31 research outputs found

    The Embryonic Protein Nodal Supports Metastatic Phenotypes in Breast Cancer

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    Metastasis is the process by which tumour cells disseminate to distant organ sites. Aberrant expression of stem cell-associated proteins within tumours is associated with metastasis and poor patient prognosis. One example of a stem cell factor that is associated with cancer progression is Nodal, a member of the TGF-β superfamily. Nodal is normally limited to pluripotent stem cells during embryonic development, and to specialized dynamic adult tissue (such as the cycling endometrium), but is aberrantly re-expressed in multiple cancer types, including melanoma, glioma, prostate cancer, and pancreatic cancer. The central objective of this thesis is to determine the role of Nodal during various aspects of the metastatic cascade in breast cancer. First, I determined that Nodal inhibition in aggressive breast cancer cell lines impairs tumour growth in an orthotopic nude mouse model, concomitant with reduced proliferation and enhanced apoptosis. Furthermore, in an experimental metastasis assay in NOD/SCID/MPSVII mice, I determined that Nodal knockdown prevents the transition from lung micrometastases to macrometastes, by supporting a positive ratio of proliferation to apoptosis. Using numerous animal models, I then discovered that Nodal promotes angiogenesis, and that knocking down its expression in established tumours reduces vascularization and causes necrosis. Notably, Nodal protein was positively correlated with vascular density in human breast cancer lesions. Mechanistically, Nodal induced a pro-angiogenic profile in breast cancer cells by upregulating VEGF and PDGF. Finally, I investigated the role of Nodal in the regulation of EMT and invasion; phenotypes that are classically associated with this morphogen. Specifically, since Nodal is implicated in mammary gland remodeling and placentation, I examined its effects on cellular invasion in these contexts. Nodal overexpression in poorly metastatic breast cancer and choriocarcinoma cell lines enhanced invasion and EMT-associated changes in gene expression, and this effect was in part mediated by ERK signaling. Nodal inhibition in metastatic breast cancer cell lines reduced spontaneous metastasis to the liver (but not the lung) in NOD/SCID/IL2γR- mice. The results presented herein suggest that Nodal promotes several pro-metastatic processes. Given its restriction to embryonic or highly specialized adult contexts, targeting Nodal in breast cancer poses an exciting avenue for therapeutic intervention

    Integrative and perturbation-based analysis of the transcriptional dynamics of TGFβ/BMP system components in transition from embryonic stem cells to neural progenitors

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    Cooperative actions of extrinsic signals and cell-intrinsic transcription factors alter gene regulatory networks enabling cells to respond appropriately to environmental cues. Signaling by transforming growth factor type β (TGFβ) family ligands (eg, bone morphogenetic proteins [BMPs] and Activin/Nodal) exerts cell-type specific and context-dependent transcriptional changes, thereby steering cellular transitions throughout embryogenesis. Little is known about coordinated regulation and transcriptional interplay of the TGFβ system. To understand intrafamily transcriptional regulation as part of this system's actions during development, we selected 95 of its components and investigated their mRNA-expression dynamics, gene-gene interactions, and single-cell expression heterogeneity in mouse embryonic stem cells transiting to neural progenitors. Interrogation at 24 hour intervals identified four types of temporal gene transcription profiles that capture all stages, that is, pluripotency, epiblast formation, and neural commitment. Then, between each stage we performed esiRNA-based perturbation of each individual component and documented the effect on steady-state mRNA levels of the remaining 94 components. This exposed an intricate system of multilevel regulation whereby the majority of gene-gene interactions display a marked cell-stage specific behavior. Furthermore, single-cell RNA-profiling at individual stages demonstrated the presence of detailed co-expression modules and subpopulations showing stable co-expression modules such as that of the core pluripotency genes at all stages. Our combinatorial experimental approach demonstrates how intrinsically complex transcriptional regulation within a given pathway is during cell fate/state transitions

    Towards cardiac cell therapy

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    Tese de doutoramento em Biologia (Biologia Molecular), apresentada à Universidade de Lisboa através da Faculdade de Ciências, 200

