Dr Ruman Rahman
email: ruman.rahman@nottingham.ac.uk

Rachel Heath

Richard Grundy

Invited Review for BBA-Molecular Basis of Disease: Title : Cellular Immortality in Brain Tumours: An Integration of the Cancer Stem Cell Paradigm

Keywords: Neurogenesis, Neural Stem Cell, Telomerase, Telomeres, Brain Tumour Stem Cell

Brain tumours are a diverse group of neoplasms that continue to present a formidable challenge in our attempt to achieve curable intervention. Our conceptual framework of human brain cancer has been redrawn in the current decade. There is a gathering acceptance that brain tumour formation is a phenotypic outcome of dysregulated neurogenesis, with tumours viewed as abnormally differentiated neural tissue. In relation, there is accumulating evidence that brain tumours, similar to leukaemia and many solid tumours, are organized as a developmental hierarchy which is maintained by a small fraction of cells endowed with many shared properties of tissue stem cells.

Proof that neurogenesis persists throughout adult life, compliments this concept.

Although the cancer cell of origin is unclear, the proliferative zones that harbour stem cells in the embryonic, post-natal and adult brain are attractive candidates within which tumour-initiation may ensue. Dysregulated, unlimited proliferation and an ability to bypass senescence are acquired capabilities of cancerous cells. These abilities in part require the establishment of a telomere maintenance mechanism for counteracting the shortening of chromosomal termini. A strategy based upon the synthesis of telomeric repeat sequences by the ribonucleoprotein telomerase, is prevalent in ~90% of human tumours studied, including the majority of brain tumours. This review will provide a developmental perspective with respect to normal (neurogenesis) and aberrant (tumourigenesis) cellular turnover, differentiation and function. Within this context our current knowledge of brain tumour telomere/telomerase biology will be discussed with respect to both its developmental and therapeutic relevance to the hierarchical model of brain tumourigenesis presented by the cancer stem cell paradigm.

A C C E P T E D M A N U S C R I P T Introduction 1. Characteristics of intracranial neoplasms

Of all solid cancers, brain tumours have the poorest survival and highest morbidity rates. Although frequently considered collectively, brain tumours represent a diverse range of tumour types affecting both children and adults. Tumours of the brain account for less than 2% of all malignancies, yet now approach leukaemia as the leading cause of cancer-related death in children and the fourth leading cause in adults [START_REF] Kleihues | The WHO classification of tumors of the nervous system[END_REF]. The diversity of brain tumour type is a reflection of the histological complexity of the central nervous system (CNS) and can present with glial or neuronal phenotypes or a mixture of cell types.

Medulloblastoma and supratentorial primitive neuroectodermal tumours (sPNET) are World Health Organisation (WHO) grade IV embryonal tumours, resulting from the transformation of primitive neuroectodermal cells. The medulloblastoma is a malignant and invasive tumour with a relatively poor prognosis and represents the most common malignant brain tumour in children. It is predominately neuronal in nature and typically located in the cerebellum. In contrast the rarer sPNETs are more commonly found in the cerebral hemispheres and consist of poorly differentiated neuroepithelial cells which confer poor prognosis, especially in children. Despite both being classified as Grade IV tumours, sPNETs have a significantly worse prognosis [START_REF] Packer | Medulloblastoma[END_REF].

Also classified as a grade IV tumour by the WHO, glioblastoma multiforme (GBM) represents the most malignant and aggressive form of glioma, arising in the subcortical white matter as either a primary neoplasm, or from the malignant progression of a low grade glioma [START_REF] Behin | Primary brain tumours in adults[END_REF]. Typically seen in adults, prognosis is

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extremely poor as the diffuse distribution throughout the brain makes surgical resection difficult [START_REF] Holland | Glioblastoma multiforme: the terminator[END_REF].

Ependymoma are typically classified as glial tumours, however it is currently debated as to whether ependymoma may actually reflect a separate entity. Composed of neoplastic ependymal cells, the ependymoma is usually located along the ventricular system of the paediatric or adolescent brain whereas spinal sites are more commonly seen in adults. Prognosis is varied and is dependent on surgical resection [START_REF] Nejat | Initial management of childhood brain tumors: neurosurgical considerations[END_REF][START_REF] Ruda | Ependymomas of the adult: molecular biology and treatment[END_REF].

Although an understanding of human brain cancer pathogenesis has increased in recent decades, insufficient knowledge of the molecular, genetic and cellular alterations and mechanisms is at the root of treatment failure and relatively limited diagnostic and prognostic capabilities. As a result, bio-centred and target-specific brain tumour therapy has not yet been forthcoming.

Neuro-developmental biology

The CNS is a highly complex structure which develops from a restricted number of extensively plastic cells that proliferate and acquire regional identities in space and time to produce a diverse repertoire of cell types. Collectively, these cells are defined as neural stem cells (NSCs) on the basis of their potential to differentiate into mature neurons and glia (astrocytes and oligodendrocytes) and their ability to self-renew in vitro. This operational definition does not however, necessarily reflect the function of NSCs in vivo and it remains uncertain whether the cell type(s) exhibiting 'stemness' in vitro faithfully represents the cell exhibiting 'stemness' in vivo.

