Implication de MMP-9 dans le phénotype invasif des cellules souches cancéreuses et dans l'endothélium vasculaire cérébral tumoral

Le traitement des tumeurs cérébrales représente un défi d'envergure, puisqu'elles sont isolées du reste du corps par la barrière hémato-encéphalique. Il est donc impératif d'accroître nos connaissances sur les cellules composant ces tumeurs. L'une des hypothèses émergentes concerne la présence d'une sous-population spécifique de cellules au sein de la tumeur, les cellules souches cancéreuses (CSC), impliquées dans l'initiation et la récurrence des cancers. Les marqueurs de surfaces des CSC varient largement selon le tissu d'intérêt, mais il semble que CD133, une glycoprotéine membranaire, soit une signature commune à plusieurs CSC. S'il est courant de traiter le cancer en ciblant les cellules cancéreuses elles-mêmes, cibler le microenvironnement dans lequel évolue la tumeur est une avenue thérapeutique prometteuse. En effet, les cellules endothéliales (CE) au sein d'une tumeur sont connues pour avoir des propriétés différentes des CE normales. Ces cellules endothéliales tumorales (CET) peuvent donc constituer une cible supplémentaire pour inhiber sélectivement la croissance tumorale. À l'aide d'une lignée de médulloblastome (DAOY), nous avons identifié et évalué de nouvelles caractéristiques cellulaires et moléculaires impliquées dans la régulation du phénotype invasif associé à la formation de structures comparables aux neurosphères formées par les CSC CD133(+). De plus, nous avons apporté de nouvelles informations concernant les propriétés chimiopréventives du sulforaphane (SFN), un composé naturel retrouvé principalement dans le brocoli, en ciblant spécifiquement les CET cérébrales possiblement impliquées dans la cooption vasculaire. En utilisant la technologie de \ud l'ARN interférant, nous avons montré que MMP-9 et MT1-MMP, deux métalloprotéinases matricielles importantes pour l'invasion cellulaire, les métastases et la résistance à la radiation, avaient un rôle crucial dans la formation par les DAOY de \ud structures apparentées aux neurosphères CD133(+). De plus, nous avons mis en évidence une inhibition presque complète (> 90 %) de la migration des HBMEC (human brain microvascular endothelial cell) activées par un carcinogène, le phorbol 12-myristate 13-acétate (PMA). Ainsi, nos résultats suggèrent que les molécules présentes dans notre diète pourraient cibler les CET cérébrales composant la masse tumorale. En somme, nous proposons que MMP-9 constitue une cible de choix pour le traitement du cancer, que ce soit au niveau du compartiment tumoral ou vasculaire. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Cellule souche cancéreuse (CSC), Cellule endothéliale tumorale (CET), Sulforaphane (SFN), Métalloprotéinase matricielle-9 (MMP-9), Tumeur cérébrale

Standardised Nomenclature, Abbreviations, and Units for the study of Bone Marrow Adiposity: Report of the Nomenclature Working Group of the International Bone Marrow Adiposity Society

