La fonction majeure des cellules musculaires lisses (CML) est la contraction. Dans certaines situations pathologiques, une dérégulation des CMLs conduit à une hyperréactivité et à leur dédifférenciation menant à un remodelage tissulaire. La protéine Rac1 régule in vitro des fonctions essentielles aux CMLs. L’objectif de ma thèse a été de déterminer le rôle de Rac1 in vivo dans les CMLs pulmonaires (bronchiques et vasculaires) et son importance dans le développement de pathologies. La mise en place de modèles expérimentaux m’a permis de mettre en évidence une hyperactivité de Rac1 dans les CMLs bronchiques de souris asthmatiques et dans les CMLs d’artères pulmonaires (AP) de souris atteintes d’hypertension artérielle pulmonaire (HTAP). Par des approches in vivo et in vitro, j’ai démontré que Rac1 joue un rôle essentiel dans la contraction et la prolifération des CMLs bronchiques. A l’inverse, mes résultats démontrent que Rac1 dans les CMLs vasculaires pulmonaires est impliquée dans la production d’espèces réactives de l’oxygène induisant la prolifération des CMLs et le remodelage des AP associé à l’HTAP. Mes résultats démontrent un rôle différent de Rac1 en fonction de l’origine des CMLs mais identifient Rac1 comme une cible thérapeutique pour les pathologies associées à une hyperréactivité et/ou à un remodelage des CMLs. Un screening in silico, des tests in vitro et ex vivo, nous ont permis d’identifier un inhibiteur de Rac1 : la molécule A41. La nébulisation de l’A41 prévient l’hyperréactivité ainsi que le remodelage bronchique chez les souris asthmatiques, confirmant nos résultats sur le rôle de Rac1 dans l’asthme allergique et son intérêt thérapeutique. / Contraction is the major function of smooth muscle cells (SMC). In certain pathological situations, SMCs deregulation leads to hyperreactivity and dedifferentiation promoting tissue remodeling. The Rac1 protein regulates in vitro essential SMCs functions. The objective of my thesis was to determine the role of Rac1 in vivo in pulmonary SMCs (bronchial and vascular) and its importance in the development of pathologies. The implementation of experimental models allowed me to highlight Rac1 hyperactivity in bronchial SMCs from asthmatic mice and in pulmonary artery (PA) SMCs from mice with pulmonary hypertension (PAH). Through in vivo and in vitro approaches, I have demonstrated that Rac1 plays an essential role in the contraction and proliferation of bronchial SMCs. Conversely, my results show that Rac1 in pulmonary vascular SMCs is involved in the production of reactive oxygen species inducing SMCs proliferation and PA remodeling associated with PAH. My results demonstrate a different role of Rac1 depending on the origin of SMCs but identify Rac1 as a therapeutic target for pathologies associated with hyperreactivity and/or remodeling of SMCs. An in silico screening, in vitro and ex vivo tests, allowed us to identify a Rac1 inhibitor: the A41 molecule. Nebulization of A41 prevents hyperreactivity and bronchial remodeling in asthmatic mice, confirming our results on the role of Rac1 in allergic asthma and its therapeutic interest.

BRAF V600E is the most common driver mutation in human cutaneous melanoma and is frequently accompanied by loss of the tumor suppressing phosphatase PTEN. Recent evidence suggests a co-operative role for RAC1 activity in BRAF V600E -driven melanoma progression and drug resistance. However, the underlying molecular mechanisms and the role of RAC1 downstream targets are not well explored. Here, we examine the role of the NCKAP1 subunit of the pentameric cytoskeletal SCAR/WAVE complex, a major downstream target of RAC1, in a mouse model of melanoma driven by BRAF V600E; PTEN loss. The SCAR/WAVE complex is the major driver of lamellipodia formation and cell migration downstream of RAC1 and depends on NCKAP1 for its integrity. Targeted deletion of Nckap1 in the melanocyte lineage delayed tumor onset and progression of a mutant Braf ; Pten loss driven melanoma mouse model. Nckap1 depleted tumors displayed fibrotic stroma with increased collagen deposition concomitant with enhanced immune infiltration. Nckap1 loss slowed proliferation and tumor growth, highlighting a role in cell cycle progression. Altogether, we propose that NCKAP1-orchestrated actin polymerization is essential for tumor progression and maintenance of tumor tissue integrity in a mutant Braf ; Pten loss driven mouse model for melanoma.

