes agressifs ont un niveau élevé d’AT alors que les tumeurs de bonpronostic, ont peu ou pas d’AT. En effet, les NB de stade 4S ayant la capacité de régresserspontanément ont un très faible niveau d'AT. Ces observations suggèrent que la télomérase peutjouer un rôle crucial dans le développement des NB.Afin de mieux comprendre l’implication de la télomérase dans le phénotype agressif desneuroblastes malins et dans la chimiorésistance, nous avons caractérisé les modificationsphénotypiques et génotypiques induites par l’inhibition de la télomérase via l’expression ectopiqued’un mutant dominant négatif catalytiquement inactif (DN-hTERT) dans la lignée IGR-N-91 induit unedifférenciation cellulaire de type stromale et une sensibilisation à l’apoptose en réponse à trois agentscytotoxiques (cisplatine, staurosporine, TRAIL). Cette chimiosensibilisation n’est pas la conséquenced’un raccourcissement des télomères mais probablement celui d’une modulation de l’expression decertains gènes impliqués dans la réponse apoptotique (ré-expression de la caspase 8 et de p53sauvage), suggérant une fonction non canonique de la télomérase. De plus, nous avons montréqu’hTERT régule activement l’expression de N-Myc. En effet, l’expression ectopique du mutanthTERT-DN entraîne une perte des copies surnuméraires de N-Myc conduisant à l’extinction del'expression de la protéine alors que la surexpression d’hTERT sauvage augmente au contraire lenombre de copies du gène. Cette élimination de la protéine N-Myc pourrait être le signe d’une pertedu caractère agressif des cellules tumorales comme en témoigne la diminution de l’expression de laNSE (marqueur de mauvais pronostique des NB) et l’induction du CD44 dans les cellules hTERT-DN.L’ensemble de nos résultats démontre donc un nouveau rôle majeur de la télomérase,indépendant de sa fonction canonique d’élongation des télomères, dans l’acquisition du phénotypemalin et dans la chimiorésistance des NB. Ces résultats sont importants en termes de connaissancede la biologie du NB et des possibilités thérapeutiques. En effet, ces résultats suggèrent quel’inhibition de la télomérase comme stratégie anti-cancéreuse est une approche qui présente un intérêttout particulier dans les cas de NB de stade 4 dans lesquels le taux de survie des patients reste trèsinsuffisant malgré les thérapeutiques les plus intensives. / Telomerase is a ribonucleoprotein consisting of an RNA component (hTR) that serves as atemplate for the addition of telomeric sequences at the ends of chromosomes and a proteincomponent catalytic activity of reverse transcriptase (hTERT). The reactivation of telomerase in 90%of cancers compensates the shortening of telomeres, allowing the immortalization and survival oftumor cells. This canonical role of telomerase is now well documented. However recent studiessuggest that telomerase may have other functions beyond its role in maintaining telomere length intumorigenesis and / or tumor progression.In neuroblastoma (NB), increased levels of telomerase activity (TA) is associated withadvanced disease and poor prognosis. Indeed, several studies have shown that aggressiveneuroblastomas have a high level of TA while favourable tumors, have little or no TA. Therefore, lowtelomerase activity appears to be linked with regression or maturation of NB as it can be seen in theparticular group of 4S stage neuroblastoma. These observations suggest that telomerase may play acrucial role in the development of NB.To better understand the involvement of telomerase in the aggressive phenotype of malignantneuroblasts and drug resistance, we characterized the phenotypic and genotypic changes induced byinhibition of telomerase via ectopic expression of a mutant dominant negative catalytically inactive(DN-hTERT) in a metastatic chemoresistant NB cell line IGR-N-9. Our results show that theexpression of this mutant induces a stromal-type cell differentiation a sensitization to apoptosis inresponse to three cytotoxic agents (cisplatin, staurosporine, TRAIL). The chemosensitization is not theresult of telomere shortening but probably f a modulation of the expression of certain genes involved inthe apoptotic response (re-expression of caspase 8 and wild-type p53), suggesting a noncanonicalfunction of telomerase Furthermore, we showed that hTERT actively regulates the expression ofMYCN. Indeed, ectopic expression of the inactive mutant causes a loss of supernumerary copies ofMYCN leads to the extinction of the expression of the protein, whereas overexpression of wild hTERTincreases the number of copies of the MYCN gene. The elimination of MYCN protein could be a signof a loss of the aggressiveness of the tumor cells as evidenced by the decreased expression of NSE(a marker of poor prognosis of NB) and induction of CD44 in DN-hTERT cells.Overall, our findings thus demonstrate a new role of telomerase independent of its canonicalfunction of telomere elongation in the acquisition of the malignant phenotype and drug resistance inNB. These results are important in terms of knowledge of the biology of NB and therapeuticpossibilities. Indeed, our data suggest that inhibition of telomerase as an anticancer strategy is anapproach that has a particular interest in cases of stage 4 NB in which the survival rate of patientsremains very inadequate despite the therapeutic more intensive.

