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Neueste Arbeiten zeigen, dass die Selbsterneuerung und Pluripotenz von Stammzellen durch hochkonservierte Signalwege kontrolliert werden. Störungen dieser Prozesse verursachen Tumoren, die von Zellen mit stammzellähnlichen Eigenschaften ausgehen, den sog. Krebsstammzellen. Diese Arbeit zeigt, dass erhöhte Wnt/beta-Catenin- und erniedrigte Bmp-Signalgebung eine wichtige Rolle in der Entstehung humaner Plattenepithel-Karzinome der Speicheldrüsen spielen. Es wurde ein Mausmodell hergestellt, bei dem die Aktivierung der Wnt/beta-Catenin und der Verlust der Bmp-Signalgebung ebenfalls Speicheldrüsenkarzinome verursachen. Die Speicheldrüsen-Tumoren der Maus enthielten eine erhöhte Zahl von CD24+CD29+-Stammzellen, von denen bereits 500 Zellen transplantierbare Tumoren in NOD/SCID-Mäusen induzierten, d. h. dass sie Krebsstammzellen darstellen. Die Veränderung nur eines Signalweges, Wnt/beta-Catenin oder Bmp allein, war nicht tumorigen, resultierte aber in einer erhöhten Anzahl von CD24+CD29+-Stammzellen in der Speicheldrüse und einer fünffach schnelleren Geweberegeneration nach Verwundung. Die Krebsstammzellen der Speicheldrüsen zeigten eine erhöhte Expression von Pluripotenzgenen und globale Veränderungen von trimethyliertem Lysin 4 und 27 des Histons 3. Diese Veränderungen korrelieren häufig mit einer Zunahme aktiven und einer Abnahme repressiven Chromatins. Die Krebsstammzellen wuchsen in vitro als undifferenzierte Salisphären und konnten durch Inhibierung des Wnt/beta-Catenin-Signalweges in drüsenartige Strukturen differenziert werden. Dieser Prozess war von repressivem Chromatin abhängig, da DNA-Methylierungs- oder Histon-Deazetylase-Inhibitoren den ursprünglichen Krebsstammzell-Status wieder reaktivieren konnten. Diese Arbeit zeigt, dass ein aktiver Wnt/beta-Catenin-Signalweg die Transformation von normalen Stammzellen zu Krebsstammzellen durch einen epigenetischen Mechanismus fördert. Die Ergebnisse eröffnen neue Strategien für die Tumortherapie beim Menschen. / Little is known about the processes by which cancer stem cells arise in the different tissues. Our analysis of aggressive squamous cell carcinomas (SCCs) of the salivary gland in human patients suggested a link to the Wnt/beta-catenin and Bmp signaling systems. Using a genetically modified mouse strain in which Wnt signaling is up-regulated and Bmp is suppressed, we found that Wnt/beta-catenin promotes the transformation of normal stem cells into cancer stem cells through an epigenetic mechanism. Mouse SCCs of the salivary gland contained high numbers of CD24+CD29+ cancer stem cells. As few as 500 of these cells sufficed to cause tumors when transplanted into NOD/SCID mice. Mice in which only one of the signaling systems was altered had higher numbers of stem cells in the salivary gland, more efficient tissue regeneration, and no apparent tumors. We discovered that the difference of normal compared to cancer stem cells in the salivary gland is an up-regulation of specific pluripotency genes, e.g. Dppa5, as well as global changes in trimethylated Lysine 4 and 27 of histone 3. This indicates an increase of active chromatin and a decrease in the repressive form, which suggests a mechanistic explanation for the change of cell fate. Cancer stem cells of the salivary gland grew as non-adherent spheres and retained the capacity for differentiation if beta-catenin is inhibited. This depended on repressive chromatin, as shown by the fact that 5-azacytidine or HDAC inhibitors restored stemness. Our data opens new strategies for future cancer therapies in humans.