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Mammalian developmental neuro-biology now entertains an integrated view of NSCs whereby neuroepithelial cells comprising the neural tube of the developing embryo, give rise first to radial glia, which then transform into astrocyte-like adult multipotent stem cells or directly into mature ependymal cells lining the walls of the lateral ventricles, the latter of which are post-mitotic in the adult brain [START_REF] Spassky | Adult ependymal cells are postmitotic and are derived from radial glial cells during embryogenesis[END_REF]. Radial glia arise from neuroepithelial cells throughout the primitive CNS at the beginning of neurogenesis and represent the predominant neuronal progenitor [START_REF] Mori | The novel roles of glial cells revisited: the contribution of radial glia and astrocytes to neurogenesis[END_REF]. These cells characteristically display a radial morphology and a mixed primitive cell/glial immunophenotype. In mammals, radial glial cells disappear from the brain soon after birth, with a closely-related astrocyte-like cell taking the role as NSC thereafter. The belief that the genesis of new CNS cells is a rare event in the adult mammalian brain was established dogma until recently. Two constitutively active germinative layers are now known to reside in the adult brain: the subventricular zone (SVZ) associated with the anterior part of the forebrain lateral ventricles and the subgranular zone (SGZ) within the hippocampus, corresponding to the inner layer of the dentate gyrus [START_REF] Alvarez-Buylla | Neurogenesis in adult subventricular zone[END_REF][START_REF] Merkle | Neural stem cells in mammalian development[END_REF][START_REF] Peretto | The subependymal layer in rodents: a site of structural plasticity and cell migration in the adult mammalian brain[END_REF].

New neurons and glia continue to be generated within these restricted regions of the adult mammalian brain [START_REF] Zhao | Integration of adult generated neurons during epileptogenesis[END_REF]. Evidence that neurogenesis persists in the CNS of the adult mammalian brain (albeit in restricted domains) has changed our perception of the cell of origin with respect to neural neoplastic transformation [START_REF] Gage | Mammalian neural stem cells[END_REF][START_REF] Gross | Neurogenesis in the adult brain: death of a dogma[END_REF].

Telomere and telomerase status during normal and dysregulated neurogenesis

In virtually all types of malignant cells, unlimited replicative potential is conferred in considerable part, by the maintenance of telomere length above a critical minimum threshold. Telomeres (derived from the Greek telos, meaning end and meros, a component) are a complex of guanine-rich repeat sequences and associated

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proteins that form a loop structure which caps all eukaryotic chromosome termini [START_REF] De Lange | Shelterin: the protein complex that shapes and safeguards human telomeres[END_REF] and prevents chromosomes from improper recombination, nuclease degradation and end fusions, thereby contributing to genomic stability. Due to incomplete replication of lagging-strand synthesis at each mitotic division ('end-replication problem') [START_REF] Hayflick | The serial cultivation of human diploid cell strains[END_REF],

telomeres progressively shorten, a phenomenon that provides a molecular basis of cellular ageing and hints at a primitive tumour-suppressor mechanism [START_REF] Wright | Telomere biology in aging and cancer[END_REF]. In germ cells and cancer cells, unlimited proliferative capacity is achieved through the addition of telomeric repeats by the telomerase holoenzyme. Telomerase is a ribonucleoprotein consisting of an RNA subunit (TR) which provides an intrinsic template for the synthesis of de novo telomeric repeats in a reaction catalyzed by its enzymatic component (TERT) [START_REF] Greider | A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis[END_REF].

NSCs from different regions of the brain have different proliferative capacities and varying abilities to generate astrocytes, oligodendrocytes and neurons [START_REF] Horiguchi | Neural precursor cells derived from human embryonic brain retain regional specificity[END_REF][START_REF] Moses | Murine embryonic EGF-responsive ventral mesencephalic neurospheres display distinct regional specification and promote survival of dopaminergic neurons[END_REF].

This may be reflected in the level of telomerase activity and telomere length reserve within cells from different neuro-anatomical compartments, in particular regions of high neurogenic activity such as the SVZ and SGZ [START_REF] Caporaso | Telomerase activity in the subventricular zone of adult mice[END_REF]. Telomerase-mediated telomere homeostasis is therefore a likely key factor in brain plasticity throughout development and adulthood.

There is a suggestion here that a fuller understanding of the replicative histories of cells within tumour hierarchical compartments may provide clues as to the cancer cell of origin for a given brain neoplasm. Indeed telomerase activity has been detected in a growing number of brain tumours in the past decade [START_REF] Demasters | Differential telomerase expression in human primary intracranial tumors[END_REF][START_REF] Didiano | Telomere maintenance in childhood primitive neuroectodermal brain tumors[END_REF][START_REF] Hiraga | Telomerase activity and alterations in telomere length in human brain tumors[END_REF][START_REF] Langford | Telomerase activity in human brain tumours[END_REF][START_REF] Ridley | Multifactorial analysis of predictors of outcome in pediatric intracranial ependymoma[END_REF][START_REF] Sano | Telomerase activity in 144 brain tumours[END_REF]. With preclinical and clinical advances in telomerase-therapeutics for cancer, there is anticipation that this will impact upon paediatric and adult tumours of the CNS.

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Brain tumourigenesis and tumour heterogeneity

A developmental relationship between tissue stem cells and cancer cells has been proposed over 50 years ago [START_REF] Becker | Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells[END_REF][START_REF] Till | A direct measurement of the radiation sensitivity of normal mouse bone marrow cells[END_REF]. However the pioneering demonstration of 'cancer stem cells' in human cancer was first made in leukaemia [START_REF] Bonnet | Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell[END_REF][START_REF] Lapidot | A cell initiating human acute myeloid leukaemia after transplantation into SCID mice[END_REF]. From this initial discovery in hematopoietic tumours, evidence from most solid cancers studied defines restricted subsets of tumour cells that share characteristics of the corresponding normal tissue stem cells [START_REF] Al-Hajj | Prospective identification of tumorigenic breast cancer cells[END_REF][START_REF] Collins | Prospective identification of tumorigenic prostate cancer stem cells[END_REF][START_REF] Fang | A tumorigenic subpopulation with stem cell properties in melanomas[END_REF][START_REF] O'brien | A human colon cancer cell capable of initiating tumour growth in immunodeficient mice[END_REF][START_REF] Pinto | Wnt, stem cells and cancer in the intestine[END_REF]. This paradigm includes prospective isolation of a minority population of brain tumour cells, based on the expression of the cell surface antigen CD133, known to be highly expressed in normal neural stem cells. These putative brain cancer stem cells were isolated from medulloblastoma, pilocytic astrocytoma, glioblastoma multiforme and anaplastic ependymoma and possessed a marked capacity for self-renewal (evidenced by the formation of floating aggregates termed tumourspheres), proliferation and differentiation [START_REF] Galli | Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma[END_REF][START_REF] Hemmati | Cancerous stem cells can arise from pediatric brain tumors[END_REF][START_REF] Singh | Identification of a cancer stem cell in human brain tumors[END_REF][START_REF] Taylor | Radial glia cells are candidate stem cells of ependymoma[END_REF].