L’épithélium mammaire est organisé en bicouche : une couche de cellules luminales sécrétrices et une couche de cellules basales myoépithéliales. Des cellules souches multipotentes capables de régénérer l’épithélium mammaire après transplantation résident dans le compartiment basal tandis que les deux couches épithéliales mammaires contiennent des cellules souches/progénitrices à propriétés clonogéniques. De nombreuses études suggèrent que les cellules souches et progénitrices de la glande mammaire pourraient être les cibles d’une transformation oncogénique menant à des cancers du sein, justifiant l’attention particulière portée à ces populations cellulaires mineures.Pour étudier les rôles du proto-oncogène Myc, ou du suppresseur de tumeurs Trp53, dans le contrôle des fonctions des cellules souches et progénitrices, nous avons généré des souris transgéniques présentant une invalidation de Myc ou Trp53 dans la couche basale de l’épithélium mammaire. Les souris déficientes en Myc présentent des glandes hypoplasiques et les cellules basales dépourvues de Myc perdent complètement leur capacité régénérative. En revanche, la perte de p53 induit une augmentation des populations de cellules souches et progénitrices avec un potentiel d’autorenouvellement accru, suggérant que p53 restreint la propagation des cellules souches/progénitrices dans l’épithélium mammaire. Ces résultats montrent que Myc et p53 jouent un rôle essentiel dans le contrôle des fonctions des cellules souches et progénitrices mammaires.Les souris transgéniques K5Ncat générées précédemment dans notre laboratoire, développent des carcinomes mammaires de type basal avec des composants métaplastiques, induits par l’expression d’une forme activée de la -caténine dans la couche basale mammaire. Nous avons trouvé que la population de cellules souches fonctionnelles est augmentée dans l’épithélium pré-néoplasique des souris K5Ncat. Pour étudier les mécanismes cellulaires et moléculaires du développement de ces tumeurs, les souris déficientes en Myc ou Trp53 ont été croisées avec des souris K5Ncat. Nous avons constaté une inhibition complète de la tumorigenèse induite par la -caténine en absence de Myc et une accélération de celle-ci en absence de p53. Nos résultats suggèrent que les cellules souches/progénitrices basales pourraient être à l’origine des tumeurs mammaires de type basal avec des caractéristiques métaplastiques et que les rôles joués par Myc et p53 dans la tumorigenèse sont liés à leurs fonctions régulatrices des cellules souches mammaires. / Numerous studies suggested that mammary stem and progenitor cells could be targets of the oncogenic transformation in breast cancer, attracting a particular attention to these cell populations. Mammary epithelium is organized as bilayer, with a layer of luminal secretory cells and a basal myoepithelial layer. Multipotent stem cells able to regenerate mammary epithelium upon transplantation reside in the basal compartment, whereas both mammary epithelial cell layers contain clonogenic stem/progenitor cells.To study the roles played by a proto-oncogene Myc and a tumor suppressor p53 in the control of mammary stem and progenitor cell function, we generated mouse mutants presenting conditional deletion of Myc and Trp53 genes in the basal layer of the mammary epithelium. The Myc-mutants presented hypoplastic glands, and basal cells depleted of Myc were unable to regenerate mammary epithelium. In contrast, deletion of p53 led to increased stem and progenitor cell activity with enhanced capacity to self-renew suggesting that p53 restricts the propagation of the stem/progenitor cell populations in the mammary epithelium. These data clearly demonstrate that Myc and p53 play a central role in the control of the mammary stem cell function. Transgenic mice K5Ncat obtained previously by our team develop basal-type mammary carcinomas with metaplastic component, induced by the expression of activated (N-terminally truncated) -catenin in mammary epithelial basal layer. We found that stem cell activity is increased in preneoplastic K5Ncat mammary epithelium. To study molecular and cellular mechanisms underlying tumor development, Myc- and Trp53-mutants were bred to K5Ncat mice. The deletion of Myc completely inhibited tumorigenesis, whereas in the absence of p53, tumor development was significantly accelerated. Our data suggest that basal stem/progenitor cells might be at the origin of basal-type breast tumors with metaplastic characteristics and that roles played by Myc and p53 in the tumorigenesis are associated with their regulatory functions in stem cells.

