Increased local tumor tissue expression of versican in breast cancer patients is predictive of relapse and has a negative impact on survival rates. It is recognized that bone is a common anatomic site of breast cancer metastasis. The C-terminal G3 domain of versican influences local and systemic tumor invasiveness in pre-clinical murine models. However, the mechanism(s) by which G3 influences breast tumor growth and metastasis is not well characterized. We exogenously expressed a G3 construct in mouse breast cancer cell line 66c14, and found that G3 expression enhanced breast cancer cell proliferation and migration, and spontaneous metastasis to bone in an orthotopic model by upregulating the EGFR-mediated signaling pathway. Possessing anti-apoptotic and drug resistant properties, overexpression of versican was accompanied by selective sensitization to several chemotherapeutic agents. The dual roles of G3 in modulating breast cancer cell resistance to chemotherapeutic agents may, in part, explain breast cancer cell resistance to chemotherapy and EGFR therapy. The apoptotic effects of chemotherapeutics depend upon the activation and balance of down stream signals in the EGFR pathway. New knowledge gained by our experiments includes the understanding that GSK-3β (S9P) appears to function as a key check-point in this balance. In addition, versican G3 enhanced breast cancer cell self-renewal in vitro and in vivo. Versican was expressed at high levels in breast cancer mammosphere cells, which contained a high percentage of SP cells. Reduction of versican’s functionality through anti-versican shRNA or knocking out the EGF-like motifs using G3ΔEGF reduced the effect of versican on enhancing mammosphere and colony formation. Versican promoted breast cancer cell self-renew appears to play a role in enhanced chemotherapeutic drug resistance (including Docetaxel, Doxorubicin, and Epirubicin), which relates partly to its upregulated expression of EGFR signaling. Versican enhances breast cancer bone metastasis not only by enhancing tumor cell mobility, invasion, and survival in bone tissues, but also through mechanisms inhibiting osteoblast cell growth and differentiation, affording favorable microenvironments for tumor metastasis.

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Increased local tumor tissue expression of versican in breast cancer patients is predictive of relapse and has a negative impact on survival rates. It is recognized that bone is a common anatomic site of breast cancer metastasis. The C-terminal G3 domain of versican influences local and systemic tumor invasiveness in pre-clinical murine models. However, the mechanism(s) by which G3 influences breast tumor growth and metastasis is not well characterized. We exogenously expressed a G3 construct in mouse breast cancer cell line 66c14, and found that G3 expression enhanced breast cancer cell proliferation and migration, and spontaneous metastasis to bone in an orthotopic model by upregulating the EGFR-mediated signaling pathway. Possessing anti-apoptotic and drug resistant properties, overexpression of versican was accompanied by selective sensitization to several chemotherapeutic agents. The dual roles of G3 in modulating breast cancer cell resistance to chemotherapeutic agents may, in part, explain breast cancer cell resistance to chemotherapy and EGFR therapy. The apoptotic effects of chemotherapeutics depend upon the activation and balance of down stream signals in the EGFR pathway. New knowledge gained by our experiments includes the understanding that GSK-3β (S9P) appears to function as a key check-point in this balance. In addition, versican G3 enhanced breast cancer cell self-renewal in vitro and in vivo. Versican was expressed at high levels in breast cancer mammosphere cells, which contained a high percentage of SP cells. Reduction of versican’s functionality through anti-versican shRNA or knocking out the EGF-like motifs using G3ΔEGF reduced the effect of versican on enhancing mammosphere and colony formation. Versican promoted breast cancer cell self-renew appears to play a role in enhanced chemotherapeutic drug resistance (including Docetaxel, Doxorubicin, and Epirubicin), which relates partly to its upregulated expression of EGFR signaling. Versican enhances breast cancer bone metastasis not only by enhancing tumor cell mobility, invasion, and survival in bone tissues, but also through mechanisms inhibiting osteoblast cell growth and differentiation, affording favorable microenvironments for tumor metastasis.

