Publikationsserver der Universitätsbibliothek Marburg

Titel:Establishment and characterization of preclinical mouse models for evaluation of oncogenic and tumor-suppressive properties of p53 family members
Autor:Fuchs, Jeannette
Weitere Beteiligte: Stiewe, Thorsten (Prof. Dr.)
Veröffentlicht:2016
URI:https://archiv.ub.uni-marburg.de/diss/z2017/0285
URN: urn:nbn:de:hebis:04-z2017-02859
DOI: https://doi.org/10.17192/z2017.0285
DDC:570 Biowissenschaften, Biologie
Titel(trans.):Präklinische Mausmodelle zur Untersuchung tumorrelevanter Faktoren der p53 Protein Familie
Publikationsdatum:2017-04-27
Lizenz:https://creativecommons.org/licenses/by-nc-sa/4.0

Dokument

Schlagwörter:
p53 p73 tumor biology, p53 p73 Tumorbiologie, p53 p73 Tumorbiologie

Summary:
Cancer development is a multistep process which leads to tumors composed of diverse cell populations originating from one cell which underwent differential genetic changes over time. Within one tumor, such heterogeneity provides distinct subpopulations with selection advantages promoting metastasis as well as therapy resistance. In the course of targeted cancer therapies tumor composition is meanwhile monitored via circulating tumor DNA from blood samples during treatment and accordingly allows adjustment of therapy. In experimental models of solid tumor xenografts in mice, however, this method is not applicable as required blood volumes exceed blood volume of animals, hence limiting investigation of tumorrelevant genes in preclinical mouse models. Here, a method was developed which facilitates simultaneous monitoring of growth dynamics of two distinct tumor cell populations within one tumor xenograft. Therefore, cells were labelled by stable expression of either Gaussia luciferase (GLuc) or Cypridina luciferase (CLuc) prior to injection. Both luciferases are secreted into the blood stream of transplanted mice. This allows asessment of tumor composition by enzyme activity of both luciferases requiring only very low blood volumes. Moreover, to facilitate investigation of the impact of targeted genetic manipulations luciferases were linked with (non)-targeting shRNAs. To establish this method, shRNAs were used targeting the p53 familiy members p53 and p73. Whereas p53 is acknowledged as the most important tumor suppressor, p73 can occur in two N-terminally different isoforms with opposing attributes: the tumorsuppressive full length isoform TAp73 and the N-terminally truncated tumorpromoting isoform Np73. The dominant negative function of Np73 includes its ability to form hetero-oligomers with its family members p53 and TAp73, thereby interfering with their transcriptional activity. Linking the luciferases to (non)-targeting shRNAs, the differential growth properties of transplanted cells in presence and absence of p53 (or p73) can be monitored simultaneously. The here established method was successfully validated in a model of experimental metastasis as well as under therapeutic conditions. Moreover, it could be demonstrated that the growth behaviour of p73-high-expressing cells Hs 766T is highly dependent on the relative abundance of both N-terminal isoforms. The shRNA-mediated reduction of both isoforms strongly reduces tumorigenicity of these cells. In accordance with previous publications, the reintroduction of Np73 rescued this growth defect, whereas ectopic expression of TAp73 further attenuates proliferation. In order to further investigate Np73´s role in tumor development, an inducible Np73 transgenic mouse model was characterized. Whereas mere overexpression of Np73 exhibited no tumorrelevant properties, the combination with heterozygous knockout of p53 entailed earlier and accelerated tumor development particularly of lung tumors and lymphoma. The loss of the second p53 allele in lung tumors suggests that the dominant negative effect of Np73 rather impacts TAp73 than p53. Accordingly, the observed fertility and embryonic developmental defects in this transgenic model rather pointed towards a TAp73-dependent effect of Np73 as, in contrast to p53-deficient mice, severe and partially comparable defects have been described in TAp73- and complete p73-deficient mice. Finally, transcriptomewide analysis of Np73-overexpressing murine embryonic fibroblasts revealed positive regulation of metastasis-promoting factors like ITGB4, JAG1 and 2. This tumorpromoting property of Np73 goes in line with accelerated dissemination of lymphoma into lungs of Np73;p53+/- mice. Taken together, these results clearly demonstrate growth- as well as metastasis-promoting traits of Np73. However, the specific virtue of Np73 are largely cell context-dependent.