    Targeting TGFβ signaling pathway in fibrosis and cancer

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    Cancer and fibrosis are devastating diseases of high mortality rate and with limited curative therapies available. A better understanding of the biological drivers of these diseases is fundamental in order to develop effective therapeutics. At the molecular level, signaling pathways control cell growth, differentiation or apoptosis during development and adult life of the organism ensuring homeostasis. Paradoxically, the same signals are often implicated or even drive disease progression. One of the signaling pathways with key regulatory functions in homeostasis, tissue fibrosis and cancer in many organs is the TGFβ/BMP pathway. In this thesis we addressed the role and therapeutic potential of TGFβ/BMP pathway inhibition using different drug compounds that are currently towards the clinic or being tested in clinical trials. Three distinct types of inhibitors were used; small molecule inhibitors of the ALK4, 5 and 7 TGFβ receptor kinases, an antisense oligonucleotide interfering with ALK5 mRNA splicing and an ALK1 ligand trap; a peptide that contains the extracellular domain of ALK1 fused to Fc and sequesters BMP9 and BMP10. These inhibitors were used in an ex vivo human fibrosis model and in vivo mouse models of various human diseases (acute liver failure/ liver regeneration, Dupuytren's fibrosis) and cancer (prostate, liver).UBL - phd migration 201

    Epigenetic events underlying somatic cell reprogramming

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    Although differentiated cells normally retain cell-type-specific gene expression patterns throughout their lifetime, cell identity can sometimes be modified or reversed in vivo by transdifferentiation, or experimentally through cell fusion or by nuclear transfer. Several studies have illustrated the importance of chromatin remodelling, DNA demethylation and dominant transcriptional factor expression for changes in lineage identity. Here the epigenetic mechanisms required to “reset” genome function were investigated using experimental heterokaryons. To examine the epigenetic changes that are required for the dominant conversion of lymphocytes to muscle, I generated stable heterokaryons between human B-lymphocytes and mouse C2C12 myotubes. I show that lymphocyte nuclei adopt an architecture resembling that of muscle and initiate the expression of musclespecific genes in the same temporal order as developing muscle. The establishment of this novel gene expression program is coordinated with the shutdown of several lymphocyte-associated genes. Interestingly, inhibition of histone deacetylase (HDAC) activity during reprogramming selectively blocks the silencing of lymphocyte-specific genes but does not prevent the establishment of muscle-specific gene expression. In order to reprogram somatic cells to pluripotency, I fused human Blymphocytes and mouse embryonic stem (ES) cells. The conversion of human cells is initiated rapidly, occurring in heterokaryons before nuclear fusion. Reprogramming of human lymphocytes by mouse ES cells elicits the expression of a human ES-specific gene expression profile in which endogenous hSSEA4, hFgf receptors and ligands are expressed while factors that are characteristic of mouse ES cells, such as Bmp4 and Lif receptor are not. Using genetically engineered mouse ES cells I demonstrate that successful reprogramming requires the expression of Oct4, but importantly, does not require Sox2, a factor implicated as critical for the induction of pluripotency. Following reprogramming, mOct4 becomes dispensable for maintaining the multi-potent state of hybrid cells. Finally, I have examined the reprogramming potential of embryonic germ (EG), embryonic carcinoma (EC) and ES cells deficient for the Polycomb repressive complex 2 (PRC2) proteins Eed, Suz12 and Ezh2. While EC and EG cells share the ability to reprogram human lymphocytes with ES cells, the lack of Polycomb proteins abolishes reprogramming. Thus, the repressive chromatin mark (H3K27 methylation) catalysed by PRC2 play a crucial role in keeping ES cells with full reprogramming capacity. Collectively my results underscore the importance of chromatin events during cell fate reprogramming