Research into bone marrow adiposity (BMA) has expanded greatly since the late 1990s, leading to development of new methods for the study of bone marrow adipocytes. Simultaneously, research fields interested in BMA have diversified substantially. This increasing interest is revealing fundamental new knowledge of BMA; however, it has also led to a highly variable nomenclature that makes it difficult to interpret and compare results from different studies. A consensus on BMA nomenclature has therefore become indispensable. This article addresses this critical need for standardised terminology and consistent reporting of parameters related to BMA research. The International Bone Marrow Adiposity Society (BMAS) was formed in 2017 to consolidate the growing scientific community interested in BMA. To address the BMA nomenclature challenge, BMAS members from diverse fields established a working group (WG). Based on their broad expertise, the WG first reviewed the existing, unsystematic nomenclature and identified terms, and concepts requiring further discussion. They thereby identified and defined 8 broad concepts and methods central to BMA research. Notably, these had been described using 519 unique combinations of term, abbreviation and unit, many of which were overlapping or redundant. On this foundation a second consensus was reached, with each term classified as “to use” or “not to use.” As a result, the WG reached a consensus to craft recommendations for 26 terms related to concepts and methods in BMA research. This was approved by the Scientific Board and Executive Board of BMAS and is the basis for the present recommendations for a formal BMA nomenclature. As an example, several terms or abbreviations have been used to represent “bone marrow adipocytes,” including BMAds, BM-As, and BMAs. The WG decided that BMA should refer to “bone marrow adiposity”; that BM-A is too similar to BMA; and noted that “Ad” has previously been recommended to refer to adipocytes. Thus, it was recommended to use BMAds to represent bone marrow adipocytes. In conclusion, the standard nomenclature proposed in this article should be Standardised Nomenclature, Abbreviations, and Units for the Study of Bone Marrow Adiposity: Report of the Nomenclature Working Group of the International Bone Marrow Adiposity Society Nathalie Bravenboer, Miriam A. Bredella, [...], and William P. Cawthorn Additional article information Associated Data Supplementary Materials Abstract Research into bone marrow adiposity (BMA) has expanded greatly since the late 1990s, leading to development of new methods for the study of bone marrow adipocytes. Simultaneously, research fields interested in BMA have diversified substantially. This increasing interest is revealing fundamental new knowledge of BMA; however, it has also led to a highly variable nomenclature that makes it difficult to interpret and compare results from different studies. A consensus on BMA nomenclature has therefore become indispensable. This article addresses this critical need for standardised terminology and consistent reporting of parameters related to BMA research. The International Bone Marrow Adiposity Society (BMAS) was formed in 2017 to consolidate the growing scientific community interested in BMA. To address the BMA nomenclature challenge, BMAS members from diverse fields established a working group (WG). Based on their broad expertise, the WG first reviewed the existing, unsystematic nomenclature and identified terms, and concepts requiring further discussion. They thereby identified and defined 8 broad concepts and methods central to BMA research. Notably, these had been described using 519 unique combinations of term, abbreviation and unit, many of which were overlapping or redundant. On this foundation a second consensus was reached, with each term classified as “to use” or “not to use.” As a result, the WG reached a consensus to craft recommendations for 26 terms related to concepts and methods in BMA research. This was approved by the Scientific Board and Executive Board of BMAS and is the basis for the present recommendations for a formal BMA nomenclature. As an example, several terms or abbreviations have been used to represent “bone marrow adipocytes,” including BMAds, BM-As, and BMAs. The WG decided that BMA should refer to “bone marrow adiposity”; that BM-A is too similar to BMA; and noted that “Ad” has previously been recommended to refer to adipocytes. Thus, it was recommended to use BMAds to represent bone marrow adipocytes. In conclusion, the standard nomenclature proposed in this article should be followed for all communications of results related to BMA. This will allow for better interactions both inside and outside of this emerging scientific community

The NAD-Booster Nicotinamide Riboside Potently Stimulates Hematopoiesis through Increased Mitochondrial Clearance

It has been recently shown that increased oxidative phosphorylation, as reflected by increased mitochondrial activity, together with impairment of the mitochondrial stress response, can severely compromise hematopoietic stem cell (HSC) regeneration. Here we show that the NAD(+)-boosting agent nicotinamide riboside (NR) reduces mitochondrial activity within HSCs through increased mitochondrial clearance, leading to increased asymmetric HSC divisions. NR dietary supplementation results in a significantly enlarged pool of progenitors, without concurrent HSC exhaustion, improves survival by 80%, and accelerates blood recovery after murine lethal irradiation and limiting-HSC transplantation. In immune-deficient mice, NR increased the production of human leucocytes from hCD34+ progenitors. Our work demonstrates for the first time a positive effect of NAD(+)-boosting strategies on the most primitive blood stem cells, establishing a link between HSC mitochondrial stress, mitophagy, and stem-cell fate decision, and unveiling the potential of NR to improve recovery of patients suffering from hematological failure including post chemo- and radiotherapy.Peer reviewe

Effet du stress prolifératif sur la fonction des cellules souches hématopoïétiques : rôles des gènes Scl, E2A et Heb

