Phosphoinositide dependent kinase 1. PI3K. Phosphoinositide 3-kinases ..... 2.5.5.1 Mechanism of CIP2A-mediated inhibition of Myc degradation. ..... CSCs, for example, express the neural stem cell marker and cell surface ...... kinases (such as mTOR or MAPK signalling pathway members, not shown), which results in.
Helsinki University Biomedical Dissertations no. 117
Neural stem cell characteristics affected by oncogenic pathways and in a human motoneuron disease
Laura Kerosuo Medical Biochemistry and Developmental Biology Institute of Biomedicine Faculty of Medicine University of Helsinki HBGS
ACADEMIC DISSERTATION To be presented, with the permission of the Faculty of Medicine of the University of Helsinki, for public criticism and debate in Auditorium 3, Biomedicum Helsinki, on March 20th at 12 o’clock noon.
Helsinki 2009
Supervised by: Kirmo Wartiovaara, MD., PhD. and Professor Hannu Sariola, MD., PhD. Medical Biochemistry and Developmental Biology Institute of Biomedicine Faculty of Medicine University of Helsinki
Reviewed by: Professor Johanna Ivaska, PhD. Cell Adhesion and Cancer Laboratory VTT Medical Biotechnology and University of Turku and Professor Dan Lindholm, MD., PhD. Unit of Neuroscience The Minerva Foundation Institute for Medical Research, Helsinki
Opponent: Professor Eero Castrén, MD., PhD. Neuroscience Center University of Helsinki
ISSN 1457-8433 ISBN 978-952-10-5312-2 (paperback) ISBN 978-952-10-5313-9 (pdf) http://ethesis.helsinki.fi Yliopistopaino Helsinki 2009
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To Klara and Hugo. You two have convinced me that despite the fascination that science at its best can provide, the challenges of practical developmental biology are even more demanding and, most importantly, more rewarding.
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List of original publication This thesis is based on the following publications, which will be referred to in the text by the Roman numerals I-III. Additional unpublished results are referred to as unpublished in the text.
I Kerosuo Laura, Piltti Katja, Fox Heli, Angers-Loustau Alexander, Häyry Valtteri, Eilers Martin, Sariola Hannu, Wartiovaara Kirmo, (2008). Myc increases self-renewal in neural progenitor cells through Miz-1. Journal of Cell Science. Dec 1;121:3941-50.
II Kerosuo Laura, Fox Heli, Perälä Nina, Westermarck Jukka, Sariola Hannu, Wartiovaara Kirmo, (2009). CIP2A increases self-renewal and is linked to Myc expression in neural progenitor cells. Cell Research, in revision January 2009.
III Pakkasjärvi Niklas, Kerosuo Laura, Nousiainen Heidi, Gentile Massimiliano, Saharinen Juha, Suhonen Satu, Sariola Hannu, Peltonen Leena, Kestilä Marjo, Wartiovaara Kirmo, (2007). Neural precursor cells from a fatal human motoneuron disease differentiate despite aberrant gene expression. Developmental Neurobiology Feb 15;67:270-84.
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Abbreviations AJ Akt ALS aPKC ARF ASPM BDNF bHLH bHLH/LZ Bcl BLBP BMP BrdU CDK C/EBP CIP2A CNTF CSC DG Dkk1 E EGF EGFR ERBB ERK ES FCS FGF Fox G GFAP GMC GSK3 HAT HER Hes HNSCC Hox HSC ICM IL Insp6 iPS Isl1 JAK2 LCCS Lgl Lhx LT-HSC LIF LIM LIM-HD MAPK MB MEF MEK Mibp1 Miz-1
Adherence junction Cellular homolog of the acute transforming retrovirus in mice Amyotrophic lateral sclerosis Atypical protein kinase C ADP ribosylation Factor Spindle –like microencephaly associated protein Brain derived neurotrophic factor Basic helix-loop-helix Basic helix-loop-helix leucine zipper B-cell lymphoma gene family Brain lipid binding protein Bone morphogenetic proteins 5-bromo-2-deoxyuridine Cyclin dependent kinase CAAT/enhancer binding protein Cancerous inhibitor of PP2A Ciliar neurotrophic factor Cancer stem cell Dentate gyrus Dickkopf1 Embryonic day in mouse Epidermal growth factor EGF-receptor EGFR family receptor, also called HER Extracellular signal related kinase Embryonic Stem (cell) Foetal calf serum Fibroblast growth factor Forkhead-box Gap, a phase in cell cycle Glial fibrillary acidic protein Ganglion mother cell Glycogen synthase kinase 3 Histone acetyltransferases Human epidermal growth factor receptor Hairy and enhancer of split Head and neck squamous cell carcinoma Homeodomain transcription factor Hematopoietic stem cell Inner cell mass Interleukin Inositol hexakisphosphate 6 Induced pluripotent stem cell LIM-HD transcription factor Islet 1 Janus kinase 2 Lethal Congenital Contracture Syndrome Lethal gigantic larvae LIM homeobox gene Long term haematopoietic stem cell Leukaemia inhibitory factor Lin11 and Mec3 domain LIM-Homeodomain Mitogen activated protein kinase Myc homology box Mouse embryonic fibroblast Mitogen activated protein kinase kinase Myc intron I binding protein Myc-interacting Zn finger protein-1
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MSC mTORC2 Myc Myc V394D NRSF NGF Ngn NOD/SCID NPC NPM Oct Par Pax PDGF PDPK1 PI3K PIG3 PIK3 PIP3 PIPK1 PKB PP2A PtdIns PTEN PTK PUMA Ras Rb REST RGC Rex RTK RT-PCR SCM SGZ Shh SMAD SNARE Sox SRY STAT3 SVZ TA TAD TCF TCR TGF TRRAP Tuj-1 ZFP VAV VZ WB Wnt
Mesenchymal stem cell The mammalian target of Rapamycin complex 2 Family of oncogenes that are homologues to the avian Myelocytomatosis virus Myc Neuron-restrictive silencer factor Neural growth factor Neurogenin Non-obese diabetic, severe combined immunodeficient (mice) Neural Progenitor cell Nucleophosmin Octamer Protease-activated receptor
Paired box Platelet derived growth factor Phosphoinositide dependent kinase 1 Phosphoinositide 3-kinases p53-induced gene 3 Phosphatidylinositol-3 kinase phosphatidylinositol (3,4,5)-trisphosphate Phosphatidylinositol-4-phosphate 5-kinase, type I, gamma Protein kinase B, also called Akt Protein phosphatase 2A Phosphoinositides Phosphatase and Tensin homolog Non-receptor protein tyrosine kinase p53 upregulated mediator of apoptosis Responsible for activities in sarcoma Retinoblastoma tumour suppressor Repressor element-1 silencing transcription factor Radial glial cell Reduced expression Receptor tyrosine kinase Reverse transcription polymerase chain reaction Stem cell medium Subgranular zone Sonic hedgehog A combination of Mothers against decapentaplegic (MAD in Drosophila) and SMA in C. elegans soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor SRY-box Sex determining region Y Signal transducer and activator of transcription 3 Subventricular zone Transit amplifying (cell) Transcriptional activation domain Transcription factor 7-like T cell receptor Transforming growth factor Transformation/transcription domain-associated protein -III-tubulin Zink finger protein An oncoprotein family named after the sixth letter of the Hebrew alphabet Ventricular zone Western blotting Wingless and Int
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Table of Contents: ABSTRACT......................................................................................................................................9 1. INTRODUCTION ...................................................................................................................... 10 2. REVIEW OF THE LITTERATURE ......................................................................................... 12 2.1 STEM CELLS ................................................................................................................... 12 2.1.1 Embryonic stem cells ................................................................................................. 12 2.1.2 Tissue specific stem cells............................................................................................ 13 2.1.3 Cancer stem cells....................................................................................................... 14 2.2 NEURAL DEVELOPMENT .................................................................................................. 15 2.2.1 Development of the mammalian nervous system............................................................... 15 2.2.2 The cell cycle.................................................................................................................. 18 2.2.3 Symmetric and asymmetric cell divisions......................................................................... 20 2.2.4 Neurogenesis in adult CNS.............................................................................................. 24 2.2.5 The neural stem cell niche............................................................................................... 25 2.2.6 Neural and glial differentiation ....................................................................................... 27 2.2.6.1 Neurogenesis ......................................................................................................................... 27 2.2.6.2 Gliogenesis ............................................................................................................................ 28 2.2.6.3 Signalling cascades that promote neuronal, glial or stem cell fate............................................. 29 2.2.6.4 -Motoneuron development.................................................................................................... 34
2.3 STUDYING NEURAL STEM CELLS ............................................................................................... 36 2.3.1 Neurospheres.................................................................................................................. 36 2.3.2 Using neurospheres as a model to study neurological disease.......................................... 38 2.3.2.1 LCCS .................................................................................................................................... 39
2.4 REGULATION OF SELF-RENEWAL IN NPCS ................................................................................ 41 2.4.1 Rb...................................................................................................................................41 2.4.2.1 Cell cycle regulation............................................................................................................... 41 2.4.2.2 Self-renewal regulation........................................................................................................... 43
2.4.2 Bmi-1.............................................................................................................................. 44 2.4.3 p53 ................................................................................................................................. 45 2.4.4 Akt, PTEN....................................................................................................................... 46 2.4.5 Sox 1-3 ........................................................................................................................... 48 2.5 MYC ............................................................................................................................... 48 2.5.1 The Myc family members and their general expression patterns....................................... 48 2.5.2 The transcription factor Myc ........................................................................................... 50 2.5.2.1 Max and E-Box...................................................................................................................... 50 2.5.2.1 Max and E-Box...................................................................................................................... 51 2.5.2.2 Myc homology boxes ............................................................................................................. 51 2.5.2.3 Miz-1..................................................................................................................................... 52 2.5.2.4 Identification of Myc-specific binding sites............................................................................. 53 2.5.2.5 What regulates Myc?.............................................................................................................. 54
2.5.3 Cellular functions of Myc................................................................................................ 55 2.5.3.1 CELL CYCLE ACTIVATION AND GROWTH............................................................... 55 2.5.3.2 Adhesion ............................................................................................................................... 57 2.5.3.3 Apoptosis............................................................................................................................... 57 2.5.3.4 The oncogenic role of Myc ..................................................................................................... 58
2.5.4 Myc in stem cells............................................................................................................. 60 2.5.4.1 Myc in epidermal stem cells ................................................................................................... 60 2.5.4.2 Myc in haematopoietic stem cells ........................................................................................... 62 2.5.4.3 Myc in intestinal stem cells..................................................................................................... 63 2.5.4.4 Myc in neural stem cells......................................................................................................... 64 2.5.4.5 Myc in embryonic stem cells .................................................................................................. 66
2.5.5
CIP2A ....................................................................................................................... 67
2.5.5.1 Mechanism of CIP2A-mediated inhibition of Myc degradation ................................................ 67 2.5.5.2 The oncogenic function of CIP2A........................................................................................... 67 2.5.5.3 PP2A..................................................................................................................................... 69
3. AIMS OF THE STUDY.............................................................................................................. 72
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4. MATERIALS AND METHODS ................................................................................................ 73 5. RESULTS ................................................................................................................................... 75 5.1 VERIFICATION OF THE SUCCESSFUL GENETIC MODIFICATION OF NPCS (I, II).............................. 75 5.2 ACTIVATION OF ONCOGENIC PATHWAYS INCREASE PROLIFERATION (I, II, III) ............................ 75 5.2.1 Myc and CIP2A increase NPC proliferation in neurospheres........................................... 75 5.2.2 LCCS pathway results in increased proliferation ............................................................. 75 5.3 ACTIVATION OF MYC AND CIP2A INCREASE SELF-RENEWAL (I, II) ........................................... 76 5.4 DIFFERENTIATION CAPACITY IS NOT BLOCKED BY ONCOGENES OR LCCS (I, II, III) .................... 76 5.4.1 Myc or CIP2A do not block differentiation although Myc NPCs are affected.................... 76 5.4.2 Myc and CIP2A delay cell cycle exit (I,II)........................................................................ 77 5.4.3 LCCS derived neurospheres differentiate normally in vitro (III)....................................... 77 5.5 APOPTOSIS IS NOT AFFECTED BY MYC OR LCCS BUT MYC MAY CAUSE CELL PLOIDY (I, III)....... 78 5.6 SELF-RENEWAL MAINTENANCE DURING DIFFERENTIATION : MYC CAUSES RE-SPHERING (I, UNPUBLISHED).............................................................................................................................. 78 5.7 NPC SELF-RENEWAL IS REGULATED VIA MIZ-1 BINDING AND IT CORRELATES WITH THE ACTIVITY OF CIP2A EXPRESSION AND THE MEK/ERK PATHWAY (I, II, UNPUBLISHED) .................................. 80 5.7.1 Myc induced self-renewal is regulated through Miz-1 binding (I)..................................... 80 5.7.2 CIP2A (II)....................................................................................................................... 80 5.7.3 CDKs and MAPK pathways (unpublished) ...................................................................... 81 5.8 NPC MARKER EXPRESSION (I, UNPUBLISHED) ........................................................................... 82 5.9 ENDOGENOUS EXPRESSION OF MYC AND CIP2A (I, II).............................................................. 83 5.10 MICROARRAY ANALYSIS OF LCCS VERSUS CONTROL NEUROSPHERES REVEALED INCREASED EGFR LEVELS IN PATIENT NPCS (III) ............................................................................................ 84 6. DISCUSSION.............................................................................................................................. 86 6.1 MYC INCREASES NEURAL STEM CELL SELF-RENEWAL ............................................................... 86 6.2 MYC INCREASES THE POOL OF SELF-RENEWING STEM CELLS, PROGENITORS OR BOTH ................. 87 6.3 MYC INDUCES SELF-RENEWAL THROUGH MIZ-1 AND MYC EXPRESSION IS LINKED TO CIP2A ..... 88 6.4 MYC CHANGES THE WAY NPCS INTERPRET ENVIRONMENTAL CUES ........................................... 89 6.5 MYC DOES NOT PREVENT DIFFERENTIATION ............................................................................. 90 6.6 SELF-RENEWAL AND PROLIFERATION ARE SEPARATELY REGULATED CELLULAR FUNCTIONS ....... 90 6.7 IT ALL COMES DOWN TO MYC .................................................................................................. 91 6.8 NEUROSPHERE CULTURES PROVIDE INFORMATION ON CELL-AUTONOMOUS FUNCTIONS OF LCCS NEURAL DEVELOPMENT ................................................................................................................ 92 7. CONCLUSIONS......................................................................................................................... 95 ACKNOWLEDGEMENTS............................................................................................................ 96 REFERENCES............................................................................................................................. 101
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ABSTRACT Stem cells provide the self-renewing cell pool for developing or regenerating organs. The mechanisms underlying the decisions of a stem or progenitor cell to either selfrenew and maintain multipotentiality or alternatively to differentiate are incompletely understood. In this thesis work, I have approached this question by investigating the role of the proto-oncogene Myc in the regulatory functions of neural progenitor cell (NPC) self-renewal, proliferation and differentiation. By using a retroviral transduction technique to create overexpression models in embryonic NPCs cultured as neurospheres, I show that activated levels of Myc increase NPC self-renewal. Furthermore, several mechanisms that regulate the activity of Myc were identified. Myc induced self-renewal is signalled through binding to the transcription factor Miz-1 as shown by the inhibited capacity of a Myc mutant (MycV394D), deficient in binding to Miz-1, to increase self-renewal in NPCs. Furthermore, overexpression of the newly identified proto-oncogene CIP2A recapitulates the effects of Myc overexpression in NPCs. Also the expression levels and in vivo expression patterns of Myc and CIP2A were linked together. CIP2A stabilizes Myc protein levels in several cancer types by inhibiting its degradation and our results suggest the same function for CIP2A in NPCs. Our results also support the conception of self-renewal and proliferation being two separately regulated cellular functions. Finally, I suggest that Myc regulates NPC self-renewal by influencing the way stem and progenitor cells react to the environmental cues that normally dictate the cellular identity of tissues containing self-renewing cells. Neurosphere cultures were also utilised in order to characterise functional defects in a human disease. Neural stem cell cultures obtained post-mortem from foetuses of lethal congenital contracture syndrome (LCCS) were used to reveal possible cell autonomous differentiation defects of patient NPCs. However, LCCS derived NPCs were able to differentiate normally in vitro although several transcriptional differences were identified by using microarray analysis. Proliferation rate of the patient NPCs was also increased as compared to NPCs of age-matched control foetuses.
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1. INTRODUCTION Stem cells are the starting material for differentiating cells of continuously self-renewing adult tissues or, during embryogenesis, for building all organs of the developing individual. Like all cells, also a stem cell is highly specialised for its specific functions. A stem cell is able to maintain the capacity to self-renew and thus avoid differentiation. It can also generate a large amount of progeny in an extended period of time. In this thesis work, I have investigated the molecular mechanisms that regulate the maintenance of a self-renewing stem cell identity in embryonic neural stem and progenitor cells. The proteins encoded by proto-oncogenes and tumour suppressors are critical regulators of cellular proliferation and cell cycle activation, metabolism, migration, angiogenesis and apoptosis as well as several other functions that cancer needs in order to grow and survive. Many cancer genes, that in cancer have due to mutations lost their ability to read the regulatory cues provided by the cell and its surroundings, are important regulators of normal development in the embryo. In this study, I have investigated the role of several proto-oncogenes, or developmental genes as they also can be called, in the regulation of neural stem cell identity. The understanding of the regulatory mechanisms behind normal stem cell functions is essential in order to restrain the actions of misregulated oncogenes in terms of cancer therapy. Also, the possibility of exploiting neural stem cells for therapy purposes in patients with a degenerative neural disease such as Parkinson’s or Alzheimer’s disease, Multiple Sclerosis or a trauma-based neurological dysfunction, respectively, has raised high hopes in the stem cell field. Despite various promising studies and trials, more research is needed in order to guarantee the proper actions of the transplanted stem cells in the host including maintenance of self-renewal, correct differentiation ability as well as prevention of tumour formation. 10
Lastly, in vitro neural stem cell cultures can also be used to study pathogenesis of neurological diseases. In this work, neural stem cells from post-mortem foetuses with a lethal motoneuron disease Lethal Congenital Contracture Syndrome (LCCS), which belongs to the Finnish disease heritage, were used to study functional defects of patient neural stem cells in order to understand the cause of the disease.
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2. REVIEW OF THE LITTERATURE 2.1
Stem Cells
2.1.1 Embryonic stem cells All cells of an individual derive from the fertilized oocyte in which the penetration of the sperm induces vast nuclear reprogramming. These events include demethylation and histone acetylation changes as well as the finalization of the meiotic division in the oocyte pronucleus followed by organization of the maternal and paternal chromosomes to the mitotic spindle. The fertilized egg starts to divide. In humans, cells up to the 8cell stage are totipotent as all of these daughter cells have the capacity to form a new individual as well as the extraembryonic tissues needed during the gestation period (Sadler 2006). Once the blastocyst encavity is formed, the cells of the inner cell mass (ICM) are determined to become the developing embryo and the rest of the cells develop into extraembryonic structures of the placenta, chorion, allantois, the yolk sack and the amnion. The ICM cells are pluripotent as they have the capacity to promote formation of all the embryonic layers, namely the endoderm, the mesoderm and the ectoderm, that are induced in gastrulation (Sadler, 2006). Human or mouse embryonic stem cells (ES) are detached for in vitro research purposes either as totipotent from the 8-cell stage embryo or more frequently from the pluripotent ICM. Activation of genes such as Octamer-4 (Oct-4), Nanog, SRY (sex determining region Y)-box 2 (Sox2), Forkhead box d3 (Foxd3) and the Zinc finger protein 42 (ZFP42), also called Reduced expression-1 (Rex1), is essential for ES cells in order to keep them in a self-renewing, undifferentiated state (Hoffman and Carpenter, 2005; Smith, 2001). The capacity of ES cells to in vitro form various different tissues for transplantation therapy purposes has been 12
under intensive study for the last twenty years, although problems have come up concerning the alloantigenic rejection and tumorigenic potential of the cells. The customized reprogramming of patients own fibroblasts into ES-cell like cells (induced pluripotent stem cells, iPS) has given new hope to the field (Nishikawa et al., 2008; Wernig et al., 2007).
