Le récepteur de la progestérone (PR) est un acteur majeur du développement de la glande mammaire. Dans les cellules épithéliales normales, PR est exprimé sous deux isoformes PRA (94 kDa) et PRB (116 kDa) de façon équimolaire. Il est établi que celles-ci ont un impact important sur le développement des cancers du sein, mais leurs rôles dans l’évolution métastatique reste très mal connus. Le ratio d’expression PRA/PRB étant souvent déséquilibré dans les tumeurs mammaires, nous avons analysé le turnover des deux isoformes. Nous avons démontré que PRA et PRB sont les cibles de modifications post-traductionnelles dirigées par les MAPK qui tendent à stabiliser l’une ou l’autre isoforme de manière sélective. Ainsi, Erk1/2 (p42/44) inhibe la dégradation de PRB tandis que la p38 stabilise PRA. Il en résulte que le ratio PRA/PRB varie de façon importante en fonction des signalisations extracellulaires impliquant les facteurs de croissance et les cytokines inflammatoires souvent exacerbés dans les cancers. Pour mieux étudier les effets différentiels de PRA et PRB, nous avons établi un modèle cellulaire original exprimant de manière bi-inductible l’une ou l’autre isoforme de PR, à partir de la lignée cellulaire MDA-MB 231 provenant d’une métastase de cancer du sein. En étudiant les variations induites par PRA ou PRB sur le transcriptome de ces cellules, nous avons identifié les gènes cibles spécifiques de ces isoformes. Parmi-eux se trouvent de nombreux gènes impliqués dans les cancers, notamment agissant sur la prolifération, la survie et la motilité cellulaires comme uPA et PAI-1. De plus, en analysant la migration cellulaire, nous avons mis en évidence un effet pro-migratoire de PRB particulièrement important en absence d’hormone. En recherchant la cause de ces effets, nous avons découvert que PRB était colocalisé et interagissait avec la kinase d’adhésion focale (FAK) qu’il active au niveau des points d’adhésion focaux. Ces travaux soulignent l’incidence du ratio PRA/PRB et du statut du ligand sur les métastases du cancer du sein, aussi bien au niveau de la sélectivité transcriptionnelle que celui des régulations non génomiques impactant la migration cellulaire. Nous suggérons la possibilité de cibler les tumeurs mammaires par des antagonistes sélectifs de PR et des inhibiteurs des voies de signalisation des MAPK ou de FAK. / Progesterone receptor (PR) is a major actor of mammary gland development. PR is equally expressed as two main isoforms PRA (94 kDa) and PRB (116kDa) in the mammary gland epithelium. However, breast cancer progression has been associated with abnormalities of their expression ratio through undefined mechanisms. In this study, using a stably transfected cell line, we showed that PRA and PRB stabilizations are differentially regulated by Erk1/2 (p42/44) and p38 MAPKs respectively, leading to strongly influence PRA/PRB ratio. These results highlight the impact of growth factors and inflammatory cytokines on PRA/PRB imbalance in cancer cells. To study the differential effect of PR isoforms, we established an original bi-inducible cell line expressing either PRA and/or PRB. By analyzing variations induced by PRA and PRB on such cell transcriptomes, we identified the isoform-specific targets genes by DNA microarrays. Most of them are implicated in cancers, notably acting on cell proliferation, survival and motility. Furthermore, focusing our studies on cell migration, we showed that PRB acts as a pro-migratory factor particularly powerful in the absence of ligand. We discovered that PRB colocalized and interacted with the focal adhesion kinase (FAK) that was activated in focal adhesion points. Our results highlight the impacts of both PRA/PRB ratio and ligand status on metastatic evolution, in the contexts of transcriptional regulation as well as non-genomic events. We suggest the possibility to target mammary tumors by PR-selective antagonists and/or inhibitors of MAPK and FAK signalings.

