The therapeutic potential of a novel multi-targeted small-interfering RNA (siRNA) was investigated in human breast cancer cells. Previous studies had identified an siRNA that specifically and potently inhibited expression of thymidylate synthase (TS) by directly targeting human TS mRNA. TS is a folate-dependent enzyme that catalyzes the key reaction involved in synthesizing nucleotide precursors for DNA biosynthesis, and as such, it plays a critical role in maintaining cell growth. The goal of this thesis was to design and develop a novel siRNA molecule that targeted TS mRNA as well as a cellular mRNA that encodes a different cellular protein involved in cancer cell growth and proliferation, such as a member of the ErbB family. Gene sequence analysis was performed and identified an overlapping sequence between TS and ErbB2 mRNAs. An siRNA duplex was then designed to simultaneously target human TS and ErbB2 mRNA. Transfection of the multi-targeted siRNA (TS1M17) revealed that both ErbB2 and TS proteins were significantly suppressed in a time and dose-dependent manner in ErbB2-overexpressing human breast cancer SKBR3 cells. The corresponding mRNA levels, as determined by RT-PCR, were also decreased. Protein levels of other ErbB family members, including ErbB1 and ErbB3, remained unchanged with siRNA treatment. An ErbB2-specific siRNA (B2450) inhibited ErbB2, but had no effect on TS expression demonstrating the specificity of the multi-targeted siRNA against both TS and ErbB2. Mismatched (TS1-Mismatch) and control (GL2) siRNAs had no inhibitory effects on expression of the two target proteins. Suppression of activated ErbB2, as determined by expression of phosphorylated ErbB2 protein, was observed with transfection of TS1M17 siRNA. In addition, the expression of downstream signaling proteins, such as phosphorylated mitogen activated protein kinase (p-MAPK), p27Kip1, p21Cip1, cyclin D1, and survivin were significantly changed. In contrast, control siRNAs did not exert any inhibitory effects on downstream signaling. Taken together, these findings suggest that TS1M17 siRNA inhibits signaling of the ErbB2 pathway. The effect of TS1M17 siRNA on cytotoxicity was analyzed by WST-1 assay. Upon transfection into SKBR3 cells, the TS1M17 siRNA significantly suppressed cell proliferation with an IC50 value of 0.65 nM, which is 154-fold more potent than ErbB2- and TS-specific siRNAs. This study suggests that targeting expression of ErbB2 and TS, two key proteins involved in distinct and critical pathways for cancer growth and proliferation, with a single siRNA molecule may provide a novel approach for cancer chemotherapy.

Meningioma and glioma are the most common primary brain tumors, but their etiologies are largely unknown. Although meningioma is usually benign, their intracranial location can lead to lethal consequences, and despite progress in surgery, radiotherapy, and chemotherapy the prognosis for patients with glioma remains poor. The only well-established environmental risk factor for meningioma and glioma is ionizing radiation. Evidence for inherited predisposition to meningioma and glioma is provided by a number of rare inherited syndromes where collectively these diseases account for only a small proportion of the twofold increased risk of brain tumors seen in first-degree relatives for meningioma and glioma patients. It is very possible that much of the excess familial risk is a consequence of co-inheritance of multiple low-risk genetic variations. With this in mind, the aims of the studies in this thesis were to discover genetic risk variants influencing the probability of acquiring the disease and to identify the association between risk variants on the tumor phenotype. To identify genetic variants influencing meningioma risk, a comprehensive tagging of the selected genes in a case-control study was performed. We identified nine risk variants in EGF, ERBB2, and LRIG2 genes. However, these findings could not be confirmed in another larger independent dataset. In addition, the study identified a correlation between LRIG2 protein expression and ER status when analyzed with different parameters. In a separate study