Le système hématopoïétique est un tissu en constant renouvellement et les cellules souches hématopoïétiques (CSHs) sont indispensables pour soutenir la production des cellules matures du sang. Deux fonctions définissent les CSHs; la propriété d’auto-renouvellement, soit la capacité de préserver l’identité cellulaire suivant une division, et la multipotence, le potentiel de différenciation permettant de générer toutes les lignées hématopoïétiques. Chez l’adulte, la majorité des CSHs sont quiescentes et l’altération de cet état corrèle avec une diminution du potentiel de reconstitution des CSHs, suggérant que la quiescence protège les fonctions des CSHs. La quiescence est un état réversible et dynamique et les réseaux génétiques le contrôlant restent peu connus. Un nombre croissant d’évidences suggère que si à l’état d’homéostasie il y a une certaine redondance entre les gènes impliqués dans ces réseaux de contrôle, leurs rôles spécifiques sont révélés en situation de stress. La famille des bHLHs (basic helix-loop-helix) inclue différentes classes des protéines dont ceux qui sont tissu-spécifiques comme SCL, et les protéines E, comme E12/E47 et HEB. Certains bHLHs sont proposés êtres important pour la fonction des cellules souches, mais cela ne fait pas l’unanimité, car selon le contexte cellulaire, il y a redondance entre ces facteurs. La question reste donc entière, y a-t-il un rôle redondant entre les bHLHs d’une même classe pour la fonction à long-terme des CSHs? Les travaux présentés dans cette thèse visaient dans un premier temps à explorer le lien encore mal compris entre la quiescence et la fonction des CSHs en mesurant leurs facultés suite à un stress prolifératif intense et dans un deuxième temps, investiguer l’importance et la spécificité de trois gènes pour la fonction des CSHs adultes, soit Scl/Tal1, E2a/Tcf3 et Heb/Tcf12. Pour répondre à ces questions, une approche cellulaire (stress prolifératif) a été combinée avec une approche génétique (invalidation génique). Plus précisément, la résistance des CSHs au stress prolifératif a été étudiée en utilisant deux tests fonctionnels quantitatifs optimisés, soit un traitement basé sur le 5-fluorouracil, une drogue de chimiothérapie, et la transplantation sérielle en nombre limite. Dans la mesure où la fonction d’un réseau génique ne peut être révélée que par une perturbation intrinsèque, trois modèles de souris, i.e. Scl+/-, E2a+/- et Heb+/- ont été utilisés. Ceci a permis de révéler que l’adaptation des CSHs au stress prolifératif et le retour à l’équilibre est strictement contrôlé par les niveaux de Scl, lesquels règlent le métabolisme cellulaire des CSHs en maintenant l’expression de gènes ribosomaux à un niveau basal. D’autre part, bien que les composantes du réseau puissent paraître redondants à l’équilibre, mes travaux montrent qu’en situation de stress prolifératif, les niveaux de Heb restreignent la prolifération excessive des CSHs en induisant la sénescence et que cette fonction ne peut pas être compensée par E2a. En conclusion, les résultats présentés dans cette thèse montrent que les CSHs peuvent tolérer un stress prolifératif intense ainsi que des dommages à l’ADN non-réparés, tout en maintenant leur capacité de reconstituer l’hématopoïèse à long-terme. Cela implique cependant que leur métabolisme revienne au niveau de base, soit celui trouvé à l’état d’homéostasie. Par contre, avec l’augmentation du nombre de division cellulaire les CSHs atteignent éventuellement une limite d’expansion et entrent en sénescence.The hematopoietic system is constantly replenished by hematopoietic stem cells (HSCs) that are essential to sustain mature blood cells production. Two key functions characterize HSCs; their capabilities to self-renew, i.e. maintenance of cellular identity following cell division, and their multipotencies, i.e. their potentials to generate all hematopoietic lineages. In adults, most HSCs are quiescent and alterations to this state correlate with decreased reconstitution potential, thus suggesting that quiescence protects HSC functions. Quiescence is a reversible and dynamic state, and genetic networks controlling these characteristics are poorly described. Recent evidence suggests that during steady-state hematopoiesis, genes controlling HSC functions are highly redundant, whereas stress conditions may reveal their specific roles. Transcription factors of the basic helix-loop-helix (bHLHs) family include tissue-specific subclasses (e.g SCL) and more ubiquitous E proteins (e.g. E12/E47 and HEB). Several bHLH members have been described as important for HSC functions, however this question is still highly debated in the field due to functional redundancies. How different bHLHs from a same subclass can uniquely affect long term HSC functions is still an open question. The work presented in this thesis aimed to address the question how three bHLH transcription factors specifically Scl/Tal1, E2a/Tcf3 and Heb/Tcf12 control HSC functions after an important proliferative stress to eventually re-establish steady state conditions typified by quiescence in adult HSCs. . To this end, we used three converging approaches, at the cellular level, by imposing a proliferative stress on HSCs, a genetic approach, by deleting genes of interest and genome-wide transcriptomics. More precisely, HSC resistance to proliferative stress has been evaluated under two extreme conditions; i.e. by consecutive treatments with the chemotherapeutic drug 5-fluorouracil (5-FU), mimicking a clinical situation in cancer chemotherapy, and by serial transplantation assays with limited cell numbers. Moreover, to test if a genetic network regulates HSCs functions, we also used three mouse models, i.e. Scl+/-, E2a+/- et Heb+/-. Using these tools, we showed that HSC adaptation to proliferative stress and return to steady state is strictly regulated by Scl expression levels that restricts ribosomal gene expression. Moreover, despite some degree of redundancy within this network, Heb expression levels restrain the excessive proliferation of HSC upon stress conditions by inducing senescence, a function that cannot be compensated for by E2a. To conclude, our results show that HSCs can tolerate both proliferative stress and unrepaired DNA damages without affecting their primary function to replenish the hematopoietic system. This is especially true if their metabolism can come back to basal levels. However, with increased numbers of cell divisions, HSC will sooner or later reach their expansion limit and enter senescence

