The biology of cancer stem cells : part 1: nuclear translocation of CD44 : part 2: IMP2 in glioblastoma cancer stem cells

SummaryCancer stem cells (CSC) are poorly differentiated, slowly proliferating cells, with high tumorigenic potential. Some of these cells, as it has been shown in leukemia, evade chemo- and radiotherapy and recapitulate the tumor composed of CSC and their highly proliferative progeny. Therefore, understanding the molecular biology of those cells is crucial for improvement of currently used anti-cancer therapies.This work is composed of two CSC-related projects. The first deals with CD44, a frequently used marker of CSC; the second involves Imp2 and its role in CSC bioenergetics. PART 1. CD44 is a multifunctional transmembrane protein involved in migration, homing, adhesion, proliferation and survival. It is overexpressed in many cancers and its levels are correlated with poor prognosis. CD44 is also highly expressed by CSC and in many malignancies it is used for CSC isolation.In the present work full-lenght CD44 nuclear localization was studied, including the mechanism of nuclear translocation and its functional role in the nucleus. Full-length CD44 can be found in nuclei of various cell types, regardless of their tumorigenic potential. For nuclear localization, CD44 needs to be first inserted into the cell membrane, from which it is transported via the endocytic pathway. Upon binding to transportinl it is translocated to the nucleus. The nuclear localization signal recognized by transportinl has been determined as the first 20 amino acids of the membrane proximal intracellular domain. Nuclear export of CD44 is facilitated by exportin Crml. Investigation of the function of nuclear CD44 revealed its implication in de novo RNA synthesis.PART 2. Glioblastoma multiforme is the most aggressive and most frequent brain malignancy. It was one of the first solid tumors from which CSC have been isolated. Based on the similarity between GBM CSC and normal stem cells expression of an oncofetal mRNA binding protein Imp2 has been investigated.Imp2 is absent in normal brain as well as in low grade gliomas, but is expressed in over 75% GBM cases and its expression is higher in CSC compared to their more differentiated counterparts. Analysis of mRNA transcripts bound by Imp2 and its protein interactors revealed that in GBM CSC Imp2 may be implicated in mitochondrial metabolism. Indeed, shRNA mediated silencing of protein expression led to decreased mitochondrial activity, decreased oxygen consumption and decreased activity of respiratory chain protein complex I. Moreover, lack of Imp2 severely affected self-renewal and tumorigenicity of GBM CSC. Experimental evidence suggest that GBM CSC depend on mitochondrial oxidative phosphorylation as an energy producing pathway and that Imp2 is a novel regulator of this pathway.RésuméLes cellules cancéreuses souches sont des cellules peu différentiées, à proliferation lente et hautement tumorigénique. Ces cellules sont radio-chimio résistantes et sont capable reformer la tumeur dans sont intégralité, reproduisant l'hétérogénéité cellulaire présent dans la tumeur d'origine. Pour améliorer les therapies antitumorales actuelles il est crucial de comprendre les mécanismes moléculaires qui caractérisent cette sous-population de cellules hautement malignes.Ce travail de thèse se compose de deux projets s'articulant autour du même axe :Le CD44 est une protéine multifonctionnelle et transmembranaire très souvent utilisée comme marqueur de cellules souches tumorales dans différents cancers. Elle est impliquée dans la migration, l'adhésion, la prolifération et la survie des cellules. Lors de ce travail de recherche, nous nous sommes intéressés à la localisation cellulaire du CD44, ainsi qu'aux mécanismes permettant sa translocation nucléaire. En effet, bien que principalement décrit comme un récepteur de surface transmembranaire, le CD44 sous sa forme entière, non clivée en peptides, peut également être observé à l'intérieur du noyau de diverses cellules, quel que soit leur potentiel tumorigénique. Pour passer ainsi d'un compartiment cellulaire à un autre, le CD44 doit d'abord être inséré dans la membrane plasmique, d'où il est transporté par endocytose jusqu'à l'intérieur du cytoplasme. La transportai permet ensuite la translocation nucléaire du CD44 via une « séquence signal » contenue dans les 20 acides aminés du domaine cytoplasmique qui bordent la membrane. A l'inverse, le CD44 est exporté du noyau grâce à l'exportin Crml. En plus des mécanismes décrits ci-dessus, cette étude a également mis en évidence l'implication du CD44 dans la synthèse des ARN, d'où sa présence dans le noyau.Le glioblastome est la plus maligne et la plus fréquente des tumeurs cérébrales. Dans ce second projet de recherche, le rôle de IMP2 dans les cellules souches tumorales de glioblastomes a été étudié. La présence de cette protéine oncofoetale a d'abord été mise en évidence dans 75% des cas les plus agressifs des gliomes (grade IV, appelés glioblastomes), tandis qu'elle n'est pas exprimée dans les grades I à III de ces tumeurs, ni dans le cerveau sain. De plus, IMP2 est apparue comme étant davantage exprimée dans les cellules souches tumorales que dans les cellules déjà différenciées. La baisse de l'expression de IMP2 au moyen de shRNA a résulté en une diminution de l'activité mitochondriale, en une réduction de la consommation d'oxygène ainsi qu'en une baisse de l'activité du complexe respiratoire I.L'inhibition de IMP2 a également affecté la capacité de renouvellement de la population des cellules souches tumorales ainsi que leur aptitude à former des tumeurs.Lors de ce travail de thèse, une nouvelle fonction d'un marqueur de cellules souches tumorales a été mise en évidence, ainsi qu'un lien important entre la bioénergétique de ces cellules et l'expression d'une protéine oncofoetale.

