During development of epithelial organs, epithelial cells collectively migrate and invade into their surroundings to form complex 3D structures, such as the tubular ducts and alveoli of the mammary gland. A prerequisite for coordinating such collective movement is apicobasal cell polarity. This polarity divides the plasma membrane of epithelial cells into an apical domain towards the lumen of structures and a basal domain facing the matrix. Polarity is essential for the functionality of epithelial tissues and signals from the surrounding matrix are known to participate in its establishment. In contrast, loss of polarity has been associated with progression of diseases such as cancer. During epithelial tubulogenesis, the pro-invasive enzyme MT1-MMP is regulated according to apicobasal polarity. This regulation is essential for tubulogenesis to occur and restricts MT1-MMP activity to the tip of protruding tubules by ensuring that the enzyme only localises to the basal, matrix-abutting cell surface in this location. Signals from fibrillar collagen I contribute to regulating the polarised distribution of MT1-MMP. How such signals are transmitted and influence polarised trafficking is however not understood. In this study, I found that inhibition of the collagen receptor DDR1 disturbed the apicobasal distribution of MT1-MMP in MDCK cells. In 3D environments, DDR1 inhibition blocked MT1-MMP-dependent tubulogenesis of epithelial cells, which instead formed compact, multi-layered aggregates. Furthermore, polarisation of the epithelial cell membrane into an apical and a basal domain failed in absence of DDR1 signalling, suggesting that DDR1 affects establishment of epithelial polarity. In support of this, the effects of DDR1 signalling on apicobasal polarity were not limited to MT1-MMP- dependent morphogenesis, but also proved essential for polarisation of cells during 3D morphogenesis that did not require ECM degradation. An investigation of signalling downstream of DDR1 in establishment of apicobasal polarity revealed this to involve modulation of cytoskeletal tension. Inhibition of DDR1 in 2D culture of MDCK cells thus increased ROCK-dependent phosphorylation of MLC along cell-cell junctions, suggesting that DDR1 can suppress ROCK activity. Importantly, the ROCK-suppressing function of DDR1 contributed to establishment of polarity in MDCK cells in 3D matrices, where inhibition of ROCK activity rescued the formation of polarised cysts in absence of DDR1 signalling. The role of DDR1 in epithelial organisation was reflected in the epithelium of the mammary gland of lactating DDR1-null mice, which had smaller alveoli with a diffuse distribution of basement membrane components compared to wild type mice. Furthermore, DDR1 inhibition attenuated formation of milk-producing mammospheres during lactogenic differentiation of mammary epithelial cells in vitro in a ROCK- dependent manner. This suggests that the ROCK-suppressing function of DDR1 observed in MDCK cells is important for morphogenesis of other epithelial cell types as well. Overall, this study suggests that DDR1 signalling contributes to epithelial polarisation and morphogenesis in a manner involving regulation of cytoskeletal organisation, at least partly through regulation of ROCK activity.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

LIM Kinase 1 (LIMK1), a serine/threonine kinase, modulates actin polymerization and microtubule assembly. The function of LIMK1 is regulated by kinases that are activated by Rho and Rac GTPases. LIMK1 is overexpressed in various cancerous cell types and tissues and its overexpression promotes increased invasion and metastasis of breast and prostate cancer cells. Membrane-Type Matrix Metalloproteinase 1 (MT1-MMP) is a member of the zinc-binding collagenase family, which is involved in extracellular matrix breakdown and activation of secreted MMP-2. The balance between activation and inhibition of MT1- MMP and MMP-2 helps maintaining normal extracellular matrix turnover. However, it has been shown that elevated MT1-MMP expression can cause excessive ECM digestion and promote tumor invasion and metastasis. Since RhoA and Rac1 have been implicated in metastasis and invasion along with LIMK1 activation, we investigated a possible link between LIMK1 and MT1-MMP. Our results show that the level of MT1-MMP expression is correlated with that of LIMK1 and LIMK1 acts as a transcriptional regulator of MT1-MMP. Additionally, we show that LIMK1 physically associates with MT1-MMP and promotes its translocation to the plasma membrane.

