Der Tumorsupressor APC ist in der Mehrzahl aller Fälle kolorektaler Karzinome bereits in der initialen Phase der Karzinogenese mutiert. Diese Mutationen führen zu einer aberranten Aktivierung des Wnt-Signalweges sowie zu weiteren die Karzinogenese vorrantreibenden Aktivitäten, beispielsweise einem veränderten Migrationsverhalten. Dieser Dissertation zu Grunde liegt die Idee, dass durch die Trunkierung des APC-Proteins aber auch Abhängigkeiten von Genaktivitäten entstehen, die zuvor entbehrlich waren. Solche synthetisch letalen Gene sollten in einem high-content shRNA-Screen gefunden werden. Für die Durchführung des Screens wurde ein von der SW480 Kolonkarzinomzelllinie abgeleitetes, isogenes Zellsystem generiert, welches durch Induktion mit Doxyzyklin das vollständige APC-Allel (FL-APC) exprimiert. Infolge dieser Expression zeigen die Zellen einen weniger malignen Phänotyp. Dies spiegelt sich darin wider, dass die Zellen durch FL-APC Expression in ihrer Wnt-Signalwegsaktivität eingeschränkt werden. Doxyzyklininduzierte Zellen sind schlechter in der Lage ohne Adhäsion zu proliferieren als nicht induzierte Zellen. Andererseits ist ihre Fähigkeit einem FKS-gradienten entlang zu migrieren verbessert. Der shRNA-Screen wurde mit der Decipher shRNA-Bibliothek durchgeführt. Diese enthält 27.500 verschiedene shRNAs mit Interferenzaktivität gegen 5.000 mRNAs, die potentiell pharmakologisch inhibierbare Proteine kodieren. Die besten zwei Kandidaten für eine synthetisch letale Interaktion mit trunkiertem APC, BCL2L1 und EIF2B5 wurden im Verlauf einer Masterarbeit bzw. direkt in dieser Disseration validiert. EIF2B5 zeigte in vitro nach Depletion durch unterschiedliche shRNAs einen di erentiellen Proliferationse ekt bei FL-APC induzierten im Vergleich zu kontrollbehandelten Zellen. Dieser di erentielle E ekt konnte in einem weiteren Modellsystem, SW480 Zellen mit konstitutiver FL-APC Expression, ebenfalls validiert werden. Durch Expression einer shRNA mit Aktivität gegen EIF2B5 werden in beiden Zellsystem die unfolded protein response (UPR) Gene DDIT3 und splXBP1 aktiviert. Interessanterweise werden durch die Expression von FL-APC diese Gene reprimiert. Im Promotor der EIF2B5-mRNA be ndet sich eine Bindestelle für MYC. Es ist denkbar, dass durch die Expression von FL-APC eine globale Veränderung der Genexpression vorgenommen wird, die einerseits eine Repression von EIF2B5 nach sich zieht aber andererseits eine hierdurch ausgelöste ER-Stress Antwort verhindert. Eine Inhibition von EIF2B5 ohne diese Adaption andererseits führt nach diesem Model zu einer UPR-aktivierten Apoptose. In einem zweiten Projekt wurde das überraschende Verhalten von Kolonkarzinomzellen untersucht, die nach Zugabe von BEZ235, einem dualen PI3K/mTOR Inhibitor, trotz gegenteiliger Erwartungen MYC-Proteinmengen erhöhen. Eine Repression wurde erwar- tet, weil die Inhibition von PI3K einerseits zu einer proteasomalen Destabiliserung und andererseits die mTOR Inhibition zu einer verringerten Synthese von MYC führen sollte. Während bereits gezeigt werden konnte, dass durch einen FOXO-vermittelten Mechanismus MAPK-abhängig die MYC-Expression verstärkt wird, wurde in dieser Dissertation die erwartete Translationsinhibition untersucht. BEZ235 inhibiert zwar CAP-abhängige Translation, das MYC Protein wird jedoch aufgrund einer IRES-vermittelten Translation weiterhin exprimiert. Silvestrol, ein Inhibitor der Helikase eIF4A andererseits interveniert mit CAP- und IRES-abhängiger Translation und kann die MYC-Proteinkonzentrationen verringern. Wir konnten zudem feststellen, dass die Applikation von Silvestrol auch in vivo möglich und wirksam ist und zudem tolleriert wird. Dies gibt Anlass zur Ho nung, dass eine Intervention der Translation auch im Menschen eine valide Strategie zur Behandlung MYC-getriebener Tumore sein könnte. / The tumorsupressor APC is mutated in initiating colorectal cancers. These mutations lead to an aberrant actiavation of the wnt-signaling pahtway and to further carcinogenic activities such as altered migration behaviour. The idea that novel dependencies upon previously expendable genes are generated through APC-mutations form the basis of this disseration. These so called synthetic lethal Genes were searched for harnessing a high content shRNA screen. We generated an isogenic cell system which was deviated from the colorectal cancer cell line SW480. These cells naturally express truncated APC. The generated system expresses a full-lenght allele upon doxycycline exposure. SW480 cells which are induced partially revert their malignancy. Ancorage independend growth is compromised and migration along a gradient of fetal calf serum is improved. The Decipher shRNA library was used for screening. It consists of 27.500 di erent shRNAs intefering with the activity of 5.000 genes which are potantially drugable. The two best candidates scoring as hits in the screen were EIF2B5 and BCL2L1. BCL2L1 was validated in a cooperating masterthesis and EIF2B5 could be validated in the course of this diseration. Following EIF2B5 depletion using di erent shRNA constructs, we were able to see di erential behaviour in pTRE-APC cells as well as in a second model system in which FL-APC was expressed constitutively. Interestingly an activation of the ER-Stress genes DDIT3 and splXBP1 can be seen after EIF2B5 depletion. These genes are repressed, when FL-APC ist expressed. The EIF2B5 promotor has a MYC-binding site and we speculate, that FL-APC expression induces a genetic program which represses EIF2B5 on the one hand, however prohibits the ER-Stress reaction which follows this trigger. Inhibtion of EIF2B5 without this global adaption in genexpression on the other side initiates UPR-mediated apoptosis. In a second project, the suprising behaviour of colon carcinoma cell lines, which upregulate MYC upon BEZ235 exposure was examined. The dual inhibitor was thought to downregulate MYC through its PI3K and mTOR inhibitory acitivites which were thought to destabilise and MYC and prohibit it's expression, respectively. Whereas former work could demonstrate a FOXO-mediated, MAPK-dependend positive MYC-gene expression clue the aim of this thesis was to analyse the expecte protein tranlational inhibition. Indeed, BEZ235 inhibits CAP-dependend translation, however the MYC protein is still translated through IRES-dependend translation. The eIF4A-inhibitor Silvestrol intervenes with both CAP- and IRES-dependend translation and can therefore reduce MYC protein levels

Thesis (Ph.D.). / Written for the Dept. of Biomedical Engineering. Title from title page of PDF (viewed 2009/06/11). Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 96-113).