Thérapie cellulaire en cardiologie: bilan des premiers essais cliniques randomisés [Cell therapies in cardiology: results from the first randomized clinical trials]

Following acute myocardial infarction, necrotic cardiac tissue is replaced by scar leading to ventricular remodeling and pump failure. Transplantation of autologous bone marrow-derived cells into the heart, early post-infarct, aims to prevent ventricular remodeling. This strategy has been evaluated in four controlled, randomized clinical trials, which provided mixed results. A transient improvement in ventricular function was observed in one trial, and a modest improvement (the duration of which remains to be determined) in an additional trial, whereas two trials showed negative results. A modest benefit of bone marrow cell transplantation was also observed in patients with chronic ischemic heart disease. Despite mixed results reported so far, cell therapy of heart disease still is in its infancy and has considerable room for improvement.

mTORC2 regulates PGE(2)-mediated endothelial cell survival and migration

Prostaglandin E-2 (PGE(2)) promotes angiogenesis by in part inducing endothelial cell survival and migration. The present study examined the role of mTOR and its two complexes, mTORC1 and mTORC2, in PGE(2)-mediated endothelial cell responses. We used small interfering RNA (siRNA) to raptor or rictor to block mTORC1 or mTORC2, respectively. We observed that down-regulation of mTORC2 but not mTORC1 reduced baseline and PGE(2)-induced endothelial cell survival and migration. At the molecular level, we found that knockdown of mTORC2 inhibited PGE2-mediated Rac and Akt activation two important signaling intermediaries in endothelial cell migration and survival, respectively. In addition, inhibition of mTORC2 by prolonged exposure of endothelial cells to rapamycin also prevented PGE2-mediated endothelial cell survival and migration confirming the results obtained with the siRNA approach. Taken together these results show that mTORC2 but not mTORC1 is an important signaling intermediary in PGE2-mediated endothelial cell responses.

Les complications de la cirrhose hépatique: varices oesophagiennes, ascite et carcinome hépato-cellulaire [Complications of liver cirrhosis: oesophageal varices, ascites and hepato-cellular carcinoma]

The principal treatment for bleeding oesophageal varices is endoscopic ligation. Non-cardioselective beta-blockers are the gold-standard of primary prophylaxis. The principal treatment for ascites is a salt-free diet and diuretics, mainly spironolactone, if necessary associated with a loop diuretic. In refractory ascites, paracentesis or installation of a transjugular intrahepatic portosystemic shunt (TIPS) are two possible treatment options. Cirrhosis patients are at higher risk of developing hepato-cellular carcinoma. Surgery is only possible in a small number of cases. Percutaneous destruction techniques have nearly the same survival rate as that obtained by surgery and should be proposed to patients where surgery is not an option.