BACKGROUND:The invasion of glioblastoma cells into regions of the normal brain is a critical factor that limits current therapies for malignant astrocytomas. Previous work has identified roles for the Rho family guanine nucleotide exchange factors Trio and Vav3 in glioblastoma invasion. Both Trio and Vav3 act on the small GTPase RhoG. We therefore examined the role of RhoG in the invasive behavior of glioblastoma cells.RESULTS:We found that siRNA-mediated depletion of RhoG strongly inhibits invasion of glioblastoma cells through brain slices ex vivo. In addition, depletion of RhoG has a marginal effect on glioblastoma cell proliferation, but significantly inhibits glioblastoma cell survival in colony formation assays. We also observed that RhoG is activated by both HGF and EGF, two factors that are thought to be clinically relevant drivers of glioblastoma invasive behavior, and that RhoG is overexpressed in human glioblastoma tumors versus non-neoplastic brain. In search of a mechanism for the contribution of RhoG to the malignant behavior of glioblastoma cells, we found that depletion of RhoG strongly inhibits activation of the Rac1 GTPase by both HGF and EGF. In line with this observation, we also show that RhoG contributes to the formation of lamellipodia and invadopodia, two functions that have been shown to be Rac1-dependent.CONCLUSIONS:Our functional analysis of RhoG in the context of glioblastoma revealed a critical role for RhoG in tumor cell invasion and survival. These results suggest that targeting RhoG-mediated signaling presents a novel avenue for glioblastoma therapy.

Aberrant activation of the canonical Wnt signaling pathway accounts for the vast majority of colorectal cancers. The Rac1 GTPase is overexpressed in colon cancer, and its splice variant, Rac1b, is preferentially expressed in colon tumours. Rac1 and Rac1b have both been previously shown to crosstalk with the canonical Wnt signaling pathway in colon cancer; however, the specific means by which this crosstalk occurs were unclear. This study examines the molecular mechanisms of Rac1/1b in the cooperation with canonical Wnt signaling in colon cancer. In a colon cancer cell line with dysregulated Wnt signaling, the constitutively active Rac1 mutant, V12Rac1, was observed to transcriptionally upregulate the expression of a gene set associated with cellular migration. Further, V12Rac1-mediated promotion of cell migration was dependent on its nuclear localization. Previous work in our lab has shown a Rac1-specific activator, Tiam1, is present in the nucleus at the promoter of Wnt target genes upon Wnt3a stimulation; and that exogenous introduction of Tiam1 increased the expression of a Wnt-responsive reporter (TopFlash). Given the importance of nuclear localization of Rac1 in the promotion of tumourigenic processes, we demonstrated that knockdown of endogenous Tiam1 reduced TopFlash expression, proving reverse specificity and strengthening the evidence of a nuclear role for Rac1. Since some functional differences exist between Rac1 and Rac1b, we also examined Rac1b for transcriptional targets following induction, and identified the RhoA effector, ROCK2, which has been previously associated with cell migration. ROCK2 demonstrated a positive correlation with Rac1b transcript expression in primary colon tumours as compared to matched normal tissue specimens. Interestingly, the observed induction in ROCK2 transcript did not translate into a detectable change in protein expression or kinase activity. Like Rac1, Rac1b also promotes cellular motility, which is dependent on nuclear localization. Cell migration can be negatively regulated by E-cadherin. Following Rac1b knockdown in HT29 cells, we show that Rac1b might contribute to motility through upregulation of the E-cadherin-repressor, Slug. Taken together, we provide greater insight into the mechanistic roles of Rac1 and Rac1b in transcriptionally regulating target genes to promote cellular processes, such as cell migration, in colon cancer with dysregulated canonical Wnt signaling.