Crucially, only the CD133+ fraction was capable of tumour initiation upon orthotopic transplantation into non-obese diabetic severe combined immunodeficient (NOD-SCID) mouse brain [START_REF] Singh | Identification of human brain tumour initiating cells[END_REF]. The CD133+ fraction produced a tumour that generated a phenocopy of the original tumour and could be serially transplanted into secondary recipients, providing evidence of self-renewal capacity in vivo [START_REF] Singh | Identification of human brain tumour initiating cells[END_REF]). Our growing understanding of the hierarchical organization of the functional compartments in renewing brain tissues provides a theoretical and technical framework upon which to base falsifiable questions regarding brain tumour initiation and cell of origin.

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Developmental Neurobiology

Neuroepithelial cells, radial glia and early neurogenesis

The origin of all CNS cells can be traced to a single layer of embryological cells, which constitute the neuroepithelium. The edges of this sheet fold together to form the neural tube, which later gives rise to the ventricular system and spinal canal.

Initially, neuroepithelial cells divide symmetrically in order to increase the stem cell pool; thereafter these highly plastic cells proliferate and acquire regional identities in a spatial-temporal manner [START_REF] Merkle | Neural stem cells in mammalian development[END_REF]. It is likely that neuroepithelial cells differentiate directly into radial glial cells in the embryonic ventricular zone [START_REF] Noctor | Neural stem and progenitor cells in cortical development[END_REF], although this has

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not been confirmed experimentally. Some recent supporting evidence is presented by the derivation of both neuroepithelial cells and radial glia from human embryonic stem cells; the course of differentiation (with respect to morphology change and lineage-specific marker expression) followed a temporal manner with neuroepithelial cells generated first, followed later by radial glial cells and finally mature neurons [START_REF] Nat | Neurogenic neuroepithelial and radial glial cells generated from six human embryonic stem cell lines in serum-free suspension and adherent cultures[END_REF].

The fate and function of a radial glial cell varies considerably from region to region in the CNS. It had long been accepted that radial glia are both astroglial progenitors [START_REF] Levitt | Coexistence of neuronal and glial precursor cells in the cerebral ventricular zone of the fetal monkey: an ultrastructural immunoperoxidase analysis[END_REF][START_REF] Schmechel | Arrested proliferation of radial glial cells during midgestation in rhesus monkey[END_REF] and oligodendrocyte progenitors [START_REF] Choi | On the origin of oligodendrocytes[END_REF][START_REF] Levine | Spatial and temporal patterns of oligodendrocyte differentiation in rat cerebrum and cerebellum[END_REF], but recent evidence has demonstrated that radial glia produce most of the cortical neurons [START_REF] Hartfuss | Characterization of CNS precursor subtypes and radial glia[END_REF][START_REF] Noctor | Neurons derived from radial glial cells establish radial units in neocortex[END_REF][START_REF] Wu | Pyramidal neurons of upper cortical layers generated by NEX-positive progenitor cells in the subventricular zone[END_REF]. Radial glial cells thus serve as neural stem/progenitor cells for the majority of neurogenesis and gliogenesis in the developing brain and gives rise to the astrocytic-like neural stem cells of the adult brain [START_REF] Hartfuss | Characterization of CNS precursor subtypes and radial glia[END_REF][START_REF] Noctor | Neurons derived from radial glial cells establish radial units in neocortex[END_REF][START_REF] Wu | Pyramidal neurons of upper cortical layers generated by NEX-positive progenitor cells in the subventricular zone[END_REF][START_REF] Anthony | Radial glia serve as neuronal progenitors in all regions of the central nervous system[END_REF][START_REF] Gotz | Radial glial cells defined and major intermediates between embryonic stem cells and CNS neurons[END_REF]. Whether radial glia transform directly into an adult multipotent neural stem cell or progenitor, or whether they first differentiate into an intermediate cell type, is unknown (see question marks, Figure 1). This concept of early neurogenesis is in dramatic contrast from the long-held view that embryonic, post-natal and adult NSCs are discrete unrelated populations.

Multipotent neural stem cells and neurogenesis in the mature CNS

Although widespread neurogenesis is restricted to the embryonic period, a limited number of neural stem cells persist throughout adulthood and are restricted mainly to two neurogenetic domains [START_REF] Gage | Mammalian neural stem cells[END_REF][START_REF] Gross | Neurogenesis in the adult brain: death of a dogma[END_REF][START_REF] Bonfanti | Radial glial origin of the adult neural stem cells in the subventricular zone[END_REF]. The largest of these regions is the mammalian SVZ situated throughout the lateral walls of the forebrain lateral ventricles [START_REF] Peretto | The subependymal layer in rodents: a site of structural plasticity and cell migration in the adult mammalian brain[END_REF]. A similar hierarchical neurogenetic system exists in the SGZ, an area corresponding to the dentate gyrus of the hippocampal granular layer [START_REF] Eriksson | Neurogenesis in the adult human hippocampus[END_REF][START_REF] Roy | In vitro neurogenesis by progenitor cells isolated from the adult human hippocampus[END_REF]. Stem

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cell progeny generated within these regions populate the growing CNS parenchyma, later differentiating into mature neuronal and glial fates, specified in a precise spatialtemporal manner [START_REF] Marin | Cell migration in the forebrain[END_REF]. Cells that express the intermediate filament astroglial marker, glial fibrillary acidic protein (GFAP), have been identified as putative adult neural stem cells in this region, also known as Type B cells [START_REF] Doetsch | Regeneration of a germinal layer in the adult mammalian brain[END_REF][START_REF] Vescovi | Brain tumour stem cells[END_REF] (Figure 1). The embryonic origin of Type B cells has been confirmed using in vivo lineage tracing techniques and shows that adult subventricular cells are not related to embryonic subventricular precursors, but embryonic ventricular radial glia [START_REF] Malatesta | Radial glia and neural stem cells[END_REF][START_REF] Merkle | Radial glia give rise to adult neural stem cells in the subventricular zone[END_REF].