Le cancer colorectal représente la deuxième cause de mortalité par cancer en France. Dans l’intestin, l’initiation et la progression tumorale sont corrélées à l’accumulation séquentielle de mutations génétiques et épigénétiques au niveau du compartiment de renouvellement de l’épithélium. Ces altérations ont pour conséquence une croissance incontrôlée de l’épithélium et, à terme, la formation de lésions cancéreuses. En tenant compte du nombre croissant de cas de cancers colorectaux, la découverte de facteurs de prédisposition de cette pathologie reste d’un intérêt majeur. Les données de séquençage du génome humain ne suffisant pas à expliquer la prévalence de la maladie à l’échelle de la population, nous nous intéressons aux mécanismes permettant le contrôle de l’expression de certains gènes : les mécanismes épigénétiques. Dans notre équipe nous disposons de modèles animaux génétiquement modifiés nous permettant d’étudier, dans des conditions proches de la pathologie humaine, les phases précoces de l’initiation tumorale. Ces souris, bien que génétiquement identiques, développeront pourtant un nombre de tumeurs intestinales très variable. En comparant les profils moléculaires de souris développant peu ou beaucoup de tumeurs intestinales, nous souhaitons mettre en évidence les facteurs épigénétiques mis en jeu pour expliquer cette différence de susceptibilité à la tumorigenèse. Pour ce faire, nous avons mis en place une stratégie visant à prélever un échantillon intestinal, par chirurgie, sur de jeunes souris qui ont ensuite été suivies jusqu’à l’âge adulte et à l’apparition des tumeurs. Cette stratégie innovante nous a permis de corréler les profils d'expression et de methylation d'ADN d’intestins développant peu ou beaucoup de tumeurs, ouvrant la possibilité de disposer de nouveaux marqueurs prédictifs quant aux chances de développer un cancer. Ces données sont complétées par une étude sur les conséquences de la perte du gène suppresseur de tumeur Apc, un gène couramment muté dans les cancers colorectaux humain. A l’aide de modèles de souris d’invalidation inductible, nous avons déterminé les conséquences de la perte d’Apc sur les profils de méthylation de l'ADN des cellules souches intestinales, et leur capacité à initier une tumeur. L’ensemble de ces différentes projets développés dans le cadre de ma thèse nous ont permis de mieux comprendre les mécanismes cellulaires liés à la prédisposition et à l’initiation tumorale et de proposer des nouvelles stratégies diagnostiques et d’évaluation du risque individuel. / Cancer initiation and progression represent the outcome of the progressive accumulation of genetic and epigenetic alterations. Global changes in the epigenome are now considered as a common hallmark of malignancies. However, most of our present knowledge represents the result of the comparison between fully established malignancies and their surrounding healthy tissue. Such comparison is not informative about the epigenetic contribution to the very early steps of cancer onset. By performing DNA methylation and gene expression profiling of the intestinal epithelium of relevant in vivo models we aim at shedding light on the correlation between the interindividual epigenetic polymorphisms within the population and the relative risk to develop malignancies, and establish the existence of a molecular signature associated with an increased susceptibility to develop intestinal cancer. Our results confirm that a considerable degree in the variability associated to cancer susceptibility cannot be ascribed to major genetic changes and that such heterogeneity seems to correlate with distinct molecular profiles associated to classes of poorly or highly susceptible isogenic animals.We also investigated in vivo the timing at which the remodeling occur at the epigenomic scale by analyzing the alterations in the DNA methylation and gene expression profiles of intestinal stem cells upon the loss of the Apc gene, the most common genetic lesion associated with human colorectal cancer initiation. We found that the loss of function of Apc in the Lgr5-positive intestinal stem cell compartment is rapidly accompanied by a reprogramming of the DNA methylation profiles resulting in altered gene expression and impaired fate determination in those cells. The results show that part of the phenotype resulting from the constitutive activation of the Wnt pathway upon Apc loss is acquired via differential epigenetic regulation of key biological processes controlling the balance between self-renewal and differentiation. By using conditional genetic ex vivo models we found part of these oncogenic effects to be reversible via the modulation of the machinery responsible for de novo methylation of the DNA.Overall, this work confirms that the epigenetic remodeling is an early event in tumorigenesis that might even precede actual cell transformation. The functional impact of our findings on cancer initiation is currently under investigation.