Atorvastatin-loaded hydrogel affects the smooth muscle cells of human veins

L'hyperplasie intimale est la cause majeure de sténoses de pontages veineux. Différents médicaments tels que les statines permettent de prévenir les sténoses mais leur administration systémique n'a que peu d'effet. Nous avons développé une matrice d'hydrogel d'acide hyaluronique qui permet d'avoir un relargage contrôlé d'atorvastatine sur un site désiré. L'enjeu de ce projet de recherche est de démontrer que l'atorvastatine relarguée par l'hydrogel a un effet similaire sur les cellules musculaires lisses de veines saphènes humaines comparé à l'atorvastatine directement diluée dans le milieu de culture. La recherche a été conduite conjointement par le laboratoire de médecine expérimentale du département de chirurgie thoracique et vasculaire du Centre Hospitalier Universitaire Vaudois et de l'Ecole de sciences pharmaceutiques des Universités de Lausanne et de Genève. On a incorporé de l'atorvastatine calcium (Chemos GmbH, Regenstauf Allemagne) dans des gels d'acide hyaluronique (Fortelis extra) à des concentrations déterminées afin de pouvoir analyser le relargage de l'Atovastatine dans le milieu de culture cellulaire par rapport aux concentrations d'atorvastatine directement ajoutées dans le milieu. Des cellules musculaires lisses primaires ont été cultivées à partir d'expiants de veines saphènes humaines. Elles ont été identifiées grâce à l'immunohistochimie par des anticorps contre la desmine et l'alpha-smooth muscle actine. La prolifération et la viabilité de ces cellules ont été analysées à l'aide du test MTT, leur transmigration avec le test de la chambre de Boyden et leur migration avec le principe de cicatrisation de plaies (wound healing assey). L'expression de gènes connus pour participer au développement de l'hyperplasie intimale, tels que la gap junction protein Connexin43 (Cx43), l'inhibiteur du plasminogène PAI-1, Thème oxygénase HO-1, la métalloproteinase-9 et l'inhibiteur de l'activateur du plasminogène tissulaire tPA, a été déterminée par niveau de mRNA exprimé en PCR. Leur expression en protéines a été analysée en utilisant la méthode par Western blots ainsi que l'immunohistochimie. Les expériences ont été effectuées à triple reprise en duplicats en parallèles avec de l'atorvastatine calcium directement ajoutée dans le milieu de culture et avec l'atorvastatine relarguée par l'hydrogel d'acide hyaluronique. Conclusions L'atorvastatine est relarguée par l'hydrogel de façon contrôlée. L'hydrogel contenant l'atorvastatine diminue la viabilité et la transmigration des cellules musculaires lisses de veines saphènes humaines de façon similaire à l'atorvastatine directement introduite dans le milieu de culture. L'hydrogel contenant l'atorvastatine module de façon sélective l'expression de marqueurs de la différentiation cellulaire de cellules musculaires lisses de veines saphènes humaines avec un retard de 24 heures comparé avec les effets de l'atorvastatine directement ajoutée au milieu de culture, sans néanmoins changer la distribution intra-cellulaire des protéines Cx43, HO-1 et PAI-1. Perspectives Il s'agit d'un projet d'importance clinique majeure permettant de réaliser des améliorations du traitement des artériopathies occlusives, ainsi que de relevance pharmacologique permettant de réaliser des dépôts de molécules avec un relargage stable et contrôlé à un site spécifique.

A new class of isothiocyanate-based irreversible inhibitors of macrophage migration inhibitory factor.