Genomic instability and acquisition of invasiveness through the basement membrane extracellular matrix (ECM) are two major processes for epithelial cell malignancy in breast cancer. DNA double-strand break repair (DSBR) is one of the processes that get misregulated during breast cancer progression. In addition, radiation induced breaks such as those induced during radiation therapy to treat breast cancer patients are repaired by DSBR, rendering this pathway relevant for therapy as well. DSBR can occur either by homologous recombination (HR) or non-homologous end-joining (NHEJ). HR is accepted as the more error-free pathway. HR is regulated by the cell cycle status such that an increase is observed in G2/M, whereas NHEJ is observed throughout the cell cycle. Previous data show that ECM signaling regulates HR, as well as the kinetics of ionizing radiation (IR) induced complex formation at break sites, or foci kinetics. Both human breast epithelial cell lines and primary mouse mammary epithelial cells were used to show that the ECM receptor β1-integrin is necessary and sufficient in down regulating HR, as well as IR induced foci formation kinetics for the DSBR proteins RAD51, MRE11, and γ-H2AX in single mammary epithelial cells. RAD51 is required for most HR, whereas MRE11 and γ-H2AX function in HR as well as DNA damage signaling. Interestingly, ECM signaling up-regulates HR in cells that have “correct” in vivo-like cell-cell junctions. Based on the observation that single cells and junctioned cells respond to ECM in exact opposite manner, I hypothesized that ECM signaling may interact with cell-cell junction signaling pathways in regulating DNA repair. To test this hypothesis, I asked whether the main breast epithelial adherens junction cadherin, E-cadherin, is involved. I blocked E-cadherin function using a monoclonal antibody MB2. The function blocking was demonstrated by the loss of cell-cell junction interactions and observation of increased cell scattering using phase microscopy. I then asked whether blocking E-cadherin altered the expression and localization of proteins related to DNA repair. Indirect immuno-fluorescence showed that in the E-cadherin blocked non-tumorigenic breast epithelial cell line HMT-3522 S1 there is an up-regulation of nuclear γ-H2AX and RAD51, as well as an increase in the proliferation marker Ki67. In non-proliferative MB2 blocked cells there is an upregulation of γ-H2AX and reduced Ki67. Furthermore, in these proliferative and non-proliferative blocked cells we were able to see lower levels of β-catenin near the cell membrane and an increase in its levels inside the cell especially in the nucleus. The latter has been confirmed also by western blot technique comparing the nuclear and cytoplasmic fraction expression. In addition, western blots showed that total RAD51 level was down-regulated by E-cadherin blocking and γ-H2AX levels were found to be higher in proliferative and non-proliferative MB2 treated cells. MB2 treated cells have a higher frequency of HR in the absence of ECM and in the presence of ECM, MB2 blocking abolishes the ECM effect on HR. Furthermore, in the absence of ECM, RAD51 siRNA treated cells down-regulated HR but the absence of RAD51 did not down regulate HR in the presence of ECM. I was not able to see any difference in the phosphorylated forms of β-catenin such as Tyr-142, Ser-45 and Tyr-86 that has the ability to enter into the nucleus. Therefore, E-cadherin was found to block nuclear β-catenin, RAD51 and γ-H2AX in a proliferation-independent manner. E-cadherin also was necessary for ECM to up-regulate HR. The up-regulation of HR by ECM was only slightly dependent on RAD51 suggesting a novel E-cadherin-dependent and RAD51-independent HR component in breast epithelial cells in contact with ECM as they are in vivo in the normal breast tissue. These experiments will help us to understand the role of E-cadherin and β-catenin in DNA double-stand break repair directly, as well as in combination with ECM signaling. Both alterations in integrin mediated signaling and cell-cell junction integrity contribute to breast cancer progression by rendering breast epithelial cells more invasive. My project will shed light on whether these invasive processes also alter DNA repair and contribute to genome stability. Understanding of the interrelationships among integrin signaling, cell-cell junctions, and genome stability will contribute to understanding normal breast cell processes and open up investigations on how these may go awry in cancer progression.