2.1.2 Tissue specific stem cells Tissue specific stem cells, or adult stem cells as they are called in adult tissues, have the potential to form all the cell types for their specific organs and are thus multipotent. Stem cells are found at least in the adult skin, bone marrow, gut, and to some extent the brain (Gage, 2000; Li and Neaves, 2006; Moore and Lemischka, 2006). The bone marrow (BM) contains stem cells that give rise to several tissues in addition to blood and the immune system. These so called mesenchymal stem cells (MSCs) can differentiate into endothelial cells, hepatocytes, cartilage, bone, and the myofibrils of muscle and heart. MSC are also derived from the placenta or umbilical cord, adipose tissue, skin, and thymus (Musina et al., 2005). Several human trials have shown the possibilities of using mesenchymal stem cells in tissue repair (as reviewed by Valtieri and Sorrentino, 2008). Stem cell maintenance is dependent on the various environmental cues that are provided and regulated by the stem cell niche (Li and Neaves, 2006; Moore and Lemischka, 2006; Scadden, 2006). Under normal conditions the stem cell pool and regeneration is kept stable by asymmetric stem cell division. However, in case of an injury or other non-physiological condition, many tissues are able to respond by either increasing or slowing down their regeneration. In order to permit dynamic control of the stem cell pool the self-renewing cells need additional strategies that can be utilized in case the rate of regeneration aught to be changed. Several studies show that many stem cells are
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capable of symmetrical stem cell division to maintain appropriate numbers of progeny (as reviewed by Morrison and Kimble, 2006, and the symmetric vs., asymmetric cellular divions will be further discussed later).
2.1.3 Cancer stem cells Even though a tumour can be clonally derived and thus originate from a single transformed cell it has a structure that at least partially mimics normal organ organization. The mixture of cells in a tumour contains selfrenewing stem cells and their more differentiated progeny. Cancer stem cells (CSCs) have been during the last few years characterized from a growing list of cancer types such as e.g. brain tumours, leukaemia, melanoma, colon, breast, and prostate cancer (Al-Hajj et al., 2003; Collins et al., 2005; Fang et al., 2005; Huntly and Gilliland, 2005; Singh et al., 2004). They share many characteristics with normal stem cells and their isolation is based on the expression pattern of the same markers that apply for their normal counterparts in the specific tissue. The glioma CSCs, for example, express the neural stem cell marker and cell surface antigen CD133 (Tamaki et al., 2002; Uchida et al., 2000). Transplantation of only a hundred CD133-positive cells into the brain of non-obese diabetic, severe combined immunodeficient (NOD/SCID) mice initiated tumours that can be serially transplanted and are a phenocopy of the patient’s original tumour, which thus contained a majority of non-CD133 expressing cells. At the same time injection of 105 CD133-negative cells resulted in engraftment of the cells but they were not able to cause a tumour (Singh et al., 2004). The hypothesis that cancers would arise from mutated stem and progenitor cells that already posses the ability to self-renew is supported by several studies (reviewed by Lobo et al., 2007). A recent study also showed that the gene expression signature in poorly differentiated and aggressive breast cancers and gliomas was similar to ES cells (Ben-
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Porath et al., 2008). Of the brain tumours, medulloblastomas can arise from the developing cerebellar external granular layer and gliomas can derive from the subventricular zone (SVZ) where the neural stem and progenitor cells are located (Sanai et al., 2005; Wechsler-Reya and Scott, 2001). Similar results are obtained with a mouse model of gliomas caused by a loss of p53 (Gil-Perotin et al., 2006a). An extra challenge to cancer therapy is revealed from studies with gliomas and breast cancer, which suggest that CSCs may possess innate resistance mechanisms against radiation and chemotherapy induced cancer cell death (Eyler and Rich, 2008). Understanding the normal mechanism of self-renewal and stem cell maintenance are crucially important in terms of development of future cancer treatment and the specific targeting of the CSCs.
2.2
Neural development
2.2.1 Development of the mammalian nervous system After gastrulation, when the three embryonic layers are formed, the neural plate develops from the uppermost ectodermal layer. The neural plate folds to form the neural tube. The cells of the border of the neural plate that are left over from the closure of the neural tube on the dorsal side become neural crest cells, which give rise to the sympathetic nervous system, some cranial bones, melanocytes and parts of the adrenal medulla (Sadler 2006). The central nervous system (CNS) predominantly develops from a population of proliferating primitive neuroepithelial cells that line the neural tube as a single layer. This apical area around the lumen of the neural tube is called the ventricular zone (VZ). The apical neural tube later forms into the ventricles around which the brain develops and the posterior part of the neural tube forms the spinal cord. The pluripotent neuroepithelial cells give rise to all the different neural and macroglial (astrocytes and oligodendrocytes) cell types of the CNS (Huttner and Kosodo, 2005).
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The function of neurons is to receive, process and transmit information from other cells and by electrochemical signalling in the nervous system send responses to other parts of the body for action. Astrocytes perform many functions. These include the provision of nutrients to the nervous tissue, support of endothelial cells which form the blood-brain barrier and a principal role in the repair and regeneration in the brain. Oligodendrocytes are responsive for the insulation of the axons of neurons in the CNS to enable rapid passing of the electrical signal in the neuronal web. The brain also contains microglial cells that display phagocytic capacity and are of a mesodermal origin (Purves et al. 2001). A fundamental feature that applies to all vertebrates and also to all different parts of the mammalian CNS is that different cell types are generated in a precise sequence, first neurons, then oligodendrocytes and finally astrocytes (Purves et al. 2001). Specific mechanisms behind these complex events have been most extensively studied in the telencephalon of rodents (Guillemot, 2007). In the mouse CNS, neurogenesis starts around embryonic day (E) 10. At this point, a majority of the neuroepithelial cells undergo changes in some of their epithelial associated adhesive properties and polarity and instead adopt a more glial fate and thus become Radial glial cells, RGCs (Gotz and Huttner, 2005). This includes glycogen granules and expression of several glial antigens and transformation into astrocytes perinatally once neurogenesis is, for the most part, over (Choi and Lapham, 1978; Schmechel and Rakic, 1979; Doetsch, 2003). RGCs are thus the most abundant type of neural stem cells of the developing mouse cerebrum and they line the VZ of the lateral ventricles from E12 until birth (Gotz and Huttner, 2005; Huttner and Kosodo, 2005). Despite the glial characteristics of RGCs, they are multipotent neural progenitor cells and they also retain several features present already in neuroepithelial cells such as expression of the intermediate filament protein Nestin (Hartfuss et al., 2001) and some important features of apical-basal polarity such as
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the apical location of centromeres and prominin-1 (Chenn et al., 1998; Weigmann et al., 1997; Gotz and Huttner, 2005). By asymmetric division, the RGCs give rise to one identical daughter stem cell and one neurogenic daughter cell, or alternatively an intermediate (also called basal) progenitor cell of the subventricular zone (SVZ), which still divides and the progeny then differentiates into cortical neurons. The differentiating neural progenitors migrate towards the cortical plate and their final destination. Migrating committed neural progenitors are scaffolded by the projections of the RGCs that reach from the somas attached to the ependymal cells, which line the lumen, all the way to the pial surface (Doetsch, 2003; Noctor et al., 2004; Parnavelas, 2000). The progenitors in the developing spinal cord and retina differ from those in the brain in that they mainly retain neuroepithelial features (with a more modest extent of glial properties) during neurogenesis and interestingly, they also maintain a broader developmental potential throughout neurogenesis (Gotz and Huttner, 2005; Leber and Sanes, 1995; Turner and Cepko, 1987). The polarized structure of the neuroepithelial cells as well as the RGCs is very important for the functionality and orientation of the neural stem cells. The composition of the apical plasma membrane (only a small fraction of the plasma membrane that lines the lumen of the neural tube) is very different from the basolateral plasma membrane around the rest of the cell (Gotz and Huttner, 2005), as discussed later. As in many types of epithelial cells, these two types of plasma membrane are mainly kept separate by adherence junctions (AJ), which also join the plasma membranes of neighbouring cells together by cadherins and anchore the junctions to the actin cytoskeleton of the respective cell. Several other proteins, like catenins, vinculin and
-actinin are involved in the
organisation of the correct structure of AJs (Alberts et al., 2002; Gotz and Huttner, 2005).
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2.2.2 The cell cycle
M
G1
G2
S
G0
Figure 1. The cell cycle. The cell cycle is divided into four phases: Gap1 (G1), DNA synthesis (S), G2 and mitosis (M). Additionally the cell can enter a quiescent G0 phase and remain nonproliferative for long periods, or, for ever. The G1, S and G2 all take place during interphase (grey) when the chromosomes are not yet organised and condensed in order to go through mitosis (Black).
The highly polarized neural stem cells are rapidly proliferating epithelial cells that predominantly by symmetric divisions increase the pool of pluripotent neural stem cells. In eukaryotes, cellular divisions are controlled by the cell cycle machinery that divides the process in four phases as shown in figure 1. In Gap 1 (G1) the cells increase in size, are highly metabolic and various proteins needed for DNA synthesis are transcribed. The G1 checkpoint control mechanism ensures that everything is ready for DNA synthesis. In the synthesis (S) phase DNA is replicated. Next phase is G2, during which the rate of protein synthesis (especially synthesis of microtubules needed for the mitotic spindle) and metabolia is again high and the cells continue to grow. The G 2 checkpoint control mechanism ensures that everything is ready to enter the mitosis (M) phase and divide. The M phase also has one checkpoint in the middle of mitosis (Metaphase Checkpoint), which ensures that the cell is ready to complete cell division. In non-prolific cells the cell cycle can be kept quiescent for long periods of time, which is referred to as a resting phase G0. The G0 phase can be induced due to a terminally differentiated cellular status in which the cell is otherwise fully functional (such as neurons). Alternatively, DNA damage can induce cellular senescence, which drives cells to G0 and thereby ensures nonviable progeny. Various
proteins regulate the cell cycle and cyclins and cyclin dependent kinases (CDKs) are especially important in controlling the G1 checkpoint. The migration of the nucleus during different phases of the cell cycle is a special feature of the neural stem cells, which is shown in figure 2. The nuclei migrate across the cytoplasm during the cell cycle phases in a manner in which S-phase takes place next to the basal lamina, and on the other hand, mitosis occurs when the nucleus is again in the apical surface
Pial surface
Subventricular zone
Figure 2. The migration of the nucleus in the polarised neural stem cells. The migration of the nucleus across the cytoplasm according to the different phases of the mitotic cycle is a typical feature for all neural stem cell types (neuroepithelial cells, Radial glial cells, RGCs, and Basal progenitor cells) during development. During G1-phase the nucleus is in the apical end of the cell and during DNA-synthesis it is in the other side, the basal end of the cytoplasm. When the cell divides, the nucleus is again in the apical side of the cell. Note that the nucleus of the neuroepithelial cells (picture a) migrates across the whole cytoplasm whereas it does not reach the basal lamina in RGCs, which project up to the pial surface (picture b). Also in the basolateral progenitors, the nucleus only migrates to the surface of the ventricular zone (where the subventricular zone begins). The basolateral cells loose their contact to the apical surface (and their polarity) during the cell division and the daughter cells are not anymore pluripotent as they can only give rise to neurons. The polarized neural stem cells have two different types of plasma membrane, the apical and basolateral plasma membrane, which consist of different lipid and protein complexes. The two types of plasma membranes are segregated by adherence junctions and the apical plasma membrane is always attached to the apical surface that lines the neural tube or ventricle. The schematic figure is modified from Gotz and Huttner, 2005.
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of the cell. The migration of the mitotic cycle is typical also for other types of neural progenitors, the radial glial cells (RGC) and the basal progenitors that derive from the neuroepithelial cells later in development (Gotz and Huttner, 2005).
2.2.3 Symmetric and asymmetric cell divisions Neurogenesis occurs through a combination of several modes of cellular divisions, which include 1) symmetrical divisions of neuroepithelial or intermediate progenitor cells that expand the progenitor pool, 2) asymmetrical division of the RGCs (and neuroepithelial cells) that give rise to one developing neuron and one identical progenitor cell 3) symmetrical divisions where both daughter cells divide to produce neurons, thus depleting the pool of progenitor cells (Cai et al., 2002; Noctor et al., 2004; Takahashi et al., 1996). Figure 3 illustrates the last two options during neurogenesis in the E16 mouse brain (Noctor et al., 2004). The apical-basal polarity of neuroepithelial and RGCs is an important basis for their symmetric versus asymmetric division. The unequal versus equal distribution of the cellular contents between the two daughter cells defines the identity of the progeny. Several determinants are known, many of which often also regulate each other, and the combination of different regulatory cues ultimately leads to either symmetric or asymmetric division. The key players, as discussed below, include at least the orientation of the mitotic spindle, inheritance of the apical plasma membrane, polarised expression of several proteins, as well as the cell cycle length. The molecular machinery controlling these cell divisions is beginning to be unravelled (Gotz and Huttner, 2005; Huttner and Kosodo, 2005).
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Figure 3. Asymmetric and symmetric neural divisions. Asymmetric division (star) of radial glial cells (RGCs) in the ventricular zone (VZ) generates one daughter RGC and one neuron (red cell pathway). Alternatively, RGCs can by asymmetrical division give rise to a basal progenitor cell (also called an intermediate progenitor cell), which then by symmetrical divisions (dagger) generates neurons in the subventricular zone (SVZ) in the developing E16 mouse brain (blue cell pathway). The schematic figure is modified from Noctor et al., 2004.
The Abnormal spindle –like microencephaly associated protein (ASPM) is concentrated at the poles of the mitotic spindle in the neuroepithelial cells and is crucial in maintaining the cleavage plain orientation that allows symmetrical divisions of the neuroepithelial stem cells during brain development (Bond et al., 2002; Fish et al., 2006). A horizontal cleavage plane results in asymmetric division of the apical/basal directional polarized stem cells. However, horizontal cleavages are rather rare and most asymmetric and symmetric neural stem cell divisions are cleaved vertically (Huttner and Kosodo, 2005; Kosodo et al., 2004; Smart, 1973). Asymmetric vertical cleavage is enabled by unequal distribution of the apical plasma membrane to the two daughter cells. The cell that inherits the apical plasma membrane remains proliferate (Kosodo et al., 2004) as illustrated in figure 4A. The apical plasma membrane constitutes only a tiny fraction (1-2%) of the total plasma membrane and has several specific features different from the rest of the plasma membrane. It expresses prominin-1, which interacts with membrane cholesterol (Corbeil et al., 2001; Weigmann et al., 1997). Also, proteins associated with AJs which localize in the apical side of the cell, like the PAR-proteins and -catenin, are concentrated beneath the apical plasma membrane (Gotz and Huttner, 2005; Zhadanov et al., 1999).
21
Also, the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor SNARE proteins are involved in the control of asymmetric or symmetric division by controlling membrane fusion in the end of the cell division phase. In symmetric division, the apical plasma membrane is inherited symmetrically by the daughter cells and heterophilic membrane fusion takes place (SNARE-proteins join the apical plasma membrane of the daughter cell to basolateral plasma membrane during the inclusion of the membrane). Alternatively, in the asymmetric division, membrane fusion is homophilic and the SNAREproteins join basolateral plasma membrane together, which results in asymmetric inheritance of the whole apical plasma membrane only to one (the RGC remaining) daughter cell (Gotz and Huttner, 2005). The length of the cell cycle and especially of the G1 phase is increased in association with the reduced potency of the cell in the VZ and SVZ: neuroepithelial cells, which have the highest pluripotency, have the longest G1 phase as compared to radial glial cells and further compared to basal progenitor cells, which are not pluripotent anymore and have the shortest cell cycle (Takahashi et al., 1995). Also, mouse embryonic telencephalic neural progenitor cells that express Tis21, a marker specific for the daughter cells of neurogenic determined neuroepithelial cells (Iacopetti et al., 1999), show a significantly longer cell cycle than their neighbouring cells that lack Tis21 expression. The authors hypothesis is that lengthening of the cell cycle gives the cell time to induce a new cellular fate (Calegari and Huttner, 2003; Calegari et al., 2005). Several molecules are known to regulate the asymmetric divisions and the polarized structures of the RGCs. Asymmetric inheritance of Notch1 was one of the earliest discoveries segregating the two different daughter cells on RGC division. The Notch signalling pathway determines various events during embryonic development. The Notch receptor binds to its ligand that is attached to the plasma membrane of the neighbouring cell,
22
A
B MS
basal
APM
Symmetric
Asymmetric
apical
Figure 4. Asymmetric division is associated with the orientation of the mitotic spindle, inheritance of the apical plasma membrane and asymmetric inheritance of several cellular fate determining proteins. A) Orientation of the mitotic spindle (MS) ultimately determines the symmetric or asymmetric distribution of the apical plasma membrane (APM) into daughter cells of mammalian neuroepithelial and RGCs. An exactly perpendicular (vertical) cleavage results in symmetric inheritance of the apical plasma membrane (which only comprises of a few percentages of the whole plasma membrane) to both daughter cells and thus symmetric division of the cell. As a horizontal cleavage plane is rare during neural development, a majority of asymmetrical cell divisions are also cleaved vertically. This is possible by a small deviation of the angle of the perpendicular orientation of the mitotic spindle and results in asymmetric inheritance of the whole apical plasma membrane only into one daughter cell. B) Polarized expression of Baz, Par6, aPKC in the apical part of the dividing Drosophila neuroblast as well as basal expression of Numb are determinants the daughter cell fate in asymmetric neural stem cell division. Self-renewal is maintained in the new neuroblast becoming daughter cell and the ganglion mother cell adopts a neural fate. Pictures are modified from A: Huttner and Kosodo, 2005 and B: Kim and Walsh, 2007.
which triggers a signalling cascade in the Notch-expressing cell (as shown later in Figure 6). Because of the attachment of the ligand to the neighbour cell plasma membrane, a phenomenon called lateral inhibition is special for Notch signalling. The Notch-expressing cell can bind several ligand expressing cells nearby and send an inhibiting message to all the neighbours to not adopt a certain cellular fate, which the Notchexpressing cell in the middle itself will adopt. Notch-signalling inhibits neural differentiation and promotes the glial identity of neural stem cells (Chenn and McConnell, 1995; Gaiano et al., 2000; Mizutani and Saito, 2005). It was later shown that polarized expression of another protein, Numb, in the neuron becoming basal daughter cell is needed for Notch inhibition and that the similarly polarized expression pattern of a third 23
protein, lethal gigantic larvae (Lgl1), for one’s part regulates the expression of Numb (Klezovitch et al., 2004; Shen et al., 2002). In addition to Notch inhibition in the basal part of the cell, Numb is involved in the correct targeting and trafficking of components of AJ to the neuroepithelium to maintain the apical/basal polarity. Several AJ proteins including E- and N-cadherins as well as -E and -catenin can be coimmunopresipitated with endogenous Numb (Doe, 2008; Kim and Walsh, 2007; Rasin et al., 2007). Also several other polarity proteins have been identified e.g. Baz, Par6 and the atypical protein kinase C (aPKC) that all localize to the apical part of the neuroepithelial cells (reviewed by Kim and Walsh, 2007). The homologues of the same proteins (Numb, lgl, Par-6) function to a large extent similarly in the Drosophila fly as reviewed by Kim and Walsh (2007). The neuroblast divides asymmetrically and forms a new neuroblast as well as a neuronal fate determined ganglion mother cell (GMC). Lgl, Numb and Miranda all locate to the basal GMC side of the dividing cell (Figure 4B). The tumour suppressor Brat is a binding partner of Miranda and also a segregating cell fate determinant of the neuroblast. Interestingly, Brat is a posttranscriptional inhibitor of dMyc and mutant neuroblasts lacking Brat loose their ability of asymmetric division. Thus, also Myc regulates asymmetric division in the Drosophila neural stem cells (Betschinger et al., 2006).
2.2.4 Neurogenesis in adult CNS Most of the RGCs are terminally differentiated into astrocytes in the end of embryogenesis and the progenitor cells of the neuroepithelium are thereby lost even though some astroglial cells that produce multilineage neural precursors are still left 4 weeks postnatal in the mouse brain (Ganat et al., 2006; Kim and Walsh, 2007). Although mammalian neurogenesis mostly takes place during embryonic development and early childhood some renewing activity is maintained through adulthood.
24
Several in vivo mouse studies have reported neural and glial regeneration by using genetically labelled NPCs under several different promoters (such as Gli, Doublecortin or Nestin) specific for NPCs or newborn neurons (Ahn and Joyner, 2005; Carlen et al., 2006; Couillard-Despres et al., 2006; Imayoshi et al., 2006). Regeneration of the mouse hippocampal neurons has been associated with possible maintenance and integration of new memories (Imayoshi et al., 2008). But according to a study based on detectable levels of 14C (derived from nuclear bomb tests during the cold war 1955-1963), which has integrated into DNA in people that have been exposed to it as adults, cortical neurons are not regenerated in adults in humans (Bhardwaj et al., 2006; Spalding et al., 2005).