Macrophage migration inhibitory factor (MIF) is a homotrimeric multifunctional proinflammatory cytokine that has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. Current therapeutic strategies for targeting MIF focus on developing inhibitors of its tautomerase activity or modulating its biological activities using anti-MIF neutralizing antibodies. Herein we report a new class of isothiocyanate (ITC)-based irreversible inhibitors of MIF. Modification by benzyl isothiocyanate (BITC) and related analogues occurred at the N-terminal catalytic proline residue without any effect on the oligomerization state of MIF. Different alkyl and arylalkyl ITCs modified MIF with nearly the same efficiency as BITC. To elucidate the mechanism of action, we performed detailed biochemical, biophysical, and structural studies to determine the effect of BITC and its analogues on the conformational state, quaternary structure, catalytic activity, receptor binding, and biological activity of MIF. Light scattering, analytical ultracentrifugation, and NMR studies on unmodified and ITC-modified MIF demonstrated that modification of Pro1 alters the tertiary, but not the secondary or quaternary, structure of the trimer without affecting its thermodynamic stability. BITC induced drastic effects on the tertiary structure of MIF, in particular residues that cluster around Pro1 and constitute the tautomerase active site. These changes in tertiary structure and the loss of catalytic activity translated into a reduction in MIF receptor binding activity, MIF-mediated glucocorticoid overriding, and MIF-induced Akt phosphorylation. Together, these findings highlight the role of tertiary structure in modulating the biochemical and biological activities of MIF and present new opportunities for modulating MIF biological activities in vivo.

Migration Flows and Quality of Life in a Metropolitan Area: the Case of Barcelona-Spain

The phenomenon of human migration is certainly not new and it has been studied from a variety of perspectives. Yet, the attention on human migration and its determinant has not been fading over time as confirmed by recent contributions (see for instance Cushing and Poot 2004 and Rebhun and Raveh 2006). In this paper we combine the recent theoretical contributions by Douglas (1997) and Wall (2001) with the methodological advancements of Guimarães et al. (2000, 2003) to model inter-municipal migration flows in the Barcelona area. In order to do that, we employ two different types of count models, i.e. the Poisson and negative binomial and compare the estimations obtained. Our results show that, even after controlling for the traditional migration factors, QoL (measured with a Composite Index which includes numerous aspects and also using a list of individual variables) is an important determinant of short distance migration movements in the Barcelona area.

Sedimentation and electrophoretic migration of DNA knots and catenanes.

Various site-specific recombination enzymes produce different types of knots or catenanes while acting on circular DNA in vitro and in vivo. By analysing the types of knots or links produced, it is possible to reconstruct the order of events during the reaction and to deduce the molecular "architecture" of the complexes that different enzymes form with DNA. Until recently it was necessary to use laborious electron microscopy methods to identify the types of knots or catenanes that migrate in different bands on the agarose gels used to analyse the products of the reaction. We reported recently that electrophoretic migration of different knots and catenanes formed on the same size DNA molecules is simply related to the average crossing number of the ideal representations of the corresponding knots and catenanes. Here we explain this relation by demonstrating that the expected sedimentation coefficient of randomly fluctuating knotted or catenated DNA molecules in solution shows approximately linear correlation with the average crossing number of ideal configurations of the corresponding knots or catenanes.

The Escherichia coli RuvB branch migration protein forms double hexameric rings around DNA.

The RuvB protein is induced in Escherichia coli as part of the SOS response to DNA damage. It is required for genetic recombination and the postreplication repair of DNA. In vitro, the RuvB protein promotes the branch migration of Holliday junctions and has a DNA helicase activity in reactions that require ATP hydrolysis. We have used electron microscopy, image analysis, and three-dimensional reconstruction to show that the RuvB protein, in the presence of ATP, forms a dodecamer on double-stranded DNA in which two stacked hexameric rings encircle the DNA and are oriented in opposite directions with D6 symmetry. Although helicases are ubiquitous and essential for many aspects of DNA repair, replication, and transcription, three-dimensional reconstruction of a helicase has not yet been reported, to our knowledge. The structural arrangement that is seen may be common to other helicases, such as the simian virus 40 large tumor antigen.