<i>SCL</i>, <i>LMO1</i> and <i>Notch1</i> Reprogram Thymocytes into Self-Renewing Cells

<div><p>The molecular determinants that render specific populations of normal cells susceptible to oncogenic reprogramming into self-renewing cancer stem cells are poorly understood. Here, we exploit T-cell acute lymphoblastic leukemia (T-ALL) as a model to define the critical initiating events in this disease. First, thymocytes that are reprogrammed by the SCL and LMO1 oncogenic transcription factors into self-renewing pre-leukemic stem cells (pre-LSCs) remain non-malignant, as evidenced by their capacities to generate functional T cells. Second, we provide strong genetic evidence that SCL directly interacts with LMO1 to activate the transcription of a self-renewal program coordinated by LYL1. Moreover, LYL1 can substitute for SCL to reprogram thymocytes in concert with LMO1. In contrast, inhibition of E2A was not sufficient to substitute for SCL, indicating that thymocyte reprogramming requires transcription activation by SCL-LMO1. Third, only a specific subset of normal thymic cells, known as DN3 thymocytes, is susceptible to reprogramming. This is because physiological NOTCH1 signals are highest in DN3 cells compared to other thymocyte subsets. Consistent with this, overexpression of a ligand-independent hyperactive <i>NOTCH1</i> allele in all immature thymocytes is sufficient to sensitize them to SCL-LMO1, thereby increasing the pool of self-renewing cells. Surprisingly, hyperactive <i>NOTCH1</i> cannot reprogram thymocytes on its own, despite the fact that <i>NOTCH1</i> is activated by gain of function mutations in more than 55% of T-ALL cases. Rather, elevating <i>NOTCH1</i> triggers a parallel pathway involving <i>Hes1</i> and <i>Myc</i> that dramatically enhances the activity of <i>SCL-LMO1</i> We conclude that the acquisition of self-renewal and the genesis of pre-LSCs from thymocytes with a finite lifespan represent a critical first event in T-ALL. Finally, <i>LYL1</i> and <i>LMO1</i> or <i>LMO2</i> are co-expressed in most human T-ALL samples, except the cortical T subtype. We therefore anticipate that the self-renewal network described here may be relevant to a majority of human T-ALL.</p></div

Model of the collaboration between the SCL, LMO1 and <i>Notch1</i> oncogenes.

<p>(<b>A</b>) <i>SCL</i> and <i>LMO1</i> interact to upregulate <i>Lyl1</i> gene expression and create a feed forward loop that activates self-renewal in DN3 thymocytes. DN3 cells are prone to <i>SCL-LMO1</i> self-renewal activity due to higher physiological NOTCH levels. (<b>B</b>) The <i>Notch1</i> oncogene drastically enhances <i>SCL-LMO1</i>-induced self-renewal activity to expand the pool of target cells to DN1-4 and ISP8 in a parallel pathway via <i>Hes1</i> and <i>c-Myc</i>. <i>SCL-LMO1</i> initiated cells (A) subsequently acquire gain of function <i>Notch1</i> mutations (B), causing target cell expansion and escape from thymic environmental control.</p

Transcription activation driven by SCL-LMO1 interaction is critical for thymocyte reprogramming and T-ALL induction.

