Les maladies rénales ont un impact socio-économique majeur sur la santé publique nécessitant le développement de nouvelles stratégies thérapeutiques. Le Récepteur à Domaine Discoïdine de type 1 (DDR1) est un récepteur non-intégrine des collagènes, à activité tyrosine-kinase. Son expression anormale est un facteur clé de la pathologie rénale qui promeut le développement de l’inflammation et de la fibrose.Ces travaux de thèse nous ont permis de démontrer que l'inhibition de DDR1 freinait la progression des maladies rénales dans trois modèles, dont l'un d'évolution aiguë, l'ischémie-reperfusion (I/R). Après I/R, les cellules épithéliales tubulaires proximales (CETP) exprimaient anormalement DDR1 et l'inhibition de ce récepteur empêchait l'acquisition d'un phénotype pro-inflammatoire par ce type cellulaire. Nous avons démontré in vitro que le stress du réticulum endoplasmique (RE), secondaire à l'hypoxie, était responsable de l'induction de DDR1, via l'activation du facteur de transcription CHOP. De plus, le profil d'expression de DDR1 dans des biopsies de patients transplantés était similaire à celui obtenu dans l'I/R expérimentale.Enfin, les résultats préliminaires obtenus dans un nouveau modèle de souris triples transgéniques ont montré l'installation d'une inflammation et d'une fibrose rénales secondaires à la surexpression génétiquement définie de DDR1 durant 4 semaines dans les cellules épithéliales tubulaires.En conclusion, nos résultats suggèrent que la surexpression de DDR1 joue un rôle délétère dans les néphropathies chroniques et aiguës, ce qui renforce l’intérêt du développement d’inhibiteurs spécifiques de DDR1 capables de bloquer la fonction de ce récepteur. / Renal diseases lead to severe long-term complications of kidney function and only few preventive and therapeutic options exist. Discoidin Domain Receptor 1 (DDR1) is a non-integrin collagen receptor expressed in several cell types within the kidney. Its abnormal expression has a deleterious role in experimental chronic kidney diseases (CKD) by promoting renal inflammation and fibrosis.The inhibition of DDR1 stopped the progression of renal disease in two models of experimental CKD and protected renal function and structure in a model of acute kidney disease, ischemia-reperfusion (I/R). DDR1 expression was strongly induced in proximal epithelial tubular cells (PETCs) after I/R. Moreover, isolated PETCs from DDR1 heterozygous mice after I/R did not acquire the pro-inflammatory phenotype displayed by PETCs from WT mice. Endoplasmic reticulum (ER) stress was responsible for DDR1 pathological expression in hypoxic PETCs after I/R through the activation of CHOP transcription factor. Interestingly, biopsies of transplant patients with prolonged ischemia during transplantation had a very similar expression profile of DDR1 in proximal tubules as in experimental I/R.Finally, DDR1 overexpression in epithelial tubular cells for four weeks, in a new conditional transgenic mouse model, led to the development of renal inflammation and fibrosis.To conclude, our results suggest that the genetically-induced or the pathological overexpression of DDR1 promotes renal inflammation and fibrosis. Thus, targeting DDR1 can be a promising strategy in the treatment of renal diseases.