with a larger sample of meningioma patients, the same correlation between LRIG2 and ER status was observed. To explore the potential association between reported germline risk variants and somatic genetic events, matched tumor and blood samples from glioma patients were analyzed by SNP array. The results identified correlations between EGFR gene variants and somatic aberrations within the EGFR locus and CDKN2A/B locus. To further study the relationship between germline risk variants and tumor phenotype, the same patient material was used and analyzed by three different techniques: SNP array, IHC, and FISH. The results revealed EGFR risk variants effecting copy number variation of the EGFR gene and the expression of the IDH1 and p53. Further comparison between different techniques such as SNP array and FISH analysis revealed the difficulty in achieving consistent results with different techniques. To summarize, the glioma studies show a link between genotype and phenotype where genetic risk variants in the EGFR gene were found to be associated with specific somatic aberrations. These associations are biologically interesting because EGFR is involved in multiple cellular processes. Additional studies of the direct functional role of these observations need to be conducted to elucidate the molecular mechanisms underlying the identified association between germline gene variants and somatic aberrations. For the meningioma studies, no significant risk variants influencing the disease were found but a correlation between LRIG2 and ER status was observed. This result suggests a potential role for the LRIG protein in the pathogenesis of meningioma, but more studies are needed to confirm this hypothesizes. / <p>Cancer research foundation in northern Sweden and Lions cancer research foundation at Umeå university</p>

Untersucht wurden MCF7-neuT-Zellen bezüglich ihrer optischen Verformbarkeit im Optical Stretcher, zeitabhängig nach Beginn einer ErbB2-Überexpression. Es konnte gezeigt werden, dass sich Brustkrebszellen 24 Stunden nach Beginn einer induzierten ErbB2-Überexpression deutlich von Kontrollzellen unterscheiden, indem sie sich aktiv gegen äußeren Zug zusammenziehen. Dieses als Mechanoaktivierbarkeit bekannte Phänomen ist ein Zeichen von Malignität. Nach zwei Tagen ErbB2-Überexpression zeigt sich diese Mechanoaktivierbarkeit allenfalls noch residuell, nach vier Tagen gibt es keinen Unterschied mehr zu den Kontrollzellen ohne ErbB2-Überexpression. Eine mögliche Deutung dieser Ergebnisse wäre ein Zweiphasen-Modell der Entstehung der ErbB2-überexprimierenden Brusttumore und ihrer frühen Mikrometastasen: In der ersten Phase werden die Zellen aktiv und kontraktil und sind so in der Lage, sich aus dem Zellverband zu lösen und ihren Ursprungsort zu verlassen. Bereits nach zwei bis vier Tagen sind sie aber nicht mehr von unveränderten, benignen Zellen zu unterscheiden und können unerkannt und unerreichbar für Chemotherapeutika als „dormant cells“ viele Jahre überdauern.:1 Abkürzungsverzeichnis 5 2 Einleitung 6 3 Biologische Grundlagen 10 3.1 Der ErbB2-Rezeptor 10 3.1.1 Rezeptoren der epithelialen Wachstumsfaktoren 10 3.1.2 ErbB2-Überexpression im Rahmen der Kanzerogenese 13 3.1.3 Zeitliche Veränderungen nach induzierter ErbB2-Rezeptor-Überexpression 18 3.2 Das Zytoskelett 19 4 Physikalischer Hintergrund 23 4.1 Viskoelastizität semiflexibler Polymere als zelluläre Strukturbasis 23 4.2 Überblick der bisherigen Zellelastizitätsmessungen 24 4.3 Der Optical Stretcher 25 4.3.1 Vergleich mit herkömmlichen Verfahren 25 4.3.2 Berechnung des Spannungsprofils 27 5 Aufgabenstellung 33 6 Material und Methoden 34 6.1 Verwendete Zellen 34 6.1.1 Zelllinien 34 6.1.2 Zellkultur 35 6.1.3 Zellvorbereitung 36 6.2 Elastizitätsmessungen am Optical Stretcher 37 6.3 Methodik Auswertung 41 6.3.1 Zellstatistik 41 6.3.2 Fluoreszenzmessungen 43 6.3.3 Bildauswertung 44 6.3.4 Verformungskurven und initiale Integrale 46 7 Ergebnisse 49 7.1 Motivation und Vorgehen 49 7.2 Reproduzierbarkeit der Daten 50 7.3 Entwicklung der optischen Verformbarkeit der Vektorkontrollzellen in Abhängigkeit von der Doxicyclinzugabe 53 7.3.1 Vektorkontrollzellen nach einem Tag