Tumour development in Raf-driven cancer mouse models [Elektronische Ressource] = Tumor-Entwicklung in Raf-transgenen Mausmodellen / vorgelegt von Katharina Lütkenhaus

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• Biologie

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• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2010
• Nombre de lectures: 21
• Langue: English
• Poids de l'ouvrage: 3 Mo

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BAYERISCHE JULIUS-MAXIMILIANS-UNIVERSITÄT WÜRZBURG
FAKULTÄT FÜR MEDIZIN
INSTITUT FÜR MEDIZINISCHE STRAHLENKUNDE UND ZELLFORSCHUNG




Tumour development in Raf-driven cancer mouse models

Tumor-Entwicklung in Raf-transgenen Mausmodellen







Dissertation
zur Erlangung des naturwissenschaftlichen Doktorgrades
der Bayerischen Julius-Maximilians-Universität Würzburg


vorgelegt von
Katharina Lütkenhaus
geboren in Regensburg


Würzburg 2010


Eingereicht am:



Mitglieder der Prüfungskommission:
1. Gutachter: Prof. Dr. U.R.Rapp
2. Gutachter: Prof. Dr. T.Rudel



Tag des Prüfungskolloqiums:


Doktorurkunde ausgehändigt am:









Ich versichere, dass die vorliegende Arbeit nur unter Verwendung der angegebenen
Hilfsmittel angefertigt und von mir selbständig durchgeführt und verfasst wurde.

Diese Dissertation hat weder in gleicher noch in ähnlicher Form in einem anderen
Prüfungsverfahren vorgelegen.

Neben dem akademischen Grad „Diplom-Biologin Univ.“ habe ich keine weiteren
akademischen Grade erworben oder zu erwerben versucht.


Würzburg,




Katharina Lütkenhaus


2
ACKNOWLEDGEMENTS


At this point it is a pleasure for me to convey my deepest gratitude to all people, who made
this thesis possible.

First of all, I would like to thank Prof. Dr. U.R.Rapp for giving me the opportunity to do my
PhD thesis at the MSZ of the University of Würzburg, for providing working space and
funding, as well as for his excellent scientific guidance during my work at the MSZ. His wide
knowledge and his professional environment have been of great value for me and have
provided me a good basis for my thesis.

Furthermore, I would like to thank Prof. Dr. T.Rudel for taking up the role as the second
referee of this thesis and for providing lab space during the last month of my work.

Special thanks go also to PD Dr. R.Götz for welcoming me in his group and for his
continuous support, detailed constructive comments and for his support during this work.

I would like to thank Prof. Dr. M.Wegener for providing the Sox10 antibody and Prof. Dr.
K.Bister for providing the chicken c-Myc antibody.

I would like to thank Ellen Leich for performing the microarray and the appropriate analyses.
Furthermore, I would like to thank her for the fruitful discussions about the results and the
fast answers to any questions.

A great thank you also goes to the scientists and technicians of the group of PD Dr. R.Götz
and all members of the mouse club and the animal caretakers. Especially I would like to thank
Dr. Christiaan Karremann, Dr. Fatih Ceteci and Emanuele Zanucco for their continuous
support, advice and helpful comments and Semra and Ines for not only being more than
cooperative colleagues but also friends encouraging me in hard times during my thesis.

I am grateful to many other colleagues from the MSZ.

I would like to thank Dr. B.Bergmann and all members of her group for accepting me as an
associate member of her group during the last months of my work and the helpful discussions
during this time.

I would like to express my deepest gratitude to all my friends, especially Frank and Kerstin,
for understanding me during hard periods of my PhD work and for supporting me and
bringing me back to motivation. I am indebted to thank particularly my former colleague and
friend Sandra for all the building up messages and phone calls and the unforgettable evenings
with her, and Victor for his support during the final period of this thesis.

Finally, I would like to thank my parents Elisabeth and Paul, my grandma Betty and my sister
Barbara for understanding me not to be with them a lot of times and for encouraging me
during hard times not to give up. Without their support this thesis would not have been
possible.