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The Streptococcus Anginosus Group (SAG) is a group of Gram-positive cocci which require carbon dioxide to grow. They are commensal members of the healthy upper respiratory, gastrointestinal and female urogenital tract; however, they are most commonly known as major pathogens in brain and liver abscesses, forming both mono- and polymicrobial infections. The Streptococcus invasion locus (sil), first identified as a virulence factor in Group A Streptococcus (GAS), has recently been identified in the SAG. The sil locus in GAS is a two component quorum-sensing system composed of three operons: silAB, coding for a two component system; silE/D/CR, coding for an ABC transporter and a signal peptide, and silC, which overlaps silCR on the opposite strand. The presence of exogenous SilCR activates SilA, which in turn upregulates the transcription of the silE/D/CR operon. In the SAG, however, silCR and silED have distinct promoters, and the SAG sil system lacks the silC gene. In this study, I examined the transcriptional dynamics of the sil system in S. intermedius B196. I determined that SilA is the major regulator of the genes in the sil system, being one of the first genes of the system to be expressed, and likely upregulates its own transcription. I also found evidence suggesting that, despite having its own promoter, silCR transcription may still be driven by the silED promoter. I also found evidence that suggests silED may be responsible for the export and/or processing of bacteriocins targeting closely related species or strains. / Thesis / Master of Science (MSc) / The Streptococcus are a group of bacteria known for causing diseases ranging from strep throat to flesh-eating disease; however, many species of Streptococcus are usually non-pathogenic, and live in our bodies without causing harm. One group of these bacteria, the Streptococcus Anginosus Group (SAG), is commonly found in our mouths and gut and usually cause no harm; however, in some cases it can cause infections. How these organisms switch from being nonpathogenic to pathogenic is unknown, but recently a gene network that appears to play a role in infection, the Streptococcus invasion locus (sil), was identified in the SAG. This gene network senses the signals released by other bacteria with the network, and only turns on when enough signal is present. The goal of this study is to examine how the system works in the SAG in order to determine how these bacteria coordinate sil gene expression.

Les glioblastomes sont des tumeurs fatales du fait de leur agressivité et du manque de traitements efficaces. La prolifération accrue, le caractère invasif et la résistance à la mort cellulaire leur confèrent une croissance rapide et une invasion du parenchyme cérébral environnant, à l’origine de leur systématique récidive. Exprimée par la composante tumorale en hypoxie mais également par la composante vasculaire, l’AM participe de façon autocrine et paracrine au développement des glioblastomes en favorisant la croissance des cellules tumorales et l’angiogenèse tumorale.Il a été montré que des anticorps polyclonaux dirigés contre les récepteurs de l’AM inhibent in vitro la croissance, la migration et la formation de pseudo-capillaires des cellules endothéliales, suggérant une neutralisation par ces anticorps de certaines étapes de l’angiogenèse. De même, il a été montré in vivo que ces anticorps inhibent la croissance tumorale en supprimant l’angiogenèse et la croissance des cellules tumorales suggérant ainsi que les récepteurs de l’AM constitueraient une bonne cible thérapeutique. Des anticorps capables de reconnaître et neutraliser à la fois l’AM, les CLR, RAMP2 et RAMP3 agissant de la même manière sur la croissance tumorale et l’angiogenèse représenteraient un bénéfice thérapeutique majeur. Des anticorps dirigés contre un peptide chimérique constitué de l’enchainement de séquences peptidiques des protéines CLR, RAMP2, RAMP3 et du peptide AM sont en cours. Le traitement par ces anticorps diminue la croissance des cellules tumorales ainsi que leurs migration et invasion. Ces résultats très encourageants nous permettent pour le moment de valider la faisabilité du concept d’anticorps développés à partir d’un peptide chimérique pour neutraliser le système AM/AMR dans le but d’envisager dans le futur une application thérapeutique. / Glioblastoma are fatal tumors because of their aggressiveness and lack of effective treatments. The increased proliferation, the invasiveness and resistance to cell death gives them a rapid growth and invasion of brain parenchyma surrounding the origin of their systematic recurrence. Expressed by the tumoral component in hypoxia but also by the vascular component, the AM participate by an autocrine and paracrine way, the development of glioblastoma by promoting tumor ell growth and tumor angiogenesis.It was shown that polyclonal antibodies directed against the AM receptor inhibit in vitro growth, migration and the formation of pseudo-capillary of endothelial cells, suggesting neutralization by theses antibodies in certain stages of angiogenesis. Similarly, it has been shown in vivo that these antibodies inhibit tumor growth by suppressing angiogenesis and tumor cell growth, suggesting that the AM receptor would be a good therapeutic target. Antibodies that recognized and neutralized both the AM, the CLR, RAMP2 and RAMP3 acting the same way on tumor growth and angiogenesis represent a major therapeutic benefit. Antibodies against a chimeric peptide consisting of peptide sequence of CLR, RAMP2, RAMP3 and AM peptide are in progress. Treatment with these antibodies decreases the growth of tumor cells, their migration and invasion. These encouraging results allow us the time to validate the feasibility of the concept of antibodies developed from a chimeric peptide to neutralize the AM/AMR system in order to consider in the future therapeutic application.