Further support of the astrocytic nature of NSCs has been confirmed in experiments in which GFAP+ cells were conditionally ablated in the brain of adult mouse, resulting in near complete loss of neurogenesis [START_REF] Garcia | GFAPexpressing progenitors are the principal source of constitutive neurogenesis in adult mouse forebrain[END_REF][START_REF] Imura | The predominant neural stem cell isolated from postnatal and adult forebrain but not early embryonic forebrain expresses GFAP[END_REF]. Type B cells are proposed to be relatively quiescent with a cell cycle duration of ~24 hours and constitute a small fraction of the total astrocytic population in the SVZ [START_REF] Doetsch | Regeneration of a germinal layer in the adult mammalian brain[END_REF].

Pioneering experiments retrospectively viewed, adds support to this notion; Reynolds and Weiss in 1992, isolated a small population of cells (<0.1% of total cells) from the adult striatum that could proliferate and generate multiple clones of cells in freefloating clusters in vitro called neurospheres [START_REF] Reynolds | Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system[END_REF]. It is worth noting that even with current refined methods to isolate neural stem cells using the neurosphere assay, the stem cell content is variable; although Type B and Type C cells can produce neurospheres, not all cells within the neurosphere express stem cell markers [START_REF] Vescovi | Brain tumour stem cells[END_REF][START_REF] Doetsch | EGF converts transit-amplifying neurogenic precursors in the adult brain into multipotent stem cells[END_REF].

Multipotent Type B astrocytes within the SVZ give rise to fast-cycling transiently amplifying precursor cells called Type C precursors, which in turn generate either mitotically active Type A neuroblasts or terminally differentiated mature glia (astrocytes, oligodendrocytes). Type A neuroblasts migrate towards the olfactory bulb where they integrate as mature neurons (Figure 1). Similarly, Type B

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT astrocytes in the SGZ produce intermediate Type D precursors which give rise to

Type G granule neurons [START_REF] Doetsch | The glial identity of neural stem cells[END_REF]. Over the course of development, NSCs not only feature altered morphology but also exhibit changes to gene expression profiles in a temporalspecific manner [START_REF] Abramova | Stage-specific changes in gene expression in acutely isolated mouse CNS progenitor cells[END_REF]. This expression profile may underlie an intrinsic developmental program as progenitors grown in culture generate progeny at certain times, similar to progenitors in vivo [START_REF] Shen | The timing of cortical neurogenesis is encoded within lineages of individual progenitor cells[END_REF]. In addition to GFAP, Type B cells express other intermediate filaments such as vimentin and nestin. As NSCs differentiate along developmental pathways, neuronal-specific (polysialylated neural cell adhesion molecule (PSA-NCAM), microtubule-associated protein 2 (MAP2), neurofilament protein, tyrosine hydroxylase) and glial-specific markers (O4 monoclonal antibody that recognizes the sulfatides and myelin basic protein (MBP)) are expressed in late-amplifying and mature cell compartments [START_REF] Dell'albani | Stem cell markers in gliomas[END_REF] (Figure 1).

The role of telomeres and telomerase during neurogenesis

Most studies aimed at understanding the nature of NSCs with respect to selfrenewal, proliferation and differentiation, focus on external cues such as secreted growth factors [START_REF] Taupin | FGF-2-responsive neural stem cell proliferation requires CCg, a novel autocrine/paracrine cofactor[END_REF][START_REF] Tropepe | Transforming growth factor-alpha null and senescent mice show decreased neural progenitor cell proliferation in the forebrain subependyma[END_REF]. Few studies have addressed the role of intrinsic mechanisms regulating NSC behaviour [START_REF] Groszer | Negative regulation of neural stem/progenitor cell proliferation by the Pten tumor suppressor gene in vivo[END_REF]. Chromosome integrity is essential for cell viability, therefore highly proliferative cell types such as tissue stem cells, require active telomere maintenance strategies to proliferate indefinitely or at least long enough for the host organism to reach reproductive age. The role of telomere and telomerase dynamics in NSC compartments has scarcely been addressed. In the mouse, high levels of TERT and TERC (murine telomerase RNA subunit) mRNA are present in the developing neural tube as early as E10.5 [START_REF] Martin-Rivera | Expression of mouse telomerase catalytic subunit in embryos and adult tissues[END_REF]. Both telomerase subunits continue to be expressed in different regions of the developing murine CNS and correlates with