No description available.

Yes / The dysregulation of Membrane - type 1 matrix metalloproteinase (MT1-MMP) has been extensively studied in numerous cancer types, and plays key roles in angiogenesis, cancer progression, and metastasis. MT1-MMP is a predictor of poor prognosis in osteosarcoma (OS), yet the molecular mechanisms of disease progression are unclear. This review provides a summary of the literature relating to the gene and protein expression of MT1-MMP (MMP-14) in OS clinical samples, evaluates the expression in cell lines and experimental models, and analyses its potential role in the progression and metastasis of OS. In addition, the therapeutic potential of MT1-MMP as a drug target has been assessed. Due to the biological complexity of MMPs, inhibition has proven to be challenging. However, exploiting the expression and proteolytic capacity of MT1-MMP could open new avenues in the search for novel, safer and selective drugs for use in OS. / This work was supported by the Bone Cancer Research Trust (No. BCRT 6218).

La dérégulation des tyrosine kinases (TK) joue un rôle majeur dans la tumorigénèse et la progression tumorale des cancers du sein. Dans ce contexte, mon travail de thèse s'est focalisé sur la caractérisation des mécanismes moléculaires régulés par les TK associés à l'invasion cellulaire à travers deux axes d'études distincts : la régulation de l'invasion cellulaire par la protéine TOM1L1 dans les cancers du sein ERBB2-positifs et le rôle des TK ABL/ARG dans l'invasion cellulaire des cancers du sein triple-négatifs (n'exprimant ni ERBB2, ni les récepteurs hormonaux) (TN). TOM1L1 est une protéine du trafic vésiculaire à domaine GAT initialement caractérisée au laboratoire comme un régulateur négatif de l'activité mitogénique de la TK Src. Cependant, nos travaux indiquent que les gènes codant pour TOM1L1 et le récepteur TK ERBB2, localisés au niveau du chromosome 17q, sont co-amplifiés dans environ 50% de ces cancers et associés à un mauvais pronostic suggérant une activité oncogénique non-anticipée de TOM1L1. Mon travail de thèse montre que le récepteur ERBB2 régule indirectement la phosphorylation de la sérine 321 de TOM1L1 afin de permettre son association avec la protéine du trafic vésiculaire TOLLIP au niveau d'endosomes Rab7/MT1-MMP-positifs. Ainsi localisée, TOM1L1 favorise le recrutement membranaire de la métalloprotéase MT1-MMP au niveau de structures d'invasion cellulaire spécifiques, les invadopodes, amplifiant ainsi la capacité invasive des cellules ERBB2+. Des données récentes indiquent que les TK ABL/ARG sont aussi impliquées dans la régulation des invadopodes des cellules des cancers TN. Grace à une approche préliminaire d'inhibition pharmacologique (Imatinib, Nilotinib), mes travaux de thèse ont permis de dévoiler un rôle anti-invasif non-anticipé de ABL dans certaines lignées de cancers du sein TN, dont nous recherchons actuellement le mécanisme. / Deregulation of tyrosine kinases (TK) plays crucial role in breast cancer tumorigenesis and metastatic progression. In this context, my work tried to decipher the molecular mechanisms of tyrosine kinases-induced cell invasion via two distinct research axes: the regulation of cell invasion by TOM1L1 in ERBB2-positive breast cancers and the role of ABL/ARG TK in triple-negative (TN) breast cancer cells (negative for ERBB2 and hormonal receptors expression). The GAT domain-containing vesicular trafficking protein TOM1L1 was first identified in the lab as a negative regulator of Src mitogenic activity. Here, we show that ERBB2 and TOM1L1, located on chromosome 17q, are co-amplified in 50% of breast tumor samples and define a subgroup of ERBB2-positive tumors with poor prognosis. We then studied TOM1L1 pro-oncogenic function and found that TOM1L1 regulates exclusively ERBB2-driven cell invasion. This effect requires indirect ERBB2-induced TOM1L1 phosphorylation on serine 321 and interaction with the endosomal sorting protein TOLLIP at Rab7/MT1-MMP-positive endosomes. Thus, TOM1L1 promotes membrane recruitment of MT1-MMP into specialized invasive structures called invadopodia and, as such, promotes cell invasion. Recent observations show that ABL/ARG TK are also involve in invadopodia regulation of TN breast cancers. With a preliminary pharmacological-inhibition strategy (Imatinib,Nilotinib), my work shows an unanticipated anti-invasive function of ABL in some TN cell-lines. We are currently looking for ABL kinases molecular mechanisms involved in this effect.