Migrations sans frontières mais...barrières des représentations [Migration without borders, but...barriers of meaning].

Immigration, a political, economic, demographic, social and ethic, as well as a medical issue, continues. Among migrants, asylum seekers, refugees and undocumented immigrants are characterised by their vulnerability, particularly related to their health status. Western physicians are more and more frequently confronted to "colorful" and often vulnerable patients. They face diseases related to international migrations; and at the same time have to integrate the differences in representations and meanings given to illness by patients of diverse origins. A bio-psychosocial and spiritual approach coupled with an evaluation of pre-migration, migration and post-migration trajectories is therefore useful for the clinician; these complementary approaches have all been integrated in the learning of cultural competencies.

Migration et santé : analyse des besoins dans le canton de Vaud

[Table des matières] 1. Introduction. 2. Besoin en matière de programmes offrant une égalité des chances. 3. Programmes de promotion de la santé et de prévention évalués. 4. Mesures prises pour garantir l'égalité des chances d'accès lors de la conception et de la mise en oeuvre des programmes. 5. Participation aux programmes. 6. Quels obstacles à la participation des migrants et quels facteurs susceptibles de l'améliorer?: avis des personnes consultées. 7. Mesures recommandées. 8. Annexes. 9. Références.

Mechanisms of CYR61 mediated breast cancer metastasis to the lung

CYR61 (Cysteine-rich angiogenic inducer 61) is a matricellular protein that regulates cell proliferation, adhesion, migration and cell survival through interaction with various types of integrin cell adhesion receptors. At tissue level it is implicated in the regulation of embryonic development, wound healing and angiogenesis. CYR61 has also been involved in cancer progression, however its role appears to be diverse and complex depending on the cancer type and stage. Its contribution to metastasis formation is still unclear. Previous findings reported by our laboratory demonstrated that CYR61 cooperates with avßs integrin to promote invasion and metastasis of cancers growing in a pre-irradiated microenvironment. In this work, we used an orthotopic model of breast cancer to show for the first time that silencing of CYR61 in breast cancer cells suppresses lung metastasis formation. Silencing of MDA-MB-231 reduced both local growth and lung metastasis formation of tumor cells implanted in a pre-irradiated mammary fat pad. CYR61 silencing in tumors growing in non-irradiated mammary fat pads did not impact primary tumor growth but decreased lung metastasis formation. The effect of CYR61 on spontaneous lung metastasis formation during natural cancer progression was further examined by using an experimental model of metastasis. Results from these experiments indicate that CYR61 is critically involved in promoting cancer cells entry into lung parenchyma rather than later steps of colonization. In vitro experiments showed that CYR61 promotes tumor cell spreading, migration and transendothelial migration. CYR61 also supported colony formation under anchorage-independent condition and promotes resistance to anoikis through the involvement of ß1 and ß3 integrin. These results indicate that CYR61 promotes lung metastasis of breast cancer by facilitating extravasation into lung parenchyma through enhanced motility, transendothelial migration and resistance to anoikis. - CYR61 (Cysteine-rich angiogenic inducer 61) est une protéine matricellulaire qui régule la prolifération, l'adhérence, la migration et la survie des cellules par son interaction avec différents types de récepteurs d'adhésion cellulaire de la famille des intégrine. Au niveau des tissus, CYR61 est impliquée dans la régulation du développement embryonnaire, de la cicatrisation et de l'angiogenèse. CYR61 a également été impliquée dans le cancer, mais son rôle semble être divers et complexe en fonction du type du cancer et de son stade. Son rôle dans la formation des métastases n'est pas encore clair. Des résultats antérieurs rapportés par notre laboratoire ont montré que CYR61 coopère avec l'intégrine avß5 pour favoriser l'invasion et la métastase de tumeurs se développant dans un micro-environnement pré-irradié. Dans ce travail, nous avons utilisé un modèle orthotopique de cancer du sein pour démontrer pour la première fois que l'extinction (silencing) du gène CYR61 dans le cancer du sein réduit la formation de métastases pulmonaires. L'extinction de CYR61 dans la lignée cellulaire de cancer du sein humain MDA-MB- 231 réduit à la fois la croissance local ainsi que la formation de métastases pulmonaires à partir de cellules implantés dans les coussinets adipeux mammaires pré-irradié. L'extinction de CYR61 dans des tumeurs grandissant dans les coussinets adipeux mammaires non irradiées n'a pas d'incidence sur la croissance tumorale primaire mais réduit la formation des métastases pulmonaires. Par la suite nous avons examiné l'effet de CYR61 sur la formation de métastases pulmonaires en utilisant un modèle expérimental de métastase. Les résultats de ces expériences indiquent que CYR61 est impliquée de manière cruciale dans les étapes précoces de la formation de métastases, plutôt que dans les étapes tardives de colonisation du poumon. Des expériences in vitro ont montré que CYR61 favorise l'étalement, la migration et la transmigration endothéliale des cellules tumorales. CYR61 favorise également la formation de colonies dans des conditions indépendante de l'ancrage et la résistance à l'anoïkis par l'engagement des intégrines ß1 et ß3. Ces résultats indiquent que CYR61 favorise les métastases pulmonaires du cancer du sein en facilitant l'extravasation dans le parenchyme pulmonaire grâce à la stimulation de la motilità, de la migration transmigration endothéliale et de la résistance à l'anoïkis.