    The role of hypoxia signalling pathways in normal and leukaemic haemopoiesis

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    Although haemopoietic stem cells (HSCs) represent one of the best-defined stem cell systems, the pathways regulating HSC development and maintenance are not fully understood. HSCs reside in the hypoxic niche and maintain intracellular hypoxia. Hypoxia and hypoxia signalling pathways are thought to play a vital role in HSC maintenance. Hypoxia inducible factors (Hifs) are evolutionarily conserved and are the key regulators of hypoxia. Hifs consist of an unstable, oxygen-dependent α-subunit and an oxygen-independent stable β-subunit. The two main isoforms of Hif-α, namely Hif-1α and Hif-2α, are critical for the response to hypoxia. Hif-mediated pathways have been extensively studied and have been shown to regulate metabolic adaptation and to influence various cellular mechanisms, including cell growth, survival, differentiation and apoptosis, erythropoiesis and angiogenesis. Hif-1α has been shown to be essential for maintenance of HSC functions under stressful conditions of serial transplantation and aging, but the role of Hif-2α and the interplay between Hif-1α and Hif-2α in regulating HSC functions and their niche is not known. Hence, in this study, I have investigated the role of Hif-α in HSC functions. Furthermore, published evidence suggested that leukaemic stem cells (LSC) share the hypoxic properties with HSCs. Cited2, a hypoxia-inducible Hif-1α and Hif-2α target gene, is critical for embryonic and adult haemopoiesis and possesses oncogenic properties. I have investigated the role of Cited2 in AML generation. The results demonstrate that Hif-2α is not essential for maintenance of HSC functions in a cell-autonomous manner under steady state and stressful conditions of serial transplantation and aging. It is also evident that HSCs lacking Hif-2α together with Hif-1α successfully maintain normal haemopoiesis. However, the data in this thesis show that Hif-2α is essential for non-cell-autonomous maintenance of HSC functions, particularly in males and current work also indicate that a previously unappreciated complex interplay between Hif-1α- and Hif-2α-dependent signalling is required for adult HSC maintenance in a non-cell-autonomous manner. Additionally, the data demonstrate that haemopoietic stem and progenitor cells (HSPCs) lacking Cited2 display reduced transformation potential and failure to generate transplantable AML in vivo. Overexpression of Mcl-1 (an anti-apoptotic gene), in Cited2Δ/Δ cells bypassed their defective transformation potential forming transformed colonies in vitro. Hence, the data in this thesis provide evidence that Cited2 is essential for leukaemic transformation at least in part via Mcl-1 regulation

    Role of the BMP9/ALK1 pathway in the regulation of pathological and VEGF-mediated angiogenesis

