This work explored features of canine mast cell tumours (MCT) to improve prognosis and to discover potential therapeutic targets. Subcutaneous MCT - a subset of these tumours arising in the subcutis - are usually grouped with cutaneous MCT, but there is evidence that they may be clinically different. The first objective was to develop a grading scheme for subcutaneous MCT. Over 300 canine subcutaneous MCT were evaluated retrospectively and parameters were correlated with clinical outcomes, making this the largest retrospective survival study of these tumours to date. The results of the study showed that the majority of subcutaneous MCT had excellent outcomes and key prognostic markers were identified (mitotic index, surgical margins and degree of infiltration). A subset of the subcutaneous MCT from the retrospective study was further evaluated to assess the cellular localization of KIT - a receptor tyrosine kinase (RTK) which is dysregulated and constitutively activated in some cutaneous MCT - as well as Ki67, a proliferation marker. In addition, evaluation of mutations of c-KIT, the gene for KIT, was determined for each MCT. Cytoplasmic KIT localization and high Ki67 values were predictive of decreased survival time and time to local reoccurrence, but no c-KIT mutations were detected. The majority of canine MCT do not appear to depend solely upon KIT for tumour progression and few other RTK targets have been studied in canine MCT. Based on evidence that vascular endothelial growth factor receptors (VEGFR) and platelet-derived growth factor receptors (PDGFR) - may play a role in the progression of canine MCT; the expression and distribution of these RTK were evaluated. The results showed that canine MCT have unique expression profiles and activity of KIT, VEGFR2 and PDGFR. Two novel mast cell tumour cell lines were generated and used to assess signalling of KIT and VEGFR2 in vitro. Stimulatory and inhibitory responses were assessed and found to be different in both cell lines. Both had autophosphorylated VEGFR2 and an autocrine VEGF/VEGFR2 signalling pathway existed for both cell lines. These findings are unique and the first that identify autocrine VEGF signalling as a possible survival mechanism for canine MCT. / Pet Trust Foundation, Ontario Veterinary College

La sclérose systémique (ScS) est une maladie auto-immune dévastatrice d'étiologie inconnue. Le dysfonctionnement immunitaire, la fibrose et la vasculopathie sont les trois principales caractéristiques de cette maladie. Une récente étude a révélé un nouveau lien entre l'auto-immunité et la fibrose, par la présence d'auto-anticorps stimulant le récepteur du facteur de croissance dérivé des plaquettes (PDGFR) des fibroblastes. Ces auto-anticorps sont capables de stimuler les espèces réactives de l'oxygène et d’activer la kinase régulée par un signal extracellulaire (ERK1/2). L’hypothèse que nous formulons est que les cellules musculaires lisses vasculaires (VSMCs) exprimant conjointement les PDGFR, répondront elles aussi aux autoanticorps anti-PDGF-R. Le travail présenté ici vise à valider la présence d'auto-anticorps PDGFR dans les sérums de patients ScS, et à caractériser ensuite la réponse de VSMCs exposées à de l'immunoglobuline G (IgG) de ces sérums, en mesurant l’activation des cascades de signalisation spécifiques, ainsi que l'induction des gènes impliqués dans la réponse fibrotique. Nos résultats démontrent la présence d'une fraction IgG stimulant une réponse phénotypique dans les cultures de VSMCs. Notamment, d’importantes régulations positive et négative des gènes pro-fibrotiques tgfb1 et tgfb2 respectivement, ont été observées dans les VSMCs exposées à des fractions de ScS-IgG. Les fractions de IgG positives pour l'activation de ERK étaient présentes dans la plupart, mais pas dans tous les échantillons de SSc (68%, 19/28), et moins présentes dans les contrôles 27% (11/3). Bien que, les fractions de SSc-IgG ont pu considérablement immunoprécipiter le PDGFR, l'utilisation d'un inhibiteur spécifique des récepteurs au PDGF (AG1296), n'a pas inhibé l'activation de ERK médiée par les fractions de SSc-IgG. Globalement, nos résultats indiquent la présence d'autoanticorps stimulants avec activité pro-fibrotique