Die Mehrheit chronischer Nierenerkrankungen wird durch glomeruläre Defekte hervorgerufen. In dieser Arbeit wurde deshalb im Mausmodell die Bedeutung der Kollagenrezeptoren DDR1 (Discoidin Domain Rezeptor 1) und ITGA2 (Integrin Alpha 2) in der Pathogenese von glomerulären Nierenerkrankungen untersucht. Von zentralem Interesse waren neben der Betrachtung des renalen Phänotyps, die Analyse der glomerulären Basalmembran sowie die Prüfung auf Vorhandensein nierenschädigender Faktoren. Zur Orientierung angefertigte H.E.-Färbungen waren lichtmikroskopisch unauffällig, jedoch ließ sich mittels Gelelektrophorese eine Mikro-, Makro- und Albuminurie mit einem Maximum zum Zeitpunkt von 100 Lebenstagen nachweisen, die mit 200 Tagen wieder stark sank. Auf dem Boden der nierenschädigenden Proteinurie, zeigten die Western-Blot-Analysen das Vorhandensein der Zytokine TGF-ß und CTGF auf. Die zur Detektion von Narbengewebe durchgeführte Fibronektinfärbung, erbrachte keinerlei weiterführende Anhaltspunkte. In der Elektronenmikroskopie ließ sich vereinzelt eine Mehrschichtung der GBM nachweisen, was als Ausreifungsstörung interpretiert wurde. Der Wegfall der beiden Kollagenrezeptoren ITGA2 und DDR1 scheint somit die Interaktion der Podozyten mit der GBM zu stören. Dies hat eine Proteinurie zur Folge. In Folge dessen werden profibrotische Zytokine sezerniert. Das Fehlen der beiden Kollagenrezeptoren DDR1 und ITGA2 führte jedoch nicht zur Ausbildung einer renalen Fibrose, wie in der Fibronektin-Färbung gezeigt werden konnte. Gross und Girgert zeigten, dass nierenkranke Mäuse nach dem Verlust von DDR1 oder ITGA2 einen verzögerten Verlauf der Nierenfibrose entwickelten. Vielversprechend scheinen Untersuchungen z.B. am Mausmodell Col4A3/DDR1/ITGA2 -/- oder an einer diabetischen ITGA2/DDR1 -/- Maus. Gesetzt dem Fall, dass eine renale Fibrose im Vergleich zum Einzelknockout noch später eintritt, eignen sich diese beiden Kollagenrezeptoren als therapeutisches Ziel. Aktuell stehen nur wenige nephroprotektive Medikamente, wie ACE-Hemmer, zur Verfügung. Anti-Integrine und Inhibitoren gegen Tyrosinkinase-Rezeptoren, wie DDR1, haben bereits Einzug in den klinischen Alltag gehalten und stellen eventuell einen wirksamen Ansatzpunkt zur Verhinderung einer renalen Fibrose dar.

Background: Glioblastomas (GBM) are characterised by genetic and epigenetic alterations in resistance-mediating genes and destructive infiltration of the surrounding normal brain. Cell adhesion molecules play an important role for intrinsic and acquired therapy resistances. Among the group of adhesion molecules, the collagen-binding discoidin domain receptor 1 (DDR1) is considered as potential, druggabale and promising cancer target owing to its own tyrosine kinase activity and its overexpression in various cancer types. This study evaluates the so far unknown role of DDR1 in GBM invasion and response to radiochemotherapy and investigates the underlying molecular mechanisms. Materials and Methods: Expression of DDR1 and GBM stem cell markers was evaluated in 13 biopsies of patients with primary GBM and correlated with overall survival. The effects of DDR1 inhibition by either DDR1-siRNA or DDR1 inhibitor (DDR1-IN-1), Temozolomide (TMZ) or a DDR1-IN-1/TMZ combination on GBM invasion capacity and cancer cell survival upon irradiation were evaluated in a panel of 7 GBM cell lines, 5 GBM stem-like cell cultures and an orthotopic GBM mouse model. Changes in underlying signal transduction mecha-nisms after DDR1 inhibition were assessed by Western blot and broad-spectrum phosphopro-teome analysis. Direct DDR1 binding partners were evaluated by immunoprecipitation, mass spectrometry, FRET analysis, truncated and mutated DDR1 variants and GST-pulldown. Results: DDR1 was found to be co-expressed with GBM stem cell markers in clinical GBM samples. High DDR1/Nestin co-localisation correlated with poor patient outcome. In GBM models a pharmacological DDR1 inhibition enhanced sensitivity and prolonged survival in response to radiochemotherapy with Temozolomide compared to conventional therapy. Moreover, a 50 % reduced invasion was determined in GBM cell cultures. Mechanistically, DDR1 inhibition impaired Akt/mTOR