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    L’angiogenèse est définie comme la formation de nouveaux capillaires à partir des vaisseaux sanguins pré-existants. Elle contribue à l’extension du réseau vasculaire et assure ainsi l’efficacité des échanges gazeux et du transport des cellules, nutriments, métabolites et molécules de signalisation vers les tissus. L’angiogenèse par bourgeonnement passe par la spécification d’une cellule endothéliale en cellule meneuse et la formation d’un réseau de cellules suiveuses à la base du bourgeon vasculaire. Toute perturbation de ce mode de néovascularisation génère des vaisseaux fortement tortueux, immatures et non-étanches qui soit affectent les fonctions physiologiques des organes en causant ainsi des pathologies potentiellement fatales, ou accélèrent la progression des conditions telles que le cancer. Dans l’oeil, l’angiogenèse pathologique des vaisseaux choroïdiens et rétiniens cause une perte de la vue. Particulièrement, la dégénérescence maculaire liée à l’âge (DMLA) de type néovasculaire, une maladie oculaire caractérisée par le bourgeonnement anormal de la choroïde dans l’espace sous-rétinien, représente la cause majeure de cécité au sein des populations des pays industrialisés. Les thérapies conventionnelles contre la DMLA humide reposent sur l’usage des médicaments qui ciblent la signalisation du facteur de croissance de l’endothélium vasculaire (VEGF). Bien que démontrant des résultats cliniques, ces traitements anti-VEGFs sont invasifs et présentent multiples effets secondaires. Par ailleurs, ils n’induisent aucun effet chez une portion des patients traités. De ces faits, il existe présentement un grand besoin de thérapies alternatives aux anti-VEGFs. De façon intéressante, la protéine de morphogénèse osseuse 9 (BMP9), qui active son récepteur “activin receptor-like kinase 1” (ALK1), régule l’angiogenèse développementale des vaisseaux rétiniens de l’oeil de la souris. Par ailleurs, les mutations au sein du BMP9, de son récepteur ALK1 ou de ses intermédiaires de signalisation sont associées à la morphogénèse anormale des vaisseaux qui contribue ultimement à la pathogénèse de diverses maladies néovasculaires. De façon additionnelle, le récepteur ALK1 au BMP9 est restreint à la cellule endothéliale; contrairement à ceux des ligands angiogéniques tels que le VEGF, exprimés par une diversité de cellules. Par ailleurs, au sein de cette cellule, le BMP9 contribue à la régulation des phénotypes meneur et suiveur qui sont induits par le VEGF et requis pour le déroulement de l’angiogenèse par bourgeonnement. De ces faits qui précèdent, nous avons émis l’hypothèse du rôle du BMP9 dans la régulation de la néovascularisation pathologique relative à la DMLA humide. Ainsi, les travaux de la présente thèse déterminent spécifiquement l’effet du BMP9 sur l’angiogenèse pathologique à l’aide des modèles oculaires pertinents à la DMLA humide et examine aussi sa base mécanistique. Les travaux de cette thèse démontrent l’effet anti-angiogénique du BMP9 sous les conditions expérimentales de néovascularisation choroïdienne induite au laser (CNV) et de rétinopathie induite par l’oxygène (OIR). Ils montrent aussi les effets régulateurs de la signalisation du BMP9 sur les voies de signalisation endothéliales du VEGF et de Notch, respectivement de façon dépendante de VEGFR1 et de JAG1. En somme, les présentes études démontrent les effets anti-angiogéniques du BMP9 sur la néovascularisation pathologique relative à la DMLA humide et identifient les facteurs moléculaires qui contribuent à son action inhibitrice du bourgeonnement vasculaire induit par le VEGF.Angiogenesis is defined as the formation of new capillaries from existing blood vessels. It extends the vasculature and thereby sustains the efficient exchange of gases and transport of cells, nutrients, metabolites and signalling molecules to tissues. Sprouting angiogenesis proceeds through the selective specification of an endothelial cell into a leading tip cell and the formation of stalk cells at the base of the sprout. A disturbance in this modality of neovascularisation leads to highly tortuous, immature and leaky vessels that either impair the physiological functions of organs, thereby causing life-threatening diseases, or accelerate the progression of conditions such as cancer. In the eye, the pathological angiogenesis of choroidal and retinal vessels specifically results in vision loss. Particularly, the neovascular form of the age-related macular degeneration (AMD), an ocular disease characterized by the abnormal sprouting of the choroidal network into the subretinal space, represents the leading cause of blindness in populations of industrialized countries. Conventional therapies against wet AMD are based on drugs that target the signaling of the vascular endothelium growth factor (VEGF). Despite their clinical achievements, the anti-VEGFs treatments are invasive and show multiple adverse effects. Moreover, they are not effective in a portion of treated patients. Thus, there currently is a substantial need of therapy alternatives to anti-VEGFs. Interestingly, the bone morphogenetic protein 9 (BMP9), that activates its activin receptor-like kinase 1 (ALK1) transducer, regulates the developmental angiogenesis of the mouse eye retina vasculature. Moreover, mutations in BMP9, its receptor ALK1 or its signaling mediators correlate with the abnormal vessel morphogenesis that ultimately drives the pathogenesis of various neovascular diseases. Additionally, the BMP9-specific receptor ALK1 is restricted to endothelial cells; in contrast to those of neovascularisation-inducing ligands such as VEGF, expressed by a range of cells. Particularly within these cells, BMP9 contributes to regulate the VEGF-induced tip/stalk phenotypes required for sprouting angiogenesis. Given the aforementioned, we hypothesized the role of BMP9 in regulating the pathological angiogenesis associated with wet AMD. Thus, the studies from the current thesis specifically determine the effect of BMP9 on pathological NV using ocular models relevant to AMD and further investigate its mechanical basis. The current work demonstrates the antiangiogenic effects of BMP9 under experimentally induced oxygen-induced retinopathy (OIR) and laser-induced choroid neovascularisation (CNV) conditions. Moreover, this thesis shows the regulatory effects of BMP9 signaling on the VEGF and Notch endothelial pathways, respectively in VEGFR1 and JAG1 -dependent manners. Collectively, the current studies demonstrate the anti-angiogenic effects of BMP9 on pathological NV associated with wet AMD and identify the molecular players that mediate its inhibitory action on VEGF-mediated sprouting