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the proliferation of neural progenitors [START_REF] Klapper | Differential regulation of telomerase activity and TERT expression during brain development in mice[END_REF][START_REF] Ostenfeld | Human neural precursor cells express low levels of telomerase in vitro and show diminishing cell proliferation with extensive axonal outgrowth following transplantation[END_REF][START_REF] Prowse | Developmental and tissue-specific regulation of mouse telomerase and telomere length[END_REF]. This suggests a role for telomerase during specification of neural cell types. Consistent with our knowledge of neurogenetic regions of the adult brain, telomerase activity has been shown in neural precursor cells isolated from the SVZ and hippocampus in adult mice [START_REF] Caporaso | Telomerase activity in the subventricular zone of adult mice[END_REF]. With reference to stem cell populations in other tissues, the current working hypothesis states that telomerase activity in NSCs (and actively proliferating early neural precursors) may combat induced telomere shortening upon mitotic division. However, human progenitor cells isolated from the developing cortex which exhibit low/absent levels of telomerase activity, undergo decreased neurogenesis and eventually enter replicative senescence in vitro [START_REF] Wright | Human progenitor cells isolated from the developing cortex undergo decreased neurogenesis and eventual senescence following expansion in vitro[END_REF]. These findings are corroborated by studies in other tissues where low levels of telomerase activity have been found in tissue stem cells such as haematopoietic, skin, intestinal crypt and pancreas [START_REF] Brummendorf | Telomere length dynamics in normal hematopoiesis and in disease states characterized by increased stem cell turnover[END_REF][START_REF] Hiyama | Telomerase activity in human breast tumors[END_REF][START_REF] Moriscot | Human bone marrow mesenchymal stem cells can express insulin and key transcription factors of the endocrine pancreas developmental pathway upon genetic and/or microenvironmental manipulation in vitro[END_REF][START_REF] Sugihara | Decreased expression of telomeraseassociated RNAs in the proliferation of stem cells in comparison with continuous expression in malignant tumors[END_REF]. In all these cases, telomerase activity is insufficient to completely prevent telomere loss and senescence. Rather, telomerase in tissue stem cells may slow the rate of telomere shortening and maintain a replicative reserve pre-determined for the tissue stem cell in question. It is difficult to argue that this phenomenon is not a primitive tumoursuppressor mechanism and perhaps one that predates protein-mediated tumour suppression.

Telomere attrition dramatically impairs the in vitro proliferation of adult NSCs isolated from the SVZ of telomerase-deficient mice, but not that of embryonic NSCs, even though the latter exhibited critically shortened telomeres. This is consistent with a recent report describing telomerase levels as high in embryonic cortical neural progenitor cells, but low in newly generated neurons and mature neurons [START_REF] Cheng | Telomere protection mechanisms change during neurogenesis and neuronal maturation: newly generated neurons are hypersensitive to telomere and DNA damage[END_REF]. Collectively these results hint at intrinsic differences in the protective states between adult and embryonic NSCs, with respect to chromosomal stability and

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response to dysfunctional telomeres [START_REF] Ferron | Telomere shortening and chromosomal instability abrogates proliferation of adult but not embryonic neural stem cells[END_REF]. A comprehensive understanding of how the regulation of telomeres/telomerase is tightly linked to cell cycle regulation in tissue stem cells will be crucial to determining how cancer may arise when stem cell regulation is perturbed.

Cellular Immortality and Tumour Initiation

Telomeres and telomerase in brain tumours

Tumourigenesis involves multiple oncogenic insults which collectively define the tumour phenotype. Experimental data from cultured human fibroblasts indicate that cells must bypass two checkpoints, mortality stage 1 (M1), regarded as cellular senescence and mortality stage 2 (M2), characterised by widespread apoptosis, prior

to immortalization and unlimited replicative potential [START_REF] Shay | Defining the molecular mechanisms of human cell immortalization[END_REF]. Reactivation hTERT is a crucial determinant of cellular immortalization and if accompanied by inactivation of tumour-suppressor genes and/or activation of cellular oncogenes, can subsequently result in neoplastic formation. Telomerase-mediated telomere maintenance is evident in virtually all types of malignant cells, and ~90% of tumours show evidence of upregulated telomerase [START_REF] Bryan | Telomerase and the maintenance of chromosome ends[END_REF][START_REF] Shay | A survey of telomerase activity in human cancer[END_REF]. Brain tumours in which telomerase activity has been detected include glioblastoma, anaplastic astrocytoma, oligodendroglioma, primitive neuro-ectodermal tumours, medulloblastoma and ependymoma [START_REF] Didiano | Telomere maintenance in childhood primitive neuroectodermal brain tumors[END_REF][START_REF] Hiraga | Telomerase activity and alterations in telomere length in human brain tumors[END_REF][START_REF] Langford | Telomerase activity in human brain tumours[END_REF][START_REF] Ridley | Multifactorial analysis of predictors of outcome in pediatric intracranial ependymoma[END_REF]. These data indicate that hTERT reactivation or overexpression is an essential event for tumour progression in CNS tumours. The role of hTERT may not be restricted to addition of telomere repeats as mouse TERT has been shown to promote cell survival (prevents apoptosis) in neurons, a function that does not require the telomerase RNA subunit [START_REF] Mattson | Emerging roles for telomerase in neuronal development and apoptosis[END_REF]. This extracurricular TERT function may contribute to cancer development and aging independently of telomere lengthening and telomerase activity.

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In a comprehensive analysis of telomere length, the majority of grade II astrocytomas, anaplastic astrocytomas and glioblastomas with telomerase activity, exhibited significantly shorter mean telomere lengths relative to corresponding tumours with undetectable telomerase activity and to normal brain tissue. Similarly, all primitive neuroectodermal tumours exhibited shorter telomere lengths relative to normal brain tissue [START_REF] Hiraga | Telomerase activity and alterations in telomere length in human brain tumors[END_REF]. Recent findings from our own laboratory reveal that paediatric glioblastoma multiforme and ependymoma, tumours of glial origin, have significantly longer mean telomere length than those of supratentorial neuroectodermal tumours and medulloblastoma, tumours of neuroectodermal origin.

This highlights the consideration required for telomere length with respect to duration/course of treatment when using anti-telomerase drugs which are predicted to exert their effects upon telomere shortening (Rahman R et al. unpublished data). The concurrent shortening of telomeres with telomerase reactivation/overexpression supports a hypothesis that telomerase is required to maintain both minimum length and a terminal capping structure at a subset of critically shortened telomeres [START_REF] Counter | Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity[END_REF].