Les modifications post-transrationnelles avec les molécules ubiquitine et ubiquitine-like sont des mécanismes essentiels pour la régulation des fonctions protéiques et des voies de signalisations. Les ubiquitine-like molécules comme par exemple NEDD8 jouent un rôle majeur dans la fonction des protéines et sont impliqués dans des maladies humaines comme par exemple les maladies neuro dégénératives. NEDD8 est essentiel pour la survie, la croissance et le développement. C’est pour ceci, l’élucidation de leurs mécanismes d’action est très importante.Dans notre laboratoire, nous nous intéressons à l’ubiquitine-like molécule NEDD8. Le mécanisme de modification avec cette protéine est dit NEDDylation et il nécessite une intervention enzymatique médiées par E1, E2 et E3. Le rôle majeur de NEDD8 est la régulation de l’activité des Cullins, des E3 ubiquitine ligases. Les Cullins sont des scaffolds dont le rôle est de faciliter la dégradation des substrats en assemblant tout le complex enzymatique. La dérégulation de l’activité des Cullins a été montrée de contribuer à l’oncogenèse suite à l’accumulation des oncoprotéines ou suite à la dégradation excessive des suppresseurs de tumeurs. Récemment, la voie de NEDDylation a été ciblée pour des interventions thérapeutiques ; MLN4924 (Provenodistat) (Millenium Pharmaceuticals) bloque la voie de NEDDylation en formant une liaison covalente avec l’enzyme E1 de la voie de NEDDylation. L’identification des mécanismes de régulation des fonctions protéiques par NEDD8 fournira plus d’informations sur le mécanisme d’action de NEDD8 d’un point de vu moléculaire, ce qui permettra l’identification de nouvelles cibles pour de nouvelles interventions thérapeutiques. Dans 50% des tumeurs, le gène p53 est muté. Dans les 50% qui restent, le gène p53 est intact mais la protéine est non fonctionnelles dû à des dérégulations des voies de signalisations. Nous avons trouvé que l’inhibition de la NEDDylation via MLN4924 bloque l’invasion des cellules de mélanomes métastatiques. D’un point de vu moléculaire, MLN4924 provoque la dégradation de Mdmx, l’homologue de Mdm2, tous les deux sont des majeurs régulateurs négatifs de p53. Ceci est dû à la formation d’un complexe entre Mdmx, Mdm2 et Cullin4A inactif. On a démontré que la dégradation de Mdmx est essentielle pour l’activation de la voie RhoA/ROCK capable de bloquer l’invasion tout en modulant le cytosquelette d’actine.De plus, Mdmx a été identifié en tant que régulateur de l’activité de RhoA. Mdmx se lie à la forme inactive de RhoA (GDP-RhoA) et contrôle son activation. Durant les travaux de recherche, nous avons aussi essayé de développer une méthode nous permettant d’utiliser MLN4924 d’une façon spécifique dans le but de minimiser les effets secondaires normalement accompagnant les traitements de chimiothérapies. MLN4924 comme tout agent chimio-thérapeutique, est toxique envers les cellules normales. Dans le but d’utiliser MLN4924 d’une manière spécifique nous permettant de cibler uniquement les cellules cancéreuses sans affecter les cellules normales, nous avons testé cette molécule dans une approche basée sur le statut de p53 appelé « cyclotherapie ».Nos résultats montrent que l’activation de p53 avec des concentrations minimes d’Actinomycine D, induit un arrêt du cycle cellulaire dans la phase G1, protégeant ainsi les cellules de l’effet toxique de MLN4924. Ceci n’est pas le cas pour les cellules cancéreuses dont p53 est déficient ou sans p53. Celles-ci progressent en