Kulturdauer 53 7.3.2 Vektorkontrollzellen nach zwei Tagen Kulturdauer 55 7.3.3 Vektorkontrollzellen nach vier Tagen Kulturdauer 57 7.4 Entwicklung der optischen Verformbarkeit der MCF-7/neuT-Zellen in Abhängigkeit von der Doxicyclinzugabe 59 7.4.1 NeuT-Zellen nach einem Tag Kulturdauer 59 7.4.2 NeuT-Zellen nach zwei Tagen Kulturdauer 61 7.4.3 NeuT-Zellen nach vier Tagen Kulturdauer 63 7.5 Vergleich MCF-7/neuT und Vektorkontrollzellen 65 7.5.1 Veränderung durch Expression des neuT-Genes 65 7.5.2 Veränderungen durch die Kulturdauer 68 7.6 Zusammenfassung 70 8 Diskussion und Ausblick 71 8.1 Statistische Relevanz 72 8.2 Biphasischer Verlauf der ErbB2-induzierten Zellveränderungen im Vergleich zu anderen Untersuchungen 72 8.3 Interpretation des biphasischen Musters 74 8.4 Ausblick 76 9 Zusammenfassung der Arbeit 78 10 Referenzen 81 10.1 Weblinks 91 11 Anlagen 92 11.1 Selbstständigkeitserklärung 92 11.2 Lebenslauf 93 11.2.1 Allgemeine Angaben 93 11.2.2 Schulische Ausbildung 93 11.2.3 Studium 93 11.2.4 beruflicher Werdegang 94 11.3 Publikation 95 11.4 Danksagung 96

Oxidative stress is a documented factor in the pathogenesis of inflammation and cancer. Vitamin E with its antioxidant properties holds promise for use in clinical practice. There are two main forms of vitamin E, tocopherols and tocotrienols. Palm oil contains almost 70% of tocotrienols. Tocotrienols exerts its antiproliferative activity against malignant cells but not on normal cells. Tocotrienols play an important role in counteracting cellular inflammatory response secondary to oxidative stress, thus exerting an anticancer property. Tocotrienols mediate function of NF-kappa B, STAT3 (signal transduction and activators), and COX-2. In addition to its role as an antioxidant and anti-inflammatory agent, tocotrienols also mediate multiple cell cycle pathways. More work needs to be done on animal models and in genetic models of pancreatic cancer to gather more data to eventually consider phase III clinical trial in human subjects.

The use of peptides in drug development has been hampered by their poor pharmaceutical properties, most notably their inability to reliably permeate biological membranes and lack of targeting. To overcome these disadvantages, the AMino acid Intracellular Delivery SysTem (AMIDST) was developed. This modular peptide-based delivery system confers cellular permeability and organelle-specific targeting for therapeutic peptides. As demonstrated in this study, the delivery of a HER2-binding peptide to the secretory organelles of breast cancer cells resulted in intracellular sequestration, a reduction in downstream signalling, and reduced viability compared to the delivery of a control peptide. Given its modular design and ease of production, AMIDST has the potential to enhance the use of peptides as therapeutic agents.

Die Charcot-Marie-Tooth-Krankheit 1A (CMT1A) gehört zur Familie der erblichen Neuropathien des peripheren Nervensystems (PNS) und ist mit einer Prävalenz von 1 zu 2500 die häufigste Subform. Die Patienten leiden an einer distal ausgeprägten Muskelatrophie und sensorischen Beeinträchtigungen. Die Histopathologie zeigt sich im PNS durch Myelinisierungsdefekte, die Bildung von Zwiebelschalenformationen, eine langsam fortschreitende Demyelinisierung und einen axonalen Verlust in späteren Krankheitsstadien, die für die klinische Symptomatik verantwortlich sind. Bis heute gibt es keine Behandlungsmöglichkeiten. Die genetische Duplikation des Gens, das für das periphere Myelinprotein von 22kDa (PMP22) kodiert, ist Hauptursache der Erkrankung und betrifft speziell die myelinisierenden Gliazellen des PNS, die Schwannzellen. Erkrankte Schwannzellen induzieren eine de novo Expression des glialen Wachstumsfaktors Neuregulin 1-Typ I (NRG1-I). Die chronische NRG1-I Überexpression wurde als ein maßgeblicher Faktor der Krankheitspathogenese der CMT1A identifiziert. Aberrantes NRG1-I überstimuliert den ERBB2 Rezeptor, was zu einer Hyperaktivierung des MEK-ERK Signalwegs führt. Die genetische Ablation von NRG1 spezifisch in Schwannzellen verbesserte die klinische Präsentation und Histopathologie in CMT1A Mäusen. Damit einhergehend waren die