E4F1 est un facteur de transcription liant l'ADN, exprimé de façon ubiquitaire par tous les tissus, et qui possède une activité E3 ubiquitine ligase atypique dirigée contre le suppresseur de tumeur p53. La protéine E4F1 interagit directement avec plusieurs suppresseurs de tumeurs cellulaires et des oncogènes viraux (p53, pRb, DRAL, RASSF1A, p19ARF, BMI1, HBX et GAM1...), suggérant qu’elle est elle-même impliquée dans la tumorigenèse. La perte d’E4F1 dans des fibroblastes embryonnaires (Mefs) transformés ou dans des cellules de sarcomes histiocytaires déficientes pour la voie p53, entraine la mort de ces cellules. La même inactivation d'E4F1 dans les cellules normales n'affecte pas leur survie mais entraine un arrêt de prolifération. Des analyses transcriptomiques et de liaison à l'ADN à l'échelle du génome entier (ChIP-seq et analyses différentielles des transcriptomes de cellules E4F1 WT et KO) nous ont permis d’identifier une centaine de gènes liés et régulés directement par E4F1. Ces gènes codent notamment pour des protéines mitochondriales impliquées dans le métabolisme et l'homéostasie de cette organelle, dont plusieurs composants et régulateurs de l'enzyme multimérique, pyruvate déshydrogénase. Un second groupe de gênes cibles d’E4F1 est impliqué dans la réponse aux dommages à l'ADN, dont le gène codant pour la kinase CHK1 qui joue un rôle essentiel dans le contrôle de la stabilité du génome. En accord avec la fonction de ces gènes cibles, la perte d’E4F1 entraine des perturbations du métabolisme cellulaire et des checkpoints de réponse aux stress génotoxiques. Dans les cellules déficientes pour la voie p53, ces perturbations conduisent à des stress oxydatifs (surproduction de ROS mitochondriaux) et énergétiques, suivis de dommages aux protéines et à l'ADN, et in fine, à une mort cellulaire massive. Dans les cellules compétentes pour la voie p53 ces altérations sont fortement atténuées et conduisent à un arrêt de la prolifération. Une partie des effets protecteurs de p53 est due à sa capacité à stimuler l'expression du gène ALDH4a1 qui code pour une aldehyde dehydrogenase impliquée dans le catabolisme de la proline et dont l'activité possède des propriétés anti-oxydantes.Mes travaux mettent également en évidence que le niveau d'expression de la protéine E4F1 et son activité augmentent lors de la transformation cellulaire ainsi qu'en réponse à des stress énergétiques, génotoxiques ou oxydatifs. Dans ces trois dernières conditions, la phosphorylation d'E4F1 est également augmentée au niveau de plusieurs sérines qui ont été identifiées par spectrométrie de masse. En résumé, tous ces éléments indiquent qu'E4F1 est un acteur important du contrôle de l'homéostasie métabolique et de la réponse aux stress, particulièrement essentiel pour la survie des cellules cancéreuses déficientes pour la voie p53. Mes observations suggèrent également qu'E4F1 est activé en réponse à différents stress et qu'il pourrait jouer un rôle essentiel dans la capacité des cellules cancéreuses à s'adapter aux multiples stress environnementaux auxquels elles sont exposées au cours de la tumorigenèse. / The ubiquitously expressed E4F1 protein acts as a transcription factor that binds a consensus DNA sequence at promoters, and as an atypical E3-ligase for the tumor suppressor p53. E4F1 physically interacts with several bona fide cellular tumor suppressors and viral oncoproteins (including p53, pRb, DRAL, RASSF1A, p19ARF, BMI1, HBX and GAM1...), suggesting that it might itself be involved in tumorigenesis. E4F1 genetic inactivation in transformed mouse embryo fibroblasts (Mefs) and in hematopoietic tumors deficient for the p53 pathway, results in massive cell death. Importantly, inactivation of E4F1 in normal cells does not affect cell survival. Genome wide approaches (ChIP-seq profiling and comparative transcriptomics performed on E4F1 WT and KO cells) identified a limited