The most common malignant glioma, glioblastoma multiforme (GBM), remains an intractable tumor despite advances in therapy. Its proclivity to infiltrate surrounding brain tissue contributes greatly to its treatment failure and the grim prognosis of patients. Radiation is a staple in modern therapeutic regimens, though cells surviving radiation become more aggressive and invasive. Consequently, it is imperative to define further the cellular mechanisms that control GBM invasion and identify promising novel therapeutic targets. Melanoma differentiation associated gene-9 (MDA-9/Syntenin) is a highly conserved PDZ domain-containing scaffolding protein that promotes invasion and metastasis in human melanoma models. We show that MDA-9/Syntenin is robustly expressed in GBM cell lines and patient samples, and expression increases by tumor grade. These findings are confirmed through database analysis, which revealed MDA-9/Syntenin expression correlates with shorter survival times and patient tumors high in MDA-9/Syntenin have a worse prognosis when undergoing radiotherapy. Modulating MDA-9/Syntenin levels produced changes in invasion, angiogenesis, and signaling, indicating MDA-9/Syntenin enhances glioma pathogenesis. Overexpression of MDA-9/Syntenin enhances invasion, while knockdown inhibits invasion, migration, and anchorage-independent growth in soft agar. MDA-9/Syntenin increases activation of c-Src, P38MAPK, and NF-kB, leading to elevated MMP2 expression and IL-8 secretion. Through an orthotopic tumor model, we show that shmda-9 tumor cells formed smaller tumors and had a less invasive phenotype in vivo. Knockdown of MDA-9/Syntenin radiosensitizes GBM cells and significantly reduces post-radiation invasion gains through abrogation of radiation-induced Src and EphA2 activity. In efforts to pharmacologically inhibit MDA-9/Syntenin, we describe the effects of a novel small molecule, PDZ1i, which targets the PDZ1 domain of MDA-9/Syntenin and successfully reduces invasion gains in GBM cells following radiation. While it does not effect astrocyte radiosensitivity, PDZ1i radiosensitizes GBM cells. PDZ1i inhibits crucial GBM signaling including FAK and mutant EGFR, EGFRvIII, and can negate gains in secreted proteases, such as MMP2 and MMP9, following radiation. In a model of glioma, PDZ1i treatment combined with radiation results in less invasive tumors and extends survival. Our findings indicate that MDA-9/Syntenin is a novel and important mediator of GBM pathogenesis, and further identify it as a targetable protein that enhances radiotherapy for treatment in glioma.

Résumé confidentiel / Confidential abstract

The goal of this study was to identify novel drug repurposing opportunities in cancer by utilizing the off-target profiles of clinically relevant kinase inhibitors. This was based on the observation that the global target profiles of compounds are largely ignored and that many compounds have activity that cannot be explained by their cognate target alone. Additionally, by utilizing clinically relevant compounds, any results would hold a high potential for eventual clinical implementation. We utilized a systems pharmacology approach utilizing cell viability-based drug screening to identify compounds with beneficial off-target activity and then using chemical and phosphoproteomics in order to elucidate the mechanisms of action of these compounds. We found that tivantinib has off-target activity in NSCLC cells through inhibition of GSK3. Based on tivantinib’s ability to inhibit GSK3, we hypothesized that tivantinib would therefore have activity in acute myeloid leukemia (AML). We found that tivantinib had potent activity in AML through inhibition of GSK3. We also identified a highly synergistic combination with ABT-199 by drug synergy screening which was effective in HL60 cells and patient derived AML cells. We also found that the anaplastic lymphoma kinase (ALK) inhibitor, ceritinib, had activity across several ALK-negative lung cancer cell lines. We utilized integrated functional proteomics to identify the new targets and network-wide signaling effects. Combining pharmacological inhibitors and RNA interference revealed a polypharmacology mechanism involving the noncanonical targets IGF1R, FAK1, RSK1 and RSK2. Mutating the downstream signaling hub YB1 protected cells from ceritinib. Consistent with YB1 signaling being known to cause taxol resistance, combination of ceritinib with paclitaxel displayed strong synergy, particularly in cells expressing high FAK autophosphorylation, which we show to be prevalent in lung cancer. Together, we present a systems chemical biology platform for elucidating multikinase inhibitor mechanisms, synergistic drug combinations, mechanistic biomarker candidates and identifying novel drug repurposing opportunities.