Zusammenfassung:
Die Tumorentwicklung ist ein mehrstufiger Prozess, bei dem sich aus einer einzigen Ursprungszelle durch Akkumulierung diverser Mutationen Tumoren entwickeln, die aus mehreren Populationen unterschiedlichen genetischen Status bestehen. Diese Heterogenität führt zu Selektionsvorteilen einzelner Subpopulationen innerhalb eines Tumors, die unter anderem Metastasierung sowie Therapieresistenz begünstigen. Zur Verbesserung der gezielten Krebstherapie von Patienten kann mittlerweile während des Therapieverlaufs die Zusammensetzung der Tumoren durch im Blut zirkulierende Tumor DNA verfolgt und die Therapie demensprechend angepasst werden. In experimentellen Ansätzen, wie Transplantationsmodellen solider Tumoren in Mäusen, kann diese Methode allerdings nicht angewandt werden, da das Blutvolumen der Versuchstiere zu klein ist. Dies limitiert die Untersuchung tumorrelevanter Faktoren in präklinischen Studien im Mausmodell. In dieser Arbeit wurde eine Methode entwickelt, mit deren Hilfe die Wachstumsdynamiken zweier unterschiedlicher Zellpopulationen in einem einzigen Tumorzellimplantat simultan verfolgt werden können. Hierzu wurde vor der Transplantation jeweils eine Zellpopulation durch stabile Expression von Gaussia Luziferase (GLuc) oder Cypridina Luziferase (CLuc) markiert. Beide Luziferasen werden aus den Zellen sezerniert und ins Blut der Versuchstiere abgegeben. Kleinste Blutproben sind bereits ausreichend, um die Tumorkomposition mittels Messung der Enzymaktivität beider Luziferasen zu bestimmen. Um die Auswirkungen gezielter genetischer Manipulationen untersuchen zu können wurden die Luziferasen zusätzlich an (un)spezifische shRNAs gekoppelt. Zur Etablierung wurden shRNAs verwendet, die sich gegen die p53 Familienmitglieder p53 und p73 richten. Während p53 als der wichtigste Tumorsuppressor bekannt ist, gibt es von p73 zwei N-terminal unterschiedliche Isoformen, die entgegesetzte Funktionen besitzen: das ebenfalls tumorsuppressive TAp73 und das tumorfördernde Np73. Die dominant negative Wirkung von Np73 liegt unter anderem in der Komplexierung seiner Familienmitglieder p53 und TAp73, wodurch diese die Fähigkeit verlieren an ihre Zielgene zu binden. Durch Kopplung der Luziferasen an unspezifische bzw. experimentelle shRNAs konnte die unterschiedliche Wachstumsdynamik transplantierter Zellen in An- bzw. Abwesenheit von p53 (oder p73) simultan verfolgt werden. Die in dieser Arbeit etablierte Methode wurde sowohl in einem Modell der experimentellen Metastasierung als auch unter therapeutischen Bedingungen erfolgreich validiert. Darüberhinaus konnte gezeigt werden, dass das Wachstumsverhalten p73-hochexprimierender Hs 766T Zellen abhängig vom relativen Verhältnis beider N-terminaler Isoformen zueinander ist. Eine shRNA-vermittelte Reduktion beider Isoformen hemmt die Tumorigenität dieser Zellen. In Übereinstimmung mit dem aktuellen Wissensstand wurde dieser Effekt durch ektopische Expression von Np73 wieder aufgehoben, wohingegen die Wiedereinführung von TAp73 die Proliferation noch weiter reduzierte. Um die Rolle von Np73 während der Tumorentwicklung genauer zu untersuchen, wurde zudem ein induzierbares Np73-transgenes Mausmodell charakterisiert. Obwohl die alleinige Überexpression von Np73 keinen tumorigenen Effekt aufwies, führte die Kombination mit heterozygotem Verlust von p53 zu einer früheren und verstärkten Tumorentstehung, insbesondere von Lungentumoren und Lymphomen. Der Verlust des zweiten p53 Allels in den Lungentumoren lässt eher auf eine dominant negative Wirkung von Np73 auf TAp73 als auf p53 schließen. Auch die beobachteten Fertilitäts- und embryonalen Entwicklungsdefekte dieser transgenen Mäuse weisen vielmehr auf einen TAp73-abhängigen Effekt von Np73 hin, da im Gegensatz zu p53-defizienten Mäusen bereits ähnliche Defizite in TAp73- und gesamt-p73-defizienten Mäusen beschrieben wurden. Letztlich wurde durch die transkriptomweite Analyse Np73-überexprimierender muriner embryonaler Fibroblasten eine positive Regulation metastasierungsrelevanter Faktoren (ITGB4, JAG1, JAG2) festgestellt. Diese onkogene Eigenschaft von Np73 geht einher mit der Beobachtung dass Lymphome aus Np73;p53+/- Mäusen verstärkt in die Lungen disseminierten. Insgesamt zeigen diese Ergebnisse, dass Np73 sowohl wachstums- als auch metastasierungsfördernde Eigenschaften besitzt, die genauen Wirkungsmechanismen allerdings abhängig vom Zellkontext sind.

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