3
Abstract
Metastasis is the cause of death in 90% of cancer-related deaths in men. Melanoma and
Non-Small-Cell Lung Cancer (NSCLC) are both tumour types with poor prognosis,
lacking appropriate therapeutic possibilities, not least because of their high rate of
metastasis. Thus understanding the process of metastasis might unravel therapeutic
targets for developing further therapeutic strategies.
V600EThe generation of a transgenic mouse model expressing B-Raf in melanocytes, a
mutation that is found in about 60% of all melanoma, would result in an ideal tool to
study melanoma progression and metastasis. In this work, a doxycycline-inducible
V600Esystem was constructed for expression of B-Raf and transgenic animals were
generated, but the expression system has to be improved, since this strategy didn’t give
rise to any viable, transgene carrying mice.
Furthermore, since it was shown in the work of others that the metastatic behavior of
tumour cell lines could be reversed by an embryonic microenvironment and the
influence of a tumourigenic microenvironment on melanocytes lead to the acquisition of
tumour cell-like characteristics, the question arose, whether B-Raf is as important in
melanocyte development as it is in melanoma progression. In this work, the embryonal
melanocyte development in B-Raf-deficient and wildtype mouse embryos was examined
and there were no differences observed in the localization and number of neural crest
stem cells as well as in the localization of the dopachrome-tautomerase positive
melanoblasts in the embryos and in cultured neural tube explants.
The expression of oncogenic C-Raf in lung epithelial cells has yielded a model for
NSCLC giving rise to adenomas lacking spontaneous progression or metastasis. The co-
expression of c-Myc in the same cells accelerates the tumour development and gives rise
to liver and lymphnode metastases. The expression of c-Myc alone in lung epithelial cells
leads to late tumour development with incomplete penetrance. A mutation screen in this
work resulted in the observation that a secondary mutation in KRas or LKB1 is
necessary for tumour formation in the c-Myc single transgenic animals and suggested
metastasis as an early event, since the corresponding metastases of the mutation-prone
primary lung tumours were negative for the observed mutations. Furthermore, in this
work it was shown that the expression of chicken c-Myc in a non-metastatic NSCLC cell
line leads to metastatic clones, showing that c-Myc is sufficient to induce metastasis.
Additionally a panel of metastasis markers was identified, that might serve as diagnostic
markers in the future.
Zusammenfassung
In 90% der Todesfälle aufgrund von Krebserkrankungen sind Metastasen für den Tod
des Patienten verantwortlich. Sowohl Melanom, als auch nicht-kleinzelliges
Lungenkarzinom (Non-Small Cell Lung Cancer, NSCLC) sind beides Tumortypen, die
eine schlechte Prognose haben und für die sich wenige Therapiemöglichkeiten bieten,
nicht zuletzt aufgrund ihrer häufigen Metastasierung. Somit würde ein besseres
Verständnis des Metastasierungsprozesses neue therapeutische Angriffspunkte
aufdecken und damit die Möglichkeit zur Entwicklung neuer Therapieansätze bieten.
V600EDie Entwicklung eines transgenen Mausmodells, in dem B-Raf , eine Mutation die
man in 60% der Melanompatienten findet, melanocyten-spezifisch exprimiert wird,
würde ein geeignetes Werkzeug ergeben, um die Entstehung und die Metastasierung von
Melanom zu untersuchen. Im Rahmen dieser Arbeit wurde ein Konstrukt zur
V600EDoxycyclin-abhängingen Expression von B-Raf erzeugt und mit diesem wurden
transgene Tiere generiert. Da dieser Ansatz nicht zu lebensfähigen, das Transgen
tragenden Linien führte, muss das Expressionssystem weiter verbessert werden.
Da in der Arbeit von anderen gezeigt wurde, dass das metastasierende Verhalten von
Tumor-Zelllinien durch eine embryonale Mikroumgebung aufgehoben werden konnte,
und dass der Einfluss einer tumorähnlichen Mikroumgebung in Melanocyten zur
Erlangung von Tumorzell-Charakteristika führte, kam die Frage auf, ob B-Raf eine
ähnlich wichtige Rolle in der Entwicklung von Melanocyten wie in der Entstehung von
Melanomen spielt. Im Rahmen dieser Arbeit wurde die embryonale
Melanocytenentwicklung in B-Raf-defizienten sowie in wildtypischen Mausembryonen
untersucht. Es konnten keine Unterschiede in der Lokalisation und Anzahl von
Stammzellen des Neuralrohres und in der Lokalisation von Dopachrome-tautomerase
positiven Melanoblasten in den Embryonen und in kultivierten Explantaten des
Neuralrohres festgestellt werden.
Die Expression von oncogenem C-Raf in Lungenepithelzellen von Mäusen ist ein Modell
für NSCLC und führt zur Ausbildung von Adenomen ohne spontane Weiterentwicklung
oder Metastasen. Die Koexpression von C-Raf mit c-Myc in denselben Zellen
beschleunigt die Entwicklung von Tumoren und führt zu Metastasen in Leb