2.2.5 The neural stem cell niche A stem cell niche provides appropriate circumstances for stem cells to maintain their self-renewal and thereby tissue homeostasis. These specific anatomic areas located in the renewing organ regulate the actions of stem cells and the way they participate in the regeneration, maintenance and repair of the tissue. The niche also shelters stem cells from differentiative, apoptotic or other stimuli that would challenge stem cell reserves (Moore and Lemischka, 2006; Scadden, 2006). During embryogenesis the whole neuroepithelium can be considered as a neural stem cell niche, the anatomy of which changes along the phases of neural development. As discussed above, early developmental phases contain neuroepithelial cells as a monolayer lining the apical lumen of the neural tube. The neuroepithelium later forms a layered structure that predominantly contains RGCs in the VZ once neurogenesis has started (Doe, 2008; Doetsch, 2003). The multipotent NSCs in adult are restricted to the SVZ lining of the lateral ventricle wall as well as to the dentate gyrus (DG) in the hippocampus (Altman and Das, 1965; Bhardwaj et al., 2006; Gage, 2000;
25
Posterior
Anterior
Figure 5. Schematic sagittal section through the adult mouse brain depicting the two principal sites of adult neurogenesis. The subventricular zone (SVZ), which lies along the lateral ventricle (LV, light grey), and the subgranular zone (SGZ) of the hippocampal formation are the neural stem cell niches in the adult brain. The SVZ born neuroblasts migrate into the olfactory bulb (OB) through the rostral migratory stream (RMS) where they fully differentiate into neurons. In contrast, neurons in the dentate gyrus (DG) are born locally in the SGZ. This figure is modified from Doetsch 2003.
Morshead et al., 1998; Reynolds and Weiss, 1992; Suhonen et al., 1996; Taupin and Gage, 2002). The adult NSCs are slowly dividing glial fibric acidic protein (GFAP) -expressing astrocytes (or astrocyte-like cells) that give rise to transit amplifying progenitor cells which, in turn, generate neuroblasts. The identity of the adult neural stem cells is, however, currently controversial. A Swedish study shows that the ciliated ependymal cells that line the ventricular wall are the adult neural stem cells (Johansson et al, 1999) even though the theory of the glial identity of NSCs is predominant in the field. Neuroblasts of the SVZ migrate through a network of pathways extending along the SVZ and join the rostral migratory stream that leads to the olfactory bulb, where they differentiate into interneurons (Ahn and Joyner, 2005; Lledo et al., 2008). Newborn hippocampal neurons of the DG, on the other hand, are generated from astrocyte stem cells in the subgranular zone (SGZ) from which intermediate progenitors are formed. The differentiating neurons move only a short distance from the SGZ into the granule cell layer (Doetsch, 2003). Despite the predominant glial
26
nature of NPCs the expression of the proneural factor Neurogenin2 (Ngn2) and Mash1, for example, are also essential for the formation of the DG (Galichet et al., 2008). The location of adult neural stem cells in the mouse brain is illustrated in figure 5. The NSC niche in SVZ is a unique structure in which the astrocyte stem cells, the progenitor cells and the neuroblast are in contact with each other and with the ciliated ependymal cells. Occasionally a SVZ astrocyte extends a process between the ependymal cells to contact the lateral ventricle (Doetsch, 2003). A recent study shows that the stem and transit amplifying cells of the SVZ niche are in direct contact with the vasculature at sites that lack astrocyte endfeet and pericyte coverage, a modification of the blood brain barrier unique to the SVZ. This structure thereby allows the cells to receive spatial cues and regulatory signals from the vascular system (Tavazoie et al., 2008). Also, the laminin receptor
1 integrin is important for tethering SVZ progenitors to the
vascular niche (Shen et al., 2008).
2.2.6 Neural and glial differentiation 2.2.6.1 Neurogenesis Terminal differentiation of cells is associated with cell cycle inhibition, permanent epigenetic changes such as methylation and histone modification as well as vast changes in the protein expression profile to promote the new cellular state (Hamby et al., 2008). The proneural factors comprise of basic-helix-loop-helix (bHLH) transcription factors that are evolutionary conserved (Bertrand et al., 2002; Ross et al., 2003). The proneural genes expressed in the mammalian telencephalon include Ngn1, Ngn2 and Mash1. Activation of their target genes instructs the cell towards a neural fate by promoting neural maturation and migration, neural subtype specification as well as inhibition of the astroglial identity, respectively (Guillemot, 2007). Ngn1 and Ngn2 promote cell cycle exit
27
through upregulation of the Cyclin dependnt kinase (CDK) inhibitor p27Kip1 (Farah et al., 2000; Nguyen et al., 2006) and by inducing the expression cascade of neuronal genes such as NeuroM, NeuroD and -IIItubulin (Tuj-1; Fode et al., 1998; Ma et al., 1998; Farah et al., 2000). The transcription factor Paired box 6 (Pax6) also induces neural fate, which in cortical progenitors is partially induced by activation of Ngn2,
but
induction of neurogenesis in postnatal astrocytes is bHLH independent (Heins et al., 2002; Scardigli et al., 2001). Also, expression of Tis21 in neuroepithelial progenitor cells is a determinant of neural fate of the daughter cell (Gotz and Huttner, 2005; Iacopetti et al., 1999). 2.2.6.2 Gliogenesis The Olig1 and Olig2 genes that also are transcription factors of the bHLH family are major determinants of oligodendroglial fate and their expression inhibits astrogliogenesis. In the developing spinal cord Olig1 and 2 are expressed by common progenitors for oligodendrocytes and motoneurons although motoneuron specification only requires transient Olig2 expression whereas constant Olig expression is needed throughout oligodendrogliogenesis and in terminally differentiated oligodendrocytes (Guillemot, 2007; Lu et al., 2002; Zhou and Anderson, 2002). The function of Olig genes in the embryonic telencephalon is less well understood but is likely to be similar (Guillemot, 2007). In addition to Olig expressing cells, oligodendrocyte progenitor cells in the VZ alternatively arise from cells that express the homeobox genes Nkx (2.1 and 2.2) but later on both progenitor types co-express both genes and become indistinguishable (Fu et al., 2002). Also the SoxE genes promote oligodendrogliogenesis in the spinal cord. Deletion of Sox9 during embryogenesis results in a severe decrease of oligodendrocyte progenitors and an increase of motoneurons (Stolt et al., 2003), whereas Sox8 and Sox10 are expressed in fully mature oligodendrocytes (Guillemot, 2007; Stolt et al., 2003).
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As mentioned earlier, the development of the nervous system during embryogenesis takes place in a certain order and neurons are first formed, then oligodendrocytes and finally astrocytes. Thus, astroglial identity is a default fate of neural progenitor cells, which leads to differentiation of astrocytes postnatally unless it is not inhibited by expression of proneural or oligodendrocyte differentiation promoting factors (Doetsch, 2003; Zhou and Anderson, 2002). Activation of the astrocyte-specific genes GFAP and/or S100 promote astrocyte differentiation. The Sox9 gene is also involved in specification of certain astrocyte subtypes in the spinal cord (Guillemot, 2007; Stolt et al., 2003). 2.2.6.3 Signalling cascades that promote neuronal, glial or stem cell fate The complexity of developmental programs is substantial and the roles of different signalling cascades are by far not understood. However, there are five signal transduction pathways (as illustrated in Figure 6.) namely the Wingless and Int (Wnt), Notch, Tyrosine kinases (which include receptor tyrosine kinases, RTKs, and non-receptor protein tyrosine kinases, PTKs), Transforming growth factor (TGF)
superfamily and the
Sonic hedgehog (Shh) family that basically are responsible for all signalling in an individual (Sadler 2006). This is possible through several aspects: 1) All of these signalling families contain, on each step of the signal transduction cascade, several closely related but functionally separate members (e.g. the 19 different Wnt ligands) that operate on the same complex signalling cascade and are also expressed in different combinations in different tissues. 2) The outcome of the signalling depends dramatically on the concentration of the signalling molecule as well as the developmental time window. 3) A cell simultaneously receives various signalling cues and also members of different signalling pathways continuously crosstalk. The composition of all the messages together dictates the identity of the cell. Thus, these aspects aught to be kept in mind when interpreting the functions of certain molecules, which
29
TK
HH
TGFsuperfamily
Receptor tyrosine kinase
Sonic Heghehog ligand
Wnt
BMP, activin
Wnt ligand
Serine/ threonine kinase
P
Gli
MAPK -signalling pathway
Ras
P
P
Activin receptor -like kinase
Smad
Dishevelled
----------
GSK APC
Smad
Ras
Notch
Frizzled
Smoothened P
neighbouring cell
Jagged or Delta
Ligand e.g. TGF- ,
Mitogen ligand
Patched
Notch
P
P
degradation Raf Raf
P
Smad
MEK
P
MEK
Smad4
P
cytoplasm
Erk P Erk P
-cat
nucleus
P
Activated Gli
Lef1/TCF
Hes
P P P
P
Smad
Smad4
-cat Lef1/TCF
Csl
Activated Transcription factor
Fig 6. A schematic figure of the five signal transduction pathways responsible for all signalling in an individual. BLUE: Sonic Hedgehog is a ligand of the Hedgehog (HH) signalling family. When Shh is bound to its receptor Patched, Patched no longer inhibits Smoothened, which therefore activates a transcription factor of the Gli-family. Activated Gli enters the nucleus and activates transcription of target genes. RED: The large family of tyrosine kinases (TKs) includes the subfamilies of Receptor tyrosine kinases (RTKs) and nonreceptor protein tyrosine kinases (PTKs) that initiate the signalling. An example of a RTK signalling pathway is illustrated here: The mitogen activated (e.g. EGF or FGF as ligands) signalling is mediated by the MAPK kinase signalling pathway that includes several alternative kinases (which in this example are Ras/Raf/Mek1,2/Erk1,2) that induce a phosphorylation cascade to finally activate the downstream transcription factor. GREEN: The ligands of TGF- superfamily signal through a complex formed by two different receptors. The ligand is recognized by type II receptor that is a serine/threonine kinase. The signal is mediated further by a type I receptor (an Activin receptor –like kinase) by phosphorylating an intracellular mediator of the Smad family (Smad 1,2,3,5 or 8). Smad 4 then binds the phosphorylated protein and the complex enters the nucleus and acts as a transcription factor to regulate transcription of the target gene. YELLOW: The Wnt signalling family comprises of several different Wnt ligands that bind to the Frizzled receptor, which leads to activation of the Dishevelled-protein. Activated Dishevelled inhibits the action of the glycogen synthase kinase GSK and the interaction with APC, a protein complex that otherwise phosphorylates -catenin and marks it for degradation in the cytoplasm. When the Wnt ligand is bound, -catenin is stabilized and enters the nucleus and then binds to and activates the transcription factor Lef1/TCF. BROWN: The Notch receptor is a transmembrane protein, which is activated by binding to its ligand (Delta or Jagged), that also is a transmembrane protein and thus attached to the plasma membrane of the neighbouring cell. Once Notch is bound to the ligand, its intracellular tail is cleaved off and enters the nucleus and activates a transcription factor of the Csl-family and thereby activates trancription of the Hes-family genes. The illustration is based on information obtained from Sariola et al, 2003 and Junttila et al., 2008.
30
might have totally opposite roles at different developmental time points or in different cell types (Sadler 2006). Main neurogenesis pathways Wnt signalling promotes neural stem cell progenitor self-renewal and proliferation (at early stages of cortical development) and neural differentiation (at later stages after E13.5) in the mouse embryo and promoters of the Ngn1 and Ngn2 proneural genes are activated by the catenin/Transcription factor 7-like (TCF) complex (Hirabayashi et al., 2004; Zechner et al., 2003). Many growth factors and thereby ligands of the RTK signalling family, like the Platelet derived growth factor (PDGF) or the neurotrophins neural growth factor (NGF) or the Brain derived neurotrophic factor (BDNF) or NT 3,4,5 promote neuronal differentiation, survival and growth (Purves et al., 2001). One of the intracellular signalling pathways activated by ligand binding of RTKs is the Responsible for activities in sarcoma (RAS) / Mitogen activated protein kinase kinase (MEK) / Extracellular signal related kinase (ERK) pathway, which directly promotes neuronal differentiation by activating neural genes by phosphorylation of the CAAT/Enhancer binding protein C/EBP family members (Menard et al., 2002). The same RTK family ligands, such as PDGF, can also promote neural progenitor cell survival mediated by a distinct pathway like the phosphatidylinositol-3 kinases (PI3K)-Akt (a cellular homolog of the Acute Transforming retrovirus in mice) -pathway (Barnabe-Heider and Miller, 2003), which again reminds of the complexity of the signalling systems and the crosstalk between different pathways. Main astrogliogenesis pathways Notch signalling is essential in maintaining the astroglial identity of RGCs and NPC survival as well as in the differentiation of RGCs to astrocytes later on in development. At least the Brain lipid binding
31
protein (BLBP) and the neuregulin receptor ErbB2 (also known as Human Epidermal growth factor Receptor 2, HER2/neu) are direct Notch signalling targets in RGCs (Anthony et al., 2005; Mizutani and Saito, 2005; Oishi et al., 2004; Schmid et al., 2003) and Notch signalling also promotes astrogliogenesis in the adult mouse brain progenitor cells (Gaiano et al., 2000; Tanigaki et al., 2001). Many activities of the Notch signalling pathway are mediated by the Hairy and enhancer of split (Hes) family of transcriptional repressors which inhibit expression of the proneural genes. Notch also induces astrogliogenesis through a cross talk with the Janus kinase 2 ( JAK2) / Signal transducer and activator of transcription 3 (STAT3) non-receptor PTK signalling pathway, which can also be activated by PTK ligands such as the Interleukin 6 (IL-6) related cytokines such as Ciliar neurotrophic factor (CNTF) and Leukaemia inhibitory factor (LIF). Also Notch can directly and/or via Hes activation activate the GFAP promoter (Bonni et al., 1997; Guillemot, 2007; Hatakeyama et al., 2004; Nakamura et al., 2000). The astrogliogenesis induced by Notch and the JAK/STAT pathway is coactivated by signalling of the bone morphogenetic proteins (BMPs), which belong to the TGF
super family, through Smad transcription
factors, which bind to nearby sites in the GFAP promoter and thus participate in the synergistic activation of the target gene (Nakashima et al., 2001). A recent paper shows that phosphorylation of STAT3 is regulated by the expression of Myc and p19Arf that antagonize each others effects. During late embryogenesis, when the switch from neurogenesis promoting NPCs to gliogenesis promoting NPCs occurs, high Myc expression (that promotes self-renewal and the maintenance of neurogenic potential in NPCs) is downregulated, and in response to CNTF signalling the expression of p19Arf increases and NPCs start to predominantly give rise to glia (Nagao et al., 2009). Main oligodendrogliogenesis pathways During early embryogenesis, Shh is expressed by the notochord and is a key determinator of neurulation and thereafter ventralisation of the
32
formed neural tube (Sadler 2006). Later in embryogenesis, ventrally born oligodendrocyte progenitors are induced by Shh, which is secreted by the neuroepithelial cells near the ventral midline of the telencephalon (Alberta et al., 2001; Nery et al., 2001; Guillemot, 2007) and it induces oligodendrocytosis by induction of expression of Olig1 and Olig2 and PDGF receptor (Lu et al., 2000; Nery et al., 2001). Stem cell maintenance pathways If stem cell maintenance is promoted, differentiation is usually inhibited and vice versa. As already mentioned in several occasions above, various signalling
cascades
like
the
Wnt
signalling
pathway
promote
differentiation and thus inhibit neural stem cell maintenance (Hirabayashi et al., 2004). However, Wnt signalling is essential in the formation of the identity of dorsal neural progenitor cells of the neural tube in the early phases of embryonic development as well as in the dorsal telencephalon after the onset of neurogenesis (Backman et al., 2005; Sadler 2006). Also, localisation of
-catenin in the vicinity of AJs is associated with the
regulation of asymmetric cell divisions in the neural epithelial cells and RGCs. The inheritance of -catenin is essential for the maintenance of self-renewal capacity in the daughter cells (Gotz and Huttner, 2005). Notch signalling is important for the glial identity and stem cell maintenance of RGCs and is involved in the regulation of asymmetric stem cell division (Guillemot, 2007). Shh signalling is important for the identification of the progenitor cells derived from the ventral part of the neural tube (such as oligodendrocyte and motoneuron progenitors) but is also involved in the maintenance of at least adult NPC self-renewal and proliferation (Guillemot, 2007;Ahn et al., 2005; Lai et al., 2003; Machold et al., 2003) as also discussed earlier. The Epidermal growth factor (EGF) and Fibroblast growth factor (FGF) that bind to their receptors of the RTK family are essential mitogens for telencephalic self-renewing progenitors although at high concentrations
33
FGF promotes astrocytogenesis when NPCs have become competent for differentiation (Guillemot, 2007). The signal of EGF and FGF is mediated through the Mitogen activate protein kinase (MAPK) pathway which includes activation of the proto-oncogene Ras as well as the MEK and ERK kinases (Sadler 2006). The MAPK signalling pathway is illustrated in figure 6. Unequal distribution of EGF-receptor (EGFR) to daughter cells is involved also in the asymmetric division of cortical NPCs in a manner that the EGFRhigh cells remain RGCs and EGFRlow cells become more committed progenitors (Sun et al., 2005). The functions of the MAPK signalling pathway on NPC self-renewal are also discussed below in section 2.4. 2.2.6.4 -Motoneuron development The -motoneurons (or lower motoneurons) are cholinergic neurons that have their cell bodies located in the anterior (also called ventral) horn of the spinal cord from where they project axons outside the CNS and innervate somatic muscles. In addition, there are also branchial
-
motoneurons, which have their nuclei in the brainstem and innervate branchial muscles that motorize the face and neck in land vertebrates (and are thus derived from the branchial arches during development). The visceral motoneurons innervate involuntary muscles of the heart, visceral smooth muscles and glands and operate in coordination with the PNS. The rest of the motoric system comprises of the upper motoneurons in a complex regulatory system including several areas in the brain stem and the frontal cortex that are also controlled by the cerebellum and the basal ganglia (Purves et al 2001). The -motoneurons of the anterior horn derive from Shh-induced Olig2expressing progenitors that also produce oligodendrocytes (Lu et al., 2000; Lu et al., 2002; Zhou and Anderson, 2002). Overexpression studies in the chick embryo suggest that co-expression of Olig2 with Nkx2.2 induces oligodendrocyte differentiation but olig2 expression alone induces motoneuron differentiation (Guillemot, 2007; Sun et al., 2001).
34
The region specific expression of different homeodomain transcription factors (the Hox-genes such as Nkx or Pax6) is regulated by a Shh expression gradient in the ventral neural tube, which plays a key role in the establishment of distinct progenitor domains that give rise to the different types of neurons (Ericson et al., 1997; Briscoe et al., 2000). Pax6 is essential in the patterning of various organs during embryonic development. Pax6 has a critical role in neurogenic regulation and survival of RGCs in the human and rodent forebrain and it is a direct activator of Ngn2 (Nikoletopoulou et al., 2007; Scardigli et al., 2003). In mouse embryonic cortical and striatal cells neurogenesis is increased by overexpression of Pax6 and Pax6 promotes neurogenesis in human embryonic striatal neural stem cells (Hack et al., 2004; Heins et al., 2002; Kallur et al., 2008). Shh-induced Pax6 activity represses the expression of Nkx2.2, which controls the identity of developing motoneurons and inhibits the oligodendrocyte fate (Ericson et al., 1997). Also, early neuroectodermal cells derived from human ES cells that express Pax6 can be differentiated into spinal motoneurons by retinoic acid and Shh (Li et al., 2005). Proliferating progenitors in different progenitor domains express a unique combination of LIM homeodomain (LIM-HD) transcription factors that function to establish neuronal cell type identity as the progenitors exit the cell cycle. The motoneuron progenitor domain expresses the LIM-HD factors Lhx3 (LIM3) and Islet 1 (Isl1), which activate motoneuron specific transcription (Ericson et al., 1992; Sharma et al., 1998; Thaler et al., 2002). However, some LIM-HD proteins are expressed already in the proliferating progenitor cells and act co-operatively with the bHLH transcription factors such as NeuroM, which then induces expression of a motoneuron-specific gene HB9 (Allan and Thor, 2003; Lee and Pfaff, 2003). In addition to its role as a bHLH transcription factor, Ngn2 regulates neuronal subtype specification also in a phopshorylation dependent manner. The phosporylated form of Ngn2 interacts with the LIM-HD complex and activates motoneuron specific transcription and is
35
thus involved in the temporal specification of motoneuron differentiation (Ma et al., 2008).