dans les sérums des patients ScS. Des travaux sont en cours pour identifier l'entité moléculaire responsable de la réponse d’IgG observée dans les cultures de VSMCs. / Systemic Sclerosis (SSc) is a devastating autoimmune disease of unknown etiology. Immune dysfunction, fibrosis and vasculopathy are the three major features of the disease; however, the interactions between these components are poorly understood. A novel link between autoimmunity and fibrosis has been proposed by the presence of stimulatory autoantibodies to the platelet-derived growth factor receptor (PDGFR) on fibroblasts. These autoantibodies were capable of stimulating reactive oxygen species and subsequent activation of ERK1/2. If the anti-PDGFR autoantibodies are present in the systemic circulation of SSc patients, they will most certainly encounter vascular smooth muscle cells (VSMCs). The latter are known to express the PDGFR and response to PDGF, which is a known phenotypic modulator of VSMCs. The work presented here seeks to readdress the presence of stimulatory anti-PDGFR autoantibodies in serum derived from SSc-patients and to characterize the effects of SSc-IgG on VSMCs by measuring the activation of specific signaling cascades and the induction of genes involved in fibrotic responses. Our results demonstrate the presence of an IgG fraction stimulating a phenotypic response in cultured VSMCs. Notably, a significant up-regulation of the pro-fibrotic gene tgfb1 and a significant down-regulation of the anti-fibrotic gene tgfb2 were observed in VSMC exposed to SSc-IgG fractions. Positive IgG fractions for ERK activation were present in most, but not all, SSc samples (68%, 19/28), and they were less present in controls (27%) (3/11). Although, the SSc-IgG fractions were able to significantly immunoprecipitate the PDGFR, the use of a selective PDGFR inhibitor, AG1296, did not inhibit the activation of ERK mediated by SSc-IgG fractions. Altogether, our findings suggest the presence of stimulatory autoantibodies with profibrotic activity in serum derived form SSc patients. Work is in progress to identify the molecular entity responsible for the IgG response observed in cultured VSMCs.

Adulte ou pédiatrique, les tumeurs solides ont besoin d’oxygène et de nutriments pour se développer et métastaser. Leur apport est assuré par la néo-vascularisation tumorale issue d’un processus multifactoriel appelé l’angiogénèse. Son équilibre est maintenu par une balance entre facteurs pro- et anti-angiogéniques. Elle fait partie des principales cibles pour traiter les cancers et l’inhibition de la voie VEGF en est un facteur clé. Cependant, la réponse aux agents anti-angiogéniques a montré, malgré des résultats encourageants, un effet transitoire associé à l’apparition d’une résistance adaptative de la tumeur.Nous avons étudié l’inhibition de l’angiogénèse et les mécanismes potentiels d’échappement dans les tumeurs pédiatriques solides, et principalement dans le neuroblastome. Le neuroblastome est une tumeur originaire de la crête neurale et atteint généralement l’enfant jeune. Nous avons exploré l’effet anti-tumoral de l’inhibition sélective des récepteurs 1, 2, 3 du VEGF à l’aide de l’inhibiteur à tyrosine kinase axitinib dans divers modèles précliniques de neuroblastome. L’axitinib a montré une activité anti-tumorale modérée associée à une inhibition de la vascularisation. Néanmoins, après un traitement prolongé in vitro, les cellules tumorales IGR-N91-Luc deviennent résistantes à l’axitinib. Elles prolifèrent normalement mais secrètent de «l’ hepatocyte growth factor» (HGF) et activent la voie MAPK. In vivo, le traitement prolongé par axitinib entraîne le développement d’un phénotype plus agressif de la tumeur avec l’augmentation du nombre d’animaux présentant des métastases, associée à une activation de la voie SRC. Ceci nous a conduit à explorer l’effet d’une inhibition ciblant principalement VEGFR2 et MET (récepteur à l’HGF) avec le cabozantinib. Ainsi nous avons contrôlé le développement tumoral en inhibant la néo-vascularisation et l’activation de SRC, et induit la mort cellulaire dans le modèle orthotopique IGR-N91-Luc et inhibé le développement métastatique dans le modèle systémique IMR-32-Luc. Par