PhD Thesis Investigating the role of the PIDDosome in tumorigenesis Submitted by Lukas Peintner, MSc for the granting of the academic degree Doctor of Philosophy (PhD) at the Medical University of Innsbruck Division of Developmental Immunology Biocenter Under the supervision of Univ.-Prof. Dr. Andreas VILLUNGER 2015 ABSTRACT Caspase-2 functions as a proteolytic enzyme and was shown to act as a tumor suppressor gene in some mouse tumor models. For example, Caspase-2 was reported to have tumor suppressor functions in c-Myc driven B cell tumors, in ATM deficient T cell lymphomas or in MMTV/c-neu-driven breast cancer as well as K-Ras- driven lung cancer. Extensive efforts were put into understanding how Caspase-2 is activated and which of its postulated biological roles actually drives its tumor suppressor function. A high molecular protein complex called the PIDDosome was reported to form right before activated Caspase-2 emerges in stressed cells. The PIDDosome, consisting of Procaspase-2, the p53-induced protein with a death domain, PIDD, and the scaffold protein RAIDD is since believed to be an important activation platform for Caspase-2. However, mice lacking Pidd still show normal activation of Caspase-2 after genotoxic stress, pointing out that alternative modes of activation likely exist. In the work presented here, I report on my investigation on the role of Caspase-2 and its associated proteins Raidd and Pidd in tumorigenesis. I provide evidence that Caspase-2 does not show tumor suppressor function in tumor models driven by low dose radiation or bulky adduct formation but confirm its tumor suppressor role in the Eµ-Myc mouse model. Surprisingly, loss of Raidd does not shorten tumor latency while loss of Pidd delays tumor onset in this tumor model. The delay of tumor formation cannot be explained by defects in proliferation, differentiation, apoptosis or migration of B-cells, but mice lacking Pidd show deregulated levels of cytokines in their serum, which might influence tumor growth. Altogether, I was able to confirm the tumor suppressor function of Caspase-2 in MYC-driven B cell lymphomagenesis. The tumor suppressive effect of Caspase-2 appears to be independent of the two proteins Raidd and Pidd that were shown to interact with Caspase-2 in the PIDDosome. ii Zusammenfassung Caspase-2 ist ein proteolytisches Enzym welches eine wichtige Rolle bei der Unterdrückung von Krebs spielt. Es konnte festgestellt werden, dass Caspase-2 als „Anti-Onkogen“ die Ausbildung von MYC induzierten B Zelllymphomen, ATM- defizienten T Zelllymphomen, MMTV/c-neu gesteuerten Brustkrebs und K-Ras getriebenen Lungenkrebs unterdrücken kann. Von großem Interesse war daher in den letzten Jahren, wie Caspase-2 genau aktiviert wird und welche der verschiedenen biologischen Funktionen, die Caspase-2 zugeschrieben wurden, für die Unterdrückung von Tumorzellwachstum verantwortlich sind. Wenn Zellen DNA-schädigenden Behandlungen ausgesetzt werden, kann es zur Bildung von einem Proteinkomplex, der als das „RAIDD-PIDDosom“ bezeichnet wird, kommen. Dieser Komplex, welcher aus den Proteinen Caspase-2, PIDD und dem Gerüstprotein RAIDD besteht, könnte eine wichtige Rolle in der Aktivierung von Caspase-2 und somit bei der Tumorsuppression spielen. Überraschenderweise konnte aber bereits früh gezeigt werden, dass Caspase-2 nach genomschädigendem Stress auch in Abwesenheit von PIDD aktiviert werden kann. Die vorliegende Arbeit beschreibt meine Untersuchungen zur Rolle von Caspase-2 und deren aktivierenden Proteinen bei der Entwicklung von Krebs in unterschiedlichen Tumormodellen in der Maus. Diese Untersuchungen zeigen, dass Caspase-2 und die anderen Komponenten des PIDDosoms keine Hemmer von Tumoren sind, die z.B. durch wiederholte Bestrahlung oder chemische Mutagenese induziert wurden. Andererseits konnte die tumorunterdrückende Funktion von Caspase-2 in dem Eµ-Myc Lymphom Modell bestätigt werden. Dabei wurde ersichtlich, dass das Gerüstprotein Raidd keinen Einfluss auf die Tumorentstehung hat. Der Verlust von Pidd hingegen verzögert die Bildung von B Zell Lymphomen deutlich. Diese Verzögerung konnte aber nicht durch Fehler im Wachstum, der Differenzierung oder anhand des Zelltodes von B Zellen erklärt werden. Eine Deregulation der Zyktokinausschüttung nach onkogenem Stress, welche das Tumorwachstum eventuell indirekt beeinflusst, könnte hierfür ebenfalls von Bedeutung sein. Zusammenfassend bestätigen diese Untersuchungen die unterdrückende Funktion von Caspase-2 im Tumorwachstum. Diese Rolle von Caspase-2 scheint jedoch komplett unabhängig von den Proteinen RAIDD und PIDD zu sein, obwohl beschrieben wurde, dass diese Proteine mit Caspase-2 interagieren können. iii Chapters Introduction ........................................................................................................................... 1 Proteolytic Enzymes .......................................................................................................... 1 Family of Caspases ........................................................................................................ 2 Caspase-2 ...................................................................................................................... 5 Caspase-2 as a tumor suppressor .................................................................................11 RAIDD ...........................................................................................................................14 PIDD .............................................................................................................................17 THE PIDDosome ..............................................................................................................19 Physiological relevance of the PIDDosome .......................................................................21 Aims of the study ..................................................................................................................24 Main Results: .......................................................................................................................25 Discussion ............................................................................................................................25 Conclusions: .....................................................................................................................29 Attached Manuscripts ...........................................................................................................30 1) P53-induced protein with a death domain (PIDD) – Master of Puppets? .......................30 2) PIDDosome-independent tumor suppression by Caspase-2 .........................................38 3) Death of p53-defective cells triggered by forced mitotic entry in the presence of DNA damage is not uniquely dependent on Caspase-2 or the PIDDosome ..............................50 4) The tumor-modulatory effects of Caspase-2 and Pidd1 do not require the scaffold protein RAIDD ...................................................................................................................60 5) Loss of PIDD limits NFκB activation and cytokine production but not cell survival or transformation after DNA-damage ....................................................................................95 REFERENCES for Introduction: ......................................................................................... 108 SUPPLEMENT ................................................................................................................... 120 Abbreviations .................................................................................................................. 120 Curriculum Vitae ............................................................................................................. 122 List of Publications .......................................................................................................... 125 Conference attendances, fellowships and awards .......................................................... 126 Acknowledgements ......................................................................................................... 127 iv Introduction Introduction Thermodynamic systems have a common drive to increase entropy. Life, as we know it, uses this drive to generate energy by concentration gradients. The enclosure of biochemical reactions in compartmentalized areas was the successful foundation of life and this is only possible, when the actions within a compartment or the interactions of many components, such as cells, are tightly regulated and controlled. As usual for social systems there are forces that try to escape the generally accepted codes of practise. In biological systems some cells often disrupt the general tissue architecture, proliferate massively and build their own nutrient supply system. By this reckless behaviour they induce massive damage to the tissue and in long term may also kill the organism from which they were derived. To prevent cells of becoming tumorigenic several mechanisms evolved, closely controlling proliferation, programmed cell death, differentiation or migration of each individual cell. Loss of certain proteins may lead to deficits in this supervision. The present work focuses on the function of such a protein, Caspase-2, that has influences on various levels in the biology of a cell. Since it is that versatile its activation and mode of action is tightly controlled by various means. Proteolytic Enzymes Targeted degradation of