Membrane type-1 matrix metalloproteinase (MT1-MMP) is a metalloproteinase which becomes upregulated in prostate cancer and has been implicated in processes of prostate cancer metastasis. Here, we show that MT1-MMP is minimally expressed in nonmalignant primary prostate cells, moderately expressed in DU-145 cells, and highly expressed in invasive PC-3 and PC-3N cells. Using MT1-MMP promoter reporters and mobility shift assays, we show that Sp1 regulates MT1-MMP expression in DU-145, PC-3, and PC-3N cells and in PC3-N cells using chromatin immunoprecipitation analysis and silencing RNA. Investigation of signaling pathways in these cells showed that DU-145 cells express constitutively phosphorylated extracellular stress-regulated kinase (ERK), whereas PC-3 and PC-3N cells express constitutively phosphorylated AKT/PKB and c-Jun NH2 terminal kinase (JNK). We show that MT1-MMP and Sp1 levels are decreased in PC-3 and PC-3N cells when PI-3K and JNK are inhibited, and that MT1-MMP levels are decreased in DU-145 cells when MEK is inhibited. Transient transfection of PC-3 and PC-3N cells with a dominant-negative JNK or p85, and DU-145 cells with a dominant negative ERK, reduced MT1-MMP promoter activity. We also identified the insulin-like growth factor (IGF-1R) as an upstream regulatory component of MT1-MMP in PC-3N and LNCaP cells, which express high and low levels of the enzyme, respectively. Treatment of PC-3N cells with an IGF-1R specific inhibitor decreased MT1-MMP promoter activity, RNA and protein levels. Additionally, treatment of LNCaP cells with a synthetic androgen to increase IGF-1R levels and subsequent treatment with IGF-I increased MT1-MMP promoter activity, RNA and protein levels. Analysis of MT1-MMP and IGF-1R expression in human prostate cancer tissues demonstrated that MT1-MMP expression was high in the apical cytoplasmic regions of PIN and prostate cancer and less intense in the basalateral cytoplasmic membrane regions of benign glands. IGF-1R was expressed in normal glands and highly expressed in prostate cancer. In conclusion, we have identified several novel mechanisms regulating MT1-MMP expression in prostate cancer cell lines as well as differential localization of the enzyme in human prostate cancer tissues. These results provide insight into the complex mechanisms of prostate cancer metastasis and may be useful for developing future diagnostic procedures or therapies.