ailleurs, nous avons étendu notre exploration à d’autres facteurs jouant un rôle dans l’angiogénèse comme FGFR ou PDGFR car ils représentent, comme MET, de puissants oncogènes. Pour cibler simultanément VEGFR et PDGFR, nous avons utilisé l’inhibiteur multi-kinase regorafenib. In vivo, à des doses bien tolérées qui permettent un traitement prolongé, le regorafenib a montré une activité anti-tumorale significative. Cet effet a été associé principalement à une forte inhibition de la vascularisation mais également à l’induction de la mort cellulaire. En élargissant notre étude à d’autres modèles de tumeurs pédiatriques, nous avons observé que son activité est indépendante du type histologique. Compte tenu du caractère oncogénique de PDGFR, nous avons évalué cet inhibiteur dans des modèles présentant une amplification du gène PDGFRA, qui entraine une surexpression et une activation forte du récepteur. Combiné avec des thérapies standards capables d’induire des dommages à l’ADN telles que l’irradiation ou l’irinotecan, l’effet du regorafenib a été potentialisé, notamment dans les modèles amplifiés pour le gène PDGFRA se traduisant par des régressions tumorales. Ces évaluations précliniques soutiennent le développement d’une nouvelle stratégie thérapeutique pour les enfants atteints de tumeurs solides. / Solid tumors either adult or pediatric need oxygen and nutrients to grow and metastasize. Their input is provided by tumor neovascularization after a multifactorial process called angiogenesis. Balance is maintained by equilibrium between pro and anti-angiogenic factors. It is one of the main targets for treating cancers and the inhibition of the VEGF pathway is a key factor. However, despite encouraging results, the response to anti-angiogenic agents showed a transient effect associated with the development of an adaptive tumor resistance. We studied the inhibition of angiogenesis and potential escape mechanisms in solid pediatric tumors, mainly in neuroblastoma. Neuroblastoma is a solid tumor derived from the neural crest and it usually affects childhood. We investigated the anti-tumor effect of selective inhibition of VEGF receptors 1, 2, 3 using the tyrosine kinase inhibitor axitinib in various preclinical neuroblastoma l models. Axitinib showed a moderate anti-tumor activity associated with the inhibition of vascularization. However, after prolonged treatment in vitro, tumor cells IGR-N91-Luc become resistant to axitinib. They proliferate normally but secrete the "hepatocyte growth factor" (HGF) and activate the MAPK pathway. In vivo, prolonged treatment by axitinib results in the development of a more aggressive tumor phenotype with an increase in the number of animals exhibiting metastases associated with an activation of SRC signaling. This led us to explore the effect of inhibiting concomitant VEGFR2 and MET (HGF receptor), main cabozantinib targets. So we stabilized tumor growth by inhibiting the neovascularization and activation of SRC, induced cell death in the orthotopic model IGR-N91-Luc and inhibited metastatic development in the IMR-32-Luc systemic model. In addition, we extended our exploration of other factors that play a role in angiogenesis like FGFR or PDGFR because they represent, like MET, powerful oncogenes. To simultaneously target VEGFR and PDGFR, we used the multi-kinase inhibitor regorafenib. In vivo, at well-tolerated doses that allow prolonged treatment, regorafenib showed significant anti-tumor activity. This effect was mainly associated with a strong inhibition of vascularization, but also (with) induction of cell death. By expanding our study to other models of pediatric tumors, we observed that its activity was independent of histologic type. Given the oncogenic character of PDGFR, we evaluated the inhibitor in models which present a PDGFRA gene amplification, which results in a strong activation of the receptor. Combined with standard therapies that can induce DNA damages such as irinotecan or radiation, the effect of regorafenib was potentiated, mainly in PDGFRA gene amplified models, where tumor regressions were obtained. These preclinical evaluations support the development of a new therapeutic strategy for children with solid tumors.