2.3 Studying neural stem cells
2.3.1 Neurospheres Neural stem and progenitor cells can be isolated from the VZ and SVZ of the developing mammalian brain as well as from the SVZ or hippocampal DG of the adult brain. When these isolated stem and progenitor cells are cultured in a serum free medium supplemented with EGF and FGF, their proliferative and self-renewing multipotent capacity is maintained for several months with little change in the proliferation and differentiation capacity of early and late passage cells. The cultured stem and progenitor cells form sphere-formed aggregates called neurospheres consisting of hundreds or even several thousands of cells as shown in figure 7A (Chojnacki and Weiss, 2008; Gritti et al., 1999; Reynolds and Weiss, 1996; Vescovi et al., 1999). The neurosphere can be considered to contain an in vitro model of the NPC niche. The cells of the neurosphere consist of a heterogeneous population A
of
cells.
The
signalling
B
cues
received
from
the
1 cell/well for 7 days in EGF/FGF containing stem cell medium. (SCM) Dissociation of neurospheres into single cells. Microscopic evaluation: How many single cells were able to form new neurospheres?
Figure 7. A) A light microscopic picture of floating neurospheres in stem cell culture. B) Scheme of the neurosphere self-renewal assay.
36
microenvironment (including the neighbouring cells and the extracellular matrix) forms the identity of cells to become a multipotent neural stem cell or a committed progenitor of a certain lineage. Even cells with differentiated neural or glial morphology can be seen inside the neurospheres (Kilpatrick and Bartlett, 1993; Ray et al., 1993; Reynolds and Weiss, 1992; Reynolds et al., 1992). In addition to neurosphere cultures, neuronal stem cells can also be cultured niche-independently as symmetrically self-renewing monolayers where no differentiating progeny is generated (Conti et al., 2005; Laywell et al., 2000). The self-renewal capacity of the NPCs cultured as neurospheres can be measured by using a clonal colony forming assay (the neurosphere assay, NSA as illustrated in figure 7B) where neurospheres are dissociated and plated as single cells on cell culture dish wells. In this assay, self-renewal is counted as the percentage of single cells that are capable of forming a secondary neurosphere (Chojnacki and Weiss, 2008; Reynolds and Weiss, 1992), which consists of about 2.4% of the cells (Reynolds and Rietze, 2005). Nevertheless, not all of the 2.4% are true multipotent stem cells possessing long term potentiality, which would fulfil the criterion of producing a multipotent progeny of a magnitude much more numerous than the starting population over an extended period of time. In fact, according to recent calculation, only less than 10% of the sphere-forming cells are real multipotent stem cells and rest of them are progenitors with the capacity to only form 2-4 secondary spheres (Reynolds and Rietze, 2005). Technical details of the NSA have shown that in order to guarantee that the counted number of spheres really are clonal, the plating density (cells per well) has to be kept low and the plates should not be moved around during the formation of neurospheres to avoid chimerism (Coles-Takabe et al., 2008). As the most criticism raised against the NSA has been the concern of the inability to distinguish between the long term capacity possessing stem cells and their progeny with a smaller expansion capacity, the Reynolds lab has further evolved the NSA (Louis et al., 2008). In the advanced version the NPCs are cultured in EGF and FGF
37
containing semi-solid matrix with collagen added. The neurospheres that are formed can be enumerated into neural stem or progenitor cells according to the diameter of the sphere based on the higher proliferative potential of the stem cell population (Louis et al., 2008). Several studies have proven the capacity of in vitro cultured neurospheres to engraft and differentiate to neurons and glia in vivo (Gage et al., 1995; Suhonen et al., 1996) but a serial transplantation assay (adopted from the haematopoietic stem cell in vivo functionality test) failed to isolate NPCs from the mouse SVZ that would have been progeny of the in vitro cultured and transplanted neurospheres. In that study, all transplanted cells had differentiated and were found as migrating neuroblast in the rostral migratory stream and olfactory bulb (Marshall et al., 2006).
2.3.2 Using neurospheres as a model to study neurological disease Neurological diseases, such as Amyotrophic lateral sclerosis (ALS), Huntington disease, Alzheimer’s disease or Parkinson disease, display degeneration of discrete sets of cells in the CNS and many of them are associated with specific gene mutations. Various approaches have been used in trying to understand the link between the mutations and the underlying disease mechanisms to reveal the specific cellular failure responsible for the pathology. The advances in gene manipulation technology have allowed generation of mouse models with the same respective mutation as in the human disease. However, both behavioural and cellular changes observed in these models are often very different from those observed in the human condition. Furthermore, successful therapies in animal models are often not replicated in clinical trials. Species differences between mice and humans might be one reason for the clinical failures. In vitro studies with post-mortem brain tissue from affected patients can provide more information about the mechanisms behind the changed cellular and molecular status in the particular disease
38
(Jakel et al., 2004). However, accessibility of human neural tissue is limited and the tissue samples are often acquired at late stages of disease. Human neural stem cells derived from ES-cells as well as foetal or adult brain tissues, respectively, have been exploited in several studies, which have revealed several differences between human and rodent cells and between the developmental source of the cells (D'Amour and Gage, 2003; Hitoshi et al., 2002). If affected patient tissue is not available, successful disease models have been created by genetic manipulation of neural stem cells derived from healthy human tissue (Jakel et al., 2004). Another major aim of in vitro stem cell and differentiation culture studies is the characterisation and manipulation of these cells in order to develop stemcell based transplantation therapies for human patients with neurological disorders or injury-derived trauma (Lindvall and Kokaia, 2006). Neurosphere cultures can also be derived from tumour samples or genetically modified brain tumour animal models and are a widely used tool in brain tumour research (as discussed in a review by SanchezMartin 2008). Characterization of different signalling pathways of the tumour derived stem cell cultures and their relationship to self-renewal regulation of CSCs has provided useful information of brain tumours e.g. concerning the role of Myc, PTEN and p53 as discussed later (Zheng et al., 2008; Wang et al., 2008), which can hopefully be utilised for therapy purposes. 2.3.2.1 LCCS Lethal congenital contracture syndrome (LCCS) provides a human model to study early motoneural development. LCCS1 is a foetal motoneuron disease that is a part of the Finnish disease heritage with a prevalence of 1 in 25 000 births in Finland (Pakkasjarvi et al., 2006).
39
Figure 8. Severe atrophy of quadriceps muscles (left) as well as virtual total loss of anterior motor neurons in the spinal cord of LCCS foetuses (right). The typical features for LCCS, joint contractures and micrognatia are seen in the photograph of a LCCS foetus. The figures are obtained from Pakkasjärvi et al., 2005.
Because of total immobility, LCCS foetuses are detectable at the 13th week of pregnancy and the syndrome leads to death before the 32nd gestational week. The immobility is caused by a motoneuron defect seen as a degeneration of the anterior horn and the descending tracts of the developing spinal cord (Vuopala et al., 1996). Typical characteristics of LCCS include severe muscle atrophy, which is considered neurogenic, as well as hydrops, multiple joint contractures, pulmonary hypoplasia and facial features including micrognatia (small lower jaw) and pterygia, an abnormal tissue growth in the eye, (Herva et al., 1985), as shown in figure 8. Comparison of the mRNA expression patterns in the spinal cord of LCCS and normal age-matched foetuses by microarray analysis revealed changes in the expression of several genes (such as Pax6, Gli2 and Nkx2.2) that are involved in neuronal function as well as in the development of motoneurons and oligodendrocytes (Pakkasjarvi et al., 2005). The autosomal recessive condition LCCS1 is caused by mutations in the gene that codes a ubiquitously expressed mRNA export mediator protein GLE1 (Nousiainen et al., 2008), which is located in the chromosome locus 9q34 (Makela-Bengs et al., 1998). According to the Paircoil2 prediction programme, The LCC1 FinMajor mutation alters the secondary structure of the GLE1 protein and indicates disruption of the coiled-coil 40
domain. As the dramatic pathological changes of LCCS1 are only observed in the anterior horn motoneurons, it is likely that the predicted disruption prevents a critical interaction of GLE1 and a motoneuronspecific protein (Nousiainen et al., 2008). The gene mutations causing LCCS2 and LCCS3 have also been recently identified. LCCS2 is caused by a mutation in phosphatidylinositol-4-phosphate 5-kinase, type I, gamma (PIP5K1C) and LCCS3 has a loss-of-function mutation in ERBB3. Both identified genes code for proteins (PIPK
and HER3,
respectively) that are involved in the regulation of the phosphatidyl inositol pathway (Narkis et al., 2007a; Narkis et al., 2007b) and specifically in the synthesis of Inositol hexakisphosphate (Insp6), which also binds directly to the yeast homologue of GLE1. Insp6 together with GLE1 stimulate the ATPase activity of the DExD/H-box protein Dbp5 for nuclear mRNA export. The export of mRNA to various subcellular regions as a ready to use storage mechanism to guarantee an effective translation response to extracellular stimuli might be crucially compromised in the developing motoneurons (Nousiainen et al., 2008). Even though the exact defect in the motoneuron development is not presently known these data indicate a connection between motoneuron development and the posphatidyl inositol/PI3K/Akt signalling pathway.
2.4 Regulation of self-renewal in NPCs
2.4.1 Rb 2.4.2.1 Cell cycle regulation The family of Retinoblastoma (Rb) pocket proteins consists of three transcription factors namely pRb, p107 and p130. They all have tumour suppressor activity and they are all differentially expressed throughout the cell cycle. While pRb expression is constitutive during all phases of the cell cycle, p107 is synthesized predominantly during S phase and p130 expression predominates at G0 (Classon and Dyson, 2001). Rb 41
Figure 9 Cartoon of cell cycle regulatory activities mediated by pRb, histone deacetylases (HDAC), and E2F/DP-1 transcription factors. When Rb is bound to the E2F transcription factor complex, transcription of several cell cycle promoting genes downstream of E2F is repressed. The recruitment of HDAC by Rb to the E2F-inducible promoters further inhibits transcription, HDAC can deactylate histones and thereby keeps nucleosome composition tightly coiled. HDAC can also directly deacetylate E2F and inactivate its function. When Rb becomes phosphorylated (in G1) by cyclin D/cdk4/6 kinases, the Rb-HDAC proteins are released and E2F mediated transcription is activated. The figure is modified from Longworth and Laimins, 2004.
family members are key substrates for CDKs and cyclins, which phosphorylate it. Phosphorylated pRb is released from inhibiting the E2F transcription factors, which activate several genes needed for cell cycle initiation (in G1). Additionally, two classes of molecules can bind to CDKs and/or cyclins and inhibit their actions. The first group includes p15 INK4b, p16INK4a, p18 INK4c and p19 INK4d and the second group includes the CDK inhibitors p21 Cip1, p27 Kip1, and p57 Kip2. By forming different inhibitory binding complexes these inhibitors impair the function of cyclins and CDKs and pRb thus remains unphosphorylated as illustrated in figure 9 (Harbour et al., 1999; Pucci et al., 2000; Stiegler et al., 1998). Inhibition of Rb family proteins in embryonic NPCs increases proliferation but does not block their differentiation into neurons and astrocytes although the exit from the cell cycle is delayed (Piltti et al., 2006).
42
2.4.2.2 Self-renewal regulation The cell cycle regulatory network is, in addition to regulating cell division, involved in the regulation of the maintenance of pluripotency and stemness as well as in the induction of senescence (Clarke et al., 1992). In embryonic mice that lack both copies of the pRb gene, dividing neural precursor cells are found outside the normal neurogenic regions in both CNS and the PNS and the expression of neural differentiation markers is reduced (Clarke et al., 1992; Jacks et al., 1992; Lee et al., 1992). Our previous studies with decreased expression of the Rb family proteins (by overexpression of the viral papillomavirus oncogene HPV16 E7) show an increase in NPC self-renewal in vitro (Piltti et al., 2006). The cell cycle inhibitors p16INK4a and p19
INK4d
as well as p21
Cip1
are
important regulators of NPC self-renewal and ageing (Molofsky et al., 2003; Molofsky et al., 2005; Fasano et al., 2007). Also the downstream effectors of the MAPK signalling pathway ERK 1 and 2 induce rapid phosphorylation of pRb (Guo et al., 2005). Chemical inhibition of the MEK/ERK signalling pathway totally blocks self-renewal of embryonic neurospheres
whereas
proliferation
is
only
partially
inhibited.
Interestingly, chemical inhibition of CDKs only partially inhibits NPC self-renewal and proliferation thus strengthening the concept that proliferation and self-renewal are separately regulated cellular functions (Piltti et al., 2006). Studies with ES-cells have revealed that the mechanism of CDK regulation in these pluripotent cells is regulated differently as compared to other cell types. Proliferating ES cells have a very short G1 phase and the great majority of cells are in S phase and among other differences, the pRb and p107 are hyperphosphorylated and thus inactivated. These changes are crucial for the maintenance of pluripotency (Stead et al., 2002; White et al., 2005). Also, pluripotent stem cells are often quiescent and their cell cycle is thus in a temporary arrest and the cell cycle of these quiescent cells therefore has to be regulated in a specific manner (Galderisi et al., 2003). In leukaemia stem cells, the expression of p21Cip
43
is indispensable for the maintenance of self-renewal. In response to DNA damage induced by oncogenes, p21Cip expression leads to cell cycle arrest that provides time for DNA repair and thus prevents functional exhaustion of leukaemic cells due to an excess of mutations (Viale et al., 2009).
2.4.2 Bmi-1 Bmi-1 is a member of the polycomb group of proteins that function by epigenetic modification of chromatin. Bmi-1 knockout mice have severe developmental defects of the cerebellum, which is reduced in size due to severe loss of granular layer neurons but the development of the forebrain is relatively normal (Molofsky et al., 2003; Valk-Lingbeek et al., 2004). The expression of Bmi-1 in granular progenitor cells is activated by Shh signalling (Leung et al., 2004). Loss of Bmi-1 in E14.5 SVZ NPCs results in a reduced self-renewal capacity (as shown by the NSA), which was further decreased in P0 NPCs and dramatically reduced in P30 NPCs. The embryonic cerebral defects are thus more dramatic in vitro than in vivo. Loss of Bmi-1 also decreases the rate of proliferation, which probably also affected the self-renewal result. Proliferation of restricted, non selfrenewing neural progenitors was not affected by the depletion of Bmi-1, which distinguishes these two cellular functions. Bmi-1 represses the cyclin dependent kinase inhibitors p16ink4a and p19Arf and deletion of them partially rescued the self-renewal capacity in vitro (Molofsky et al., 2003). In vivo the deletion of p19Arf was able, but only partially, to rescue the cerebellar phenotype, which made the authors to conclude that additional downstream targets must be involved (Molofsky et al., 2005). A more recent study shows that another cell cycle inhibitor p21cip1 is repressed by Bmi-1 in NPCs isolated from an earlier developmental stage (E11). Acute loss of Bmi-1 clearly impairs NPC self-renewal, proliferation and survival in vitro and in vivo. Interestingly, loss of p21cip1 alone did not have the same effects (Fasano et al., 2007). In
44
neuroblastoma, a neural crest derived childhood tumour, Bmi-1 was also shown to be a target gene of the transcription factor E2F-1 which also activates transcription of essential cell cycle promoting genes including the transcription of N-Myc (Nowak et al., 2006). These new data reveal the complexity of the various cross reacting signalling pathways and also reminds about the various developmental stage-dependent regulatory differences (Fasano et al., 2007). The reduced expression of Bmi-1 increases levels of p16ink4a during ageing, which results in decreased numbers of NPCs in the forebrain SVZ regions but does not affect the DG of the hippocampus. The role of reduced Bmi-1 during ageing pinpoints an interesting regulatory aspect of self-renewal control and ageing, which is the balance between the risk of the development of cancer (which occurs if cell cycle inhibitor levels are reduced) and the depletion of stem cells (which occurs if cell cycle inhibitor levels are kept high). Because the balance has to be continuously re-evaluated in accordance with the increasing age of the individual, the system eventually results in an impaired renewal capacity of organs and finally aging (Molofsky et al., 2006).
2.4.3 p53 p53 is a widely studied tumour suppressor and transcription factor. It is most famous for its ability to induce apoptosis and cellular senescence but it is also involved in the regulation of neural differentiation and inhibition of self-renewal maintenance. The molecular mechanisms of p53-induced neuronal differentiation are associated with cell cycle arrest due to elevated levels of p21Cip and thereby activation of Rb (Chylicki et al., 2000; Ehinger et al., 1997; Galderisi et al., 2003). Myc also has an essential role in the regulation of NPC self-renewal and proliferation, which is discussed in the next chapter 2.5 in more detail.
45
In proliferating NPCs, p53 is barely detectable in the nuclei and after exposure of the cells to NGF to induce neural differentiation, high levels of p53 are found in the nuclei. Importantly, p53 is only visible in the cytoplasm of fully differentiated neurons (Eizenberg et al., 1996) and its role is to prevent cell cycle re-entry at early stage of neuronal differentiation (Miller et al., 2003). We and others have shown that embryonic NPCs null for p53 display increased in vitro proliferation and self-renewal capacity and they are able to differentiate normally although their cell cycle exit is delayed (Piltti et al., 2006; Zheng et al., 2008). This indicates that p53 expression is perhaps not crucial for the initiation of neural or glial differentiation but instead for the maintenance of the differentiated phenotype. Indeed, differentiated p53-null NPCs (that have all exit the cell cycle during 2 weeks in differentiation promoting culture conditions) are able to re-entry the cell cycle and self-renew when changed back to stem cell maintenance promoting culture conditions (Piltti et al., 2006). p53 is also implicated in suppression of adult NPC self-renewal (Gil-Perotin et al., 2006; Meletis et al., 2006).
2.4.4 Akt, PTEN The Akt kinase (also called protein kinase B, PKB) is a serine/threonine kinase. It is a downstream protein in a signalling cascade that belongs to the RTK family and it mediates its signal at least via receptors of the EGFR family ERBBs (Yuan and Cantley, 2008). Akt binds to phosphoinositides (PtdIns) such as the phosphatidylinositol (3,4,5)trisphosphate (PIP3) in the plasma membrane, which have been first phosphorylated by the family of phosphatidyl inositol 3 kinases (PI3Ks) which comprise of two units, a 110kDa and a 85, 55 or 50kDa protein. Once correctly positioned in the membrane via binding to PIP3, Akt can be phosphorylated by its activating kinases, phosphoinositide dependent kinase 1 (PDPK1), the mammalian target of rapamycin complex 2 (mTORC2) or other proteins like the MAPK kinases. Activated Akt can phosphorylate a myriad of substrates (Franke, 2008; Rossig et al., 2001;
46
Nave et al., 1999) and, among various other targets Akt activity also regulates the expression of Myc and Cyclin D (Gera et al., 2004). The lipid phosphatase and tumour suppressor Phosphatase and Tensin homolog, PTEN (Di Cristofano and Pandolfi, 2000) acts by negative regulation of the PI3K signalling pathway via dephosphorylation of Ptdins (Maehama and Dixon, 1998) as illustrated in figure 10.
PI3K Figure 10. A schematic illustration of the PI3K/Akt signalling pathway, which is a member of the RTK signalling family. The signal is mediated at least through EGFR family receptors ERBBs but the ligand is not known. Phosphatidylinositol (3,4,5)-trisphosphate PIP3 is phosphorylated by phosphatidyl inositol 3 kinase (PIK3, which consist of p85 and p110). Once bound to PIP3, the serine/threonine kinase Akt can be activated by several kinases (such as mTOR or MAPK signalling pathway members, not shown), which results in an increase in Akt signalling related cellular functions. The tumour suppressor PTEN dephosphorylates PIP3 and thus inhibits activation of Akt. The picture is modified from Yuan and Cantley, 2008.
Elevated levels of Akt1 in cortical neural progenitors increase selfrenewal, proliferation and survival. Only the increased self-renewal was regulated by mTOR activity (and was independent of p21cip, another known target of Akt1) which once more segregates proliferation and selfrenewal as separately regulated cellular functions (Sinor and Lillien, 2004). Deletion of PTEN in Nestin-positive neural progenitors results in increased proliferation of neural stem cells and decreased neural cell death. It also leads to a rapid brain hypertrophy and death of the mice soon after birth (Groszer et al., 2001) due to modulations of the Go-G1 cell cycle entry possibly induced by PI-3K activation dependent increase in Akt activity (Groszer et al., 2006; Morrison, 2002). E13.5 mouse NPCs that lack PTEN show a modest increase in proliferation and self-renewal capacity and are able to differentiate normally. Co-operative loss of PTEN and p53 together in NPCs, by contrast, results in highly increased self-renewal and proliferation. These cells fail to differentiate and thus obtain a more severe phenotype than that induced only by single tumour
47
suppressor depletion (Zheng et al., 2008). In line with this, our results with ectopic expression of the viral HPV16 E6/E7 oncogenes that decrease the activity of the tumour suppressor p53 and Rb family proteins are similar to this. Co-operative decrease of Rb and p53 activity in embryonic NPCs resulted in a much more dramatic increase of selfrenewal capacity than what was observed by downregulation of only one tumour suppressor (Piltti et al., 2006).
