In many ways cancer is a disease of cellular signalling disequilibrium. When the equilibrium of key signalling pathways is upset, critical biological functions such as cell growth, survival, motility, proliferation, metabolism and apoptosis are affected, and can lead to the initiation of cancer. Reversible protein phosphorylation is an extremely important mechanism by which the activity of enzymes and proteins in signalling cascades can be regulated. Dual specificity phosphatases (DUSPs) are a unique subgroup of the protein tyrosine phosphatases (PTPs) in that they can dephosphorylate both phospho-tyrosine and phospho-serine/threonine residues within the one substrate. Many DUSPs have been implicated in cancer as critical regulators of key cancer- associated signalling cascades including the mitogen activated protein kinase (MAPK) pathway. Transcript profiling of 51 primary ovarian tumours and four normal ovaries as controls identified an uncharacterised atypical DUSP, DUSP26 as being potentially down-regulated in all histological subtypes of ovarian cancer compared with normal ovaries. DUSP26 is located at 8p12, a chromosomal region previously shown to exhibit allelic imbalance in ovarian cancer. DUSP26 is predominantly expressed in neuro-endocrine tissue, with high expression also in skeletal muscle, prostate and ovary. DUSP26 mRNA expression is reduced in brain cancer, neuroblastoma, and ovarian cancer cell lines compared to normal, consistent with a role for DUSP26 as a tumour suppressor gene. Furthermore, DUSP26 can negatively affect the proliferation of epithelial cells, also consistent with a role as a tumour suppressor gene. Expression of DUSP26 in primary ovarian cancer samples is variable however, and analysis of DUSP26 protein expression is required to reconcile these results. Preliminary results suggest that DUSP26 is epigenetically regulated and that hypermethylation may contribute to its silencing in cancer. In the literature, there is great controversy in regards to the substrate specificity of DUSP26. Results presented in this thesis conclusively demonstrate that DUSP26 is not a MAPK phosphatase, despite reports to the contrary. Instead, using a substrate trapping approach, two novel potential DUSP26 substrates were identified: DNA-dependent protein kinase (DNA-PK) and nuclear mitotic apparatus protein (NuMA), which are often dysregulated in cancer. Consequently, DUSP26 may affect the pathogenesis of cancer via DNA-PK and or NuMA.

In many ways cancer is a disease of cellular signalling disequilibrium. When the equilibrium of key signalling pathways is upset, critical biological functions such as cell growth, survival, motility, proliferation, metabolism and apoptosis are affected, and can lead to the initiation of cancer. Reversible protein phosphorylation is an extremely important mechanism by which the activity of enzymes and proteins in signalling cascades can be regulated. Dual specificity phosphatases (DUSPs) are a unique subgroup of the protein tyrosine phosphatases (PTPs) in that they can dephosphorylate both phospho-tyrosine and phospho-serine/threonine residues within the one substrate. Many DUSPs have been implicated in cancer as critical regulators of key cancer- associated signalling cascades including the mitogen activated protein kinase (MAPK) pathway. Transcript profiling of 51 primary ovarian tumours and four normal ovaries as controls identified an uncharacterised atypical DUSP, DUSP26 as being potentially down-regulated in all histological subtypes of ovarian cancer compared with normal ovaries. DUSP26 is located at 8p12, a chromosomal region previously shown to exhibit allelic imbalance in ovarian cancer. DUSP26 is predominantly expressed in neuro-endocrine tissue, with high expression also in skeletal muscle, prostate and ovary. DUSP26 mRNA expression is reduced in brain cancer, neuroblastoma, and ovarian cancer cell lines compared to normal, consistent with a role for DUSP26 as a tumour suppressor gene. Furthermore, DUSP26 can negatively affect the proliferation of epithelial cells, also consistent with a role as a tumour suppressor gene. Expression of DUSP26 in primary ovarian cancer samples is variable however, and analysis of DUSP26 protein expression is required to reconcile these results. Preliminary results suggest that DUSP26 is epigenetically regulated and that hypermethylation may contribute to its silencing in cancer. In the literature, there is great controversy in regards to the substrate specificity of DUSP26. Results presented in this thesis conclusively demonstrate that DUSP26 is not a MAPK phosphatase, despite reports to the contrary. Instead, using a substrate trapping approach, two novel potential DUSP26 substrates were identified: DNA-dependent protein kinase (DNA-PK) and nuclear mitotic apparatus protein (NuMA), which are often dysregulated in cancer. Consequently, DUSP26 may affect the pathogenesis of cancer via DNA-PK and or NuMA.