<p>(<b>A</b>) Generation of transgenic mice expressing the LMO1-binding defective mutant SCLm13. The sequence coding for wild type human SCL or human SCLm13 HLH domain mutant <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004768#pgen.1004768-Lecuyer1" target="_blank">[37]</a> were cloned into the VA h<i>CD2</i> cassette to generate transgenic mice. Shown are amino acids of the HLH region of SCL or SCLm13. (<b>B</b>) Immunofluorescence of human SCL (wt or m13) by flow cytometry. Thymocytes were stained with the monoclonal antibody against human SCL (BTL73). Control cells were stained with the second antibody only. (<b>C</b>) Expression of E protein target genes is inhibited both by <i>SCL-LMO1</i> and <i>SCLm13-LMO1</i> transgenes in DN3 thymocytes. mRNA levels in purified DN3 thymocytes from the indicated transgenic mice were determined by qRT-PCR and normalized to <i>β-Actin</i> (Mean +/- SD, n = 3). (<b>D</b>) Kaplan-Meier curves of the time to leukemia for <i>LMO1^tg</i>, <i>E2a^+/-LMO1^tg</i>, <i>SCL^tgLMO1^tg</i> and <i>SCLm13^tgLMO1^tg</i> mice. (<b>E</b>) The interaction between SCL and LMO1 is required to activate the transcription of the self-renewal genes <i>Lyl1</i>, <i>Hhex</i> and <i>Nfe2</i> in DN3 thymocytes. mRNA levels in purified DN3 thymocytes from the indicated transgenic mice were determined by qRT-PCR and normalized to <i>β-Actin</i> (Mean +/- SD, n = 3). (<b>F–G</b>) SCL but not the LMO1-binding defective SCL-m13 mutant collaborates with LMO1 to induce abnormal thymic reconstitution potential to thymocytes. Pre-leukemic thymocytes (1.5×10⁷ cells) from 3-week-old mice were transplanted. Recipient mice were analysed for thymic reconstitution (CD45.2⁺Thy1⁺) after 6 weeks (F) and the proportion of DP cells in engrafted CD45.2⁺Thy1⁺ thymocytes was assessed by FACS (G).</p

Notch1 collaborates with SCL-LMO1 to increase the pool of pre-LSCs and their competitiveness independently of a functional pre-TCR.

<p>(<b>A</b>) The engraftment of <i>SCL-LMO1</i> DN3 thymocytes is abrogated by γ-secretase inhibitor (GSI) treatment prior to transplantation. DN3 thymocytes were purified from pre-leukemic <i>SCL</i>^tg<i>LMO1</i>^tg mice and co-cultured on OP9-DL1 stromal cells in the presence or absence (vehicle) of 2.5 µM DAPT (GSI) for 4 days. The total numbers of viable cells recovered per culture are shown (<i>right panel</i>). Following drug treatment, equal numbers of viable cells were transplanted (5×10⁴ per mouse, n = 5). Engrafted mice: number of positive mice showing thymic reconstitution per group. (<b>B</b>) A hyperactive <i>Notch1</i> allele is insufficient to induce aberrant self-renewal in thymocytes but significantly enhances the engraftment of <i>SCL</i>^tg<i>LMO1</i>^tg thymocytes. Total thymocytes (1.5×10⁷) from 1-week-old mice of the indicated genotype were transplanted; recipient mice were analyzed for thymic engraftment 3 weeks later. (<b>C</b>) Oncogenic <i>Notch1</i> increases the frequencies of <i>SCL-LMO1</i> pre-LSCs independently of a functional pre-TCR. Purified DN3 thymocytes from <i>SCL</i>^tg<i>LMO1</i>^tg and <i>Notch1</i>^tg<i>SCL</i>^tg<i>LMO1</i>^tg mice with (<i>Cd3ε</i>^+/+) or without (<i>Cd3ε</i>^-/-) a functional pre-TCR were transplanted in limiting dilution assays (<i>upper panel</i>). Mice were scored positive when T-cell lineage reconstitution was more than 1%; pre-LSC frequencies and confidence intervals (<i>lower panel</i>) were calculated by applying Poisson statistics using the Limiting Dilution Analysis software (StemCell Technologies). (<b>D</b>) <i>Cd3ε</i>^-/-<i>Notch1</i>^tg<i>SCL</i>^tg<i>LMO1</i>^tg pre-leukemic thymocytes outcompete <i>Cd3ε</i>^-/-<i>SCL</i>^tg<i>LMO1</i>^tg thymocytes. Reconstitution by <i>Cd3ε</i>^-/-<i>Notch1</i>^tg<i>SCL</i>^tg<i>LMO1</i>^tg (CD45.2⁺ GFP^-, closed circles) and <i>GFP</i>^tg<i>Cd3ε</i>^-/-<i>SCL</i>^tg<i>LMO1</i>^tg (CD45.2⁺ GFP⁺, open circles) thymocytes transplanted with the indicated cell numbers at 1∶1 or 1∶20 ratio. (<b>E</b>) <i>Notch1</i> expands the cellular targets of <i>SCL-LMO1</i> to DN1-4 and ISP8 cells. Pre-leukemic thymocyte subsets (DN1-4, ISP8 and DP) were purified from <i>Notch1^tgSCL^tgLMO1</i>^tg mice and transplanted at 5×10⁴ cells per recipient mouse. The absolute numbers of donor-derived DN1-4 and ISP8 cells was calculated for each transplantation.</p