list (100) of genes that are bound and directly regulated by E4F1. Several E4F1 target genes code for mitochondrial proteins involved in mitochondria metabolism and homeostasis, including several components of the pyruvate dehydrogenase complex. Another set of E4F1 target genes codes for factors involved in DNA repair and damage checkpoints, including the checkpoint kinase CHK1. Accordingly, both mitochondrial and checkpoint functions are altered in E4F1 KO cells. In proliferating cells deficient for the p53 pathway, these defects lead to energetic and oxidative stresses, protein and DNA damages, and in fine, massive cell death. In p53-proficient cells, these alterations are attenuated and lead to growth arrest. Part of this protective effect of p53 is mediated by ALDH4a1, a p53 target gene encoding an aldehyde dehydrogenase involved in proline catabolism and that exhibits antioxidant properties.In this thesis, I also demonstrate that E4F1 protein level and activity is increased during cell transformation and upon exposure to genotoxic, energetic or oxidative stresses. These stresses also lead to E4F1 phosphorylation at specific serine residues that were identified by mass spectrometry. All together this work shows that E4F1 controls cellular functions that are important for mammalian cells metabolic homeostasis and stress responses, and that are essential for the survival of p53-deficient cancer cells. This work also suggests that E4F1 is activated in response to various stresses and therefore, that it could play an essential role in allowing cancer cells to adapt to environmental stresses.

Submitted by Maria Aparecida (cidazen@gmail.com) on 2017-11-27T17:43:41Z No. of bitstreams: 1 Marta Belfort.pdf: 1659970 bytes, checksum: 7aca8a1f671f32a3e25959a2840b065f (MD5) / Made available in DSpace on 2017-11-27T17:43:41Z (GMT). No. of bitstreams: 1 Marta Belfort.pdf: 1659970 bytes, checksum: 7aca8a1f671f32a3e25959a2840b065f (MD5) Previous issue date: 2017-08-28 / FAPEMA,CNPQ. / Penile cancer (PeCa) is a rare neoplasm in developed countries; however, its incidence is high in underdeveloped countries. In Brazil, regions North and Northeast are those with the higher number of cases for the disease. Among the factors associated with this neoplasm we highlight: poor hygiene, phimosis, chronic inflammation and infection by human papilloma virus (HPV). Changes in the PI3K/AKT/PTEN cell signaling pathway have been reported for several malign neoplasms, but little is known about the involvement of this pathway in penile tumors. Thus, the aim of this study was to verify the role of HPV infection and the occurrence of copy number alterations (CNA) in genes from the signaling pathway mediated by receptors of growth factors and PI3K in a population characterized by advanced tumors and high frequency of high risk HPV. To achieve that, we collected tumor tissue samples (both fresh and in formalin-fixed paraffin-embedded tissue-FFPE) from 34 patients from two reference hospitals: Instituto Maranhense de Oncologia Aldenora Belo (IMOAB) and the Hospital Universitário Presidente Dutra from the Universidade Federal do Maranhão (HU-UFMA). Fresh tumors were submitted to detection and genotyping of HPV by Nested PCR (Polymerase Chain Reaction) and direct sequencing. CNA analyzes were carried out in FFPE tissue from a subgroup HPV positive (91.2%), of which 88.2% were at high oncogenic risk. TaqMan® Copy Number Assays (Life TechnologiesTM) and CopyCaller software version v2.0 were performed to determine copy number for EGFR, HER3, HER4, AKT1, AKT2, PI3KCA and PTEN. Increase of 3 and 4 copies was considered gains, while increase of 5 or more copies was considered amplifications. The presence of a single gene copy was referred to as loss, while the absence of two copies was named deletion. Clinical and histopathological parameters were analyzed as to the presence of HPV