2.4.5 Sox 1-3 Expression of the transcription factors Sox1, Sox2 and Sox3 (Sox1-3) maintain neural cells undifferentiated by counteracting the activity of proneural proteins in the chick embryo. Conversely, the capacity of proneural bHLH proteins to direct neuronal differentiation critically depends on their ability to suppress Sox1-3 expression in CNS progenitors (Bylund et al., 2003), which is promoted by expression of another Sox-family member Sox21 (Sandberg et al., 2005).
2.5
Myc
2.5.1 The Myc family members and their general expression patterns The Myc-family of basic helix-loop-helix-zipper (bHLHZ) transcription factors comprises of the members, Myc (C-myc), N-myc and L-myc, Bmyc and S-myc. Myc is a cellular homologue of the avian Myelocytomatosis retroviral V-myc gene (Sheiness et al., 1980; Vennstrom et al., 1982). Myc, N-myc and L-myc have oncogenic potential and have been more studied than the rest of the family members. The proteins exhibit common structural features despite the relatively low (e.g. mouse Myc and n-myc sequence homology is 35%) similarity on gene level (DePinho et al., 1986; Legouy et al., 1987; Vennstrom et al., 1982). The expression patterns of these three Myc family members are clearly distinct albeit somewhat overlapping. Generally, the highest 48
mRNA expression is detected during development and in newborns although high expression levels in specific tissues (e.g. spleen and liver) are also detected in adults (Schreiber-Agus et al., 1993; Semsei et al., 1989; Zimmerman et al., 1986). Furthermore, Myc expression in adult mice is increased with age in the brain, liver, skin and small intestine (Semsei et al., 1989). A Myc null mutation causes embryonic lethality (between E9.5 and E10.5) with generally smaller and developmentally retarded embryos as compared to their normal littermates. Abnormalities are found in the neural tube and heart (Davis et al., 1993). The N-myc knockout mice also die around midgestation (E11.5) and the affected organs include CNS, PNS, heart, mesonephros (the developing kidney), lung and gut (Charron et al., 1992; Stanton et al., 1992). In contrast, homozygous mice lacking L-myc are viable (Hatton et al., 1996). Even though L-myc mRNA is specifically expressed in the developing kidney and lung as well as in the proliferative and the differentiating zones of the brain and neural tube, compensatory expression levels of Myc or N-myc could not be detected in the L-myc mutants (Hatton et al., 1996). However, N-myc can functionally replace Myc during development. Homozygous genetically modified mice with the replacement gene survive into adulthood and reproduce (Malynn et al., 2000). Also L-myc possibly compensates for the loss of Myc in the epidermis (Zanet et al., 2005). These findings emphasize the functional similarity of the Myc family members in the regulation of the vast variety of cellular functions that they are involved in. Finally, ectopic Myc expression or reduced levels of Myc, respectively, introduced to the germ line of mice does not result in major developmental disturbances, although tumour formation is increased by Myc overexpression (Leder et al., 1986; Stewart et al., 1984; Trumpp et al., 2001).
49
2.5.2 The transcription factor Myc Myc is a transcription factor that specifically binds to the regulatory DNA of various target genes and thereby either activates or represses their transcription. Myc is estimated to regulate as many as 15% of the genes of the genome from mouse to flies (Coller et al., 2000; Grandori et al., 2000; Levens, 2003) through several mechanisms including recruitment of
histone
acetylases,
chromatin
modulating
proteins,
basal
transcriptional factors and DNA methyltransferase (Dang et al., 2006; Eilers and Eisenman, 2008).
a. Competition on binding to Max b.
Myc Max Mad Miz-1 p300
Transcription - - - CACGTG - - - of Myc target gene E-box
c. Respression of - - - CACGTG - - -Myc target gene E-box transcription
d.
Transcription of Miz-1 target -----------gene
e.
Repression of Miz-1 target -----------gene transcription
Fig. 11. The Myc/Max heterodimer. a. The transcription factors Myc and Mad both need to heterodimerize with Max in order to be able to bind to their target gene promoter areas and they compete of available Max-proteins. b. The Myc/Max heterodimer binds to the E-box (CACGTG) sequence of the target gene promoter and thereby activates transcription. c. The transcription of various target genes that is activated by Myc/Max binding is inhibited by Mad/Max binding. d. Miz-1 is a transcription factor that does not bind to E-box. e. Binding of Myc/Max to Miz-1 represses transcription of Miz-1 target genes by inhibiting binding of co-activators like p300 to Miz-1.
50
2.5.2.1 Max and E-Box Several domains of the Myc protein are critical for its function. With the C-terminal bHLH leucine zipper (bHLH/LZ) domain it heterodimerizes with its binding partner protein Max and mediates sequence specific DNA recognition of the canonical E-box (CACGTG) elements (Amati et al., 1992; Luscher and Larsson, 1999; Pelengaris et al., 2002a). Members of the Mad family (known as Mad1, Mad2, Mxi1Mad3 and Mad4) compete with Myc of binding to Max. When bound to E-box, Mad/Max heterodimers (through recruitment of other co-regulators such as Histone deacetylase HDAC) repress the same transcriptional events that are activated by Myc/Max (Alland et al., 1997; Ayer et al., 1995) as shown in figure 11. 2.5.2.2 Myc homology boxes The N-terminal transcriptional activation domain (TAD) of Myc contains evolutionarily conserved domains called Myc homology boxes (MBs). MB I is implicated in regulation of Myc protein stability through a phosphorylation-dependent
ubiquitylation-mediated
proteosomal
degradation pathway which involves multiple protein kinases (Dai et al., 2006; Moberg et al., 2004). Several essential transcriptional co-activators such as Transformation/transcription domain-associated protein TRRAP (McMahon et al., 1998), a core component of the complex with histone acetyltransferases (HATs) CGN5 and Tip60, are recruited to Myc target gene promoters through binding to MB II and thus mediate histone acetylation and subsequent facilitation of target gene transcription (Frank et al., 2003; McMahon et al., 2000; Park et al., 2001) as shown in figure 12. Myc also directly upregulates the transcription of the HAT GCN5 (Knoepfler et al., 2006). Also, components of other chromatin remodelling complexes interact with Myc at MB II (Wood et al., 2000) as well as the ribosomal protein L11 that by negative feedback inhibits Myc induced ribosomal biogenesis (Dai et al., 2007). MB III domain is involved in the regulation of Myc-mediated apoptosis, transformation and tumorigenesis by inhibition of the recruitment of TRRAP (Herbst et al.,
51
2005). The conserved MB IV domain was recently characterized and it regulates DNA binding, apoptosis, transformation and G2-arrest (Cowling et al., 2006).
Figure 12. Myc facilitates target gene transcription by recruitment of histone acetylases. Top: a canonical gene is shown with deacetylated histones in nucleosomes (cylinders) and an E-box (CACGTG) bound by Mad–Max–Sin3 complex with histone deacetylase (HDAC) resulting in inhibition of transcription of the target gene. Myc– Max is shown in lower panels, demonstrating the sequential recruitment of TRRAP and histone acetylase (HAT) to MBII, the action of which unpacks the DNA from around the nucleosomes. Additional recruitment of SNF5, and pTEFb are needed for the RNA polymerase II machinery to start transcription of the target gene. The illustration is adopted from Dang et al., 2006.
2.5.2.3 Miz-1 The Myc/Max heterodimer can also directly bind to the zinc finger proteins Sp-1 or Myc-interacting Zinc finger protein-1 (Miz-1) and interfere with the transcription of various target genes. Importantly, as shown in figure 11, Miz-1 can recruit Myc/Max to promoters that lack the E-box binding site (Herold et al., 2002; Peukert et al., 1997). The Nterminus of Miz1 bears a Pox virus and Zinc finger / Bric-a-brac Tramtrack Broad complex (POZ/BTB) domain (Bardwell and Treisman, 1994), which is essential for both transactivation and Miz1-mediated cell
52
cycle arrest through upregulation of CDK inhibitors like p15ink4b and p57kip (Seoane et al., 2001; Staller et al., 2001) or the p53 induced increase of p21cip in response to DNA damage (Seoane et al., 2001; Herold et al., 2002). In addition to Myc, several other proteins bind to and regulate Miz-1 activity, including B-cell lymphoma gene 6 (Bcl-6), the Topoisomerase II binding protein (TopBP1), the ubiquitin ligase HectH9 and Smad protein complexes containing Smad3 and Smad4 (Adhikary et al., 2005; Herold et al., 2002; Phan et al., 2005; Seoane et al., 2001). For example, Myc/Max binding to Miz-1 prevents the association of Miz-1 with its obligatory co-factors such as p300 and nucleophosmin (NPM) that are needed for activation of p15ink4b (Eilers and Eisenman, 2008; Seoane et al., 2001; Staller et al., 2001). Miz-1 and the expression of p57kip are essential for embryonic development and the knockout mice die because of gastrulation defects at E7.5 (Adhikary et al., 2003). Also, TGF -regulated Myc levels affect the expression of various adhesionrelated genes in the mouse epidermis as discussed later (Gebhardt et al., 2006; Gebhardt et al., 2007).
2.5.2.4 Identification of Myc-specific binding sites Recent studies suggest that specific histone modifications, namely signs of active methylation of silent histones H3-K4 and H3-K79 are specific determinants of Myc binding and Myc-induced transcription (Eilers and Eisenman, 2008; Guccione et al., 2006). The data implies that the general transcription machinery is already engaged at these sites prior to Myc binding and has been accessed previously by other transcription factors. So, Myc perhaps functions as a modulator of the rate of ongoing transcriptional or post-transcriptional processes (Eilers and Eisenman, 2008).
53
2.5.2.5 What regulates Myc? Several studies have concentrated on the downstream targets of Myc but less is known about the regulatory mechanisms that regulate Myc activity itself. Despite the numerous binding sites of Myc in the various target proteins expressed in every cell, the general expression level of Myc is kept low. It is rapidly degraded and the half-life of Myc is only 20 minutes short (Eilers and Eisenman, 2008). Myc protein is activated by serial phosphorylation. The ERK kinase phosphorylates Myc on its serine 62 residue followed by the GSK3 -mediated phosphorylation of Myc on threonine 58 (Arnold and Sears, 2008; Pulverer et al., 1994). Myc can then be degraded by the protein phosphatase 2A (PP2A) -B56 holoenzyme mediated dephosphorylation on its serine 62 residue (Arnold and Sears, 2006; Arnold and Sears, 2008), which leads to degradation of Myc by the ubiquitination cascade in which at least the ubiquitin ligases that belong to the F-box proteins, Fbw7 and skp2, are involved (Eilers and Eisenman, 2008; Popov et al., 2007; Welcker and Clurman, 2008). A mutant form of Myc (T58A) is not recognized by the Fbw7 ubiquitin ligase and thus keeps Myc levels stable even upon growth factor withdrawal (Cartwright et al., 2005). The dephosphorylation of Myc by PP2A is inhibited by the binding of the cancerous inhibitor of PP2A (CIP2A) to Myc (Junttila et al., 2007), which is discussed later in more detail. In medulloblastomas, activation of the E2F transcription factor drives transcription of n-Myc (Strieder and Lutz, 2003). In ES cells, Myc is activated by the LIF/STAT /JAK PTK kinase signalling pathway and withdrawal of LIF from its receptor results in a reduction of Myc mRNA levels. In epithelial cells such as in the epidermis or hepatocytes, TGFinhibits Myc by an increase of CDK inhibitors p16 INK4a, p15ink4B and P21cip as well as Smad3, Smad4 and Rb (Gebhardt et al., 2006; Seoane et al., 2001; Sheahan et al., 2007). In the DNA level, the set of transcription factors that bind the Myc promoter as well as the precise
54
definition of the promoter and enhancer elements of the Myc gene are yet incompletely described and understood (Levens, 2003).
2.5.3 Cellular functions of Myc Even though Myc behaves as a global regulator of transcription by affecting thousands of target genes, genes involved in cell cycle regulation, metabolism, ribosome biogenesis, protein synthesis, growth and mitochondrial function dominate the Myc target gene network, (Dang et al., 2006 and http://www.myccancergene.org), which is also illustrated in figure 13. Myc's role in induction of apoptosis is also widely studied. 2.5.3.1 Cell cycle activation and growth Myc promotes cell proliferation by activating transcription of the cell cycle proteins such as E2F, cyclins D1, D2 and B1, Bmi-1 as well as the cyclin dependent kinase cdk4 by binding to E-box elements in the promoters of these genes (Fernandez et al., 2003; Hermeking et al., 2000; Eilers and Eisenman 2008 ). The expression of the cell cycle inhibitors p21Cip1 and p15ink4b are repressed by Myc via Miz-1 binding (Coller et al., 2000; Staller et al., 2001; Wanzel et al., 2003). Myc is a positive regulator of the mammalian cell size in the liver (Baena et al., 2005; Kim et al., 2000) and keratinocytes, which affects the overall size of the mice (Zanet et al., 2005). Myc also affects the signals that mediate organ or body size via affecting cell division decisions without an increase in cell size (Trumpp et al., 2001). In the Drosophila, dMyc stimulates the G1/S cell cycle activating transition independently of mitosis control thus resulting in increased cell size (Johnston et al., 1999) and dMyc also regulates organ size (de la Cova et al., 2004). Similar results are obtained from human keratinocytes, which result in cell size increase upon a cell division block induced by constitutive Myc expression (Gandarillas et al., 2000). Additionally, a massive induction of
55
Figure.13 The various cellular functions of Myc. A) The known cellular functions either activated (green lines) or repressed (red lines) by Myc. The figure is obtained from Eilers and Eisenman, 2008. B) Distribution of Myc targets by gene ontology (GO). One thousand five hundred and sixty-one Myc targets (small circles) from http://www.myccancergene.org are displayed in concentric rings by the OSPREY software (http://www.biodata.mshri.on.ca/os prey/servlet/Index) with functional groups coloured and labelled. GO groups highlighted in red are statistically over-represented as determined by EASE analysis (http://www.david.niaid.nih.gov/dav id/ease.htm). The figure is obtained from Dang et al., 2006.
B
ribosome biogenesis and regulation of RNA polymerases I, II and III and thus an overall increase in protein synthesis is associated with the increased levels of Myc-induced cellular growth (Arabi et al., 2005; Dang et al., 2006; Gomez-Roman et al., 2003; Grandori et al., 2005; Kim et al., 2000; Oskarsson and Trumpp, 2005). Myc also affects many enzymes to
56
increase glucose uptake and glycolysis as well as mitochondrial biogenesis. The increase in the intracellular iron pool as well as in nucleotide biosynthesis are also associated with increased Myc expression. Taken together, Myc is involved in a myriad of cellular events but many of these functions that affect metabolism and thus facilitate the Myc-associated growth phenotypes require further investigation (Dang et al., 2006; Liu et al., 2008). 2.5.3.2 Adhesion Myc overexpression studies have revealed downregulation of a set of genes that encode proteins essential for maintenance of cellular adhesion and the cytoskeleton including the extracellular matrix proteins collagen and fibronectin as well as tropomyosin in fibroblasts (Coller et al., 2000). In keratinocytes, 30% of the downregulated genes were adhesion related proteins (Frye et al., 2003). In the epidermis, the adhesive changes of Myc are largely induced via Miz-1 binding (Gebhardt et al., 2006). The Myc-induced changes in adhesion properties are important in controlling the balance of self-renewal and differentiation in the stem cell niche (Murphy et al., 2005 as will be discussed below) and may also facilitate transformation of cells by inducing morphological changes and by promoting anchorage-independent growth (Shiio et al., 2002). 2.5.3.3 Apoptosis Myc can drive cells to undergo apoptosis but the molecular mechanism remains still somewhat unclear (Meyer et al., 2006). Acute or ectopic expression of Myc increases cell proliferation and growth but it simultaneously also activates expression of p53 and ADP Ribosylation Factor (ARF), which induce either apoptosis or cell cycle arrest (Dang et al., 2005). Myc also promotes apoptosis by inducing expression of Bim (a member of the Bcl family proteins), which in turn inhibits the antiapoptotic factor Bcl-2 (Hemann et al., 2005) as illustrated in figure 14. Myc also determines the outcome of the p53 response after DNA damage: Myc by binding to Miz-1 represses p53 induced p21Cip1
57
BCL-2 Figure 14. Illustration of how Myc promotes apoptosis and inhibits cell cycle activity through induction of p53 expression. Acute Myc expression increases cell cycle progression and cell growth. At the same time acute Myc can increase the expression of p53, Arf and the Bcl-family protein Bim, which all promote apoptosis and/or cell cycle arrest. Bim also represses the activity of Bcl-2, another member of the Bcl family that has an anti-apoptotic function. The figure is modified from Dang et al., 2005.
expression which would promote cell cycle arrest. Therefore, binding of Myc favours the other option of increased p53 expression and thus the transcriptional activation of p53 upregulated mediator of apoptosis (PUMA) and p53-induced gene 3 (PIG3), which mediate apoptosis (Seoane et al., 2002). In addition to apoptosis, reduced Myc signalling triggers senescence in fibroblast in a telomere independent manner by regulating the polycomb group repressor protein Bmi-1 and p16ink4a (Guney et al., 2006). 2.5.3.4 The oncogenic role of Myc Myc is involved in 20% of all cancers affecting about 100 000 cancer deaths in the USA each year (Cole and McMahon, 1999; Dang et al., 2006). Myc expression level correlates with the prognosis of breast, colon, gastric cancer, glioma, medulloblastoma, various types of haematological malignancies and many others (Eberhart et al., 2004; Faria et al., 2006; Robson et al., 2006; Schwab, 2004; Wang et al., 2008). Whether Myc overexpression alone is capable of transformation and initiation of malignant growth is dependent on the cell type and developmental context. As shown with inducible Myc in murine hepatocytes, they are more resistant to Myc induced tumorigeneity in 58
adults than in young animals (Beer et al., 2004). Also some cell types, like stem cells, provide favourable circumstances for cancer initiation. One example of this is the neural stem cells. Myc overexpression in NPCs or early astroglial cells predisposes mice to primary malignant gliomas (Jensen et al., 2003; Zheng et al., 2008). Also, Myc overexpression in neural stem cells of the external granule layer of the cerebellum does not produce medulloblastoma formation but coexpression
with
repressor
element-1
silencing
transcription
factor/neuron-restrictive silencer factor REST/NRSF (a transcriptional repressor of neuronal differentiation) was enough to trigger cerebellumspecific tumorigenesis (Su et al., 2006). In Drosophila, dMyc overexpression due to the inactivation of the dMyc inhibitor Brat leads to the formation of larval brain tumours in NSCs (Betschinger et al., 2006). Myc facilitates the creation of the feasible environment for transformation: e.g. induction of elevated levels of active forms of the oncogenes Ras and Akt are sufficient to induce glioma formation in NPCs but not in differentiated astrocytes. Simultaneous overexpression of Myc in GFAP-positive astrocytes changes the identity of the cells towards a more undifferentiated phenotype (expression of GFAP is lost, the progenitor marker Nestin is activated) and glioma formation is activated (Lassman et al., 2004). Also, the tumorigeneity of the induced pluripotent cells (iPS) is caused by Myc (Nakagawa et al., 2008), as discussed later. Some cells have defence mechanisms against cancer formation. As acute Myc overexpression promotes cell proliferation and an increase in general metabolic activity, it also induces activation of p53, ARF and/or Bim and thereby triggers apoptosis (Beer et al., 2004; Dang et al., 2005; Pelengaris et al., 2002b). Inhibition of apoptosis by co-expression of the anti-apoptotic factor BCLXL together with Myc induces neoplastic lesions and cancer progression in pancreatic -cells (Pelengaris et al., 2002b). Inability of a spontaneously mutated form of Myc to activate Bim is
59
found in cells of Burkitt’s lymphoma (Hemann et al., 2005). However, not all cells, such as differentiated keratinocytes, protect themselves by induction of apoptosis. Sustained overexpression of Myc in these cells is sufficient to induce papillomatosis with profound angiogenesis, which is completely reversible upon inactivation of Myc (Pelengaris et al., 1999). Studies on other cancer types have later repeated the permanent reversibility of cancer progression by inactivation of Myc (Flores et al., 2004; Jain et al., 2002; Pelengaris et al., 2002b) although tumour dormancy was reported in hepatocellular cancer (Shachaf et al., 2004). These results have encouraged development of cancer therapy models based on targeted inactivation of Myc or its direct downstream targets essential for Myc-induced tumour progression without affecting the functions of Myc in healthy, self-renewing tissues (Lawlor et al., 2006; Pompetti et al., 2003). A recently published model using a dominantinterfering Myc mutant report promising results from Ras-induced lung adenocarcinomas (Soucek et al., 2008).