O ameloblastoma é definido como um tumor odontogênico epitelial de crescimento lento e localmente invasivo, que acomete os maxilares com alta taxa de recorrência quando não removido adequadamente. Este trabalho tem por objetivo estudar as expressões imuno-histoquímicas das proteínas -catenina, p-Akt, CD44 e vimentina em ameloblastomas. Para este estudo foram selecionados 40 casos de ameloblastoma, pertencentes aos arquivos do Serviço de Patologia da Disciplina de Patologia Bucal da FOUSP. Para a realização das reações imuno-histoquímicas foi utilizada a técnica da estreptavidina-biotina e os cortes submetidos aos anticorpos anti--catenina, anti-pAkt, anti-CD44 e anti-vimentina separadamente. O padrão de marcação celular da -catenina nos ameloblastomas foi: marcação citoplasmática, nuclear e de membrana em 33 (82,5%) casos, marcação citoplasmática e de membrana em 7 (17,5%) casos; quanto à sua localização, em 21 (52,5%) casos marcações central e periférica, em 15 (37,5%) casos observou-se marcação central, em, 4 (10%) marcação periférica. Com relação à proteína p-AKT em 36 (90%) casos o padrão de marcação celular foi citoplasmático, sendo em 4 (10%) casos evidenciado o padrão citoplasmático e nuclear. De todos os casos analisados quanto a localização da marcação do p-AKT, 23 (57,5%) casos com marcação nas áreas central e periférica e 17 (42,5%) apresentaram marcação periférica. Nenhuma das lâminas estudadas apresentou marcação exclusiva em áreas centrais da lesão. Para a proteína CD44, 27 (67,5%) casos dos ameloblastomas estudados apresentou marcações citoplasmática e de membrana, enquanto 13 (32,5%) casos mostraram apenas marcações citoplasmáticas. No que diz respeito a localização, 28 (70%) casos apresentaram marcações centrais e periféricas concomitantes, 11 (27 %) casos marcações centrais, e 1 (2,5%) caso marcação periférica. 37 (92,5%) dos casos incluídos nesta pesquisa apresentou marcação citoplasmática para vimentina, sendo 3 (7,5%) casos negativos para a proteína. Dentre os casos com positividade, 22 (55%) referiram-se à região central 14 (7,5%) para as regiões central e periférica , e 1 (2,5%) caso para a região periférica. De acordo com os resultados obtidos, acredita-se que as áreas central e periféricas do tumor possuem células responsáveis pela proliferação e invasividade do tumor, assim como a presença de células tronco responsáveis pela invasividade do tumor, também estão presentes nessas áreas. / The ameloblastoma is defined as an epithelial odontogenic tumor of slow growth, and locally invasive, affecting the jaws with a high rate of recurrence if not removed properly. This study aims to study the immunohistochemical expression of the protein -catenin, p-Akt, CD44 and vimentin in ameloblastomas. For this study we selected 40 cases of ameloblastoma, from the archives of the Pathology of Oral Pathology FOUSP. To carry out the reactions immunohistochemical technique was used streptavidin-biotin and cuts subjected to anti--catenin, anti-pAkt, anti-CD44 and antivimentin separately. The pattern of cell labeling of -catenin in ameloblastomas was: cytoplasmic, nuclear and membrane in 33 (82.5%) cases, cytoplasmic membrane in 7 (17.5%) cases, as regards its location in 21 (52.5%) cases markings central and peripheral in 15 (37.5%) cases observed central marking in, 4 (10%) peripheral marking. With respect to the p-AKT protein in 36 (90%) cases, the staining pattern was cytoplasmic cell, and 4 (10%) patients demonstrated the nuclear and cytoplasmic pattern. In all cases analyzed as marking the location of the p-AKT, 23 (57.5%) cases with marking the central and peripheral areas and 17 (42.5%) had peripheral marking. None of the studied thin sections show labeling exclusively in the central areas of the lesion. For protein CD44, 27 (67.5%) cases of ameloblastomas studied showed markings and cytoplasmic membrane, while 13 (32.5%) cases showed only cytoplasmic markings. Regarding localization, 28 (70%) presented concomitant central and peripheral markings, 11 (27%) patients central markings, and 1 (2.5%), peripheral marking case. 37 (92.5%) of the cases included in this study showed cytoplasmic staining for vimentin, and 3 (7.5%) cases negative for protein. Among the positive cases, 22 (55%) referred to the central 14 (7.5%) for the central and peripheral, and 1 (2.5%) case for the peripheral region. According to the obtained results, it is believed that central and peripheral areas of the tumor cells have responsible for the proliferation and invasiveness of the tumor, as well as the presence of stem cells responsible for tumor invasiveness, are also present in these areas.