and to CNAs. Our data showed that EGFR/PI3K/AKT/PTEN signaling pathway is highly altered in PeCa. The results showed that 100% of the tumors presented an increase of the number of copies for HER3; out of those, 93.9% were amplified, with 84.4% having 10 or more copies. EGFR also showed an increase of copies in 87.8% of tumor samples, out of which 65.6% were amplifications, with 48.2% having more than 10 copies. Furthermore, HER4 and AKT1 also presented an increase in the number of copies of 20.6% and 15%, respectively. AKT1 had a higher frequency of tumors with a regular number of copies (78.8%). On the other hand, PI3KCA, HER4, PTEN and AKT2 presented a higher frequency of samples with deletions, presenting 56%, 52.9%, 39% and 36%, respectively. Loss of copies was also frequent on tumors, so that genes AKT2, PTEN and PI3KCA appeared in heterozygosis in 60.6%, 54.5% and 37.5%, respectively. Our data show the occurrence of genetic alterations that may justify the differential expression of growth factor receptors and the downstream genes of the PI3K/ AKT pathway in penile carcinoma. However, in this study there was no association between CNAs and clinical and histopathological variables. On the other hand, taking into consideration the high frequency of HPV in the evaluated tumors, we suggest that CNAs are related to HPV integration into genome host. Finally, we highlight the high frequency of tumors with amplifications in HER3 and EGFR, reinforcing these markers as targets for specific therapies in CaPe. / Câncer de pênis (CaPe) é uma neoplasia rara em países desenvolvidos, entretanto, sua incidência é elevada em países subdesenvolvidos. No Brasil, as regiões Norte e Nordeste são as regiões com os maiores números de casos da doença. Dentre os fatores de risco, destaca-se a má higiene do órgão genital, fimose, inflamação crônica e infecção pelo papilomavírus-humano (HPV). Tem sido reportada alterações na via de sinalização celular PI3K/AKT/PTEN em diversas neoplasias malignas, entretanto, pouco se sabe sobre o envolvimento dessa via nos tumores de pênis. Assim, buscou-se neste trabalho verificar o papel da infecção por HPV e a ocorrência de alteração no número de cópias (CNAs) em genes da via de sinalização controlada por receptores dos fatores de crescimento e PI3K, em uma amostra caracterizada por tumor avançado e alta frequência de HPV de alto risco. Para isso, foram coletadas amostras de tecido tumoral (fresco e de tecido fixado com formalina e embebido em parafina - FFPE) de 34 pacientes provenientes de dois hospitais de referência, Instituto Maranhense de Oncologia Aldenora Belo (IMOAB) e o Hospital Universitário Presidente Dutra da Universidade Federal do Maranhão (HU-UFMA). As amostras de tumor fresco foram submetidas a detecção e genotipagem de HPV por Nested PCR (Polymerase Chain Reaction) e sequenciamento direto. As análises de CNAs foram realizadas em amostras de FFPE, HPV positivas (91,2%), das quais 88,2% eram de alto risco oncogênico. Utilizou-se o ensaio TaqMan® Copy Number Assays (Life TechnologiesTM) e o software CopyCaller versão 2.0 para determinação do número de cópias dos genes EGFR, HER3, HER4, AKT1, AKT2, PIK3CA e PTEN. Aumento de 3 e 4 cópias foi considerado ganho, enquanto aumento de 5 ou mais cópias foi considerado amplificação. A presença de uma única cópia gênica foi nomeada perda, enquanto a ausência de duas cópias foi nomeada deleção. Os parâmetros clínicos e histopatológicos foram analisados quanto a presença de HPV e também quanto a CNAs. A via de sinalização celular EGFR/PI3K/AKT/PTEN revelou-se altamente alterada nos tumores de pênis. Observou-se que 100% dos tumores apresentaram aumento de número de cópias do gene HER3. Destes, 93,9% apresentaram-se amplificados, sendo que 84,4% apresentavam 10 ou mais cópias. O gene EGFR apresentou aumento de cópias em 87,8% das amostras, das quais 65,6% eram amplificações, sendo