<p>The proline-rich tyrosine kinase (Pyk2) together with focal adhesion kinase (FAK) define a family of non-receptor protein tyrosine kinases that are regulated by diverse stimuli. Activation of Pyk2 has been implicated in multiple signaling events, including modulation of ion channels, activation of MAP kinase cascades and apoptotic cell death. This thesis investigates the role of Pyk2 in the regulation of mitogenic signals and cell cytoskeleton.</p><p>We identified a hematopoietic isoform of Pyk2 (designated Pyk2-H)that is generated by alternative RNA splicing and is mainly expressed in thymocytes, B cells and natural killer cells. In addition, we demonstrated that engagement of antigen receptors in lymphocytes leads to rapid tyrosine phosphorylation of Pyk2-H suggesting a potential role in host immune responses. These findings were corroborated by defects in B cell-mediated immune responses of Pyk2-/- mice. </p><p>Several reports have previously indicated that Pyk2 acts as an upstream regulator of ERK and JNK MAP kinase cascades in response to numerous extracellular signals. Which MAP kinase pathway is activated by Pyk2 depends on arrays of effector proteins associated with Pyk2. We proposed a model where the formation of Pyk2-Src complexes results in phosphorylation of Shc, p130Cas and Pyk2. This creates binding sites for the SH2 domains of adaptor proteins Grb2 and Crk, which in turn recruit exchange factors for Ras and Rho GTPases that specifically activate ERK or JNK.</p><p>Integration of signaling pathways initiated by receptor tyrosine kinases and integrins is essential for growth factor-mediated biological responses. We described neuronal cellular models where activation of both growth factor receptors and integrins is required for neurite outgrowth. In these cells, Pyk2 and FAK associate with integrin-linked complexes containing EGF receptors via their C- and N-terminal domains. Inhibition of Pyk2/FAK functions was sufficient to block neurite outgrowth and effectors of the C-terminal domain of Pyk2/FAK, including paxillin, were shown to regulate neurite outgrowth independently of ERK/MAP kinase in these cells. We thus proposed that Pyk2 and FAK play important roles in signal integration proximal to the integrin-growth factor receptor complexes.</p>

The proline-rich tyrosine kinase (Pyk2) together with focal adhesion kinase (FAK) define a family of non-receptor protein tyrosine kinases that are regulated by diverse stimuli. Activation of Pyk2 has been implicated in multiple signaling events, including modulation of ion channels, activation of MAP kinase cascades and apoptotic cell death. This thesis investigates the role of Pyk2 in the regulation of mitogenic signals and cell cytoskeleton. We identified a hematopoietic isoform of Pyk2 (designated Pyk2-H)that is generated by alternative RNA splicing and is mainly expressed in thymocytes, B cells and natural killer cells. In addition, we demonstrated that engagement of antigen receptors in lymphocytes leads to rapid tyrosine phosphorylation of Pyk2-H suggesting a potential role in host immune responses. These findings were corroborated by defects in B cell-mediated immune responses of Pyk2-/- mice. Several reports have previously indicated that Pyk2 acts as an upstream regulator of ERK and JNK MAP kinase cascades in response to numerous extracellular signals. Which MAP kinase pathway is activated by Pyk2 depends on arrays of effector proteins associated with Pyk2. We proposed a model where the formation of Pyk2-Src complexes results in phosphorylation of Shc, p130Cas and Pyk2. This creates binding sites for the SH2 domains of adaptor proteins Grb2 and Crk, which in turn recruit exchange factors for Ras and Rho GTPases that specifically activate ERK or JNK. Integration of signaling pathways initiated by receptor tyrosine kinases and integrins is essential for growth factor-mediated biological responses. We described neuronal cellular models where activation of both growth factor receptors and integrins is required for neurite outgrowth. In these cells, Pyk2 and FAK associate with integrin-linked complexes containing EGF receptors via their C- and N-terminal domains. Inhibition of Pyk2/FAK functions was sufficient to block neurite outgrowth and effectors of the C-terminal domain of Pyk2/FAK, including paxillin, were shown to regulate neurite outgrowth independently of ERK/MAP kinase in these cells. We thus proposed that Pyk2 and FAK play important roles in signal integration proximal to the integrin-growth factor receptor complexes.