La dissémination des cellules cancéreuses et la formation de métastases sont des étapes cruciales dans la progression tumorale et constituent une cause majeure des décès dus au cancer. La métalloprotéase transmembranaire MT1-MMP est un acteur clé impliqué dans le franchissement des barrières tissulaires et le remodelage de la matrice extracellulaire (ECM) par les cellules cancéreuses. MT1-MMP est présente dans des vésicules intracellulaires, appelées endosomes, via lesquels elle est adressée à la membrane plasmique (PM) afin d'y dégrader la ECM. Des travaux menés au laboratoire ont identifié le complexe exocyste (CE) comme un acteur important pour la formation d'invadopodes dans la lignée d'adénocarcinome mammaire MDA-MB-231. Ce complexe multiprotéique (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 et Exo84) est impliqué dans l'arrimage des vésicules intracellulaires à la PM. Des cribles double-hybride ont identifiés la protéine WASH comme partenaire potentiel du CE (via les sous-unités Exo84 et Sec3). WASH est capable d'induire la polymérisation de l'actine en activant le complexe Arp2/3. In vitro, nous avons montré que les complexes WASH et exocyste interagissent physiquement et coordonnent le trafic intracellulaire et l'adressage de MT1-MMP à la PM. Ces résultats mettent en évidence une étroite collaboration entre le cytosquelette d'actine et les mécanismes d'exocytose lors des étapes précoces de dégradation de la ECM ainsi que dans l'invasion tumorale. / Cancer cell invasion is a prerequisite to tumor progression and metastasis. In order to disseminate, tumor cells must degrade and remodel the extracellular matrix (ECM) in a process that requires the trans-membrane matrix metalloproteinase MT1-MMP, which is a key component of the ECM remodeling apparatus of cancer cells. MT1-MMP overexpression in cancers is associated with increased invasion and metastasis. Many cellular proteins are involved in the transport and delivery of MT1-MMP-containing vesicles to the PM. Previous work from the laboratory identified the exocyst complex (EC) as a key component required for matrix proteolysis and invasion of cancer cells. This multiprotein complex (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84) plays essential roles in docking secretory vesicles at the PM for exocytosis. To better characterize this complex, a yeast two-hybrid screen was performed, identifying the protein WASH as a potential partner of Exo84 and Sec3. WASH is a Nucleation Promoting Factor (NPF) able to activate the actin nucleating Arp2/3 complex. Results of the present study showed that WASH and the exocyst complexes interact and localize on MT1-MMP-positive endosomes in MDA-MB-231 breast cancer cells. This study highlight a direct implication of WASH and exocyst complex in ECM degradation by cancer cells through the docking and exocytosis of MT1-MMP-containing endosomes at the PM through connections between these compartments and the extracellular medium. This WASH- and exocyst-dependent MT1-MMP exocytosis mechanism is required for degradation of adjacent tissue by cancer cells during tumour cell invasion.

Made available in DSpace on 2017-07-21T19:59:58Z (GMT). No. of bitstreams: 1 Kamila Caroline Camargo.pdf: 3696475 bytes, checksum: 886b21796d7054b1ce4963cb6ea656e9 (MD5) Previous issue date: 2013-03-26 / Membrane type I MMP (MT1-MMP) is involved in cellular adhesion, proliferation, migration and cell death during organogenesis and adult phase of vertebrates. However, its role in the development of the small intestinal has not been established. We formulated the hypothesis that this metalloproteinase would play a relevant role in the development of the intestinal mucosa. To assess this hypothesis was detected the labeling of MT1-MMP by immunohistochemistry in the intestinal mucosa along the pre-and postnatal development of rats and chickens so that from a comparative analysis, it were possible infer about the role of this metalloproteinase.In thepre natal phase of rats, the labeling of MT1-MMP correlates with events that lead to morphogenesis of villi and establishment of the tecidual homeostasis. In the pre natal phase of chickens, the labeling of MT1-MMP correlates with events that lead to growth of the intestine. In the postnatal phase of rats and chickens, the labeling of MT1-MMP was maintained in the intestinal mucosa, which led us to suggest that this metalloproteinase have a relevant role in the tecidual homeostasis throughout adulthood.We suggest that the role of that MT1-MMPin the remodeling and growth of the intestinal mucosa of rats and chickens is related with adhesion, proliferation, migration and death of epithelial cells and migration of mesenchymalcells. This is the first work that evidence the MT1-MMP in the small intestine of vertebrates during development, making it possible that further studies willclarify its role in the formation of this important organ. / A metaloproteinase de membrana 1 (MT1-MMP) é envolvida em processos de adesão proliferação, migração celular e morte celular durante a organogênese e na fase adulta de vertebrados. Entretanto, o seu papel no desenvolvimento do intestino delgado de vertebrados ainda não foi estabelecido. Nós formulamos a hipótese de que essa metaloproteinase de membrana teria um papel relevante no processo de desenvolvimento da mucosa intestinal. Para avaliar essa hipótese, detectou-se a marcação da MT1-MMP por imunohistoquímica na mucosa intestinal ao longo do desenvolvimento pré e pós-natal de ratos e galinhas para que, a partir de uma análise comparativa, fosse possível inferir a respeito do papel dessa metaloproteinase. No período pré-natal de ratos, a marcação da MT1-MMP correlaciona com eventos que levam à morfogênese das vilosidades e estabelecimento da homeostase tecidual. No período pré-natal de galinhas, a marcação da MT1-MMP correlaciona com eventos que levam ao crescimento do intestino. No período pós-natal de ratos e galinhas, a marcação da MT1-MMP foi mantida no epitélio intestinal, o que nos levou a sugerir que essa metaloproteinase tem um papel importante na homeostase tecidual durante toda a vida adulta.Nós sugerimos que o papel da MT1-MMPna remodelação e crescimento da mucosa intestinal de ratos e galinhas está relacionado com adesão, proliferação, migração e morte de células epiteliais e migração de células mesenquimais. Esse é o primeiro trabalho que evidencia a MT1-MMP no intestino delgado de vertebrados durante o desenvolvimento, possibilitando estudos subsequentes que venham a esclarecer o papel dessa metaloproteinase na formação desse importante órgão.