Aberrant activation of the canonical Wnt signaling pathway accounts for the vast majority of colorectal cancers. The Rac1 GTPase is overexpressed in colon cancer, and its splice variant, Rac1b, is preferentially expressed in colon tumours. Rac1 and Rac1b have both been previously shown to crosstalk with the canonical Wnt signaling pathway in colon cancer; however, the specific means by which this crosstalk occurs were unclear. This study examines the molecular mechanisms of Rac1/1b in the cooperation with canonical Wnt signaling in colon cancer. In a colon cancer cell line with dysregulated Wnt signaling, the constitutively active Rac1 mutant, V12Rac1, was observed to transcriptionally upregulate the expression of a gene set associated with cellular migration. Further, V12Rac1-mediated promotion of cell migration was dependent on its nuclear localization. Previous work in our lab has shown a Rac1-specific activator, Tiam1, is present in the nucleus at the promoter of Wnt target genes upon Wnt3a stimulation; and that exogenous introduction of Tiam1 increased the expression of a Wnt-responsive reporter (TopFlash). Given the importance of nuclear localization of Rac1 in the promotion of tumourigenic processes, we demonstrated that knockdown of endogenous Tiam1 reduced TopFlash expression, proving reverse specificity and strengthening the evidence of a nuclear role for Rac1. Since some functional differences exist between Rac1 and Rac1b, we also examined Rac1b for transcriptional targets following induction, and identified the RhoA effector, ROCK2, which has been previously associated with cell migration. ROCK2 demonstrated a positive correlation with Rac1b transcript expression in primary colon tumours as compared to matched normal tissue specimens. Interestingly, the observed induction in ROCK2 transcript did not translate into a detectable change in protein expression or kinase activity. Like Rac1, Rac1b also promotes cellular motility, which is dependent on nuclear localization. Cell migration can be negatively regulated by E-cadherin. Following Rac1b knockdown in HT29 cells, we show that Rac1b might contribute to motility through upregulation of the E-cadherin-repressor, Slug. Taken together, we provide greater insight into the mechanistic roles of Rac1 and Rac1b in transcriptionally regulating target genes to promote cellular processes, such as cell migration, in colon cancer with dysregulated canonical Wnt signaling.

No description available.

Le régulateur transcriptionnel Cux1 est impliqué dans le processus de migration des cellules cancéreuses. Dans un modèle d’inflammation intestinale de type colite expérimentale, l’absence de Cux1 entraîne une perte de la restitution de l'épithélium intestinal. Ceci nous a amenés à poser l’hypothèse que Cux1 serait fonctionnellement impliqué dans le processus de migration cellulaire lors de la régénération épithéliale en contexte de maladies inflammatoires intestinales. En utilisant des conditions de culture limitant la prolifération, une diminution de la réponse migratoire des cellules épithéliales a été observée en absence de Cux1. Cette diminution de migration a été accompagnée d’une perte du remaniement du cytosquelette d'actine et d’une diminution du nombre de lamellipodes formés au front de migration. En vérifiant l’activité des voies classiques de migration RhoA et Rac1, une modulation des modifications post-traductionnelles sur chacune des protéines terminales de ces voies a été observée. Une augmentation du niveau de phosphorylation inhibitrice sur sérine 3 de la cofiline et une diminution de la phosphorylation activatrice sur la protéine ribosomale S6 ont été observées en absence de Cux1. Toutefois, ces protéines ne sont pas des cibles transcriptionnelles du régulateur Cux1. Une analyse par micropuce a permis d’identifier de nouveaux substrats de Cux1 pertinents au processus de restitution épithéliale. Ainsi, les gènes FgfR2, Shc4 et Vav2 ont été découverts pour être impliqués autant au niveau cellulaire que physiologique en contexte inflammatoire. De plus, Cux1 semble en mesure de cibler directement ces trois promoteurs. Un essai de récupération du phénotype en inhibant la voie RhoA a permis d'observer un rétablissement partiel de la réponse migratoire des cellules n’exprimant pas Cux1, suggérant que Vav2 et la voie Rac1 auraient une importance dans cette restitution épithéliale intestinale. Des modulations de gènes impliqués dans la restitution ont été observées par séquençage des ARNs chez des souris mutantes pour Cux1.

Introduction: The Rho family of small GTPases, including Rac1 and Rac2, are key regulators of osteoclast differentiation and function; however, little is known about their roles in bone quality and aging. Methods: Male four and nine month old mice (n=10) with Rac1, Rac2 or both (DKO) isoforms deleted in osteoclast precursors were assessed using dual energy x-ray absorptiometry (DXA), scanning electron microscopy (SEM), micro computed tomography (microCT), compression, torsion and three-point bending testing, back scattered electron microscopy (BSE), Goldner’s trichrome and TRAP staining. Results: All Rac null mice demonstrated decreased cortical structural properties and improved trabecular architecture. With age, Rac null mice demonstrated the ability to attenuate age-related bone loss. Conclusions: Using an in vivo model with Rac1, Rac2 or both Rac isoforms deleted in osteoclasts, our findings demonstrate the deletion of Rac1 and Rac2 compromised cortical bone while improving trabecular bone properties and attenuated age-related bone loss.