Η FAK, οι MAPKs και ο μεταγραφικός παράγοντας Elk-1 έχει αναφερθεί ότι εμπλέκονται στις ίδιες κυτταρικές διαδικασίες, παρ’ όλα αυτά, η άμεση ή έμμεση αλληλεπίδρασή τους και οι ενδεχόμενες λειτουργίες τους δεν έχουν ακόμα τεκμηριωθεί. Σκοπός αυτής της διατριβής ήταν η περαιτέρω διερεύνηση των σηματοδοτικών μονοπατιών που ρυθμίζουν τη διαδικασία της κυτταροφαγίας και ειδικότερα την αποκάλυψη της φυσικής διασύνδεσης των παραπάνω σηματοδοτικών μορίων. Αρχικά, μελετήσαμε την υποκυτταρική κατανομή των FAK, ERK και Elk-1 στα αιμοκύτταρα της μύγας της Μεσογείου, παρουσία ή απουσία E. coli. Πειράματα ανοσοφθορισμού έδειξαν ότι οι FAK, MAPΚs και Elk-1 κατανέμονται ομοιόμορφα σε όλο το κύτταρο, όμως παρουσία E. coli, φωσφορυλιώνονται και σταδιακά εισέρχονται στον πυρήνα. Στη συνέχεια, με συνεστιακή μικροσκοπία δείχθηκε ότι παρουσία E. coli, οι FAK και ERK φωσφορυλιώνονται και συνεντοπίζονται στην περιφέρεια του κυττάρου. Βιοχημική προσέγγιση επιβεβαίωσε ότι μόνο οι φωσφορυλιωμένες FAK και ERK συμπλοκοποιούνται. Ακολούθως, η φυσική διασύνδεση της FAK και του Elk-1 διερευνήθηκε με ανοσοσυγκατακρήμνιση, αντίστροφη ανοσοσυγκατακρήμνιση, ανάλυση κατά Western και ανοσοφθορισμό. Από τα πειράματα αυτά φάνηκε συμπλοκοποίηση μεταξύ του Elk-1 ή του pSer383Elk-1 και της FAK όχι όμως και της pTyr397FAK. Το σύμπλοκο FAK/Elk-1 εντοπίζεται, κυρίως, στον πυρήνα. Τέλος, πειράματα συνεστιακής μικροσκοπίας έδειξαν ότι η ERK συν-εντοπίζεται με τον Elk-1 σε μικρό βαθμό, όμως ανοσοσυγκατακρήμνιση, αντίστροφη ανοσοσυγκατακρήμνιση και ανάλυση κατά Western δεν αποκάλυψαν κάποια φυσική διασύνδεση μεταξύ της ERK και του Elk-1. / Focal adhesion kinase (FAK), mitogen-activated protein kinases (MAPKs) and the nuclear transcription factor Elk-1 have been reported to be implicated in the same cellular processes, however, their direct or indirect interaction and potential function(s) has not been documented. The goal of this study was to explore further the signaling pathways that regulate the process of phagocytosis and specifically to show the physical association of the above signaling molecules. Initially, we explored the subcellular distribution of FAK, ERK and Elk-1 in medfly haemocytes, treated in the presence or absence of E. coli. Immunofluorescence analysis demonstrated that FAK, MAP kinases and Elk-1 appear to localize throughout the cytoplasm but in the presence of E. coli they are phosphorylated and gradually imported in the nucleus. Furthermore, confocal analysis showed that in the presence of E. coli, FAK and ERK are phosphorylated and co-localized in the periphery of the cell. Biochemical approaches confirmed that only phosphorylated FAK and ERK are physical associated. Moreover, the physical association of FAK and Elk-1 was explored, using co-immunoprecipitation, reciprocal co-immunoprecipitation, Western blot and immunofluorescence analysis. These experiments revealed an association between Elk-1 or pSer383Elk-1 and FAK but not with pTyr397FAK. The FAK/Elk-1 complex is found, mainly, in the nucleus. Finally, confocal analysis demonstrated that ERK co-localizes with Elk-1 at a low level but co-immunoprecipitation, reciprocal co-immuno-precipitation and Western blot did not reveal a physical association between ERK and Elk-1.

The ability to adhere to the extracellular matrix (ECM) is critical for numerous cell types and tissues including epithelia and muscle. Cell-ECM adhesion is primarily mediated by integrins which provide a direct link between the ECM and the actin cytoskeleton. Integrin adhesions are frequently associated with a core of 60 different proteins (integrin-associated proteins, IAPs). Integrins are required for muscle attachment and in Drosophila, loss of integrins and several IAPs results in embryonic lethality and muscle detachment. However, the IAPs FAK, RSU1, tensin, vinculin and zyxin are not required for viability or embryonic muscle attachment. Furthermore, FAK, RSU1, tensin and vinculin have been observed to localise to muscle attachment sites in Drosophila, indicating that they have some function in muscle attachment. Unlike FAK, RSU1, tensin and vinculin, it was not previously known whether zyxin is expressed in Drosophila muscles. To test this, I generated a genomic zyxin-GFP construct that should contain most of the endogenous zyxin promotor. The genomic zyxin-GFP construct was not observed at muscle attachment sites, suggesting that it is not normally expressed in muscle. I wished to know whether FAK, RSU1, tensin and vinculin are required for muscle function. Various behavioural assays were employed to test for muscle function in larvae and adult flies. The results suggest that larval muscle function was normal in flies lacking these IAPs, but that adult muscle function might be impaired, although it proved difficult to demonstrate a clear functional defect. I then tested whether the IAPs FAK, RSU1, tensin and vinculin are required for normal morphology of adult muscles, focusing on the adult indirect flight muscles (IFMs). The IFMs are fibrillar muscles which attach to the cuticle via specialised epithelial cells known as tendon cells. At the end of the myofibril, where the myofibril attaches to the tendon cell, is a dense region of actin and IAPs known as the modified terminal Z-band (MTZ). I have found that the MTZ is not a homogenous zone of proteins, but is instead organised into at least three distinct layers. Because of the similarity between the structure of the MTZ with that of a hand, I refer to the layers as ‘fingers’, ‘palm’ and ‘wrist’. I discovered that the IAPs FAK, RSU1, tensin and vinculin are each required for the proper structure of the MTZ in unique ways. The fingers were elongated in IFMs lacking FAK, RSU1, tensin or vinculin, while the palm was disrupted in IFMs lacking RSU1, tensin or vinculin. Finally, I was intrigued by the enrichment of the actin-binding protein filamin/Cheerio in the palm and wished to know if it is required for palm function. Deletion of the C-terminus of filamin/Cheerio resulted in a reduction in palm length. Filamin/Cheerio is a mechanosensitive protein which exists in a closed and open conformation. I found that filamin/Cheerio must be open in order to help form a normal palm. Furthermore, vinculin is required to convert filamin/Cheerio from and closed to an open filamin/Cheerio state so that it can perform its function in the palm.