Le mélanome uvéal constitue le cancer intraoculaire le plus fréquent chez l’adulte. Il s’agit d’un cancer très agressif puisque plus de 50% des patients développent des métastases principalement localisées au niveau du foie. Dans le but d’identifier des gènes pronostiques de développement métastatique, nous avons comparé le transcriptome de 28 tumeurs de mélanome uvéal issues de patients ayant développé des métastases dans les trois années qui ont suivi l’énucléation et 29 tumeurs issues de patients n’ayant pas développé de métastases ou ayant développé des métastases après 36 mois. Le gène PTP4A3/PRL-3 (protein tyrosine phosphatase type IV member 3/Protein of Regenerating Liver-3) a été identifié comme prédictif de l’apparition de métastases. Il code une phosphatase et sa surexpression dans des cellules de mélanome uvéal augmente leur migration in vitro et leur invasivité in vivo. Les évènements protéolytiques à la surface des cellules sont essentiels pour la migration et l’invasivité durant plusieurs processus physiologiques ou pathologiques tels que le développement de métastases. Ces évènements sont assurés par les métalloprotéases (MMPs) qui sont responsables de la dégradation et du remodelage de la matrice extracellulaire.Dans la première partie de cette thèse, nous avons observé que la métalloprotéase transmembranaire MT1-MMP est enrichie à la surface des cellules de mélanome uvéal OCM-1, des cellules MP41 issues de xénogreffes de tumeurs de mélanome uvéal humaines ou dans des tumeurs primaires de mélanome uvéal, surexprimant PTP4A3. Nous avons aussi observé que cette accumulation de MT1-MMP à la surface des cellules de mélanome uvéal est accompagnée d’une accumulation de la sécrétion de MMP2 dans le milieu extracellulaire des cellules exprimant PTP4A3. De plus, nous avons montré que PTP4A3 et MT1-MMP s’associent physiquement et que le trafic vésiculaire de MT1-MMP est accéléré dans les cellules exprimant PTP4A3 mais pas dans celles exprimant le mutant catalytique inactif PTP4A3(C104S). Enfin, nous avons démontré que l’inhibition de l’expression de MT1-MMP dans les cellules exprimant PTP4A3 diminue leur migration in vitro et leur invasivité in vivo. Pour conclure, nos résultats indiquent que PTP4A3 agit en amont de MT1-MMP à travers une accélération de son trafic vésiculaire et son accumulation à la surface des cellules afin de promouvoir la migration et l’invasivité cellulaires.Dans la deuxième partie de cette thèse, nous nous sommes intéressés au rôle de PTP4A3 pendant le développement embryonnaire. Les mélanocytes, incluant ceux de l’uvée, dérivent de la crête neurale pendant le développement embryonnaire. Nous avons alors supposé que la fonction de PTP4A3 pendant la progression métastatique pourrait refléter un rôle de la phosphatase dans la migration des cellules de crête neurale pendant le développement embryonnaire. Dans cette partie de la thèse, nous avons montré que PTP4A3 joue un rôle important dans la migration des cellules de crête neurale céphalique pendant le développement de l’embryon de Xenopus laevis. La perte de fonction de PTP4A3 provoque une réduction du territoire de la crête neurale, alors que le gain de fonction de cette phosphatase élargit les faisceaux de migration des cellules de crête neurale céphalique. De plus, des expériences d’isogreffes montrent que les explants de crête neurale dépourvus de l’expression de PTP4A3, sont incapables de migrer dans les embryons greffés. Plus encore, l’inhibition pharmacologique de PTP4A3 dans des cellules de crête neurale en culture diminue de façon significative leur vitesse