La sclérose systémique (ScS) est une maladie auto-immune dévastatrice d'étiologie inconnue. Le dysfonctionnement immunitaire, la fibrose et la vasculopathie sont les trois principales caractéristiques de cette maladie. Une récente étude a révélé un nouveau lien entre l'auto-immunité et la fibrose, par la présence d'auto-anticorps stimulant le récepteur du facteur de croissance dérivé des plaquettes (PDGFR) des fibroblastes. Ces auto-anticorps sont capables de stimuler les espèces réactives de l'oxygène et d’activer la kinase régulée par un signal extracellulaire (ERK1/2). L’hypothèse que nous formulons est que les cellules musculaires lisses vasculaires (VSMCs) exprimant conjointement les PDGFR, répondront elles aussi aux autoanticorps anti-PDGF-R. Le travail présenté ici vise à valider la présence d'auto-anticorps PDGFR dans les sérums de patients ScS, et à caractériser ensuite la réponse de VSMCs exposées à de l'immunoglobuline G (IgG) de ces sérums, en mesurant l’activation des cascades de signalisation spécifiques, ainsi que l'induction des gènes impliqués dans la réponse fibrotique. Nos résultats démontrent la présence d'une fraction IgG stimulant une réponse phénotypique dans les cultures de VSMCs. Notamment, d’importantes régulations positive et négative des gènes pro-fibrotiques tgfb1 et tgfb2 respectivement, ont été observées dans les VSMCs exposées à des fractions de ScS-IgG. Les fractions de IgG positives pour l'activation de ERK étaient présentes dans la plupart, mais pas dans tous les échantillons de SSc (68%, 19/28), et moins présentes dans les contrôles 27% (11/3). Bien que, les fractions de SSc-IgG ont pu considérablement immunoprécipiter le PDGFR, l'utilisation d'un inhibiteur spécifique des récepteurs au PDGF (AG1296), n'a pas inhibé l'activation de ERK médiée par les fractions de SSc-IgG. Globalement, nos résultats indiquent la présence d'autoanticorps stimulants avec activité pro-fibrotique dans les sérums des patients ScS. Des travaux sont en cours pour identifier l'entité moléculaire responsable de la réponse d’IgG observée dans les cultures de VSMCs. / Systemic Sclerosis (SSc) is a devastating autoimmune disease of unknown etiology. Immune dysfunction, fibrosis and vasculopathy are the three major features of the disease; however, the interactions between these components are poorly understood. A novel link between autoimmunity and fibrosis has been proposed by the presence of stimulatory autoantibodies to the platelet-derived growth factor receptor (PDGFR) on fibroblasts. These autoantibodies were capable of stimulating reactive oxygen species and subsequent activation of ERK1/2. If the anti-PDGFR autoantibodies are present in the systemic circulation of SSc patients, they will most certainly encounter vascular smooth muscle cells (VSMCs). The latter are known to express the PDGFR and response to PDGF, which is a known phenotypic modulator of VSMCs. The work presented here seeks to readdress the presence of stimulatory anti-PDGFR autoantibodies in serum derived from SSc-patients and to characterize the effects of SSc-IgG on VSMCs by measuring the activation of specific signaling cascades and the induction of genes involved in fibrotic responses. Our results demonstrate the presence of an IgG fraction stimulating a phenotypic response in cultured VSMCs. Notably, a significant up-regulation of the pro-fibrotic gene tgfb1 and a significant down-regulation of the anti-fibrotic gene tgfb2 were observed in VSMC exposed to SSc-IgG fractions. Positive IgG fractions for ERK activation were present in most, but not all, SSc samples (68%, 19/28), and they were less present in controls (27%) (3/11). Although, the SSc-IgG fractions were able to significantly immunoprecipitate the PDGFR, the use of a selective PDGFR inhibitor, AG1296, did not inhibit the activation of ERK mediated by SSc-IgG fractions. Altogether, our findings suggest the presence of stimulatory autoantibodies with profibrotic activity in serum derived form SSc patients. Work is in progress to identify the molecular entity responsible for the IgG response observed in cultured VSMCs.