Breast cancer is the most common cancer among Canadian women and 14-20% will develop lethal metastases within 5 years. A potential novel therapeutic target is Focal Adhesion Kinase (FAK), a cytoplasmic tyrosine kinase. FAK’s expression is inversely correlated with survival and is known to regulate cell migration, proliferation and invasion. While tyrosine kinase inhibitors are historically ineffective as single agents, they are commonly used as part of combination therapies. Therefore, given its central role in tumor cell biology and cell signaling, we hypothesized that inhibiting FAK in combination with pharmacological agents commonly used to treat metastatic breast cancer patients will result in enhanced anti-tumor activity. We combined a commercial FAK inhibitor (PF-562271) with a range of chemotherapeutic agents commonly used to treat metastatic breast cancer and searched for synergistic partners. Only DNA topoisomerase inhibitors showed potential to synergistically reduce cell viability when paired with low doses of the FAK inhibitor. However, the combination does not induce an increase in cell death or apoptosis. It was then discovered that both agents in isolation and in combination produce increased levels of ROS, a toxic metabolite. This, along with other more preliminary data, provides clues for a novel proposed mechanism of action for this interaction.

Stroke increases neurogenesis (birth of new neurons) through upregulation of ciliary neurotrophic factor (CNTF), a potent neurogenic cytokine made almost exclusively in the central nervous system. Previous study found that CNTF is induced and needed to stimulate neurogenesis in the subventricular zone (SVZ) of mouse brain in a stroke model. CNTF also has a neuroprotective function. Focal adhesion kinase (FAK), protein tyrosine kinase 2, is ubiquitously expressed in various cell types and mediates cell adhesion and migration. We previously discovered that systemic inhibition of FAK upregulates CNTF expression in the SVZ, making FAK a pharmacological target to increase CNTF to promote neurogenesis and neuroprotection after stroke. This study examined whether systemic FAK inhibitor treatment after stroke regulates SVZ neurogenesis and neuroprotection using a middle cerebral artery occlusion (MCAO) to induce a stroke in adult male C57BL/6 mice. A filament was inserted in the external carotid artery and then fed through the carotid bifurcation into the internal carotid artery to the base of the middle cerebral artery. After 30 minutes of occlusion, the filament was removed to restore blood flow. Mice were randomly assigned to receive 3 daily doses of saline or FAK inhibitor (FAK14, i.p., 3 mg/kg) and treatment started at 6 hours, 12 days, or 58 days after MCAO. Because CNTF has a neuroprotective function, the amount of tissue damage was analyzed to compare treatment groups. The neuroprotective role of FAK14 was examined by measuring MCAO-induced infarction. The infarct size was measured using the absence of NeuN (neuronal cell marker) and GFAP (activated astrocytes) and presence of CD68 (activated microglia). FAK14 given at 6 hours post-stroke reduced the infarct size to 38% of the uninjured side of the brain compared to 46% with saline. Proliferating cells were labeled by injecting bromodeoxyuridine (BrdU, 50 mg/kg), the mice were processed 2 h after the last BrdU injection, and proliferated cells in the SVZ were counted with unbiased stereology. There were no significant differences in the total numbers of BrdU+ cells between saline and FAK14 at 3, 14 and 60 days. Future studies are needed to confirm the levels of CNTF at the various times of treatment. If there is no difference in CNTF expression or increased expression of counteracting cytokines, no difference in neurogenesis between groups would be expected. The neuroprotective effect of FAK14 during the acute phase following injury could provide novel pharmacological options to stroke patients extending the current therapeutic treatment window.