The perspective presented by the stem cell origin of brain tumours implies that the genetic alterations that lead to cancer, accumulate in NSCs rather than mature neural cells. As NSCs would already exhibit telomerase activity, one would predict the enzyme to be overexpressed rather than reactivated. Whether telomerase activity increases during later stages of carcinogenesis is unclear [START_REF] Armanios | Telomerase and cancer stem cells[END_REF]. Similar to selfrenewing tissues where hTERT expression is insufficient to halt progressive telomere shortening, telomere shortening may continue within a proliferating transformed neural stem cell. However aberrant and sustained hTERT expression likely results in telomere stabilization both with respect to telomere length and 3' telomere capping structure (Figure 2). Elucidation of telomerase levels and telomere length within

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different neurogenetic subsets will influence strategies to target telomerase in brain cancer.

The Brain Tumour Stem Cell Paradigm

An acceptance of tumour hierarchical organisation readily leads to a developmental-centred view of tumourigenesis, where brain tumours represent aberrantly differentiated tissue, in turn reflecting dysregulated neurogenesis. The stem cell paradigm for brain tumours presents tumour-initiating events as occurring within the genome of a cellular entity with neural stem cell-like properties.

The identification of brain tumour stem cells was prospectively demonstrated by Peter Dirks and colleagues using cells derived from glioblastoma and medulloblastoma. As few as 100 cells sorted positively for the cell surface antigen CD133, were capable of initiating infiltrative tumours upon orthotopic transplantation into SCID mice whereas injection of 100,000 CD133-cells failed to initiate tumours [START_REF] Singh | Identification of human brain tumour initiating cells[END_REF]. In a later study, tumour stem cells isolated from ependymoma manifested a radial glial phenotype with CD133+/Nestin+/RC2+/BLBP+ positivity confirmed to be tumourigenic [START_REF] Taylor | Radial glia cells are candidate stem cells of ependymoma[END_REF].

It is important to note that a definitive demonstration of tumour stem cells in the brain is lacking as CD133 enriches for, rather than definitively identifies, cancer stem cells. This caveat has been emphasized with recent studies showing that CD133glioblastoma cells have tumour-initiating ability [START_REF] Beier | CD133(+) and CD133(-) glioblastoma-derived cancer stem cells show differential growth characteristics and molecular profiles[END_REF][START_REF] Wang | CD133 negative glioma cells form tumors in nude rats and give rise to CD133 positive cells[END_REF]. In addition, it is the CD133+/Nestin+/RC2-/BLBP-phenotype that exhibited a tumourigenic phenotype in medulloblastoma in contrast to the CD133+/Nestin+/RC2+/BLBP+ phenotype in ependymoma [START_REF] Taylor | Radial glia cells are candidate stem cells of ependymoma[END_REF], suggesting the characteristics of these two subsets may reflect different cell of origins. Clearly stem-like properties in a tumour cell cannot be taken

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as direct evidence for neoplastic transformation occurring in a neural stem cell that originates in a zone of active neurogenesis. The cancer stem cell hypothesis and the cellular origin of cancer are distinct paradigms. It is conceivable that a neural progenitor or mature cell could give the appearance of 'stemness' upon neoplastic transformation (Figure 2).

An alternative focus is that of molecular events that permit tumour initiation rather than on the cell type that initiated the tumour. The cancer stem cell hypothesis predicts mechanistic similarities between self-renewal of cancer stem cells and normal stem cells. In brain tumours there is particular interest in the Notch [START_REF] Maillard | Notch and cancer: best to avoid the ups and downs[END_REF][START_REF] Radtke | The role of Notch in tumorigenesis: oncogene or tumour suppressor?[END_REF] and Sonic-hedgehog [START_REF] Lai | Sonic hedgehog regulates adult neural progenitor proliferation in vitro and in vivo[END_REF][START_REF] Machold | Sonic hedgehog is required for progenitor cell maintenance in telencephalic stem cell niches[END_REF] pathways, as these have been shown to be important for normal stem cell self-renewal as well as dysregulated growth in cancer.

Overexpression of Notch receptors and their ligands Delta-like 1 (DLK1) and Jagged

(JAG1) correlates with the proliferative capacity of human glioma cells, implicating

Notch as a positive effector of self-renewal in adult neural stem cells [START_REF] Hitoshi | Notch pathway molecules are essential for the maintenance, but not the generation, of mammalian neural stem cells[END_REF].

Furthermore, overexpression of Notch-2 intracellular domain protein increases proliferation of medulloblastoma cell lines [START_REF] Fan | Notch1 and notch2 have opposite effects on embryonal brain tumor growth[END_REF], whereas medulloblastoma growth is suppressed by the known Notch inhibitor, γ-secretase [START_REF] Hallahan | The SmoA1 mouse model reveals that notch signaling is critical for the growth and survival of sonic hedgehog-induced medulloblastomas[END_REF]. Similarly Sonic Hedgehog (SHH) and its downstream effectors, glioma-associated homologue 1 (GLI1), GLI2 and GLI3 have been shown to specifically regulate self-renewal and neurogenesis within the external granular layer of the post-natal cerebellum and to control proliferation of precursors within the adult SVZ [START_REF] Palma | Sonic hedgehog controls stem cell behavior in the postnatal and adult brain[END_REF]. Mutations in this pathway are thus implicated in the initiation of medulloblastoma [START_REF] Goodrich | Altered neural cell fates and medulloblastoma in mouse patched mutants[END_REF][START_REF] Pomeroy | Molecular biology of medulloblastoma therapy[END_REF].