Résumé: L’autophagie est un processus essentiel au maintien de l’homéostasie cellulaire. Elle permet de dégrader et recycler aussi bien des organelles entières que des composants cytoplasmiques non fonctionnels. De plus, l’augmentation d’autophagie en condition de stress constitue une réponse adaptative favorisant la survie cellulaire. Chez les cardiomyocytes, l’autophagie en condition basale est indispensable au renouvellement, entre autres, des mitochondries et des protéines formant les sarcomères. De plus, les stress tels l’ischémie cardiaque ou la carence en nutriments induisent une augmentation de l’autophagie protectrice. Dans certaines conditions extrêmes, il a été suggéré qu’un surcroît d’autophagie puisse toutefois exacerber la pathologie cardiaque en provoquant la mort des cardiomyocytes. Considérant l’importance de ce processus dans la physiopathologie cardiaque, l’identification des mécanismes signalétiques régulant l’autophagie chez les cardiomyocytes a été le sujet de recherches intenses. À cet effet, l’activation des Mitogen-Activated Protein Kinase (MAPK) a été démontrée pour réguler, avec d’autres voies signalétiques, l’autophagie et l’apoptose des cardiomyocytes. Il est donc probable que les Dual-Specificity Phosphatase (DUSP), enzymes clés contrôlant l’activité des MAPK, participent aussi à la régulation de l’autophagie. Afin de vérifier cette hypothèse, nous avons induit l’autophagie chez des cardiomyocytes isolés de rats nouveau-nés en culture. L’analyse de marqueurs d’autophagie par immunobuvardage démontre que l’activation des MAPK ERK1/2 et p38 corrèle avec l’activité autophagique chez les cardiomyocytes. Dans ces conditions, la diminution d’expression de la majorité des ARNm encodant les différentes DUSP retrouvées chez les cardiomyocytes contraste de façon marquée avec l’augmentation d’expression de l’ARNm Dusp5. De plus, nous avons démontré par une étude de gain de fonction que l’activation soutenue de p38 par surexpression d’un mutant MKK6 constitutivement actif stimule l’autophagie chez les cardiomyocytes. De façon surprenante, la perte de fonction de p38 obtenue par surexpression d’un mutant p38 dominant négatif n’altère en rien la réponse autophagique initiatrice dans notre modèle in vitro. Nos résultats suggèrent que les DUSP puissent réguler, via leurs actions sur les MAPK, d’importantes étapes du processus autophagique chez les cardiomyocytes. / Abstract: Autophagy is a process essential to the maintenance of cellular homeostasis. It helps degrade and recycle whole organelles and nonfunctional cytoplasmic components. In addition, the adaptative up regulation of autophagy in stress condition promotes cell survival. In cardiomyocytes basal autophagy is essential to the renewal of, among others, mitochondria and proteins forming sarcomeres. In addition, stresses such as ischemic heart or nutrient deficiency induce an increase in protective autophagy. In extreme conditions, it has been suggested that autophagy may exacerbate cardiac disease causing the death of cardiomyocytes. Considering the importance of this process in cardiac pathophysiology, identify ing safety mechanisms regulating autophagy in cardiomyocytes has been the subject of intense research. To this end, activation of mitogen-activated protein kinase (MAPK) has been demonstrated to regulate, with other signaling pathways, autophagy and cardiomyocyte apoptosis. It is therefore likely that Dual-Specificity Phosphatases (DUSPs), key enzymes that control the activity of MAPKs, also participate in the regulation of autophagy. To test this hypothesis, we have induced autophagy in isolated cardiomyocytes of newborn rats in culture. Analysis of autophagy