que 48,2% apresentavam mais de 10 cópias. Além desses, os genes HER4 e AKT1 também apresentaram aumento de cópias gênicas, em 20,6% e 15%, respectivamente. AKT1 apresentou maior porcentagem de tumores com número normal de cópias (78,8%). Por outro lado, os genes PI3KCA, HER4, PTEN e AKT2 apresentaram maiores frequências de amostras com deleções, com 56%, 52,9%, 39% e 36%, respectivamente. As perdas também foram frequentes nos tumores, de modo que os genes AKT2, PTEN e PI3KCA apresentaram-se em heterozigose em 60,6%, 54,5% e 37,5%, respectivamente. Nossos dados mostram a ocorrência de alterações genéticas que podem justificar a expressão diferencial dos receptores de fatores de crescimento e de genes downstream da via PI3K/AKT em carcinoma peniano. No entanto, não foi encontrada associação entre CNAs e as variáveis clínicas e histopatológicas. Por outro lado, considerando-se a alta frequência de HPV nos tumores avaliados, levanta-se a possibilidade de haver uma relação entre a integração do HPV no genoma do hospedeiro e a ocorrência de CNAs em CaPe. Finalmente, destacamos a alta frequência de tumores com amplificações em HER3 e EGFR, abrindo a possibilidade desses marcadores serem utilizados como alvos para terapias específicas em CaPe.

Submitted by Maria Aparecida (cidazen@gmail.com) on 2017-11-27T18:12:10Z No. of bitstreams: 1 Juliana Macedo.pdf: 3843653 bytes, checksum: a40308c73af9a3d7ce2e665d6464831f (MD5) / Made available in DSpace on 2017-11-27T18:12:10Z (GMT). No. of bitstreams: 1 Juliana Macedo.pdf: 3843653 bytes, checksum: a40308c73af9a3d7ce2e665d6464831f (MD5) Previous issue date: 2017-08-28 / FAPEMA. / Penile cancer (PeCa) is a rare neoplasm with higher incidence in regions with low socioeconomic indexes. In Brazil, most of the men afflicted by this disease reside in the North and Northeast. Among the main risk factors are lack of hygiene, phimosis, high-risk human papillomavirus (HPV) infection and chronic inflammation. Although the role of inflammation and HPV infection is known in some cancers, the relationship between these two factors and the disruption of genes involved in the CaPe genesis is not yet well established. Thus, our main goal was to determine the expression of genes involved in the process of chronic inflammation and in the carcinogenesis mediated by HPV infection and the role of the deregulation of these genes in the establishment and progression of penile tumor. For this purpose, fresh and formalin-fixed paraffin-embedded (FFPE) tissues from 55 patients with penile cancer were evaluated. HPV detection and genotyping were carried out by nested PCR and direct sequencing in all samples. A subgroup (N = 37) was evaluated by qRT-PCR to determine COX-2, EGFR, MYC, RB and P53 gene expression. For this, sections of FFPE tissues containing more than 70% tumor cells were analyzed. Protein expression of these genes and PGE2 were determined by immunohistochemistry in the same tumor tissues. An analysis of the association between the clinical-histopathological parameters, presence of HPV, and gene and protein expression was performed. All tumors were classified as epidermoid squamous cell carcinoma. HPV DNA was detected in 80% of tumors, of which 95% had at least one high-risk subtype, and HPV16 was the most frequent subtype (63%). Among the HPV negative samples in the tumor tissue, 14% were positive in the tissue adjacent to the tumor, so that 94% of the patients, in total, were positive for the presence of HPV DNA.. Overexpression was identified in all genes involved in the inflammatory process. EGFR showed overexpression in 84% of the samples, while COX2 and PGE were overexpressed in 40% of the tumors, each. There was an associationbetween the levels of EGFR and COX2 expression, and between COX2 and PGE2. On the other hand, the genes related to HPV infection, MYC, RB and P53, were underexpressed in 97%, 85% and 