Another emerging concept is that brain tumour stem cells may crucially be supported by a specific microenvironment which mimics the neural vascular niche [START_REF] Calabrese | A perivascular niche for brain tumor stem cells[END_REF]. Differentiated neural cells or blood vessel cells have been implicated as niche

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elements. CD133+ glioblastoma cells secrete increased levels of vascular endothelial growth factor (VEGF) compared to CD133-counterparts, implicating tumour stem cells as contributing to tumour angiogenesis [START_REF] Bao | Stem cell-like glioma cells promote tumor angiogenesis through vascular endothelial growth factor[END_REF]. However, whether the tumour stem cell niche differs from that of the bulk tumour population or whether it provides signals which govern aberrant self-renewal is unclear. Future work will validate the proposition that the tumour stem cell niche represents an attractive therapeutic target.

The cancer stem cell field is at present contentious, in part due to differing semantic interpretations. The 'cancer stem cell' term is often applied to cells identified by varying methods and criteria. The danger here is the application of the term to cells with no evidence of cancer-initiating capacity. It is important to clarify what 'brain tumour stem cell' refers to in the present context: these are cells which demonstrate cancer-reinitiating ability upon orthotopic transplantation, with a capacity to generate a phenocopy of the original tumour mass (consisting both tumourigenic and non-tumourigenic cells); show extensive self-renewal ability ex vivo and in vivo; demonstrate aberrant multipotent differentiation; harbour karyotypic and/or genetic alterations. At present, this does not exclude the possibility of more committed progenitors or even mature cells reverting to a stem-like phenotype and causing tumour-initiation in contexts that differ from those imposed by the prementioned strategies. For example, it may be argued that the mouse may not represent a physiologically relevant microenvironment for engraftment and growth of human tumours, thus potentially resulting in an under-estimation of cell types capable of tumour initiation.

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Towards A New Therapeutic Outlook

The discovery of putative brain tumour stem cells and the growing understanding of neurogenesis and cellular immortality, presents both a novel cellular target and novel molecular targets within. Much larger numbers of brain tumour samples and prospective multi-parameter cell sorting are needed to determine whether the cancer stem cell hypothesis is robust as well as to delineate patient subsets within a particular brain tumour type. Do all tumours under a common nomenclature reflect a common cell of brain cancer origin? What are the implications for therapy if they do not? In addition, evidence to date does not address whether the cell type conferring self-renewal capacity through tumoursphere-formation in vitro is the same cell type that initiates the neural tumour in vivo; these studies involve tumour-initiating populations (heterogeneous) rather than (homogenous) tumour-initiating cells.

Similarly, it is important to separate tumour-initiation and tumour-propagation; this may not involve the same cell type as the tumour-propagating cell may be a much differentiated progeny of the tumour-initiating cell. It is conceivable therefore, that improved therapeutic efficacy may be achieved by targeting both the cell-type(s) which drives malignant progression as well as that which initiates -and maintains the stem cell pool of-the tumour.

Since telomerase activity has been detected in almost all advanced brain tumours, the use of telomerase inhibitors may provide an attractive approach to therapy. These may be most effective in reducing the risk of relapse by targeting cancer stem cells. As normal neural stem cells would be expected to have longer telomeres than brain cancer stem cells, it follows that telomere depletion will be spared in the normal stem cell pool [START_REF] Shay | Telomerase in cancer: diagnostic, prognostic, and therapeutic implications[END_REF][START_REF] White | Telomerase inhibitors[END_REF]. There is a practical consideration here with respect to telomere length: if anti-telomerase approaches rely on telomere

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shortening for efficacious results, then the initial telomere length in the target cancer cell must be a consideration. This invokes the brain tumour cell of origin once again, as telomere dynamics reflect the mitotic history of highly proliferating cells.

Elucidation of telomere length and telomerase activity in subsets of cells within the tumour hierarchy will enable a more targeted anti-telomerase approach, perhaps in combination therapy. The identification of a greater array of surface markers will likely aid this characterisation.

Key cell signalling pathways such as Notch and Sonic Hedgehog, that are involved in the regulation of self-renewal in both normal and cancer stem cells, also represent putative molecular targets. A more thorough understanding of the mechanisms of signal transduction in each context, particularly with respect to gene expression levels will determine whether disrupting self-renewal pathways in brain tumour stem cells will spare normal neural stem cell pool function and result in tumour growth inhibition.

In addition, better therapeutics will rely on a better understanding of the intrinsic mechanisms of resistance in brain tumour cells. The cancer stem cell hypothesis predicts that only tumourigenic cells are resistant to therapy. Recent work studying radiation resistance supports this idea as CD133+ glioma stem cells show preferential activation of the DNA damage response upon radiation treatment [START_REF] Bao | Stem cell-like glioma cells promote tumor angiogenesis through vascular endothelial growth factor[END_REF]. However, whether the same cell type that confers radioresistance is the same cell type that confers chemoresistance is unknown. Tumour resistance may also arise if brain tumour stem cells, similar to normal stem cells, generally reside in a quiescent state outside the cycle of division. Although direct evidence is lacking, if shown to be correct, this would affect the rationale and approach of drugs targeting telomerase and self-renewal pathways. In addition, brain tumour stem cells may plausibly have high Understanding the molecular and genetic basis of the cell type(s) surviving multi-modal therapy will be crucial to our unwavering hope of a curative response in brain cancer patients (Figure 3). Is each factor contributing to resistance, both necessary and sufficient for tumour resistance? Or is coercion required involving all factors of resistance? If the latter, will targeting one mechanism of resistance suffice?

Concluding Remarks

The brain cancer stem cell paradigm without reference to neurodevelopment and dysregulated cellular proliferation is perhaps reductionist and oversimplified. The emerging concept of a close relationship between developmental neurobiology, telomere/telomerase biology and brain cancer stem cells, encourages a consilient approach to develop the next generation of brain cancer therapeutic interventions. At one time disparate, these areas of investigative biology should help determine the cellular culprit of particular brain cancer types, both with respect to the cell type where neoplastic transformation occurs and to the molecular events that permit it. If these tumour-initiating cells are intrinsically resistant to conventional therapeutic strategies, then a comprehensive characterisation of this mechanism will allow more effective combination therapy to be developed in the future.