markers by immunoblotting demonstrated that the activation of MAPKs ERK1/2 and p38 correlates with autophagic activity in cardiomyocytes. Under these conditions, the decrease in expression of the majority of mRNAs encoding different DUSPs found in cardiomyocytes contrast sharply with the increase mRNA expression of Dusp5. Furthermore, we demonstrated by again of function study that sustained activation of p38 by overexpression of a constitutively active MKK6 mutant stimulates autophagy in cardiomyocytes. Surprisingly, the loss of p38 function obtained by overexpression of a dominant negative p38 mutant does not affect the autophagic response in our in vitro model, but increases the lipidation of autophagosomes marker LC3. Our results suggest that DUSPs can regulate, through their actions on MAPKs, important stages of autophagy in cardiomyocytes.

The proteins tyrosine kinases (PTKs) and the proteins tyrosine phosphatases (PTPs) are very important proteins implicated in the regulation of the cell cycle and numerous human diseases including cancer. VHR is a dual-specific phosphatase whose principles substrats are the MAPKs ERK and JNK. Previous studies of our laboratory showed that this phosphatase is regulated during the cell cycle. Its level is low during the G1 phase and increases during S and G2 phases to reach a top at the G2/M phases. The low level of VHR during the G1 phase is probably due to an alteration of the protein stability demonstrated by a decreased half-life (after treatment of the cell with cycloheximide). In addition, VHR deletion by RNA interference in HeLa cells induces a cell cycle arrest during G1/S and G2/M transitions [1]. In the first part of our work, we show that the dual-specificity phosphatase VHR is overexpressed in cervix cancer cell lines compared to primary keratinocytes. These cell lines are infected (HeLa, CaSki and SiHa) or not (C33 and HT3) by HPV, suggesting that VHR overexpression is HPV independent, virus which is responsible of cervix cancer. We show that VHR overexpression is associated with a differential subcellular localization. Indeed, VHR is localized in the cytoplasm of normal keratinocytes while it localizes in both cytoplasm and nucleus of the cell lines studied. This observed overexpression is not associated with an increased expression of its mRNA but with a stabilization of the protein. CHX chase showed us that VHR half life is about 2 hours in primary keratinocytes and longer than 8 hours in cervix cancer cell lines. The TMA technique allowed us to study a large number of preneoplasic and neoplasic cervical lesions. Interestingly, we observe that VHR is significantly overexpressed in CIN III (Cervical intraepithelial Lesions III) (n=18) and in SCCs (Squamous Cell Carcinoma) (n=12) compared to normal exocols (n=16) and although in ADCs (Adenocarcinoma) (n=12) and AISs (Adenocarcinoma in situ) (n=9) compared to normal endocols (n=19). The differential subcellular localization is also observed in CIN III and SCCs compared to normal exocols but not in ADCs and AISs compared to normal endocols. In the second part of our work, we analyzed the effect of small selectif inhibitors of VHR developped by Dr. L. Tautz from Burnham Institute in La Jolla, CA on cervical cell lines, HeLa and CaSki. We show that these small sulfonic acids induced a decreased number and a decreased proliferation of HeLa and CaSki cells. These effects are similar to those induced by RNA interference. We also show that these inhibitors induced an increased level of ERK phosphorylation. Since ERK is a specific substrat of VHR, these results suggest that the small inhibitors developped by L. Tautz et al. are specific for VHR.