phase S où elles seront éliminées par apoptose. Cette combinaison a été aussi testée in vivo, plus spécifiquement en utilisant les zebrafish comme model, ce qui nous a permis de confirmer l’effet protecteur de l’Actinomycine D contre l’effet toxique de MLN4924. Nos résultats constituent une nouvelle possibilité de combinaison de MLN4924 basé sur le statut de p53. / Post-translational modifications with ubiquitin and ubiquitin-like molecules (ubls) are essential regulatory mechanisms of protein function and signalling networks. Ubiquitin-like molecules, such as NEDD8 have emerged as major mechanisms of protein function regulation and implicated in human diseases including cancer and neurodegeneration. It is essential for viability, growth and development. Therefore, the elucidation of their mechanism of action is critical. In our lab, research is focused on NEDD8. The process called NEDDylation, involves the three enzymatic activities (E1, E2 and E3). The well-known role of NEDD8 is the regulation of the activity of E3 ubiquitin ligases called Cullin Ring Ligases through modification of Cullins. Cullins are scaffolds to facilitate E3 ligase complex assembly and mediated substrate degradation. Deregulation of Cullins activity has been shown to contribute to oncogenesis through the accumulation of oncoproteins or excessive degradation of tumour suppressors. Recently, the NEDDylation pathway has been targeted for therapeutic intervention; MLN4924 (Pevonedistat) (Millenium Parmaceuticals) forms a covalent adduct with the NEDD8 E1 enzyme and blocks the NEDDylation cascade. Identification of mechanisms of protein function regulation by NEDDylation will provide the molecular basis for the action of the NEDD8 inhibitors in clinic and potentially identify novel targets for therapeutic intervention. In 50% of tumours the TP53 is mutant. In cancer cells where TP53 is WT, upstream or downstream signalling pathways are deregulated. We found that inhibition of NEDDylation by MLN4924 dramatically blocks invasion of metastatic melanoma cells. At the molecular level, MLN4924 induces proteasomal degradation of the Mdmx oncogene product, a key negative regulator of the p53 tumor suppressor. Mechanistically, MLN4924 induces the complex formation of Mdmx with Mdm2 and inactive Cullin4A that promote Mdmx degradation. We found that the degradation of Mdmx is required for the activation of the RhoA/ROCK pathway which blocks cell invasion through changes in the cell cytoskeleton. Interestingly, Mdmx was identified as regulator of RhoA activity through binding with inactive RhoA (GDP- bound RhoA). Another question was addressed which is how to use MLN4924 in a specific manner, thus reducing side effects usually resulting from chemo treatments. MLN4924 as all chemo-agents, is toxic towards healthy cells. In order to use MLN4924 in a specific manner targeting cancer cells specifically leaving healthy cells unharmed, a p53 based cyclotherapy was tested. Our results show that low activation of wild type p53 by low doses of Actinomycin D, causes a G1 cell cycle arrest and protects normal cells from MLN4924 treatment leaving cancerous cells with mutant p53 or no p53 to progress to S-phase and selectively commit apoptosis upon MLN4924 treatment. Our protocol was tested in vivo, in zebrafish model system confirming the protective effect of LDACTD against the cytotoxic effect of MLN4924. Our results provide a possible combination therapy for MLN4924 based on the p53 status.