Les carcinomes hépatocellulaires (CHC) mutés CTNNB1 ont des caractéristiques phénotypiques propres en termes de polarité et de métabolisme (absence de stéatose). Nous avons émis l’hypothèse que ce phénotype pouvait être secondaire à l’activation du gène suppresseur de tumeurs LKB1 qui code une Ser/Thr kinase multitâches.Nous avons tout d’abord montré qu’il existait effectivement un dialogue complexe entre les voies Wnt/β-Caténine et LKB1 dans le foie. Les mutations de CTNNB1 sont en effet capables d’induire l’expression protéique de LKB1 dans des lignées hépatomateuses humaines, et les CHC mutés CTNNB1 présentent une expression protéique accrue de LKB1 et une signature transcriptionnelle d’activation de LKB1. De plus, dans deux modèles murins d’invalidation hépatospécifique de Lkb1, LKB1 est apparu comme requis pour l’activation complète du programme transcriptionnel de β-Caténine mais de façon dépendante du stade de développement et du contexte nutritionnel. Enfin, la signalisation LKB1 est apparue comme nécessaire à la survie des hépatocytes activés pour β-Caténine dans deux modèles murins différents.Nous avons aussi caractérisé les rôles métaboliques de LKB1 dans le foie. L’invalidation hépatospécifique de Lkb1 induisait une augmentation progressive de la masse grasse corporelle avec utilisation préférentielle des glucides comme substrat énergétique. Il existait une activation de la néoglucogenèse hépatique avec hyperglycémie et une lipogenèse accrue avec accumulation hépatocytaire de lipides. Enfin, nous avons mis en évidence une activation paradoxale de la signalisation AKT dans les hépatocytes, même à jeun, et une dépendance énergétique aux acides aminés. Enfin, nous avons identifié une nouvelle isoforme protéique de LKB1 délétée de son domaine N-Terminal et d’une partie de son domaine kinase. D’expression tissulaire préférentiellement musculaire et myocardique, cette isoforme catalytiquement inactive se comportait comme dominant positif sur l’activation de l’AMPK par la forme conventionnelle mais comme dominant négatif dans l’activité polarisation induite par LKB1. Enfin, elle était capable d’induire, en l’absence de la forme conventionnelle, la prolifération cellulaire et la tumorigenèse chez la souris nude. Elle pourrait exercer des rôles métaboliques particuliers dans les tissus fortement oxydatifs et des rôles oncogéniques dans certains contextes. / CTNNB1-Mutated hepatocellular carcinomas (HCC) share a specific polarity and metabolic phenotype without steatosis. We hypothesized that such phenotype could imply the tumor suppressor gene LKB1 that encodes for a multi-Task Ser/Thr kinase.We first demonstrated that a complex crosstalk indeed exists in the liver between LKB1 and the Wnt/β-Catenin pathway. LKB1 proteic expression was controlled by mutant β-Catenin in hepatomatous cell line and CTNNB1-Mutated HCCs had an enhanced LKB1 proteic expression as well a transcriptomic signature of LKB1 activation. In two mouse model of liver-Specific invalidation of Lkb1, we showed that LKB1 was required for full activation of the β-Catenin transcriptomic program, but it depended on the developmental stage and nutritional context. At least, LKB1 appeared to be required for the survival of β-Catenin activated liver cells in two other mouse models.Then, we wanted to caracterize the metabolic roles of LKB1 in the liver. Liver-Specific invalidation of Lkb1 progressively raised the body fat mass and we observed that carbohydrates were preferred as whole-Body energetic fuel. In the liver, gluconeogenesis and lipogenesis were enhanced, resulting in mild hyperglycemia and lipid accumulation in the hepatocytes. At least, we identified an aberrant activation of the AKT signaling in the liver, even during fasting, and an energetic dependence towards amino acids.At least, we identified a novel LKB1 proteic isoform that is deleted of its N-Terminal domain and part of its kinase domain. Highly expressed in the muscle and in the heart, this catalytically inactive isoform however acted as a positive dominant towards AMPK activation by full length LKB1 but as a negative dominant towards LKB1-Induced cell polarization. This isoform is also able to enhance cell proliferation and to induce tumors in a xenograft model, even when expressed alone. It could play specific metabolic roles in oxidative tissues and could be oncogenic in some contexts.