2.5.4 Myc in stem cells 2.5.4.1 Myc in epidermal stem cells Myc regulates stem cells in several tissues. Of the vast variety of Myc functions many also apply to stem cells although each tissue and stem cell type has its own specific outcome of Myc expression (Murphy et al., 2005) as summarised in table 1. In the human and mouse skin epidermal stem cells, Myc induces the self-renewing stem cells to become rapidly proliferating transit amplifying (TA) cells that are determined to differentiate (Arnold and Watt, 2001; Gandarillas and Watt, 1997). However, none of the steps of epidermal differentiation are totally blocked despite the lack of Myc expression, which suggest that the role of Myc is to enlarge the progenitor pool but also other factors are involved in the control of self-renewal and differentiation (Zanet et al., 2005). Interestingly, in keratinocytes cell cycle activation is permitted even
60
during differentiation. Continuous activation of Myc in differentiating human keratinocytes results in a block of G2/M and thereby cellular growth, endoreplication and ultimately polyploidy of the post-mitotic cells, which also occurs in wild type (WT) postmitotic keratinocytes (Gandarillas et al., 2000). A more recent study suggests that endoreplication is not needed for the terminal differentiation of the cells to take place, but that endoreplication is necessary for the cell enlargement that occurs during normal post-mitotic differentiation (Zanet et al., 2005).
Table 1. Myc functions in tissue stem cells. Transit amplifying cell, TA; haematopoietic stem cell; HSC; neural stem cell, NSC; neural progenitor cell, NPC; Species, SP; mouse, Ms; human, Hu; Drosophila, Dro. TISSUE
STEM CELL TYPE
MYC FUNCTION
SP
Epidermal stem cell
Self-renewal, proliferation and stem cell pool size maintenance
Ms
Skin Keratinocyte (TA)
Intestine
Bone marrow
Ms, Hu
SPECIAL CHARACTERISTICS
Miz-1 1-integrin
Hair (TA)
Expansion of the TA pool and exit from the niche
Intestinal stem cells (at the bottom of the crypt)
Self-renewal maintenance and crypt formation in juveniles
Long term HSC
Self-renewal, proliferation and stem cell pool maintenance
Lower Myc level than in TA cells
Short term HSC (TA)
Expansion of the TA pool and exit from the niche
High Myc level Cip (p21 down)
Ms
Ms
Wnt-signalling: Myc activated by TCF4
Arf
Brain
NSCs
Self-renewal maintenance
NPCs (TA)
Expansion of the TA pool
Ms,Rat Hu,Dro Ms
High Myc level (p19 low, p53/PTEN low during neurogenesis before gliogenesis) Miz-1 N-myc
High expression levels of 6 and 1-integrins are especially important for epidermal stem cell maintenance and their binding to the niche. In response to reduced TGF
stimuli, Myc expression through Miz-1
binding reduces adhesive interactions with the local environment and
61
thereby switches the identity of epidermal stem cells into TA cells, which then exit the stem cell compartment or niche. Myc is also needed for the rapid proliferative expansion capacity of TA cells as well as for the maintenance of an appropriate size of the stem cell pool (Gebhardt et al., 2006; Zanet et al., 2005) Thus overexpression of Myc in the basal layer (which contains the epidermal stem cells) and hair follicles under the K14 keratin promoter of mouse epidermis results in depletion of stem cells because of the driving force of Myc to push all the stem cells into TA cells and no stem cell reserve is left in the niche (Arnold and Watt, 2001). Also, as shown by direct deletion of Miz-1 under the K14 promoter, the results are in accordance with the Myc studies and show that Miz-1 is required for the correct formation of hair follicle structure and hair morphogenesis and the interfollicular epidermal areas were thickened (Gebhardt et al., 2007). On the other hand, if Myc is overexpressed in terminally differentiated keratinocytes, cell cycle gets reactivated and differentiation is severely disrupted (Pelengaris et al., 1999). Depletion of endogenous Myc in the proliferative keratinocyte progenitors of the epidermis (under the K5 promoter) results in skin defects although the differentiation ability itself is not affected. In the absence of Myc, stem cells that would normally remain self-renewing and proliferate rather rarely are now turned into proliferating TA cells to compensate for the lack of cells needed for differentiation and skin renewal. This hyperproliferation defect was emphasized in areas of high mechanical stress and in the context of wound healing. 2.5.4.2 Myc in haematopoietic stem cells The role of Myc in the adult haematopoietic system is to control the balance between self-renewing stem cells in the BM niche and their differentiation into TA cells that exit the BM. The function of Myc in the BM is similar to that in the skin although there are differences in the adhesive components of these two stem cell niches. Myc expression in the haematopoietic stem cells (HSCs) predominantly decreases the
62
expression of the BM anchoring protein N-cadherin (but also regulates the expression of several other proteins including 1-integrins), which is needed for the exit from the niche and endogenous Myc expression is induced upon onset of Long Term (LT) –HSC differentiation into committed progenitor cells (Wilson et al., 2004). Loss of Myc (under the INF -inducible Mx-Cre promoter) results in accumulation of HSCs in the BM and thus total depletion of differentiating cells. The differentiation capacity of these Myc null HSCs is not defected as they differentiate normally in vitro thus in the absence of the niche. Also proliferation and survival of the Myc null LT-HSCs was normal but Myc expression is crucial for the proliferation of the lineage committed progenitors in the BM (Wilson et al., 2004). Circulating naive T-cell blast also need Myc for re-entering the cell cycle following T cell receptor (TCR) activation (Trumpp et al., 2001). Accordingly, overexpression of Myc in HSCs leads to a loss of self-renewal activity, downregulation of N-cadherin expression and differentiation of the cells (Wilson et al., 2004). Once out of the niche, Myc is not needed for the terminal differentiation process of the cells. Based on studies on Mads, the transcriptional binding partners that compete with Myc of Max binding, Max switches from Myc to Mads during terminal differentiation of monocytes and macrophages accompanied with downregulation of endogenous Myc as well and upregulation of Mads (Ayer and Eisenman, 1993). 2.5.4.3 Myc in intestinal stem cells The role of Myc in the gut renewal and the proper formation of the intestinal mucosa are regulated by the canonical Wnt signalling pathway. Ectopic expression of the Wnt inhibitor Dickkopf1 (Dkk1), under the Villin promoter that is specifically expressed in the epithelial layer of the entire crypt-villus unit of the intestine (Pinto et al., 1999) leads to an absence of nuclear
-catenin, inhibition of Myc expression and
subsequent upregulation of p21cip. Epithelial proliferation is reduced and the crypts that contain the intestinal stem cells are lost. Interestingly,
63
enterocyte differentiation seems unaffected but secretory cell lineages are largely absent (Pinto et al., 2003). Endogenous Myc is expressed in the proliferating TA compartment of the crypts whereas N-myc expression is restricted to a single cell located near the crypt base as well as to the differentiated villus epithelium. Studies made with a tamoxifen inducible loss of Myc activity model (Mycflox under the CreEr2villin promoter) show that proliferation or fate of the villus or the crypt cells or the homeostasis of the intestinal epithelium is not dependent on Myc in adults but it is required for the formation of the intestinal crypts in juvenile mucosa (Bettess et al., 2005). 2.5.4.4 Myc in neural stem cells All Myc family members are expressed in the developing human and mouse foetal brain. High expression levels of Myc, N-myc and L-myc mRNA are reported from the mitotically active periventricular zones including the precursor cells in the neuroepithelium (although expression was seen also in some postmitotic layers) and in the granular cells of the developing cerebellum. The expression pattern of all three Myc family members is different (Ruppert et al., 1986) and the peak of N-myc expression in the ventricular zone is from E12.5 to E14.5 in the mouse, thus after the onset of neurogenesis (Hirvonen et al., 1990). Also, the expression pattern of a negative regulator of Myc, the Myc intron I binding protein (Mibp1), is restricted to opposite areas than Myc expression, namely to the postmitotic cortical plate and it is absent from the proliferative zones (Campbell and Levitt 2003). N-myc expression is needed for NPC expansion and it inhibits neural differentiation during mouse brain development. The loss of N-myc in Nestin-positive NPCs severely disrupts the expansion of progenitor cell populations and the compromised proliferation is also shown as an increased expression level of several CDK inhibitors (such as p27kip and p18ink), whereas levels of p21cip1, p15ink4b and p19ink4d are not altered. Probably because of a reduction in the amount of progenitor cells, the overall brain size and the amount of neuronal differentiation is decreased due to N-myc deficiency
64
in vivo, whereas the N-myc null NPCs differentiate more in vitro as compared to control cells (Knoepfler et al., 2002). N-myc also induces maintenance of active chromatin in NPCs and the nuclei of N-myc null cells (as well as of terminally differentiated neurons) are smaller and more condensed (Knoepfler et al., 2006). The role of Myc has also been investigated regarding neural development. Myc overexpression in the mouse CNS promotes proliferation (Fults et al., 2002). Ectopic expression in astrocytes turns the morphology back to progenitor-like cells and Nestin expression is restored, which also makes the cells responsive for oncogenic changes (Lassman et al., 2004). The Drosophila dMyc is postranscriptionally regulated by the tumour suppressor Brat, which is essential in regulating asymmetric division of the neural stem cells (neuroblasts). Ribosome biogenesis and protein translation are regulated by dMyc and when dMyc is not inhibited in the Brat mutants, self-renewal promoting conditions are allowed in both daughter cells and thus all neuroblasts only divide symmetrically and no progenitor cells destined for differentiation are formed (Betschinger et al., 2006). Five recent papers, including ours, demonstrate that Myc is essential for self-renewal in mouse, rat and human NPCs (Zheng et al., 2008; De Filippis et al., 2008; Wang et al., 2008; Kerosuo et al., 2008 and Nagao et al., 2009). Myc is a target for cooperative actions of the tumour suppressor p53 and Pten/PI3K pathways in the regulation of self-renewal and differentiation of NPCs (Zheng et al., 2008). p53 binds directly to the Myc promoter and represses Myc transcription through a mechanism that involves histone deacetylation (Ho et al., 2005). Also, p53 mediated expression of p19ARF attenuates self-renewal and pushes the fate of late stage (E18.5) rat NPCs towards a glial fate. In early phase NPCs (E13) Myc expression is high and p19ARF expression level is kept low and overexpression of Myc increases self-renewal of NPCs (Nagao et al., 2009). In the same study, Myc knockout mice (that die around E10.5, Davis et al., 2003) are shown to have a smaller forebrain which contained much less mitotic cells than
65
control brains at E9.5. Also, targeted in vivo deletion of Myc under the NestinCreER promoter causes differentiation of the Myc null cells into neurons at E15.5 and in contrast, into glial cells if the deletion is induces at E16.5 or later (Nagao et al., 2009). The downstream pathway of PI3K can also affect Myc translation and protein degradation (Gera et al., 2004; Sears et al., 2000). Myc levels were substantially increased in p53/Pten double null NPCs, which also showed increased self-renewal and a distorted capacity to differentiate. Furthermore the reduction of Myc levels with a short hairpin (shRNA) largely restored the differentiation capacity (Zheng et al., 2008). Another recent study on human NPCs also shows that Myc increases NPC selfrenewal and proliferation and that ectopic overexpression of a stabilised form of Myc (MycT58A) does not inhibit neural or glial differentiation, respectively (De Filippis et al., 2008). 2.5.4.5 Myc in embryonic stem cells The self-renewal capacity of mouse ES-cells is dependent on the presence of the leukaemia inhibitor factor LIF bound to its receptor, which activates the transcription factor STAT3 pathway, which in turn activates Myc. Overexpression of Myc inhibits differentiation and Myc thus promotes self-renewal of ES cells (Cartwright et al., 2005). Several recent studies have shed light on Myc’s capacity to regulate self-renewal as it is involved in the in vitro reprogramming of pluripotent stem cells from differentiated cells together with three other factors, the known ESmarkers Sox-2, Oct4 and the transcription factor Klf4 (Takahashi and Yamanaka, 2006). iPS are competent in forming viable chimaeras, they contribute to the germline and they are indistinguishable from ES cells in terms of gene expression and DNA methylation patterns (Knoepfler, 2008; Okita et al., 2007; Takahashi and Yamanaka, 2006; Wernig et al., 2007). However, the tumorigenic potential of iPS cells seems to be due to Myc expression (Okita et al., 2007) and from the perspective of possible therapeutic usage of these cells, ectopic retrovirally induced expression of
66
Myc is problematic. Therefore, induction of pluripotency with only two ectopic factors, Oct4 and Klf4, into somatic cells that endogenously express high levels of Myc and Sox-2, like the mouse adult NPCs, is sufficient to generate iPS and plausibly a safer approach in terms of clinical applications (Kim et al., 2008). The generation of iPS from mouse and human dermal fibroblasts without Myc is successful but much more infrequent (Nakagawa et al., 2008). Ectopic retroviral expression of Myc is silenced directly after reprogramming of fibroblasts and thus suggesting that high Myc expression level is not needed for the maintenance of pluripotency in iPS (Wernig et al., 2007).
2.5.5 CIP2A 2.5.5.1 Mechanism of CIP2A-mediated inhibition of Myc degradation CIP2A (also termed p90 or KIAA1524) is a recently discovered protooncogene (Junttila et al., 2007). The 90kDa protein is mainly expressed in the cytoplasm but is visible also in the nucleus by immunostaining. It functions by binding to Myc and thereby prevents PP2A mediated dephosphorylation of Myc on its serine 62 residue. CIP2A binding thus posttranscriptionally rescues Myc from ubiquitination, which results in Myc stabilization and activity increase without affecting the mRNA level of Myc. The interaction of CIP2A and the PP2A complex is direct, the amino acids of CIP2A between 461 and 533 are involved in the binding of CIP2A to the PR65 scaffold unit of PP2A (Junttila et al., 2007). The CIP2A binding is illustrated in figure 15. 2.5.5.2 The oncogenic function of CIP2A The function of CIP2A has only been studied in malignant cells. Overexpressed CIP2A levels have been shown in three human cancers, head and neck squamous cell carcinoma (HNSCC), colon cancer (Junttila et al., 2007) and gastric cancer (Li et al., 2008). In vitro overexpression of CIP2A in cervical cancer derived HeLa cells increases proliferation. In
67
order to induce transformation, CIP2A overexpression alone is not sufficient in non-malignant mouse embryo fibroblasts (MEFs) although foci formation is increased when co-expressed with an oncogenic
form
of
Ras
(RasV12). A set of changes needed
for
immortalization
the of
human
cells include inactivation of p53 and Rb (by SV40 large T antigen, LT), expression of an active telomerase (hTERT) and expression of an active form of Ras (H-Ras, Hahn et al., 1999). To further induce a Fig 15. CIP2A stabilizes Myc. a) Myc is activated by serial phosphorylation first on its Serine 62 residue by the ERK kinase and then on its threonine 58 residue by the GSK-3 kinase. b-d) The protein phosphatase 2A (PP2A)-B56 holoenzyme binds to Myc and dephosphorylates the serine 62 residue, which targets Myc for degradation by ubiquitynation. The action of PP2A is inhibited when CIP2A binds Myc resulting in stabilization of active Myc. The illustration is obtained from Westermarck and Hahn, 2008.
fully transformed phenotype requires suppression of PP2A activity (Hahn et al., 2002). Manipulated
human
embryonic kidney fibroblasts with all the above mentioned mutations (HEK-TERV cells) except for the ability of PP2A
inhibition
are
fully
transformed
only
by
additional
CIP2A
overexpression, thus demonstrating its oncogenic potential (Junttila et al., 2007). Depletion of CIP2A, on the other hand, reduces oncogenic potential in tumour cells. Foci formation of HeLa cells as well as cell lines of gastric and colon cancer is impaired and growth is defected. CIP2A depletion induced growth arrest rather than apoptosis and senescence was reported in one gastric cancer cell line (AGS). The effects of CIP2A depletion seem to be regulated independently of the p53/p21 and Rb/p16 signalling
68
pathways (Junttila et al., 2007; Li et al., 2008). Also, in vivo tumour growth of subcutaneously injected HeLa cells into athymic mice is significantly inhibited after CIP2A siRNA injection as compared to tumours that received scrambled siRNA (Junttila et al., 2007). Furthermore, depletion of CIP2A expression in leukemic HL60 cells triggers signs of myeloid differentiation (Li et al., 2008). Importantly, all demonstrated cellular functions of CIP2A in the several studied cell types correlate with the changes of endogenous Myc expression levels (Junttila et al., 2007). 2.5.5.3 PP2A PP2A is a family of ubiquitously expressed serine / threonine phosphatases. They comprise of three subunits that altogether allow the
Fig. 16. Structure of the trimeric holoenzyme PP2A. The structural subunit A (PR65/PPP21, either or form) provides the binding core for the B and C subunits. The subunit C (either or form) is the catalytic part of PP2A and the subunit B (which has tens of different subtypes) is responsible for the substrate specificity. The figure is obtained from Westermarck and Hahn, 2008.
generation of more than 60 different heterotrimeric PP2A holoenzymes as illustrated in figure 16. Several variable possibilities such as the holoenzyme composition as well as methylation or phosphorylation of the catalytic subunit can regulate PP2A activity (Junttila et al., 2008). PP2A 69
dephophorylates hundreds of target proteins and is thus involved in the regulation of a large number of cellular events including e.g. cell cycle, apoptosis, cell growth, genomic instability and differentiation (Janssens and Goris, 2001). The structural subunit PR65 (A or A ) provides the binding core for B and C subunits. The two isoforms A and A share a 87% sequence homology and both are generally ubiquitously expressed (Hemmings et al., 1990) although different ratio of A /A expression is associated e.g. with different embryonic stages of the xenopus embryo (Bosch et al., 1995). Mutations of both isoforms are found in human lung, breast, and colon cancer (Ruediger et al., 2001; Wang et al., 1998) and the cancerassociated mutations of A or A lead to either increased phosphorylation of Akt or the small GTPase RaIA, respectively (Westermarck and Hahn, Targets:
Subunit :
c-Myc -Catenin Bcl2
B56 B56 B56
Pim1
?
p53 Paxillin Mdm2/Hdm2 p300
B56 B56 Tumour suppressor Dopamine /cAMP regulated
Cdc25 HAND1 DARPP-32
2008). The catalytic subunits PP2AC (C or C ) are highly conserved and the two mammalian PP2AC isoforms are ubiquitously expressed and share a 97% DNA sequence identity (Stone et
al.,
1987).
The
substrate
specificity and PP2A localization
Wnt signalling
determination is, on the other hand,
Oncogene
regulated by four genetically and
Transcription Factor
Figure 17. Schematic drawing of the known targets of the PP2A-B56 holoentzymes, which include many transcription factors that also have a role in neural stem cells. PP2A-B56 is the subtype B that recognises and dephosphorylates Myc. The picture is modified from Arnold and Sears, 2008.
structurally different B subunits (B, B’, B’’ and B’’’) that are each encoded by a family of related genes and tissue specific expression has been reported for many of the different isoforms (Janssens and Goris, 2001).
70
All the five members of the holoenzyme family PP2A-B56 (B’ or PPP2R5) act via regulation of oncogenes as illustrated in figure 17 (Arnold and Sears, 2008). The PP2A-B56 inhibits the Wnt /
–catenin
signalling pathway (Seeling et al., 1999) during early neural development (Yang et al., 2003) and it also inhibits the function of Myc and the antiapoptotic protein Bcl-2 (Arnold and Sears, 2006; Arnold and Sears, 2008). Several PP2A holoenzyme forms inhibit different components of the mitogenic MAPK (Ras/Raf-1/ MEK1,2/ERK1,2) signalling pathway, which among several other targets phosphorylates Myc (Junttila et al., 2008). Inhibition of PP2A by chemical inhibition or by SV40 and polymer virus induction is a perquisite for oncogenic changes (Janssens and Goris, 2001) and malignant growth in myeloid leukaemia cells is promoted by the BCR/ABL induced expression of the PP2A inhibitor SET (Li et al., 1996; Neviani et al., 2005).