Introduction: The Rho family of small GTPases, including Rac1 and Rac2, are key regulators of osteoclast differentiation and function; however, little is known about their roles in bone quality and aging. Methods: Male four and nine month old mice (n=10) with Rac1, Rac2 or both (DKO) isoforms deleted in osteoclast precursors were assessed using dual energy x-ray absorptiometry (DXA), scanning electron microscopy (SEM), micro computed tomography (microCT), compression, torsion and three-point bending testing, back scattered electron microscopy (BSE), Goldner’s trichrome and TRAP staining. Results: All Rac null mice demonstrated decreased cortical structural properties and improved trabecular architecture. With age, Rac null mice demonstrated the ability to attenuate age-related bone loss. Conclusions: Using an in vivo model with Rac1, Rac2 or both Rac isoforms deleted in osteoclasts, our findings demonstrate the deletion of Rac1 and Rac2 compromised cortical bone while improving trabecular bone properties and attenuated age-related bone loss.

The mammary gland contains a subset of cells with regenerative capacity that is able to generate both luminal and myoepithelial mammary epithelial lineages. Those cells are described as mammary epithelial stem cells. The fate of stem cells is tightly controlled by their microenvironment and adhesion receptors on the stem cells play a vital role in the microenvironment–stem cell communication. They facilitate the interaction of stem cells with the extracellular matrix as well as adjacent cells, and they regulate stem cell homing to their niches, as well as stem cell proliferation, self-renewal, and differentiation. Stem cells express high levels of ECM binding adhesion receptors such as β1 and α6-integrins. Those integrins were used to isolate stem cells from the rest of the differentiated epithelial cells within the mammary gland. However, little is known about the role of those integrins in stem cell self-renewal and differentiation. This project aimed to understand how β1-integrin receptors contribute to stem cell behavior. To achieve this, FACS sorting method of stem cells, the organoid assay, and lentivirus knockdown of β1-integrin using shRNA were optimised. The organoid assay was used as an in-vitro test to assess for the frequency of bi-lineage and luminal progenitor cells in a given mammary epithelial population. It is known that bi-lineage cells produce solid organoids in culture while luminal progenitors produce hollow organoids. The frequency of solid and hollow organoids might therefore be an indication of the stem cells and luminal progenitor frequency respectively. My results showed that cells with the highest solid organoid forming ability were within the basal population, which is high for β1- and α6-integrin. The β1-integrin signaling pathway was shown to be important for maintaining the organoid-forming population in basal and luminal populations. Knocking out β1-integrin in MECs resulted in abolishing their solid and hollow organoid-forming activity. Downstream of β1-integrin, I found that Rac1 but not ILK is important in β1-integrin maintenance of solid organoid-forming cells. Active Rac1 was able to rescue solid organoid formation but was not able to rescue hollow organoids in the β1-integrin knockdown cells. β1-integrin and Rac1 deletion resulted in the down regulation of Wnt/β-catenin signaling, which is important for stem cells. This down regulation was rescued using active Rac1. Activating Wnt/ β1-catenin signaling in primary cells (using Wnt3a ligand or GSK3β inhibitor) resulted in an increase in solid organoid and a decrease in hollow organoid formation. When activating Wnt signaling using GSK3I in β1-integrin knockdown cells, the solid organoid activity was rescued. However, Wnt3a did not rescue solid organoid formation in the β1-integrin knockdown cells. When active Rac1 was overexpressed in β1-integrin null cells, Wnt3a was able to activate solid organoid formation. When inhibiting Rac1 in primary MECs, solid but not hollow organoid activity was significantly decreased. Wnt3a or GSK3I addition did not rescue this reduction. Taken these results together, it can be concluded that β1integrin-Rac1 signaling play a role in controlling stem cells and this is might be achieved through controlling Wnt/β-catenin signaling. These studies are important in understanding the role of integrins in mammary stem cells. They will also provide new insight on how integrins might be controlling breast cancer and thereby, help in providing new targets for cancer therapy.