Platelet-derived growth factor (PDGF) is a family of signaling molecules that stimulates cell growth, survival and migration. PDGF is recognized by specific transmembrane proteins, the PDGF receptors, which relay the signals to the cell activating the Mitogen-activated protein (MAP) kinases and other signaling pathways. Aberrant activation of these pathways is frequently detected in cancer. Hence, the study of these processes is essential for identifying potential drug targets or diagnostic markers. In paper I, we identified Receptor Subfamily 4 Group A Member 1 NR4A1 to be regulated by PDGF via MAP kinases, clarifying the role of Extracellular signal–regulated kinases (Erk) 1/2, Erk5 and Nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) in its regulation. NR4A1 was found to be important for the tumorigenic potential, measured as anchorage-independent growth, of glioblastoma cells. Since the cellular responses elicited by PDGF result from the balance between phosphorylation and dephosphorylation events, we investigated the role of the dual specificity phosphatases DUSP4/MKP-2 and DUSP6/MKP-3. In paper II, we describe the crucial role of Erk1/2 and p53 in the expression of DUSP4/MKP2. Moreover, we observed that DUSP4/MKP-2 downregulation decreases Erk5 activation and accelerates PDGFRβ internalization and downregulation resulting in a specific inhibition of Signal transducers and activators of transcription (Stat) 3, Src and protein kinase C (PKC), and partially of p38, Stat1/5 and Phoshoplipase Cγ (PLCγ). In paper III, we report that DUSP6/MKP-3 creates a negative cross-talk between Erk1/2 and Erk5 and an auto-inhibitory feedback loop on the PI3-kinase/Akt pathway. In paper IV, we identify a new regulative mechanism of the PDGF pathway. PDGF induces Erk5 expression and activation that modulates the PDGFRβ activity. After Erk5 downregulation, the receptor undergoes to a faster and stronger activation that results in a faster internalization and degradation. In conclusion, we present a mechanism through which the PDGF/MAP kinases support tumor growth, and elucidate different regulatory pathways involved in PDGF signaling.

Dual specificity phosphatases (DUSP) are enzymes that dephosphorylate both phosphoserine/threonine and phosphotyrosine residues on one substrate. Most of them specifically dephosphorylate family mitogen-activated protein kinases (MAPK). Number of DUSPs increases with complexity of organisms and in human genome there are 25 DUSPs described. Some DUSPs can dephosphorylate only one protein while other interact with wider spectrum of substrates. Except for substrate specificity DUSPs differ in expression, subcellular localization etc. Although first DUSPs were described about 20 years ago, a clear factor responsible for their substrate specificity is not known. This works uses in silico methods to discover and describe similarities and differences between DUSPs which may be important in determining DUSP specificity. Key words: phosphatase, kinase, DUSP, MAPK, substrate specificity, conservation of residues, phylogenetic tree, in silico methods

Doctor of Philosophy / Department of Biology / Alexander Beeser / Large-scale genomic approaches have demonstrated many atypical dual specificity phosphatases (DUSPs) are differentially expressed or mutated in cancer. DUSPs are proteins predicted to have the ability to dephosphorylate Ser/Thr and Tyr residues, and the atypical DUSP subgroup contains at least 16 members with diverse substrates that include proteins, nucleic acids, and sugars, and some of the atypical DUSPs function in the cell not as phosphatases but as scaffolds in signal transduction pathways. Of the atypical DUSPs, DUSP12 is one of the most evolutionarily conserved with homologs found in organisms ranging from yeast to humans. DUSP12 is of particular interest as it has been identified to be one of only two candidate genes for the target of a genetic amplification found in liposarcomas. Furthermore, DUSP12 may be an oncogene in that over-expression of dusp12 in cell culture promotes apoptosis resistance, cell motility, and the up-regulation of two established oncogenes, the hepatocyte growth factor receptor (c-met) and integrin alpha 1 (itga1). Additionally, DUSP12 may protect from apoptosis by functioning as a regulator of stress-induced translation repression and stress granule formation that may be due to its interaction with the DEAD Box RNA Helicase, DDX3.