71
3. AIMS OF THE STUDY
I
To investigate the roles of the proto-oncogenes Myc and CIP2A in neural
progenitor
cell
self-renewal,
proliferation
and
differentiation. II
To investigate the molecular regulatory pathways behind the selfrenewal control changes activated by overexpressed oncogenes.
III
To study the specific
mechanisms
of NPC self-renewal
maintenance in relation to other stem cell properties such as proliferation. IV
To develop procedures for studying patient neural stem cell characteristics and to understand the possible stem cell pathology behind a human motoneuron disease.
72
4. MATERIALS AND METHODS All methods and materials applied to this thesis are listed below and sited according to the original publications in which they appear. Method
Original publication
NPC collection
I, II, III
Neurosphere culture
I, II, III
Differentiation culture of NPCs
I, II, III
Retroviral transduction
I, II
Reverse Transcriptase PCR
I, III
Immunofluorescence (IF)
I, II, III
Protein extraction and Western Blot (WB)
I, II
Flow cytometry (FACS)
I
BrdU cell proliferation assay
I, II, III
Self-renewal assay
I, II
Cell ploidy analysis assay
I
Apoptosis assay
I, III
Re-sphering assay
(self-renewal
maintenance) I
Immunohistochemistry (IHC)
I, II
Growth measurements
I, II
In situ hybridization
II
Cell cycle exit assay
I, II
Microarray analysis
III
73
Unpublished materials and methods CDK and MAPK pathway inhibitors R-Roscovitin (10µM, ALX-350-251, Alexis Biochemicals, Switzerland), was dissolved as a 10mM stock solution in DMSO and used for inhibition of CDKs (1, 2 and 5). U0126 (50 µM, Calbiochem, Ca, USA) an inhibitor of MEK1 and 2 was dissolved as 10mM stock solution in DMSO. The inhibitors were added to the culture medium at the beginning of the experiment (self-renewal and BrdU assays) and respective concentrations of DMSO were used as a control. Stem cell marker antibody staining for FACS Several stem cell associated antigens were tested in order to find specific markers for long term self-renewal potential neural stem cells. Oct-4 (1 µg/ml, AB3209 Chemicon, USA), Sox-2 (1 µg/ml, AB5603, Chemicon), c-Kit (1 µg/ml, AB5506, Abcam. UK), CD133 (1 µg/ml, AB16518, Abcam) and Musashi (1 µg/ml, 5977 Chemicon). Re-sphering assay II The neurospheres were isolated into single cells by mechanic trituration and 200 cells/well on a 96-well cell culture plate were cultured in differentiation medium supplemented with 2% FCS. After 7 days, when all cells had attached to the bottom of the wells and differentiated, the culture medium was changed back to stem cell culture conditions supplemented with EGF and FGF. The number of newly formed neurospheres was counted after a week of stem cell medium culture.
74
5. RESULTS 5.1 Verification of the successful genetic modification of NPCs (I, II) Mouse embryonic E12 NPCs were retrovirally transduced with human MycER or CIP2A, respectively, and cloned to the pBabe vector with puromycin resistance. For controls, empty pBabe or untransduced cells were used. Expression of the transduced gene was verified by culturing the cells in selective conditions and by using Western blotting (WB) and fluorescence activated cell sorting (FACS). The expression level of the transgene in individually transduced NPC pools was verified to be similar (I/Fig 1A-B and II/Fig 2A).
5.2 Activation of oncogenic pathways increase proliferation (I, II, III) 5.2.1 Myc and CIP2A increase NPC proliferation in neurospheres The NPCs derived from the lateral ventricle area of embryonic mice were cultured in a stem cell medium containing EGF and FGF. Proliferation rate was measured by using the 5-bromo-2-deoxyuridine (BrdU) labelling method, which shows the percentage of cells entering S phase in 24h of culture. Also growth measurements to count the actual numbers of newly formed cells were used. Based on these studies, both experimental settings clearly showed that Myc and CIP2A NPCs proliferated faster than WT control cells or controls transduced with an empty vector (I/Fig 2A-B and II/Fig 2B-C).
5.2.2 LCCS pathway results in increased proliferation The NPCs derived from human LCCS patients and age-matched (from 14-20 gestational weeks) control foetuses were cultured in EGF, FGF and
75
LIF containing stem cell medium. Proliferation rate measured by BrdU was slightly increased in the patient neurospheres from 2.5% in controls to 7% in LCCS NPCs (III/Fig 2A-B).
5.3 Activation of Myc and CIP2A increase self-renewal (I, II) Self-renewal was measured by using the neurosphere assay that is based on the counting of secondary sphere formation from single cells cultured in the serum free EGF and FGF containing stem cell medium (SCM). The percentage of self-renewing cells in the neurosphere cultures increased significantly in Myc (from 4,2% in controls to 24% in Myc ) and CIP2A (from 5% in controls to 15% in CIP2A) overexpressing NPCs, respectively, as compared to the controls (I/Fig 2C and II/Fig 3B). The increased self-renewal capacity did not affect the proportion of differentiated cells inside the neurospheres as shown by FACS (I/Fig 2E). Taken together, Overexpression of Myc and CIP2A, respectively, significantly increases the proportional number of self-renewing NPCs in a neurosphere.
5.4 Differentiation capacity is not blocked by oncogenes or LCCS (I, II, III) 5.4.1 Myc or CIP2A do not block differentiation although Myc NPCs are affected Differentiation capacity of the Myc or CIP2A overexpressing NPCs was evaluated by using several methods. After in vitro differentiation in 2% serum containing medium (for 4-7 days), the morphology of the differentiated CIP2A astrocytes and neurons was normal as compared to control cells by IF. The expression of the retrovirally induced CIP2A was verified by using IF (II/Fig 4A-F). Myc NPCs also attached to the polylysine coated surface and expressed the astrocyte and neuron specific markers (GFAP and Tuj-1) but the morphology of some cells was affected and seemed less differentiated as compared to differentiated 76
control cells (I/Fig 3A). Also, the expression of stem cell markers Nestin and Bmi-1 was lost during differentiation of both Myc overexpressing and control cells (I/Fig 3B-C). CIP2A expression was downregulated to non-detectable levels in differentiated control NPCs and substantially in Myc overexpressing NPCs (II/Fig 5G-H).
5.4.2 Myc and CIP2A delay cell cycle exit (I,II) One sign of terminal differentiation is cell cycle arrest. We used BrdU labelling to analyse whether the oncogene expressing NPCs had defects in their capacity to shut down cell cycle. Both Myc and CIP2A NPCs showed significantly reduced capacity in cell cycle exit and were thus still proliferating (Myc 4% on day 3, CIP2A 0.9% by day 3 and 0.1% by day 7) at the time point when all control cells had already shut down their cell cycle (I/Fig 3D and II/Fig 4G). Based on these differentiation studies, overexpression of neither Myc nor CIP2A blocks neural or glial differentiation in NPCs even though Myc causes clear morphological changes in some cells. However, both oncogenes induce a delay in the capacity of differentiating cells to exit cell cycle.
5.4.3 LCCS derived neurospheres differentiate normally in vitro (III) Differentiation of the human LCCS and control NPCs was promoted by withdrawal of the proliferative growth factors and addition of either FCS or a Shh analogue SAG, a chlorobenzothiophene-containing Shh pathway agonist (Chen et al., 2002) to the culture medium. No major differences were detected between the two differentiation promoting culture mediums or between the potential of the LCCS NPCs to differentiate as compared to control NPCs. Both cell types differentiated into Tuj-1 positive neurons as well as islet-1 and Hb9 expressing motoneurons, respectively. The differentiation capacity of LCCS and control NPCs into GFAPpositive astrocytes and O4-expressing oligodendrocytes was also similar (III/Fig 1). So, even though LCCS foetuses have a loss of motoneurons in 77
vivo, the capacity of LCCS derived NPCs to differentiate into motoneurons and astrocytes in vitro was not affected.
5.5 Apoptosis is not affected by Myc or LCCS but Myc may cause cell ploidy (I, III) As Myc can induce genomic instability in cancer, we tested whether our transgenic NPCs had a normal cell cycle distribution and a diploid karyotype. One out of three Myc transduced NPC pools was tetraploid. Since 2/3 of the transfected Myc cell pools had a normal karyotype and all the obtained results from all of the three Myc cell pools were similar we could conclude that the karyotype change did not affect any of the results of the methods that were used in this study (I/Fig 7A). However, we did not specifically evaluate possible oncogenic advantages due to the tetraploidy (e.g. increase in growth factor receptors, increased autonomous production of mitogens or in vivo transformation capacity). Myc is known for apoptosis induction in some cells and therefore we measured apoptosis of the Myc NPCs by using annexin staining and FACS. Very low levels of apoptosis were detected in both control and Myc NPCs cultured in SCM (0.02%) and Myc did not increase apoptosis (I/Fig 7B). Apoptosis was also measured from the LCCS and control NPCs that had been cultured in differentiation promoting culture conditions but no statistically significant differences in the amount of apoptotic or necrotic cells could be shown between the two cell populations (III/Fig 3).
5.6 Self-renewal maintenance during differentiation: Myc causes Re-sphering (I, unpublished) The cell cycle exit analysis (5.4.2) showed that some, but not all, of the oncogene expressing NPCs (Myc 4%, CIP2A 0.1%) would resist the differentiation promoting cues provided by the changed culture 78
conditions from SCM to serum-containing medium and were repeatedly proliferating even after a week of culture. We then investigated whether these cells also maintained self-renewal capacity in the differentiation cultures. A small percentage (1,5%) of the Myc NPCs was able to form neurospheres in the serum-containing medium, which we named as the phenomenon of re-sphering. CIP2A NPCs would not re-sphere in the presence of the differentiation culture conditions but they were able to form new neurosphere populations after they were put back to EGF and FGF containing serum free SCM (unpublished, results not shown). In contrast, virtually all control cells had lost their ability to self-renew and they did not form a growing population of neurospheres (I/Fig 4). As Myc cells were the only ones that re-sphered already in the differentiation promoting culture conditions, we further evaluated the phenomenon by using Myc NPCs. We show that the amount of resphering cells could not be increased by time thus suggesting that the resphering cells were present already in the beginning of the experiment. The phenomenon was also density dependent, if less than 200 single cells were plated per well, no re-sphering was seen even though 10 000 cells were analysed per experiment. Thus a subsequent amount of other cells in the same well was needed in order for the re-sphering to take place (I/Fig 5A). We also wanted to rule out the possibility that the re-sphering cells were originally selected because they represent subclones of higher transduced expression level of Myc as compared to the non-re-sphering population. We analysed the progeny of the re-sphered cells by putting single spheres back to SCM and letting them form a new NPC population. The expression level of Myc was not increased in the re-sphered population and their capacity to proliferate, self-renew or differentiate was similar to the results obtained with primary Myc NPCs (I/Fig 5B-F).
79
5.7 NPC Self-renewal is regulated via Miz-1 binding and it correlates with the activity of CIP2A expression and the MEK/ERK pathway (I, II, unpublished) Several experimental setups were used to investigate molecular pathways behind the increased self-renewal capacity that was seen by the ectopic expression of Myc in NPCs.
5.7.1 Myc induced self-renewal is regulated through Miz-1 binding (I) In order to activate or inhibit transcription Myc can bind to a various number of target genes and thus regulate several cellular functions (Coller et al., 2000). By binding to another transcription factor Miz-1, Myc represses activation of Miz-1 activated genes that in the epidermal stem cell niche are associated with adhesive properties of the stem cell niche (Gebhardt et al., 2006). We overexpressed a Myc mutant (MycV394D) that can’t bind to Miz-1 (Herold et al., 2002) to investigate whether this would give the same results concerning the increased stemness associated properties that we have shown to be induced by WT Myc in NPCs. As compared to controls, overexpression of the Myc mutant did not increase NPC self-renewal in the neurosphere stem cell cultures nor were the cells capable of re-sphering when they were introduced into differentiation promoting culture conditions. Furthermore, overexpression of the mutant Myc was able to increase proliferation and thus increase cell number in the same fashion as WT Myc thus indicating that Miz-1 binding was necessary only to Myc induced self-renewal (I/Fig 6).
5.7.2 CIP2A (II) One way to increase cellular Myc activity is to inhibit its degradation by posttranscriptional regulation. CIP2A stabilizes Myc by inhibiting its dephosphorylation and ubiquitylation induced by PP2A (Junttila et al., 2007). We overexpressed CIP2A in NPCs and showed that it affects their 80
capacity to self-renew (5.3) and proliferate (5.2.1) in a similar manner than what we report on the effects of Myc overexpression, although the overall results were somewhat milder than induced by direct Myc overexpression. The morphology of the differentiated neural and glial cells was not affected and cell cycle exit was delayed in a small subpopulation of cells (5.4.1 and 5.4.2). Also, re-sphering did not take place in the differentiation cultures but only when the cells were changed back to SCM (5.6). CIP2A expression was linked to Myc expression by using several techniques. Overexpression of CIP2A increased expression of Myc, and vice versa, overexpression of Myc increased the expression level of CIP2A as shown by WB. Also, CIP2A expression was not detected in differentiated control NPCs but low expression was visible in differentiated cells overexpressing Myc. We used a direct downstream target of Myc, Nucleolin (Coller et al., 2000) to verify the linkage, and cells immunopositive for Nucleolin were also detected from the ventricle wall areas similar to CIP2A immunostaining in the E12.5 mouse brain (II/Fig 5A-F).
5.7.3 CDKs and MAPK pathways (unpublished) Especially EGF and also FGF promote NPC growth and survival and are by standard used as growth factors in the neurosphere cultures (Chojnacki and Weiss, 2008; Ciccolini and Svendsen, 1998; Pollard et al., 2006). These mitogens are ligands of the RTK family receptors and their signalling inside the cell is mediated by the MAPK signalling pathway which includes the MEK/ERK signalling pathways (Junttila et al., 2008). CDKs, on the other hand, regulate the cell cycle and they are highly regulated by the Rb family proteins and p53 through p21 (Galderisi et al., 2003). We used chemical inhibitors specific for these two groups of regulatory molecules, respectively. The MEK/ERK inhibitor U0126 (Favata et al., 1998) inhibited self-renewal totally in control NPCs and
81
almost completely in Myc NPCs. The CDK inhibitor Roscovitin (Meijer et al., 1997) reduced self-renewal by 50% in both controls and Myc NPCs (unpublished, Figure 18). We have previously shown that proliferation of control cells is reduced approximately by 50% by both inhibitors (Piltti et al., 2006) and our preliminary results by using the BrdU assay show a similar decrease also for Myc cells with both inhibitors (unpublished, not shown).
** *
*
*
Figure 18. Chemical inhibition of self-renewal. NPC self-renewal is inhibited by chemically blocking the MEK/ERK signalling cascade by using U0126 (50µM) in both control and Myc overexpressing cells. Inhibition of cyclin-dependent kinases with Roscovitin (10 µM) decreases self-renewal by approximately 50% in both cell types. The results are averages (%) of cells that are able to form neurospheres from single cells (contr SD=1,81, n=5; contr/DMSO SD=1,86, n=3; contr/Rosco SD=0,81, n=3; contr/U0126 SD=0, n=3; Myc SD=6,19, n=4; Myc/DMSO SD=10,71, n=3; Myc/Rosco SD=5,07, n=4; Myc/U0126 SD=1,18, n=4; Student’s t-test: Contr/ContrRosco p=0,0345; Contr/ContrU0126 p=0,016; Myc/MycRosco p= 0,0328; Myc/MycU0126 p=0,0056).
5.8 NPC marker expression (I, unpublished) There are not many NPC markers available except for the interfilament protein Nestin, which is expressed in proliferating NPCs and lost during differentiation of neurons and glia (Lendahl et al., 1990). Our results show that only a minority (4%) of the cells of the control neurospheres is
82
self-renewing and this percentage is increased by ectopic expression of oncogenes (5.3). Thereby the rest of the cells in neurospheres are more committed progenitors or differentiated cells. We used Myc NPCs as a tool to test whether we could find a marker that would be specific to only the self-renewing population and thus leave the more committed progenitors immunonegative. Unfortunately, none of the markers that were tested (Oct-4, Musashi, Prominin, (CD133 in humans), C-Kit or Sox-2) were useful. There was either no expression, which can be also due to technical reasons regarding the used antibody, or the results were not statistically repeatable (unpublished, results not shown). Even though we could not specify a marker of neural self-renewing stem cells, our results show that Bmi-1 can be used as a marker of mouse NPCs. The expression of Bmi-1 and Nestin was restricted to NPCs as differentiated neural or glial cells were not immunopositive. Also, Myc overexpression significantly increased the percentage of immunopositive cells stained for Nestin or Bmi-1, respectively, strengthening the result that Myc expression increases stemness associated cellular properties in NPCs (I/Figs 2D and 3B-C).
5.9 Endogenous expression of Myc and CIP2A (I, II) Finally, we investigated the endogenous expression of Myc and CIP2A in the developing brain to give insight of the in vivo relevance of these proto-oncogenes in neural development. As shown earlier by in situ hybridisation (Hirvonen et al., 1990), Myc immunopositive cells were found lining the neurogenic areas in the lateral ventricular wall in E16 mouse brain by using immunohistochemistry (IHC). Endogenous Myc expression was only faintly visible in WT neurospheres by WB and it was also verified by reverse transcription polymerase chain reaction, RT-PCR (I/Fig 1C-D),
83
CIP2A is a newly found proto-oncogene and its expression pattern in normal tissue has not been previously studied. By in situ hybridisation in E12 mouse brain, CIP2A mRNA was visible in the VZ of the lateral ventricles as well as in a more basally located cortical layer closer to the cortical plate. By IHC on E12.5 paraffin sections, CIP2A immunopositive cells were found around the lateral ventricle wall and the E13 derived neurospheres were also intensively CIP2A positive. Furthermore, CIP2A mRNA was also seen located in the whisker follicles, in the skin and in the paws in the areas that will be depleted by apoptosis between the developing toes ( II/Fig 1).
5.10
Microarray
analysis
of
LCCS
versus
control
neurospheres revealed increased EGFR levels in patient NPCs (III) A microarray analysis was performed to compare the expression profiles of the LCCS-derived and control NPCs both in stem cell cultures and differentiation promoting cultures, respectively. The transcriptome of cultured LCCS NPCs was different from controls in both set ups but no genes directly associated with defects in motoneuron or oligodendrocyte development could be identified as such. When the gene expression profile of differentiated LCCS NPCs were compared to NPCs derived from age-matched control foetuses, the approach identified 45 upregulated transcripts and 43 downregulated transcripts in the LCCS samples. To understand the biological networks underlying the transcriptional changes, the regulated genes were classified into groups according to the gene ontology. This list includes the following groups for the upregulated samples: GO:0007416 synaptogenesis (p-value 0.000106) and GO:0030111 regulation of Wnt receptor signaling pathway (p ¼ 0.000478). The downregulated groups include: GO:0004864 protein phosphatase inhibitor activity (p ¼ 0.078449), GO:0019212 phosphatase inhibitor activity (p ¼ 0.089052),
84
GO:0008366 nerve ensheathment (p ¼ 0.000213), GO:0030297 transmembrane receptor protein tyrosine kinase activator activity (p ¼ 0.000226) and GO:0043121 neurotrophin binding (p ¼ 0.000452). (III/Table 2 and Figure 4). Among the set of differently expressed genes in LCCS NPC as compared to controls in the stem cell culture conditions were two genes involved in RTK signaling pathways. Both EGFR and VAV3 were upregulated in LCCS NPCs. EGFR is a RTK family receptor that activates the mitogenic MAPK pathway. VAV3 is a member of the family of VAV oncoproteins that activate pathways leading to actin cytoskeletal rearrangements and transcriptional alterations (Bustelo 2000) and is also regulated by EGFR and PI3-K activation (Movilla and Bustelo, 1999), Also SH3KBP1, a gene encoding the protein Human Src family kinase binding protein 1 (HSB-1) that enhances tumour necrosis factor mediated apoptotic cell death (Narita et al., 2001) was upregulated (III/Table 1).