O ameloblastoma é definido como um tumor odontogênico epitelial de crescimento lento e localmente invasivo, que acomete os maxilares com alta taxa de recorrência quando não removido adequadamente. Este trabalho tem por objetivo estudar as expressões imuno-histoquímicas das proteínas -catenina, p-Akt, CD44 e vimentina em ameloblastomas. Para este estudo foram selecionados 40 casos de ameloblastoma, pertencentes aos arquivos do Serviço de Patologia da Disciplina de Patologia Bucal da FOUSP. Para a realização das reações imuno-histoquímicas foi utilizada a técnica da estreptavidina-biotina e os cortes submetidos aos anticorpos anti--catenina, anti-pAkt, anti-CD44 e anti-vimentina separadamente. O padrão de marcação celular da -catenina nos ameloblastomas foi: marcação citoplasmática, nuclear e de membrana em 33 (82,5%) casos, marcação citoplasmática e de membrana em 7 (17,5%) casos; quanto à sua localização, em 21 (52,5%) casos marcações central e periférica, em 15 (37,5%) casos observou-se marcação central, em, 4 (10%) marcação periférica. Com relação à proteína p-AKT em 36 (90%) casos o padrão de marcação celular foi citoplasmático, sendo em 4 (10%) casos evidenciado o padrão citoplasmático e nuclear. De todos os casos analisados quanto a localização da marcação do p-AKT, 23 (57,5%) casos com marcação nas áreas central e periférica e 17 (42,5%) apresentaram marcação periférica. Nenhuma das lâminas estudadas apresentou marcação exclusiva em áreas centrais da lesão. Para a proteína CD44, 27 (67,5%) casos dos ameloblastomas estudados apresentou marcações citoplasmática e de membrana, enquanto 13 (32,5%) casos mostraram apenas marcações citoplasmáticas. No que diz respeito a localização, 28 (70%) casos apresentaram marcações centrais e periféricas concomitantes, 11 (27 %) casos marcações centrais, e 1 (2,5%) caso marcação periférica. 37 (92,5%) dos casos incluídos nesta pesquisa apresentou marcação citoplasmática para vimentina, sendo 3 (7,5%) casos negativos para a proteína. Dentre os casos com positividade, 22 (55%) referiram-se à região central 14 (7,5%) para as regiões central e periférica , e 1 (2,5%) caso para a região periférica. De acordo com os resultados obtidos, acredita-se que as áreas central e periféricas do tumor possuem células responsáveis pela proliferação e invasividade do tumor, assim como a presença de células tronco responsáveis pela invasividade do tumor, também estão presentes nessas áreas. / The ameloblastoma is defined as an epithelial odontogenic tumor of slow growth, and locally invasive, affecting the jaws with a high rate of recurrence if not removed properly. This study aims to study the immunohistochemical expression of the protein -catenin, p-Akt, CD44 and vimentin in ameloblastomas. For this study we selected 40 cases of ameloblastoma, from the archives of the Pathology of Oral Pathology FOUSP. To carry out the reactions immunohistochemical technique was used streptavidin-biotin and cuts subjected to anti--catenin, anti-pAkt, anti-CD44 and antivimentin separately. The pattern of cell labeling of -catenin in ameloblastomas was: cytoplasmic, nuclear and membrane in 33 (82.5%) cases, cytoplasmic membrane in 7 (17.5%) cases, as regards its location in 21 (52.5%) cases markings central and peripheral in 15 (37.5%) cases observed central marking in, 4 (10%) peripheral marking. With respect to the p-AKT protein in 36 (90%) cases, the staining pattern was cytoplasmic cell, and 4 (10%) patients demonstrated the nuclear and cytoplasmic pattern. In all cases analyzed as marking the location of the p-AKT, 23 (57.5%) cases with marking the central and peripheral areas and 17 (42.5%) had peripheral marking. None of the studied thin sections show labeling exclusively in the central areas of the lesion. For protein CD44, 27 (67.5%) cases of ameloblastomas studied showed markings and cytoplasmic membrane, while 13 (32.5%) cases showed only cytoplasmic markings. Regarding localization, 28 (70%) presented concomitant central and peripheral markings, 11 (27%) patients central markings, and 1 (2.5%), peripheral marking case. 37 (92.5%) of the cases included in this study showed cytoplasmic staining for vimentin, and 3 (7.5%) cases negative for protein. Among the positive cases, 22 (55%) referred to the central 14 (7.5%) for the central and peripheral, and 1 (2.5%) case for the peripheral region. According to the obtained results, it is believed that central and peripheral areas of the tumor cells have responsible for the proliferation and invasiveness of the tumor, as well as the presence of stem cells responsible for tumor invasiveness, are also present in these areas.