La polyarthrite rhumatoïde (PR) est une maladie auto-immune qui mène à une inflammation chronique et à une destruction progressive des articulations. Les effecteurs principaux de cette pathologie sont les synoviocytes de type fibroblastique (FLS). Ces derniers utilisent les invadosomes, des structures riches en actine et en métalloprotéases, afin de dégrader la matrice extracellulaire (ECM). Ce phénotype pro-destructif résulte d’une activation des FLS par différents facteurs de croissance, dont le PDGF et le TGF-β. Les récepteurs à activité tyrosine kinase, dont le PDGFR, sont impliqués dans la pathogenèse de plusieurs maladies, incluant le cancer et la PR. Une activation de ces récepteurs peut mener, entre autres, à la survie, à la différenciation et à la prolifération des cellules. L’étude présentée dans ce mémoire montre que parmi les RTK les plus communs, le PDGFR est spécifiquement phosphorylé chez les cellules synoviales de patients atteints de PR, contrairement aux cellules de patients non arthritiques ou atteints d’arthrose. De plus, l’activation du PDGFR résulte en une augmentation de la formation d’invadosomes par les FLS. Nous avons aussi démontré que la formation d’invadosomes par le PDGFR nécessite l’activation de la voie de signalisation PI3K/Akt faisant intervenir les isoformes α et δ de la PI3K. De plus, l'inhibition de l’activation du PDGFR ou la neutralisation du PDGF endogène inhibe la formation des invadosomes et la dégradation de l'ECM par les synoviocytes, ce qui suggère la présence d'une boucle d'activation autocrine impliquant le PDGF. Parmi les isoformes du PDGF, nous avons démontré que le PDGF-B est exprimé de façon significativement plus élevée dans les synoviocytes provenant de patients atteints de PR. Nos données indiquent également une association entre le PDGF et le TGF-β dans la formation des invadosomes. Cette dernière implique la production autocrine de ligands du PDGFR induite par le TGFβ via la signalisation TβR1/Smad et PI3K/Akt. L’inhibition des isoformes de PI3K de classe I indique que le PI3Kα est impliquée de façon sélective dans l'expression de PDGF-B. Ces résultats démontrent que le PDGFR est un RTK nécessaire au phénotype destructeur des cellules synoviales d’arthrite. Ils fournissent aussi des preuves d'une association entre le TGF-β et le PDGFR dans la formation d’invadosomes chez les synoviocytes de patients atteints de la PR. / Abstract : Rheumatoid arthritis (RA) is an autoimmune disease that leads to chronic inflammation and progressive joint destruction. The main effectors of this pathology are fibroblast-like synoviocytes (FLS). They use invadosomes, actin-rich structures that concentrate metalloproteinases to degrade the extracellular matrix (ECM). This pro-destructive phenotype is due to the activation of FLS by various growth factors, including PDGF and TGF-β. Receptor tyrosine kinases, including PDGFR, are involved in the pathogenesis of several diseases, including cancer and RA. Activation of these receptors may lead to cell survival, differentiation and proliferation. The study presented in this thesis shows that among the most common RTKs, PDGFR is specifically phosphorylated in synovial cells of RA patients, unlike cells of non-arthritic or osteoarthritic patients. In addition, activation of PDGFR results in an increase in invadosome formation by FLS. We also shown that formation of invadosome by PDGFR requires the activation of the signaling pathway PI3K/Akt, that specifically involves the α and δ isoforms of PI3K. In addition, inhibition of PDGFR activation or neutralization of endogenous PDGF inhibits the formation of invadosomes and the degradation of the ECM by synoviocytes, suggesting the presence of an autocrine activation loop involving PDGF. Among the PDGF isoforms, we demonstrate that PDGF-B expression is significantly higher in synoviocyte cell lines from RA patients. Our data also indicates an association between PDGF and TGF-β for invadosome formation that involves autocrine production of PDGF-B induced by TGF-β through the Smad/T β R1 and PI3K/Akt pathways. Inhibition of class I PI3K isoforms indicates that PI3K α is selectively involved in the expression of PDGF-B. These results demonstrate that PDGFR is an RTK necessary for the pro-destructive phenotype of RAFLS. They also provide evidence of an association between TGF-β and PDGFR in invadosome formation by synovial cells from RA patients.