Macrophage migration inhibitory factor is critically involved in basal and fluoxetine-stimulated adult hippocampal cell proliferation and in anxiety, depression, and memory-related behaviors.

Intensive research is devoted to unravel the neurobiological mechanisms mediating adult hippocampal neurogenesis, its regulation by antidepressants, and its behavioral consequences. Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine that is expressed in the CNS, where its function is unknown. Here, we show, for the first time, the relevance of MIF expression for adult hippocampal neurogenesis. We identify MIF expression in neurogenic cells (in stem cells, cells undergoing proliferation, and in newly proliferated cells undergoing maturation) in the subgranular zone of the rodent dentate gyrus. A causal function for MIF in cell proliferation was shown using genetic (MIF gene deletion) and pharmacological (treatment with the MIF antagonist Iso-1) approaches. Behaviorally, genetic deletion of MIF resulted in increased anxiety- and depression-like behaviors, as well as of impaired hippocampus-dependent memory. Together, our studies provide evidence supporting a pivotal function for MIF in both basal and antidepressant-stimulated adult hippocampal cell proliferation. Moreover, loss of MIF results in a behavioral phenotype that, to a large extent, corresponds with alterations predicted to arise from reduced hippocampal neurogenesis. These findings underscore MIF as a potentially relevant molecular target for the development of treatments linked to deficits in neurogenesis, as well as to problems related to anxiety, depression, and cognition.

Inferring recent migration rates from individual genotypes.

We present a novel and straightforward method for estimating recent migration rates between discrete populations using multilocus genotype data. The approach builds upon a two-step sampling design, where individual genotypes are sampled before and after dispersal. We develop a model that estimates all pairwise backwards migration rates (m(ij), the probability that an individual sampled in population i is a migrant from population j) between a set of populations. The method is validated with simulated data and compared with the methods of BayesAss and Structure. First, we use data for an island model and then we consider more realistic data simulations for a metapopulation of the greater white-toothed shrew (Crocidura russula). We show that the precision and bias of estimates primarily depend upon the proportion of individuals sampled in each population. Weak sampling designs may particularly affect the quality of the coverage provided by 95% highest posterior density intervals. We further show that it is relatively insensitive to the number of loci sampled and the overall strength of genetic structure. The method can easily be extended and makes fewer assumptions about the underlying demographic and genetic processes than currently available methods. It allows backwards migration rates to be estimated across a wide range of realistic conditions.