ERBB2 und ERK Hyperaktivität in Schwannzellen normalisiert. Die Inhibition der NRG1-I-vermittelten pathologischen Signalkaskade stellt damit einen vielversprechenden therapeutischen Ansatz für die CMT1A dar. Die langfristige pharmakologische Inhibition entweder der ERBB2 oder der ERK Aktivierung verschlechterte jedoch die klinische Präsentation von adulten CMT1A Ratten. Die Verringerung der ERBB2 Aktivität verschlimmerte die axonale Funktion und bewirkte eine Zunahme von Zwiebelschalenformationen. Die Reduktion der ERK Hyperaktivität führte zu einem frühen behandlungsinduzierten Gewichtsverlust und einer Zunahme an demyelinisierten Axonen. Das Auftreten sekundär dysregulierter Signalkaskaden könnte zu dem Scheitern der Studien beigetragen haben. Das Scheitern beider Studien weist jedoch auch darauf hin, dass die Komplexität der CMT1A Pathobiologie besonders während der späten chronischen Phase noch immer unzureichend verstanden ist und weiterer Erforschung bedarf. Zusammenfassend ist die Interferenz mit der ERBB2-ERK Signalkaskade für die Behandlung der CMT1A ungeeignet. Dies weist auf die Notwendigkeit hin, ein tieferes Verständnis der Komplexität der späten chronischen CMT1A Pathobiologie und vor allem der NRG1-vermittelten Pathomechanismen zu erlangen. Beides schafft die Grundlage für die Identifizierung neuer therapeutischer Behandlungsansätze:Abbreviations 1. Introduction 1.1 The peripheral nervous system 1.1.1 Structure and functions of the nervous system 1.1.2 Neurons and glial cells 1.1.3 Signal transduction and myelination 1.1.4 Development and functions of Schwann cells 1.2 Peripheral neuropathies 1.2.1 Acquired and inherited peripheral neuropathies 1.2.2 Clinical presentation and classification of Charcot-Marie-Tooth diseases 1.2.3 The peripheral myelin protein of 22 kDa (PMP22) 1.2.4 Animal models of PMP22 – associated CMTs 1.2.5 Pathobiology and treatment options of CMT1A 1.2.6 Ablation of Schwann cell Neuregulin 1 ameliorates the phenotype of a CMT1A mouse model 1.3 The Neuregulin 1 – ERBB2 – ERK signaling axis in the Charcot-Marie-Tooth 1A disease 1.3.1 Functions of Neuregulin 1 in the peripheral nervous system 1.3.2 ERBB receptor tyrosine kinase signaling in Schwann cells 1.3.3 NRG1-I – ERBB mediated pathophysiological effects in CMT1A 1.3.4 Options for pharmacological interference with NRG1-I – mediated ERBB2 and ERK activation 1.4 Aim of the dissertation 2. Materials and Methods 2.1 Materials 2.1.1 Chemicals and Reagents 2.1.2 Consumable Supplies 2.1.3 Technical Equipment 2.1.4 Kits 2.1.5 Buffers and Solutions 2.1.6 Antibodies 2.1.7 Genotyping Primer 2.1.8 qPCR Primer 2.1.9 Enzymes 2.1.10 Software 2.2 Animal caretaking and treatment 2.2.1 Animal strains and breeding 2.2.2 Animal breeding and caretaking 2.2.3 Identification of animals 2.2.4 Herceptin® and Selumetinib treatment of CMT1A rats 2.2.5 Mechanical phenotyping 2.2.6 Electrophysiological examination 2.2.7 Euthanasia 2.2.8 Preparation and handling of peripheral nerve tissues 2.3 Histological methods 2.3.1 Embedding of isolated peripheral nerves in Agar 100 resin 2.3.2 Generation, imaging, and analysis of semi-thin sections 2.3.3 Coating of support copper grids for ultrastructure analysis 2.3.4 Generation, contrasting, imaging, and analysis of ultra-thin cross-sections 2.4 Protein Biochemistry 2.4.1 Isolation and quantification of (phospho-) proteins from frozen peripheral nerves 2.4.2 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) 2.4.3 Western blot and Fast Green staining 2.5 Molecular Biology Methods 2.5.1 Isolation of genomic DNA from tissues 2.5.2 Polymerase chain reaction of genomic DNA 2.5.3 Agarose gel electrophoresis of PCR products 2.5.4 RNA Isolation 2.5.5 Quality and quantity assessment of isolated RNA 2.5.6 cDNA Synthesis of total RNA 2.5.7 Quantitative real-time PCR analysis 2.6 Bioinformatics 2.6.1 Power analysis 2.6.2 Statistical methods 3. Results 3.1 NRG1 – ERBB2/ERBB3 – ERK signaling is increased in an adult CMT1A-resembling rat model 3.2 Late long-term blockage of ERBB2 signaling deteriorates the clinical and histopathological outcome of CMT1A rats 3.2.1 The clinical phenotype and histopathological hallmarks