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.

Deregulation of platelet-derived growth factor receptor (PDGFR) signaling is a driving event in glioblastoma, promotes tumor progression epithelial to mesenchymal transition (EMT) in multiple cancers, modulates the tumor stroma to facilitate tumorigenesis and reduces tumor uptake of chemotherapeutics. Previous studies identified the 105 kDa isoform of ankyrin3 (Ank105) as a binding partner of the PDGFR signaling machinery and demonstrated that expression of Ank105 promoted PDGFR degradation (Ignatiuk et al., 2006)(Ignatiuk et al., 2006)(Ignatiuk et al., 2006). Receptor tyrosine kinases are targeted for degradation via endocytosis and ubiquitin-dependent trafficking to the lysosome. It was hypothesized that Ank105 promoted the constitutive degradation of the PDGFR and attenuation of PDGFR signaling by facilitating endocytosis of the PDGFR and targeting the PDGFR for lysosomal degradation via an ubiquitin-dependent mechanism. The studies in this thesis characterized the effects of Ank105 expression on PDGFR signaling and protein expression levels, determined the endocytic pathways involved in Ank105-mediated PDGFR degradation and studied the role of ubiquitin binding in Ank105 function. The most robust effect of Ank105 expression on the PDGFR was constitutive degradation as PDGFR protein expression levels in Ank105-expressing cells were significantly reduced compared to NIH 3T3 cells in the absence of PDGF ligand. Low constitutive PDGFR levels resulted in attenuated pro-proliferative AKT and mitogen-activated protein kinase (MAPK) signaling in response to ligand stimulation. To determine the endocytic requirements for Ank105-mediated constitutive PDGFR degradation, a constitutive PDGFR degradation assay was developed and the effects of several small molecule endocytosis inhibitors were evaluated. Additionally, the small molecule endocytosis inhibitors were validated by determining the effects of these inhibitors on low density lipoprotein (LDL) uptake and ligand-induced PDGFR degradation in Ank105-expressing cells. Both LDL uptake and ligand induced PDGFR degradation are known to proceed by a clathrin and dynamin dependent mechanism of endocytosis. In Ank105-expressing cells, both LDL uptake and ligand incuded PDGFR degradation were dependent upon clathrin and dynamin function. Interestingly, constitutive PDGFR degradation in Ank105-expressing cells was not dependent upon CME, but required dynamin activity. Expression of Ank105 may promote clathrin-independent, dynamin-dependent, constitutive endocytosis of the PDGFR. Additionally, acute inhibition of either lysosomal or proteasomal degradation strongly impaired constitutive PDGFR degradation, whereas ligand-induced PDGFR degradation was less sensitive to protein degradation inhibitors, while LDL uptake was unaffected. It was unclear if PDGFR was degraded in the proteasome or if the proteasome was involved in sorting of PDGFR to the lysosome for degradation. Ubiquitination of receptors is required to target them for degradation. Ank105 was assayed for the ability to interact with ubiquitin and ubiquitinated proteins. Interestingly, Ank105 bound ubiquitin in vitro via the spectrin binding domain and co-immunoprecipitated with several ubiquitinated proteins, suggesting a role for Ank105 in the sorting of ubiquitinated proteins for degradation. Furthermore, Ank105 co-immunoprecipitated with a number of high and low molecular weight proteins in the absence of PDGF stimulation. Identification of Ank105 binding partners would provide further insight in the mechanism of Ank105-mediated constitutive PDGFR degradation. In summary, Ank105 promoted the attenuation of PDGFR signaling via alteration of constitutive PDGFR endocytosis and targeting of constitutive PDGFR for degradation, potentially through interaction with ubiquitin and ubiquitinated proteins. Reduction of constitutive PDGFR levels in cancers with PDGFR driver mutations, acquired PDGF responsiveness and stromal expression of PDGFR, could significantly reduce tumor proliferation, tumorigenesis and increase effectiveness of chemotherapeutics.