    Role of activin receptors in driving central nervous system regeneration of myelin

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    Myelin damage in central nervous system white matter disorders such as multiple sclerosis (MS) leads to axonal dysfunction/degeneration and clinical disability. Regeneration of myelin (termed remyelination) can occur and requires oligodendrocyte progenitor cells (OPCs) to differentiate into mature oligodendrocytes, which are then able to make contact with axons and ensheath them. However, this process fails in progressive MS. The lack of approved therapies aimed at promoting remyelination highlight the need to identify mechanisms driving this regenerative process to develop novel therapeutic strategies. Previous work in the lab identified the TGF-β superfamily member activin-A as being increased during remyelination in vivo and sufficient in stimulating activin receptor-driven OPC differentiation into mature oligodendrocytes in vitro. Here, these studies were followed up by undertaking a comprehensive assessment of the role of activin receptors and their ligands during remyelination. Using an ex vivo brain explant model of demyelination, the stimulation of activin receptors using activin-A was sufficient to enhance remyelination. Blocking activin receptors using an endogenous inhibitor (Inhibin) hindered remyelination, demonstrating the requirement of activin receptor signalling for this process. Surprisingly, blocking the binding of endogenous activin-A to activin receptors using follistatin did not impact remyelination, suggesting that other activin receptor ligands are involved in driving remyelination. As activin receptors may bind other ligands in the TGF-β superfamily, the expression and function of alternative ligands was investigated, and each was found to be important for remyelination (albeit with distinct timing/ effects). Both activin receptors and their ligands were expressed on microglia/macrophages in mouse and human disease tissue. Finally, analysis of activin receptor expression on oligodendrocytes in human tissue revealed potential functional differences between receptor subtypes. Together, these results demonstrate previously undefined roles of a subset of TGF-β superfamily members in regulating remyelination, and have implications for the development of novel approaches to enhancing remyelination in disease

    Functional analysis of the cardiogenic transcription factor mespa in mesoderm differentiation in Xenopus laevis

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    Progenitors of the heart, the vasculature, musculature, connective and supporting tissue, the gonads, the kidneys and lower urinary tract derive from the mesodermal germ layer. The knowledge about the highly complex processes and involved factors during induction and differentiation of progenitor cells are of great scientific and medical interest. It helps understand the underlying molecular genetical basics of congenital malformations on one hand and develop new therapeutic options for personalised medicine on the other hand. The bHLH transcription factor mesp1 became the focus of research as master regulator of cardiogenesis. It was shown that mesp1 is already expressed early during gastrulation and that mesp1 activates additional important regulators of cardiovascular differentiation. The knockout of the mesp1 gene in mice leads to a so-called cardia bifida, a failed fusion of the heart tube, whereas the simultaneous deactivation of mesp1 and the related mesp2 result in a complete loss of the heart anlagen. Since mesp1 and its homologue mespa of the African claw frog Xenopus laevis are activated early, further exceeding functions concerning the differentiation of further mesodermal tissues are supposed. Xenopus laevis is a particularly proper model organism due to its easy manipulability and approachability of all developmental stages. In this study, relations between mespa and mesodermal precursor cells have been examined in different developmental stages based on gene expression analysis after knockout or overexpression of mespa. It was shown, that mespa is required for the expression of early mesodermal markers eomes and xbra during gastrulation and neurulation. Moreover, mespa plays a role in skeletomyogenesis and in the development of facial structures, but is not sufficient to activate myogenic markers on its own. Furthermore, it was demonstrated that mespa is involved in the development of precursors of vasculogenesis and embryonic haematopoiesis. Formerly described effects on the epithelial-to-mesenchymal transition could not be confirmed. Likewise, no direct relation towards nephrogenesis was shown. In conclusion, it was demonstrated that mespa exerts extensive functions during the development of mesodermal tissues