worsen after long-term Herceptin treatment of CMT1A rats 3.2.2 Herceptin application inhibits NRG1-I – mediated ERBB2/ERBB3 activation and concomitant downstream signaling 3.2.3 Inhibition of ERBB2 – mediated downstream signaling does not affect CMT1A Schwann cell dedifferentiation 3.3 Late long-term application of Selumetinib deteriorates the clinical presentation of CMT1A rats 3.3.1 Late short-term Selumetinib application ameliorates NRG1-I – ERBB2/3 – mediated downstream signaling 3.3.2 Long-term Selumetinib treatment deteriorates the clinical phenotype and promotes demyelination in CMT1A rats 3.3.3 Selumetinib treatment improved the dysbalance of the ERK and AKT pathways in CMT1A 4. Discussion 4.1 NRG1-I-mediated malsignaling may not have been sufficiently inhibited to impact the clinical outcome of CMT1A rats after ERBB2 suppression 4.2 Herceptin-binding of ERBB2 may interfere with the regeneration ability of Schwann cells in CMT1A 4.3 Dysregulation of ERK activity in late CMT1A promotes demyelination 5. Abstract 6. Zusammenfassung 7. References 8. Appendix 8.1 List of figures 8.2 List of tables 8.3 Eigenständigkeitserklärung 8.4 Curriculum vitae 8.5 Publikationen 8.6 Danksagungen / The Charcot-Marie-Tooth disease 1A (CMT1A) belongs to the family of inherited neuropathies of the peripheral nervous system (PNS) and is the most frequent subform with a prevalence of 1 in 2500. Patients present with a distally pronounced muscle atrophy and sensory impairments. Histologically, peripheral nerves display developmental myelination abnormalities, onion bulb formations, slowly progressive demyelination, and axonal loss during later disease stages, which account for clinical symptomatology. No treatment options are available upon today. Genetic testing revealed a genomic duplication of the gene encoding for the peripheral myelin protein of 22kDa (PMP22) as the primary cause of disease that affects specifically the myelinating glia of the PNS, the Schwann cells. Pmp22 overexpression has been shown to dysregulate physiological axon-glia interactions. Diseased Schwann cells respond with a de novo expression of the glial growth factor, Neuregulin 1-type I (NRG1-I). While not present under physiological conditions, chronic NRG1-I signaling was identified to be a major driver of disease pathogenesis in CMT1A. On the mechanistic level, aberrant NRG1-I has been demonstrated to overstimulate the ERBB2 receptor resulting in hyperactivation of the MEK-ERK pathway. Genetic disruption of NRG1 specifically in Schwann cells strongly improved the clinical presentation and histopathological manifestation of disease hallmarks in CMT1A-resembling rodents. This beneficial outcome was accompanied by a downregulation of ERBB2 and ERK hyperactivity in Schwann cells. Therefore, interfering with NRG1-I-mediated pathological signaling was proposed to constitute a promising therapeutic approach for CMT1A. However, long-term pharmacological obstruction of either ERBB2 or ERK activation deteriorated the clinical presentation of adult CMT1A rats. ERBB2 inhibition worsened axonal function and induced an increase in onion bulb formations. Reduction of ERK hyperactivity resulted in an early treatment-induced weight loss and an increase in demyelinated axons. The development maladaptive signaling mechanisms may have caused the observed outcome of the trials. However, failing of both trials instead also demonstrates that the complexity of late chronic CMT1A pathobiology is still poorly understood and has to be further investigated. In conclusion, interfering with ERBB2-ERK signaling, downstream of NRG1-I, turned out to be not a viable approach for CMT1A treatment. This points to the necessity to gain a deeper understanding of the late chronic CMT1A intricacy and especially of NRG1-mediated pathomechanisms in neuropathy. Both may build the foundation for the identification of novel therapeutic approaches and drug targets.:Abbreviations 1. Introduction 1.1 The peripheral nervous system 1.1.1 Structure and functions of the nervous system 1.1.2 Neurons and glial cells 1.1.3 Signal transduction and myelination 1.1.4 Development and functions of Schwann cells 