81% of the samples, respectively. The gene expressions did not show any association with clinical-histopathological variables. This study describes the repression of RB and P53 activity in HPV + tumors, suggesting that there is a mechanism of control of these genes, possibly mediated by the virus. The high detection of HPV infection shows the importance of the immunization of boys in the prevention of penile cancer. Our data emphasize the need to expand the vaccine coverage to cover types of HPV present in penile cancer. The overexpression of EGFR / COX2 / PGE2, and the association found between them, support the possibility of therapeutic use of anti-EGFR and anti-COX drugs in penile tumors. / Câncer peniano (CaPe) é uma neoplasia rara com maior incidência em regiões com baixos índices socioeconômicos. No Brasil, a maior parte dos homens acometidos por essa doença residem nas regiões Norte e Nordeste. Entre os principais fatores de risco estão a falta de higiene, fimose, infecção por papilomavírus humano (HPV) de alto risco e inflamação crônica. Embora o papel d a inflamação e da infecção por HPV sejam conhecidas em alguns cânceres, ainda não é bem estabelecida a relação entre esses dois fatores e a disrupção de genes envolvidos na gênese de CaPe. Assim, neste estudo foi avaliada a expressão de genes envolvidos no processo de inflamação crônica e na infecção pelo HPV e o papel da desregulação desses genes no estabelecimento e progressão de tumor peniano. Para isto, foram avaliadas amostras teciduais frescas e fixadas em formalina embebidas em parafina (FFPE) de 55 pacientes com câncer de pênis. Foram realizadas detecção e genotipagem de HPV por nested PCR e sequenciamento direto em todas as amostras Um subgrupo amostral (N=37) foi avaliado por qRT-PCR para determinação da expressão dos genes COX-2, EGFR, MYC, RB e P53. Para isso, foram usadas secões de tecidos de FFPE contendo mais de 70% de células tumorais. A expressão proteica desses genes e de PGE2 foi determinada por imunohistoquímica em 42 amostras. Foi feita análise de associação entre os parâmetros clínico-histopatológicos, presença de HPV e expressão gênica e proteica. Todos os tumores foram classificados como carcinoma epidermóide de células escamosas. DNA de HPV foi detectado em 80% dos tumores (N=55), dos quais 95% apresentaram, pelo menos, um subtipo de alto risco, e destes, HPV16 foi o subtipo mais frequente (63%). Dentre as amostras negativas para HPV no tecido tumoral, 14% foram positivas no tecido adjacente ao tumor, de modo que 94% dos pacientes, no total, foram positivos para presença de DNA de HPV. Nas amostras nas quais foi feita análise de expressão gênica (N=37), detectou-se 94,4% de infecção, sendo 94% dos infectados possuem, pelo menos um, tipo de alto risco. Foi Identificada superexpressão em todos os genes envolvidos no processo inflamatório. EGFR mostrou superexpressão em 84% das amostras, enquanto COX2 e PGE mostraram-se, cada um, superexpressos em 40% dos tumores. Houve associação entre níveis de expressão de EGFR e COX2, e entre COX2 e PGE2. Por outro lado, os genes relacionados à infecção por HPV, MYC, RB e P53, mostraram-se subexpressos em 97%, 85% e 81% das amostras, respectivamente. A expressão dos genes estudados não mostrou associação com as variáveis clínico-histopatológicas. Este estudo descreve a repressão da atividade de RB e P53 em tumores HPV+, sugerindo que há um mecanismo de controle desses genes em câncer peniano, possivelmente mediado pelo vírus. A alta detecção de infecção por HPV mostra a importância da imunização de meninos na prevenção de câncer peniano, e ressalta-se a necessidade de ampliação da cobertura vacinal de modo a abranger tipos de vírus presentes em câncer peniano. A superexpressão de EGFR/COX2/PGE2, e a associação encontrada entre eles, sustenta a possibilidade de uso terapêutico de drogas anti-EGFR e anti-COX em tumores penianos.