85
6. DISCUSSION
6.1 Myc increases neural stem cell self-renewal The results presented in this thesis show that Myc and CIP2A regulate self-renewal in NPCs. Several stem cell types maintain self-renewal capacity and/or pluripotency by activation of Myc (Murphy et al., 2005) and regulation of the number of the self-renewing cells plays a key role in tissue homeostasis. Our results show that overexpression of Myc dramatically increases the proportion of cells that are able to self-renew in a neurosphere, thus changing the organization and cell type balance of the neurospheres, which normally consist of a variety of cells at many stages of differentiation. The role of Myc in controlling the balance of mammalian neural stem and progenitor cell identity or self-renewal has not been studied before but several newly published studies show similar results to ours. First, p53 and PTEN loss induced increased self-renewal as well as impaired differentiation capacity in mouse NPCs, which is due to increased endogenous Myc expression. In accordance, knockdown of Myc by short hairpin RNA largely restored the differentiation potential (Zheng et al., 2008). Overexpression of Myc in human NPCs also increases selfrenewal and immortalises the cells but overexpression of a stabilised mutant form of Myc (MycT58A) does not prevent neural or glial differentiation (De Filippis et al., 2008). In addition to self-renewal maintenance, Myc regulated by the expression level of p19ARF is involved in the determination of the neural or glial fate of the daughter cells in mouse and rat NPCs (Nagao et al., 2009). Lastly, Myc is also required for maintenance and self-renewal of glioma CSCs (Zheng et al., 2008; Wang et al., 2008) and the role of Myc in the developing brain seems to conserved and is similar also in the Drosophila neuroblasts (Betschinger et al., 2006).
86
Myc also affects stem cell maintenance of other tissues. Self-renewal of embryonic stem cells is maintained by the LIF/STAT3 pathway via Myc (Cartwright et al., 2005). In the epidermis and the bone marrow Myc drives proliferation of the transit amplifying progenitor cells and induces their exit from the stem cell niche towards differentiation (Arnold and Watt, 2001; Wilson et al., 2004). Myc is also needed for the formation of intestinal crypts (Bettess et al., 2005) as well as for maintenance of the homeostasis of the intestinal epithelium as a downstream target of the canonical Wnt signalling pathway (Pinto et al., 2003). Our results are in accordance with the other published studies and provide additional relevant data to Myc biology and especially to the effects and regulation of Myc in NPCs as further discussed below.
6.2 Myc increases the pool of self-renewing stem cells, progenitors or both In the epidermis, Myc has two distinct functions depending on the stem cell commitment stage. In the stem cell pool, Myc has a self-renewal promoting role and loss of Myc results in the loss of stem cells (Zanet et al., 2005). In the more committed progenitors, Myc in essential for two things, the regulation of the adhesive properties of the stem cell niche in order for the cells to exit the niche as well as the rapid proliferation and expansion of these TA cells (Arnold and Watt, 2001; Gandarillas and Watt, 1997; Gebhardt et al., 2006). We were not able to specify a marker that would only be expressed in the long term potentiality possessing neural stem cells. Therefore, we can not distinguish between the neural stem and progenitor cells, and it is known that neurospheres are heterogeneous and a large majority of the cells are more committed progenitors (Reynolds and Rietze, 2005). We show an increase in both self-renewal and proliferation but at the moment we can not specify what the specific effects in the individual cell types are. Nevertheless, by
87
comparing control neurospheres to Myc-overexpressing neurospheres, we can, by using several parameters show that the overall outcome is a general increase in the number of cells that can self-renew, but whether they derive from stem or progenitor cells is not known. Also, the number of cells expressing Nestin and Bmi-1 was increased by Myc overexpression reflecting the same general population level increase towards a more primitive identity. Furthermore, when we measured the amount of differentiated astrocytes and neurons within the neurospheres, their numbers are not decreased due to Myc expression indicating that instead the stem cell pool is increased.
6.3 Myc induces self-renewal through Miz-1 and Myc expression is linked to CIP2A In the epidermal stem cells Myc binds to Miz-1. This in turn affects the adhesive properties of the niche by repression of 1-integrin and other adhesive molecules and thereby induces stem cell exit (Gebhardt et al., 2006; Gebhardt et al., 2007). Our results suggest that Miz-1 binding site of Myc is needed for the increased self-renewal in NPCs but the exact identity of the downstream targets mediating these effects remain speculative. We also show that the newly found proto-oncogene CIP2A, which was shown to function as a Myc stabilizer by inhibition of PP2A induced Myc degradation (Junttila et al., 2007), acts in a similar manner in the NPCs as Myc. The linkage between the expression levels of Myc and CIP2A, although we have no direct evidence, suggest that CIP2A stabilises Myc also in NPCs. Also, the high expression level of endogenous CIP2A and its expression pattern in the developing mouse brain, where also Myc is expressed, further strengthens the in vivo relevance of CIP2A.
88
6.4 Myc changes the way NPCs interpret environmental cues Our results suggest that activated ectopic expression of Myc changes the way NPCs interpret the microenvironmental cues controlling their selfrenewal and differentiation. When an enhanced amount of self-renewal allowing signal is present, a larger proportion of the cells in the neurosphere adopt a stem cell identity as compared to control neurospheres. Importantly, even though the retrovirally induced signalling level of the oncogene in the clonal neurosphere is identical in every cell, a vast majority of the cells still stay as more committed progenitor cells or differentiated cells. So, the identity of the selfrenewing stem cells has to be built by other factors based on the microenvironmental cues and probably the positioning of the cell in proportion to the identity and thereby signalling of its neighbours. Our results thus suggest that the self-renewing identity can be enhanced by increasing the level of Myc but the basic building blocks for the stemness-associated functions are obtained from the surrounding microenvironment. The theory of the microenvironmental signalling cues is further strengthened by the re-sphering assay in which only a constant 1-2 % of the Myc cells showed the abnormal capacity to maintain self-renewal properties during differentiating conditions. Again, vast majority of the cells obeyed the differentiating promoting cues and acted in a similar manner as the control cells despite the constant activity of the Myc transgene. Furthermore, the re-sphering phenomenon was density dependent and thus it did not happen if too few cells were seeded on the culture plate. The environmental control was still so strong that the respheres did not further expand in the differentiation cultures by time but only when they were put back to SCM. The reports of induced pluripotent stem cells show that the reprogramming of fibroblasts into pluripotent embryonic stem cells also happens only in a small percentage of the cells. In iPS cells Myc is needed as an enhancer in the initiation of the reprogramming process and it is downregulated in the established iPS 89
(Knoepfler, 2008; Okita et al., 2007; Takahashi and Yamanaka, 2006; Wernig et al., 2007).
6.5 Myc does not prevent differentiation During
differentiation
of
NPCs
to
neurons,
astrocytes
and
oligodendrocytes they undergo irreversible changes that permanently shut down their cell cycle. Several studies have shown that the essential steps of neuronal terminal differentiation include inhibition of cyclins and CDKs by increased levels of pRb family proteins, p53 as well as other cell cycle inhibitors and on the other hand, downregulation of N-myc and other activators of the cell cycle (Galderisi et al., 2003). We have also previously shown that downregulation p53 and Rb family proteins, respectively,
increases
NPC
self-renewal
but
does
not
inhibit
differentiation (Piltti et al., 2006). Now we analysed whether upregulation of Myc, which is able to functionally replace n-Myc (Malynn et al., 2000), inhibits NPC differentiation. Majority of the transgenic NPCs were able to exit cell cycle under differentiating conditions although it was delayed as compared to the control cells and a small minority stayed in cycle. Some of the Myc cells showed a less differentiated morphology but all of them expressed neuron and astrocyte-specific differentiation markers and NPC marker Nestin was not expressed. Our results suggest that unregulated expression of Myc or CIP2A does not block neural differentiation and that the role of these proto-oncogenes is more dominant
in
self-renewal
maintenance
than
in
promotion
of
differentiation.
6.6 Self-renewal and proliferation are separately regulated cellular functions Results presented in this thesis strengthen the concept that proliferation and
self-renewal
are
separately
regulated
mechanisms.
First,
overexpression of the Myc (V394D) mutant increased proliferation of NPCs in a similar manner as WT Myc but self-renewal was not increased 90
indicating that only self-renewal maintenance needs repression of genes activated by Miz-1. Second, chemical inhibition of the MEK/ERK pathway inhibited self-renewal totally in Myc and control NPCs whereas proliferation was only 50% decreased. Roscovitin, which inhibits cyclins, affected both proliferation and self-renewal by decreasing them approximately to half of the original level in all NPC types.
6.7 It all comes down to Myc The chemical inhibition studies suggest that the MEK/ERK pathway is significant in self-renewal maintenance of NPCs and we have previously shown that the level of pERK was increased in NPCs when self-renewal is increased by simultaneous inhibition of p53 and Rb family proteins (Piltti et al., 2006). In line with this, loss of pluripotency and stemness is associated with decreased CDK activity and activation of Rb also in several other stem cell types such as in ES cells (Galderisi et al., 2006; White et al., 2005). Among phosphorylation of several other targets, pERK can activate Myc as well as inactivate Rb (Guo et al., 2005; Junttila et al., 2008) and Myc represses the cell cycle inhibitors p21Cip1, p15Ink4b and p57Kip2 via binding to Miz-1 (Adhikary et al., 2003; Seoane et al., 2002; Staller et al., 2001), all of which regulate Rb (Galderisi et al., 2006; Murphy et al., 2005). Rb, p53, p21 also all contribute to the antiproliferative effect of TGF which inactivates Myc activity via Miz-1 binding (Sheahan et al., 2007). Furthermore, p53 can also directly bind to the Myc promoter and inhibit its activity (Ho et al., 2005) and a loss of p53 and PTEN increases expression of Myc, which increases NPC selfrenewal (Zheng et al., 2008). Bmi-1 also promotes self-renewal of NPCs through repression of Rb and p53 associated cell cycle inhibitors p16ink4a and p21cip1 (Fasano et al., 2007; Molofsky et al., 2003). Rb is the key inhibitor of activation of E2F, which activates transcription of various essential cell cycle genes
91
including N-myc and Bmi-1 (Nowak et al., 2006) and Bmi-1 is also a direct transcriptional target of Myc (Guney et al., 2006). Thus Myc, that has been studied in this thesis work, as well as several other oncogenes and tumour suppressors such as p53, Rb and Bmi-1 and their various inhibitors and activators are at least partially regulated by common pathways. It is likely that most of the reports regarding selfrenewal have looked at the same regulatory pathway only from a different point of view depending on which oncogene or tumour suppressor they have focussed on. Taken together, it seems that whatever a cell needs to maintain self-renewal or proliferation, or alternatively, when the offenders are searched for the maintenance of self-renewal in CSCs, sooner or later the oncoprotein Myc shows up and will be proven guilty for the action, alone or as a member of a gang.
6.8 Neurosphere cultures provide information on cellautonomous functions of LCCS neural development Neurospheres derived post-mortem from aborted foetuses were used to reveal potential functional defects in NPC stem cell characteristics or differentiation capacity associated with the lethal motoneuron disease LCCS. However, the only major detected difference was the higher rate of proliferation. This was in accordance with the elevated expression level of EGFR and possibly VAV3. Interestingly, VAV3 can be regulated e.g. by EGFR and also the PI3-K pathway (Bustelo 2000), which seems to be involved in the pathology caused by the mutations of the LCCS1, LCCS2 and LCCS3 genes (Nousiainen et al., 2008; Narkis et al. 2007a and 2007b), The results lets us conclude that the LCCS1 finmajor mutation, which causes an altered secondary structure of the GLE1 protein (Nousiainen et al., 2008) does not inhibit cell-autonomously regulated differentiation of motoneurons or oligodendrocytes in vitro, although no studies on the
92
functionality of the differentiated cells were performed. In line with this, differences in the expression patterns of in vitro differentiated LCCSderived and control NPCs did not reveal any genes that at the moment could be directly linked to motoneuron or oligodendrocyte differentiation pathways. Severely disrupted morphology of the anterior horn motoneurons of the spinal cord is the major pathological characteristics of LCCS patients (Herva et al., 1985) and mRNA expression arrays reveal specific changes in expression of proteins related in motoneuron and oligodendrocyte differentiation in vivo (Pakkasjarvi et al., 2005). Our results thus suggest that the developmental defect of LCCS motoneuron differentiation or maintenance in the anterior horn is due to the misregulation of correct cellular signalling in the in vivo environment. The used differentiation culture conditions were not optimised for oligodendrocyte or motoneuron differentiation. As both cell types represented a clear minority of the differentiated cells (only a few percentages) the possible differences might have been better recognizable if we would have first enriched the cells for motoneurons or oligodendrocytes, respectively, either by further optimising the culture conditions for motoneuron development or by antibody based cell sorting after the differentiation. It is not known whether the absence of motoneurons in LCCS is due to a failure in differentiation of motoneurons or alternatively an increase in the apoptotic rate of these cells. Differences in the level of apoptosis were not seen in the current set up but the possible differences might thus have been lost in the heterogeneous population of the investigated cell pool. The same disadvantage of the used culture set up applies also to the differentiation experiments as there were too few motoneurons or oligodendrocytes in the cultures in order to evaluate whether there were proportional differences in the amounts of these cells between LCCS and control cultures. Furthermore, no electrophysiological studies were performed to reveal possible functional differences between the differentiated LCCS
93
and control motoneurons that by microscopic evaluation seemed to have a similar morphology.
94
7. CONCLUSIONS We have investigated the effects of overexpressed levels of the protooncogene Myc in the regulation of NPC self-renewal, proliferation and differentiation capacity. Based on the primary studies presented in this thesis work, I conclude that 1) Myc affects the regulation of NPC proliferation and self-renewal. 2) Several mechanisms that regulate the activity of Myc were identified. Myc-induced self-renewal is signalled through Miz-1 binding and the activity and expression level of the Myc protein correlates with CIP2A expression in NPCs. The MEK/ERK signalling pathway is essential to self-renewal maintenance in NPCs. 3) Myc is not a definitive inhibitor of neural or glial differentiation as its overexpression does not block differentiation. 4) Myc and CIP2A are intensively expressed in the neurogenic areas of the brain which suggests a role also in vivo. These results on normal NPC self-renewal regulation also give valuable information in order to understand the mechanisms of cancer stem cell renewal in brain tumours. 5) Neurosphere cultures were set up from post-mortem foetuses with a motoneuron disease LCCS. However, by measuring functional properties of the patient and agematched control NPCs in vitro, no major cell autonomous differences that would explain the pathology of LCCS could be shown.
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ACKNOWLEDGEMENTS This study was carried out at the Institute of Biomedicine, University of Helsinki. I want to acknowledge professor Esa Korpi, the head of the institute as well as Professors Tomi Mäkelä and Hannu Sariola, the former and present head of the Department of Medical Biochemistry and Developmental Biology, respectively, for providing the excellent working facilities. I also want to warmly thank Helsinki Biomedical Graduate School and its former and present staff Professor Tomi Mäkelä, docent Päivi Ojala, Aija Kaitera and Elina Värtö for the financial support, excellent post-graduate education and all help in practical matters. I also want to thank the Helsinki developmental biology community and especially the shepherd of the group, Professor Irma Thesleff, for the various events with a positive scientific and social spirit. I wish to warmly acknowledge my supervisor Dr. Kirmo Wartiovaara for your guidance, enthusiasm and discussions trough all these six years in the lab. You have given me free hands to work on my own and yet still support when needed, which together with your constantly encouraging attitude has actually led me to believe that I am capable of independent scientific work! I also admire your courage in carrying out challenging experiments and ideas driven by the “you can’t win if you don’t try” mentality. Working with you and your relatively good sense of humour has been enjoyable and I am more than happy for the friendship we have developed. I also want to thank my other supervisor Professor Hannu Sariola for your contribution in this thesis work. In addition to the critical scientific evaluation I am also grateful for the outstanding working equipment that you have provided. I have enjoyed the easygoing and liberal yet still scientifically enthusiastic atmosphere that you have created in the lab. It did not take me long to realize that as long as I remember to emphasize
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that nobody bicycles faster than you and that your naughty jokes have to be laughed at, everything will go smoothly. I want to thank my thesis committee members Docent Päivi Ojala and Docent Juha Partanen for your time, discussions and support concerning my thesis work. I sincerely thank Professor Martin Eilers for the valuable and highly professional input with the Myc manuscript, I am for ever grateful that you came to see my poster! I also want to thank Docent Jukka Westermarck for the fruitful collaboration concerning the CIP2A studies. I warmly thank for the opportunity to collaborate with Docent Marjo Kestilä and Niklas Pakkasjärvi in the LCCS project, which afterwards due to the strict rules of the Medical Faculty turned out to be crucial for the entity of my thesis work. I also thank Docent Johanna Ivaska and Professor Dan Lindholm for your critical comments and kindness in pre-reviewing this thesis on a tight schedule. The people in the lab make the everyday life and atmosphere. The advantage of many people is the sharing of a broad knowledge in different techniques, which has helped me in many situations. I especially want to thank Tiina Immonen for the advice and help in biochemistry. Whatever my problem has been, you always had time for it and you either know the answer directly or then you find it out. I also warmly thank our technician Agnes Viherä for your help with my experiments especially during my maternity leave when the combination of a baby crying on the lab balcony and the demands of the journal simultaneously felt almost impossible, you really are effective! I also want to thank you for your friendship and the endless willingness to have just one more beer. I also thank Lea Armassalo and Virpi Syvälahti for technical assistance and Lea especially for making thousands of litres of stem cell medium. Heli Fox is
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thanked for her joyful personality and friendship. Your eagerness made it easy for me to guide you through your baby steps in the scientific world. Without forgetting the great amount of effort you have put to the projects, getting to know you has clearly been my pleasure. Satu Kuure is thanked for her everlasting energy in organizing social activities for the lab and also for her fearless style in asking questions, a good example for all of us. Most importantly I thank you for your friendship, since you moved to New York I really have missed you and our good time in the lab! I thank Nina Perälä for collaboration with the in situ work and Madis Jakobson for his broad scientific knowledge and ideas. Nina, Madis and Marjo Hytönen are thanked for our several chat moments in the Kindergarten to ease the load of pressure concerning all the muck that has appeared especially lately. Katja Piltti is thanked for collaboration and company during our various conference trips around the world. Alexander AngersLoustau and Valtteri Häyry are also thanked for collaboration and Valtteri for his funny and enthusiastic story telling. I also thank Jukka Suokas for your patience and help with the computers. Anna Popsueva, Jetta Kelppe, Marianne Nymark, Thomas Hackelberg, Valtteri Harri, Eric Pedrono, Anita Tuomainen, Elena Arighi, Roxana Ola, Kirsi Sainio, Fares ZeidanChulia, Samer Hussein and many other past members of the lab are thanked for the nice time together in the lab. In good and in bad, this is not a normal lab, of which most of us have also had their personal share to taste. However, I am happy that of all places I ended up in Circus Sariola for my thesis work from which I have plenty of good memories to take with me! The financial support of the Biomedicum Helsinki Foundation, The Finnish Cancer Organisations, the Otto A Malm Foundation, the Academic Women of Hyvinkää Association, the K Albin Johansson Foundation and the Orion-Farmos Research Foundation are gratefully acknowledged.
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My journey towards this degree has not always been easy. The moments of success have not been in balance with the disappointments, which despite knowing the nature of scientific work, has sometimes been difficult to bear. I am really grateful for all my friends that have at times of bad luck kept my mind elsewhere from work and helped me realize my priorities. I especially want to thank Heli Uronen-Hansson for all the numerous joyful moments that we have shared during our 15 years of friendship. Your hilarious way of mocking the scientific community and giving me back the right perspective of life has helped me through various struggles during the ten long years of doing my PhD. Nina Korsisaari is thanked for her weekly “pea-soup company”, friendship and support when I was looking for a new project and and also for the high quality bead and breakfast service during my trips to California. Taru Meri is thanked for her friendship and koppi-company during the time I was doing my first PhD project in Haartman Institute. I also want to warmly thank our improvisation theatre group Roiskeläppä as well as the several projects in Ilves-Teatteri that I have participated during these years for taking my mind off science, giving me strength to go on and providing a guaranteed way of enjoying life. I am grateful for my parents in law, Citi and Nalle, who have enabled our too busy everyday schedule by taking such good and loving care of our children and the whole family. I also want to acknowledge my uncle Lauri and his late wife Pirkko for their time and love spent with Klara while I was at work in the evenings. I want to thank my sisters Saara and Noora for their love and support. I am especially grateful to Saara for being there for me whenever I have needed help or consolation (if the numerous kicks and bites of early childhood are not taken into account). You also are the only person in this world who criticizes me with cruel honesty, which for the most is an extremely good thing. To my parents Heidi and Eero I want to express my gratitude for your support in everything I have chosen to do in my
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life. Both of you have always made me feel loved and thus succeeded in the most important task that I hopefully can pass on to my children. Finally I want to express my love and deepest gratitude to my husband Johan who has waited so many nights with dinner served when my experiments just won’t finish in time. The combination of finishing my thesis work and having two small children has certainly eaten up our energy and nerves and would have never been completed without your encouragement, understanding, patience and devotion in taking care of the children. I thank you for everything that is important in my life, your love and Klara and Hugo, the combination that has given me so much more joy and happiness that I ever could have asked for.
Helsinki, March 2009, Laura
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