1.2 Peripheral neuropathies 1.2.1 Acquired and inherited peripheral neuropathies 1.2.2 Clinical presentation and classification of Charcot-Marie-Tooth diseases 1.2.3 The peripheral myelin protein of 22 kDa (PMP22) 1.2.4 Animal models of PMP22 – associated CMTs 1.2.5 Pathobiology and treatment options of CMT1A 1.2.6 Ablation of Schwann cell Neuregulin 1 ameliorates the phenotype of a CMT1A mouse model 1.3 The Neuregulin 1 – ERBB2 – ERK signaling axis in the Charcot-Marie-Tooth 1A disease 1.3.1 Functions of Neuregulin 1 in the peripheral nervous system 1.3.2 ERBB receptor tyrosine kinase signaling in Schwann cells 1.3.3 NRG1-I – ERBB mediated pathophysiological effects in CMT1A 1.3.4 Options for pharmacological interference with NRG1-I – mediated ERBB2 and ERK activation 1.4 Aim of the dissertation 2. Materials and Methods 2.1 Materials 2.1.1 Chemicals and Reagents 2.1.2 Consumable Supplies 2.1.3 Technical Equipment 2.1.4 Kits 2.1.5 Buffers and Solutions 2.1.6 Antibodies 2.1.7 Genotyping Primer 2.1.8 qPCR Primer 2.1.9 Enzymes 2.1.10 Software 2.2 Animal caretaking and treatment 2.2.1 Animal strains and breeding 2.2.2 Animal breeding and caretaking 2.2.3 Identification of animals 2.2.4 Herceptin® and Selumetinib treatment of CMT1A rats 2.2.5 Mechanical phenotyping 2.2.6 Electrophysiological examination 2.2.7 Euthanasia 2.2.8 Preparation and handling of peripheral nerve tissues 2.3 Histological methods 2.3.1 Embedding of isolated peripheral nerves in Agar 100 resin 2.3.2 Generation, imaging, and analysis of semi-thin sections 2.3.3 Coating of support copper grids for ultrastructure analysis 2.3.4 Generation, contrasting, imaging, and analysis of ultra-thin cross-sections 2.4 Protein Biochemistry 2.4.1 Isolation and quantification of (phospho-) proteins from frozen peripheral nerves 2.4.2 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) 2.4.3 Western blot and Fast Green staining 2.5 Molecular Biology Methods 2.5.1 Isolation of genomic DNA from tissues 2.5.2 Polymerase chain reaction of genomic DNA 2.5.3 Agarose gel electrophoresis of PCR products 2.5.4 RNA Isolation 2.5.5 Quality and quantity assessment of isolated RNA 2.5.6 cDNA Synthesis of total RNA 2.5.7 Quantitative real-time PCR analysis 2.6 Bioinformatics 2.6.1 Power analysis 2.6.2 Statistical methods 3. Results 3.1 NRG1 – ERBB2/ERBB3 – ERK signaling is increased in an adult CMT1A-resembling rat model 3.2 Late long-term blockage of ERBB2 signaling deteriorates the clinical and histopathological outcome of CMT1A rats 3.2.1 The clinical phenotype and histopathological hallmarks worsen after long-term Herceptin treatment of CMT1A rats 3.2.2 Herceptin application inhibits NRG1-I – mediated ERBB2/ERBB3 activation and concomitant downstream signaling 3.2.3 Inhibition of ERBB2 – mediated downstream signaling does not affect CMT1A Schwann cell dedifferentiation 3.3 Late long-term application of Selumetinib deteriorates the clinical presentation of CMT1A rats 3.3.1 Late short-term Selumetinib application ameliorates NRG1-I – ERBB2/3 – mediated downstream signaling 3.3.2 Long-term Selumetinib treatment deteriorates the clinical phenotype and promotes demyelination in CMT1A rats 3.3.3 Selumetinib treatment improved the dysbalance of the ERK and AKT pathways in CMT1A 4. Discussion 4.1 NRG1-I-mediated malsignaling may not have been sufficiently inhibited to impact the clinical outcome of CMT1A rats after ERBB2 suppression 4.2 Herceptin-binding of ERBB2 may interfere with the regeneration ability of Schwann cells in CMT1A 4.3 Dysregulation of ERK activity in late CMT1A promotes demyelination 5. Abstract 6. Zusammenfassung 7. References 8. Appendix 8.1 List of figures 8.2 List of tables 8.3 Eigenständigkeitserklärung 8.4 Curriculum vitae 8.5 Publikationen 8.6 Danksagungen