Overactivity of platelet-derived growth factor receptor (PDGFR) is a frequent event in many types of solid tumors. Therefore, it is of great importance to uncover the mechanisms that regulate PDGF/PDGFR signalling, to develop efficient inhibitors targeting this pathway. The first step of downregulation of PDGFR activity upon ligand binding is internalization; thus we investigated how endocytosis pathways affect PDGFR signaling. We showed that in Ras-transformed fibroblasts, the internalization of PDGFR is shifted from the routine clathrin-dependent endocytosis to macropinocytosis, which results in enhanced PDGFR activity and subsequent downstream signalling, promoting anchorage-independent growth. We were also interested in how intracellular trafficking regulates signalling attenuation of PDGFR. We found that His-domain containing protein tyrosine phosphatase (HD-PTP) positively regulates phosphorylation level of the ubiquitin-ligases c-Cbl and Cbl-b; consistently, silencing of HD-PTP led to a decreased level of PDGFR ubiquitination (paper II). Consequently, internalized PDGFR could not be sorted properly and escaped degradation. This resulted in enhanced activation of phospholipase C γ (PLCγ) and changed kinetics of signal transducer and activator of transcription (STAT) 3 signalling, which further increased colony formation of HD-PTP silenced cells in soft agar, indicating a tumor suppressor role of HD-PTP. Activation of PDGFR leads to stimulation of downstream pathways. We identified Fer kinase as a critical signal transducer downstream of PDGFR in a proteomic screen. We showed that Fer kinase is essential for PDGF-induced STAT3 activation; as a result (paper III), Fer depletion severely blunted the ability of PDGFR signalling to promote anchorage-independent growth in soft agar and delayed tumor initiation in a mouse model. The crosstalk between host and tumor plays a critical role in tumor progression. At present most anti-cancer drugs are targeting tumor cells; we were interested in how targeting tumor host cells affects the efficacy of anti-tumor therapy. We found that selective PDGFRβ inhibition in host cells exerted tumor inhibitory effects on growth and vascularization of tumors with autocrine PDGF signaling, whereas tumors lacking such stimulation show only minor response on tumor growth (paper IV). Meanwhile, we demonstrated that PDGF/PDGFRβ signalling promotes expression of NG2, a marker for pericytes.

Herein we describe the regulatory significance and molecular targeting of novel non-B-DNA secondary structures formed from the PDGFR-Beta core promoter nuclease hypersensitivity element.

Cancer metastasis is a highly complex process and is of great clinical importance since majority of cancer related mortality is associated with metastatic disease rather than primary tumor. The fact that cancer metastasis can develop years or even decades after primary tumor diagnosis, makes this process even more complex and therefore its understanding is of vital importance. Cervical cancer (CxC) is one of the most commonly diagnosed and cause of death among gynecologic cancers worldwide. In this chapter, our aim is to provide a broad overview of risk factors, modes of metastasis and major molecular factors and signaling pathways involved in the progression and metastasis of CxC. The understanding of these factors will enhance the knowledge of CxC pathogenesis and targeting these pathways would help combat against CxC and its metastasis.