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mutational events drive progression of the tumour. As the transformed neural stem cell (aberrantly) differentiates, telomere shortening is likely to occur; however due to hTERT acting on critically shortened telomeres, telomere length in the (mature) malignant neural cell would be expected to be stabilized. Although depicted here as arising in a neural stem cell, it remains plausible that the tumour cell of origin is a neural progenitor; in the latter scenario, the progenitor cell may acquire the capacity to self-renew through mutation, increasing the propensity to initiate a tumour.

A

  C C E P T E D M A N U S C R I P TACCEPTED MANUSCRIPT levels of ATP-binding (ABC) drug transporters, which can protect cells from cytotoxic agents[START_REF] Dean | Tumour stem cells and drug resistance[END_REF]. This may result in the efflux of cytotoxic agents delivered to tumour stem cells. If ABC transporters are shown to contribute to tumour resistance, the next generation of chemotherapy regimes may involve chemosensitisers in conjunction with agents that alter drug-transporter activity.

Figure 2 :

 2 Figure 2: Proposed role for telomerase activity and telomere stabilization in brain tumour initiation and propagation. (Normal developmental neurogenesis)The postnatal brain harbours regions that retain mitotic activity throughout adulthood, in particular the subventricular zone (SVZ) and subgranular zone (SGZ). Mature neurons and glia are generated in these regions through cell cycle re-entry and differentiation of tissue-specific stem cells, giving rise to neural lineage-restricted progenitors. Faithful cell division is safeguarded in neural progenitors and mature cell types through intact functional cell cycle and senescence checkpoints. (Dysregulated developmental neurogenesis). hTERT overexpression, loss of tumour suppressors, oncogenic insults, checkpoint failure and chromosome instability, increase the tumourigenic potential of the tissue stem cell, ultimately resulting in a transformed neural stem cell ('brain tumour stem cell'). It is unclear whether mutational events at tumour-initiation are necessary and sufficient to propagate differentiation to a brain tumour progenitor cell and 'mature' malignant neural cell, or whether additional

Figure 3 :

 3 Figure 3: Brain tumour cells exhibit intrinsic resistance to current treatment modalities. The cancer stem cell hypothesis presents an attractive framework upon which to consider a gestalt shift in treatment protocols for CNS tumours. (Top)Existing therapeutic regimes successfully achieve a substantial degree of tumour remission prior to tumour relapse fuelled by a brain tumour population(s) with intrinsic resistance to therapy. There is increasing evidence that the resistant cell type is a neural stem-like cell with dysregulated self-renewal or neural progenitor which has acquired aberrant self-renewal capabilities through mutation. Mechanisms of tumour stem cell resistance are largely based on presumptions of shared properties with neural stem cells; however evidence is beginning to emerge demonstrating that brain tumour stem/progenitor cells are intrinsically protected, relative to the bulk tumour (yellow box). (Bottom) It is likely a new therapeutic paradigm will include chemosensitizers and cyotoxic agents that target the dysregulated pathways of neural stem/progenitor cells and mechanisms that mediate resistance, such as ABC-drug transporters. Characterisation of telomere length/structure and telomerase activation levels in brain tumour sub-compartments will reveal whether anti-telomerase therapy will be an attractive proposition for CNS tumours. To accomplish a durable response

Figure 1: Stem cell theory for the role of telomeres and telomerase in postnatal neurogenesis and cancer.

  Radial glial cells (RG, green box) differentiate from embryonic neuroepithelial progenitors at the beginning of neurogenesis and are distinguished by the expression of astroglial lineage markers in the neonatal brain. Within the constitutively active germinative layers associated with the anterior part of the forebrain lateral ventricles (SVZ, shown here) and corresponding to the inner layer of the dentate gyrus within the hippocampus (SGZ, not shown), radial glia give rise to adult multipotent neural stem cells (Type B astrocytes, dark blue circle). At later developmental stages, radial glial cell division can also produce ependymal cells that constitute the epithelia lining the ventricular system of the brain and spinal cord (light blue circle). Type B neural stem cells retain markers of radial glia, have high levels of telomerase activity and may have longer telomeres relative to mature neurons or glia. During postnatal neurogenesis, neural stem cells differentiate and

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	endowed with tumour-initiating ability. These 'brain tumour stem cells' may arise in
	vivo from a transformed neural stem cell or from an early neural progenitor that
	acquires the ability to self-renew through mutation. Abbreviations: SC, stem cell; ET,
	early transit amplifying progenitor; LT, late transit amplifying progenitor; EP, ependymal cells; SVZ, subventricular zone; SGZ, subgranular zone; GFAP, glial fabrillary acidic protein; PSA-NCAM, polysialylated neural cell adhesion molecule; MAP2, microtubule-associated protein 2; O4, monoclonal antibody that recognizes the sulfatides; MBP, myelin basic protein. Blue rectangles represent stem cell/progenitor populations; Red rectangles represent terminally differentiated cell M A N U S C R I P T types.
	give rise to fast-cycling early transit amplifying progenitors (Type C putative precursors, Orange circle), which can give rise to terminally differentiated mature glia (astrocytes, oligodendrocytes) or late transit amplifying progenitors (Type A migrating neuroblasts, Yellow star) that are committed to a mature neuronal fate and that express neuronal-specific markers. As multipotent neural stem cells differentiate A C C E P T E D
	into mature functional neurons and glia, the capability to leave the stem cell
	perivascular niche and regenerate neural tissue is decreased. This is due in
	considerable part to telomere shortening across time. Decreased mobilization of stem
	cells in aged neural tissue is likely to be consequential of senescence (replicative and
	stress-induced) and apoptosis. There is increasing awareness that the growth and
	recurrence of brain tumours may be due to a transformed CNS cell type with shared
	characteristics to a somatic neural stem cell (self-renewal, differentiation) and

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