Inositolpolyphosphate (InsPs) bilden eine ubiquitäre Gruppe an hochphosphorylierten, intrazellulären Signalmolekülen in eukaryotischen Zellen. Trotz deren Beteiligung an unzähligen biologischen Prozessen bleibt die Detektion von InsPs (insb. einzelner Enantiomere) eine Herausforderung, da die momentan verfügbaren Analysemethoden immer noch limitiert sind. In der vorliegenden Arbeit wird die stabile Isotopenmarkierung von myo-Inositol (Ins) und InsPs in Kombination mit Kernspinresonanzspektroskopie (engl. Nuclear Magnetic Resonance spectroscopy, NMR) erkundet, um diese Lücke zu schließen. Die Abhängigkeit von NMR-Daten und chemischer Struktur erlaubte die Analyse komplexer Mixturen aus InsPs aus in vitro-Experimenten und biologischen Proben. Durch stereospezifische 13C-Markierung konnten sogar Enantiomere voneinander unterschieden werden. Mit Hilfe dieser Methode wurden mehrere InsP-Stoffwechselwege untersucht. Als Erstes wurde das menschliche, Phytase-artige Enzym MINPP1 (engl. Multiple Inositol Polyphosphate Phosphatase 1) detailliert in vitro und in lebenden Zellen charakterisiert. Dabei wurde ein bisher unbeschriebener InsP-Stoffwechselweg in menschlichen Zellen erstmals beschrieben. Als Zweites wurden InsP verdauende Bakterien aus der menschlichen Darmflora untersucht, sodass der Abbauweg von Inositolhexakisphosphat beleuchtet werden konnte. Als Drittes wurden DUSP-Enzyme (engl. Dual-Specificity Phosphatases) identifiziert und in vitro charakterisiert, die in der Lage sind, die Phosphoanhydrid-Bindung von Inositolpyrophosphaten (PP-InsPs) zu spalten. Die vorliegende Arbeit demonstriert, dass 13C-Markierung in Verbindung mit NMR ein mächtiges Werkzeug darstellt, um InsP-Stoffwechselvorgänge zu untersuchen. / Inositol polyphosphates (InsPs) comprise a ubiquitous group of densely phosphorylated intracellular messengers in eukaryotic cells. Despite their contributions to a myriad of biological processes the detection of InsPs remains challenging to this day, especially with regards to differentiating enantiomers, as the available analytical toolset is still limited. In this thesis the use of stable isotope labelling of myo-inositol (Ins) and InsPs is explored to address this shortcoming. Combining 13C-labelling and nuclear magnetic resonance spectroscopy (NMR) provides both enhanced sensitivity and makes use of NMR’s strong structure-data dependency. This enabled the deconvolution of complex mixtures of InsPs from in vitro experiments or biological samples. With stereo-specific 13C-labels InsP mixtures could be resolved to individual enantiomers. Using this technique several InsP metabolic pathways were examined. Firstly, the human phytase-like enzyme Multiple Inositol Polyphosphate Phosphatase (MINPP1) was characterized in depth in vitro and in living cells, establishing a hitherto undescribed inositol polyphosphate metabolic path in humans. Secondly, inositol phosphate digesting bacteria isolated from the human gut microbiome were investigated, shedding light on the metabolic fate of inositol hexakisphosphate in the digestive track. Thirdly, a set of Dual-Specificity Phosphatases (DUSPs) were identified to be able to hydrolyze the phosphoanhydride bond of inositol pyrophosphates (PP-InsPs) and characterized in vitro. The 13C-labelling approach of InsPs in junction with NMR represents a powerful tool for the study of inositol polyphosphate metabolism. In the thesis at hand, this method has facilitated our understanding of inositol polyphosphate pathways and it will be continuing doing so in the future in several biological contexts.