de migration in vitro. Les résultats de cette étude démontrent que PTP4A3 est requise pour la migration des cellules de crête neurale céphalique in vivo pendant le développement embryonnaire de Xenopus laevis. Donc, les effets pro-migratoire et -invasif reliés à l’expression de la protéine PTP4A3 peuvent refléter son rôle durant la migration des cellules de crête neurale. / Uveal melanoma (UM) is the most common intraocular malignancy in adults and is an aggressive tumor since about 50% of patients will develop metastases mostly in the liver. In order to identify metastasis prognostic genes, we compared 28 uveal melanoma tumors from patients who developed metastases within three years after enucleation to 29 tumors from patients who did not develop metastases or who developed metastases after 36 months. The PTP4A3/PRL-3 gene (protein tyrosine phosphatase type IV member of Regenerating Liver 3/Protein-3) was identified as a strong predictor of metastasis occurence. PTP4A3 encodes a dual specificity phosphatase and its expression in UM cells increases their in vitro migration and in vivo invasiveness. Proteolytic events at the cell surface are essential for cell migration and invasiveness during many physiological and pathological processes such as tumor metastasis. MMPs are responsible for the degradation and turnover of the extracellular matrix (ECM). In the first part of this thesis, We found that the membrane anchored MT1-MMP is enriched at the cell surface of OCM-1, xenograft MP41 or primary human uveal melanoma tumors expressing PTP4A3. We also found that membrane accumulation of MT1-MMP in presence of PTP4A3 in OCM-1 cells is accompanied by enhanced secretion of MMP2 in the extracellular medium. Moreover, we demonstrated that PTP4A3 and MT1-MMP physically associate and that the vesicular trafficking of MT1-MMP is accelerated in presence of active PTP4A3 but not in presence of the mutant PTP4A3(C104S). Furthermore, we found that inhibition of MT1-MMP expression in PTP4A3 expressing uveal melanoma cells impairs their migration in vitro and invasiveness in vivo. Collectively, our results indicate that PTP4A3 acts upstream of MT1-MMP through acceleration of its vesicular trafficking and accumulation at the cell surface to enhance cell migration and invasiveness of uveal melanoma cells. In the second part of this thesis, we investigated the role of PTP4A3 during embryonic development. Melanocytes, including uveal melanocytes, are derived from the neural crest during embryonic development. We therefore suggested that PTP4A3 function in uveal melanoma metastasis may be related to an embryonic role during neural crest cell migration. We show that PTP4A3 plays a role in cephalic neural crest development in Xenopus laevis. PTP4A3 loss of function resulted in a reduction of neural crest territory, whilst gain of function experiments increased neural crest territory. Isochronic graft experiments demonstrated that PTP4A3-depleted neural crest explants are unable to migrate in host embryos. Pharmacological inhibition of PTP4A3 on dissected neural crest cells significantly reduced their migration velocity in vitro. Our results demonstrate that PTP4A3 is required for cephalic neural crest migration in vivo during embryonic development.Therefore, the pro-invasive and migratory effects related to the expression of PTP4A3 protein may reflect its role during neural crest migration. Thus, understanding the mechanism of action of PTP4A3 during NC migration may provide insight into PTP4A3 related migratory and invasive phenotypes in human uveal melanoma pathology.