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ErbB2/HER2 est un récepteur tyrosine kinase de la famille d'EGFR (ErbB1) surexprimé dans plus de 20% des cancers du sein et associé à une forme particulièrement agressive de la maladie. Les récepteurs ErbBs sont actifs seulement sous forme de dimères, permettant la phosphorylation de leur queue C-terminale par leur domaine tyrosine kinase. La phosphorylation entraine l'interaction avec des protéines adaptatrices et l'activation de voies de signalisation, Ras/MAPK et PI3K/Akt principalement. Ces voies contrôlent la prolifération, la motilité cellulaire et la résistance à l'apoptose. Contrairement à ErbB1/3/4, ErbB2 dimérise en l'absence de ligand. Comprendre les autres mécanismes de régulation de la phosphorylation de ses tyrosines et de ses interactions est donc particulièrement intéressant.ErbB2 a fait l'objet de nombreuses études structurales et fonctionnelles. Elles ont permis la mise au point de traitements ciblés efficaces mais sujets à l'apparition de résistance, dont l'anticorps Trastuzumab, ciblant sa partie extracellulaire. La queue C-terminale d'ErbB2 (CtErbB2) a été très souvent ignorée dans ces études. Cette partie étant intrinsèquement désordonnée, il a fallu attendre ces dernières années pour que les concepts et les outils permettant de l'étudier émergent.Dans cette thèse, j'ai d'abord effectué la caractérisation structurale et dynamique de CtErbB2. J'ai montré que bien qu'étant dépourvue de toute structure stable, cette région riche en prolines possède plusieurs structures secondaires transitoires et un contact longue-distance participant très probablement à la régulation de ses interactions intra- et inter-moléculaires. Dans une deuxième partie je me suis intéressée à la caractérisation de la protéine adaptatrice Grb2, partenaire essentiel de ErbB2 pour l'activation de la voie des MAP kinases. L'organisation en solution des domaines de cette protéine modulaire dans sa forme libre était jusque là inconnue. J'ai ensuite étudié l'interaction entre Grb2 et CtErbB2, et montré que CtErbB2 interagit non seulement avec le domaine SH2 de Grb2 (par l'intermédiaire d'une phosphotyrosine), mais aussi avec son domaine SH3 N-terminal (grâce à un motif polyproline). Enfin, j'ai mis en place plusieurs stratégies de phosphorylation des tyrosines de CtErbB2, dans le but d'étudier plus largement l'effet des phosphorylations sur l'ensemble de cette région. / ErbB2/HER2 is a receptor tyrosine kinase of the EGFR (ErbB1) family overexpressed in 20% of breast cancers and associated to a particularly aggressive form of the disease. ErbB receptors are only active upon dimerization that enables phosphorylation of their C-terminal tail by their tyrosine kinase domain. Phosphorylation then triggers interaction with adaptor proteins and activation of signaling pathways, mainly Ras/MAPK and Akt/PI3K. Those pathways control cell proliferation, motility and resistance to apoptosis. Contrary to ErbB1/3/4, ErbB2 can dimerize without any ligand. Understanding other mechanisms of regulation of its tyrosine phosphorylation and of its interactions is thus particularly interesting.ErbB2 structure and function have been extensively studied. This has led to the development of several FDA-approved targeted drugs, that are effective but to which resistance occurs, amongst which the Trastuzumab antibody that targets ErbB2 extracellular domain. The C-terminal tail of ErbB2 (CtErbB2) has been widely ignored in these studies. Since it is intrinsically disordered, the concepts and tools to study it have only emerged in the last few years.In the present work, I have performed the structural and dynamic study of CtErbB2. I showed that despite its lack of any stable structure, this proline-rich region exhibits several transient secondary structures and a long-range contact that might participate in the regulation of its intra- and inter-molecular interactions. Then, I characterized the adaptor protein Grb2, which is a partner of ErbB2 that is essential for the activation of the MAPK pathway. The solution organization of the domains of this modular protein in its apo-form was unknown so far. I also studied the interaction between Grb2 and CtErbB2, showing that in addition to the known SH2-phosphotyrosine interaction, a polyproline motif of CtErbB2 binds to the N-terminal SH3 domain of Grb2. Finally, I implemented several strategies to phosphorylate CtErbB2 tyrosines, to study more extensively the effect of phosphorylation on the whole tail.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.