Orientador: Rebeca de Souza Azevedo / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-27T23:04:36Z (GMT). No. of bitstreams: 1 Dutra_SabrinaNogueira_D.pdf: 4132202 bytes, checksum: 8d692501f423535818a567ec7a79ed11 (MD5) Previous issue date: 2015 / Resumo: A queilite actínica (QA) é uma desordem potencialmente maligna dos lábios, resultado da exposição crônica e excessiva ao raios ultravioleta, e que pode evoluir para um carcinoma de células escamosas de lábio. A via de sinalização Wnt/?-catenina atua em genes relacionados ao ciclo e a proliferação celular e está envolvida no desenvolvimento e progressão tumoral, e algumas de suas moléculas já foram identificadas em QA. O objetivo deste estudo foi correlacionar as características histopatológicas da QA com dois sistemas de classificação para gradação histopatológica, o da OMS e do sistema binário; e avaliar a participação de marcadores da via de sinalização Wnt/?-catenina por meio de reação imunoistoquímica contra os anticorpos Wnt1, Wnt5a, ?-catenina, axina, APC e ciclina D1 em casos de QA, e relacionar esta expressão com os dois sistemas de classificação. Os resultados mostraram correlação das características histopatológicas com o aumento da displasia epitelial de forma mais evidente pelo sistema binário do que pela classificação da OMS; positividade de Wnt 1, potente ativador da via canônica, em 96,7% dos casos; marcação anormal citoplasmática com ou sem marcação nuclear de ?-catenina em 81,9% dos casos; positividade de ciclina D1 em 75,4 % dos casos; negatividade de Wnt 5a, que possui potencial inibidor da via e pode contribuir indiretamente para ativação da via canônica; positividade de APC e axina em 96,7% e 95% dos casos de QA. Além disso, os anticorpos Wnt1, ciclina D1, APC e axina apresentaram aumento do índice de marcação imunoistoquímica de acordo com o aumento do grau de displasia epitelial de forma progressiva pelo sistema binário. Pode-se, assim, sugerir uma possível relação destas proteínas da via de sinalização Wnt/?-catenina com o desenvolvimento e a progressão da QA e que o uso do sistema binário de classificação de gradação histopatológica em QA apresentou melhor correlação com as características histopatológicas e com os índices de marcação imunoistoquímica da via de sinalização Wnt/?-catenina do que o sistema de classificação da OMS / Abstract: Actinic cheilitis (AC) is a potentially malignant disorder of the lips, resulting from chronic and excessive exposure to ultraviolet radiation, which can develop into a lip squamous cell carcinoma. Wnt/?-catenin signaling pathway acts on genes related to cell cycle and proliferation and is involved in tumor development and progression, and some of its molecules have already been identified in AC. Thus, the aim of this study was to correlate the histopathological features of AC with two grading histopathological systems, the WHO and the binary system; and to evaluate Wnt/?-catenin signaling pathway involvement by means of immunohistochemistry reaction against Wnt1, Wnt5a, ?-catenin, axin, APC and cyclin D1 in AC, and to relate this expression with the two histopathological grading systems. The results showed a correlation between the histopathologic features with the increased epithelial dysplasia mainly by using the binary system than using the WHO classification; Wnt 1 positivity, a potent canonical pathway activator, in 96.7% of the cases; abnormal ?-catenin cytoplasmic staining with or without nuclear staining in 81.9% of cases; cyclin D1 positivity in 75.4% of cases; Wnt 5a negativity, which has a potential for an inhibiting the pathway and may indirectly contribute to the activation of canonical pathway; APC and axin positivity in 96.7% and 95% of AC. In addition, Wnt1, cyclin D1, APC and axin showed a progressive increased immunohistochemical staining index according to the increasing degree of epithelial dysplasia by the use of the binary system. Therefore, we were able to suggest a possible relationship of these Wnt/?-catenin signaling pathway proteins with the AC development and progression and that the use of binary grading system in the histopathological classification of AC showed a better correlation with the histopathological features and the immunohistochemical staining indexes of Wnt/?-catenin signaling pathway than the use of WHO grading system / Doutorado / Patologia / Doutora em Estomatopatologia