signalling and induced LC3b protein expression and formation of LC3b-positive autophagosomes suggesting autophagy for GBM radiochemosen-sitisation. Further, mass spectrometry on DDR1 immunoprecipitates identified a 14-3-3/Beclin-1/Akt1 protein complex as linker to the prosurvival Akt-mTOR axis. FRET analysis and GST-pulldown of truncated DDR1 variants identified complex assembly at DDR1 to be required for prosurvival Akt/mTOR signalling, regulation of autophagy and radichemosensiti-sation. Conclusion: These data suggest DDR1 targeting in GBM to be highly effective for radi-ochemosensitisation via autophagy induction, impairment of cell invasion and superior to the current conventional therapy. Based on its overexpression and key function in prosurvival mechanisms, targeting of the druggable DDR1 provides a rationale for combinatorial thera-pies with novel adjuvant autophagy-inducing agents administered with conventional radi-ochemotherapy.:Abbreviations V 1 Introduction 1 2 Background 3 2.1 Glioblastoma 3 2.1.1 Epidemiology 3 2.1.2 Molecular characteristics 5 2.1.3 GBM therapy 6 2.2 Physical and biological action of ionizing radiation 8 2.3 Therapy resistance 10 2.3.1 Tumour heterogeneity and cancer stem cells 12 2.3.2 Tumour microenvironment and cell-adhesion mediated resistance 13 2.3.3 Autophagy 16 2.4 Discoidin Domain Receptor 1 (DDR1) 17 2.4.1 Structure 17 2.4.2 Physiological role of DDR1 19 2.4.3 DDR1 in cancer 20 2.4.4 DDR1 signalling 22 3 Hypothesis and Aims 25 4 Materials and Methods 26 4.1 Materials 26 4.1.1 Devices 26 4.1.2 Further materials 28 4.1.3 Patient material 29 4.1.4 siRNA 29 4.1.5 Inhibitors 30 4.1.6 Chemotherapeutic agents 30 4.1.7 Plasmids 30 4.1.8 Primers 32 4.1.9 Polymerases, Restriction enzymes and ligases 32 4.1.10 Bacterial culture 33 4.1.11 Protein and DNA ladders 33 4.1.12 Method kits 34 4.1.13 Primary antibodies 34 4.1.14 Secondary antibodies 36 4.1.15 Solutions for cell biological applications 37 4.1.16 Solutions for protein-biochemical and molecular-biological applications 38 4.1.17 Solutions for immunofluorescence and immunohistological applications 40 4.1.18 Further solutions and chemicals 41 4.1.19 PC programs 41 4.2 Methods 42 4.2.1 Cell culture 42 4.2.2 Cell freezing and thawing 43 4.2.3 siRNA knockdown 43 4.2.4 Inhibitor treatment and chemotherapy 44 4.2.5 Radiation exposure 44 4.2.6 Colony formation assay 44 4.2.7 Sphere formation assay 46 4.2.8 Invasion Assay 46 4.2.9 Total protein extracts, SDS-PAGE and Western Blotting 47 4.2.10 Orthotopic mouse model 49 4.2.11 Histology and immunohistochemistry 51 4.2.12 Phosphoproteome analysis 52 4.2.13 Immunoprecipitation 52 4.2.14 Mass spectrometric analysis 53 4.2.15 Expression constructs, site-directed mutagenesis and transfection of plasmids 54 4.2.16 GST-Pulldown 57 4.2.17 Fluorescence microscopy 58 4.2.18 Fluorescence Resonance Energy Transfer (FRET) analysis 58 4.2.19 Autophagy analysis 59 4.2.20 Statistics 60 5 Results 61 5.1 DDR1 Expression in GBM 61 5.1.1 DDR1 expression analysis on publicly available data 61 5.1.2 DDR1 expression in GBM cell lines and GBM stem-like cells 63 5.1.3 The role of DDR1 in GBM stemness 64 5.2 The role of DDR1 in GBM radiochemoresistance and invasion 68 5.2.1 Effect of DDR1 silencing on GBM radiosensitivity 68 5.2.2 Effect of DDR1 silencing on GBM invasiveness 69 5.2.3 Pharmacological inhibition of DDR1 in vitro 69 5.2.4 DDR1 inhibition in an orthotopic GBM mouse model 73 5.3 DDR1 signalling in GBM 76 5.3.1 Akt/mTOR signalling axis 76 5.3.2 14-3-3/Beclin-1/Akt1 protein complex 79 5.4 The role of DDR1 inhibition in autophagy 85 6 Discussion 89 6.1 DDR1 expression correlates with GBM stemness and conveys radiochemoresistance and invasiveness 90 6.2 DDR1 inhibition impacts on Akt/mTOR signalling 92 6.3 A 14-3-3/Beclin-1/Akt1 complex physically connects DDR1 to the Akt/mTOR axis 93 6.4 DDR1 inhibition induces autophagy for radiochemosensitisation 94 7 Summary 97 8 Zusammenfassung 99 9 Figures 101 10 Tables 103 11 References 105 12 Acknowledgements 132 Appendix 133 Curriculum vitae 133 Veröffentlichungen 134 Anlage 1 136 Anlage 2 137 Darstellung des Eigenanteils 138

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