Neural Stem Cells and the Origin of Gliomas
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《新英格兰医药杂志》
Despite progress in research on the molecular aspects of malignant gliomas, the prognosis of these brain tumors continues to be dismal. In glioblastoma, the most common glioma variant in adults, median survival has remained at 9 to 12 months for decades.1 One reason for the lack of clinical advances is ignorance of the cellular origin of this disease, which delays the application of molecular analyses to treatment and impairs the ability to anticipate tumor behavior reliably. For nearly 80 years, the classification system for gliomas has remained largely unchanged2,3; its power to predict the course of disease is based not on identification of the tumor cell but on histologic criteria, such as endothelial proliferation and necrosis (Figure 1).4,5,6 Gliomas include tumors of presumed astrocytic, oligodendroglial, or ependymal lineage (Table 1), but this discussion will focus on the first two categories, which account for the vast majority of these tumors.
Figure 1. Grades I to IV (World Health Organization) Astrocytic Tumors.
Panels A and B show circumscribed astrocytomas. Pilocytic astrocytomas (Panel A) are typically indolent, have a limited invasive capacity, and rarely undergo anaplastic progression. These tumors may have microvascular hyperplasia and cellular pleomorphism despite their designation as grade I tumors. Pleomorphic xanthoastrocytomas (Panel B) are also relatively circumscribed and, despite their distinct, conspicuous cellular pleomorphism, tend to be low-grade (grade II) tumors with limited capacity for brain invasion. Panels C through F show diffuse-type astrocytomas, which have the capacity for dispersion into the surrounding brain and a high frequency of anaplastic progression. A grade II astrocytoma (Panel C) is well-differentiated, with mild-to-moderate nuclear pleomorphism. A grade III astrocytoma (Panel D) has a high rate of cell proliferation, as indicated by the mitotic figures. These tumors commonly have a moderate degree of cellular pleomorphism and more heterogeneous cellularity. Glioblastoma multiforme, grade IV, is the most aggressive glial tumor and has the distinctive features of palisading or geographic necrosis (Panel E) and conspicuous microvascular hyperplasia (Panel F) in addition to marked cellular pleomorphism. (Tissue samples, stained with hematoxylin and eosin, courtesy of Dr. Scott R. VandenBerg.)
Table 1. Cell Types and Associated Tumors of the Central Nervous System.
The Cell of Origin of Gliomas
The identification of the cellular origin of gliomas presents an opportunity for improving our understanding of this disease. Although the neoplastic transformation of fully differentiated glia is widely assumed to be the mechanism of gliomagenesis, this hypothesis has never been adequately tested. Furthermore, the concept of dedifferentiation of mature glia is questionable and fails to explain adequately the origin of some gliomas, such as the mixed oligoastrocytoma. The astrocytoma, another glioma variant, does possess some morphologic characteristics of mature astrocytes, but the origin of a neoplasm is not necessarily reflected in the appearance of its most common cellular component: brain tumors of apparently comparable histologic structure can exhibit vastly different behaviors. Adult glia were once thought to be the only dividing cells in the postnatal brain, making them the only brain cells susceptible to transformation. We now know that this is not the case, because other proliferative populations of cells have since been discovered in the adult human brain, as discussed below. For this reason, the classic theories regarding gliomagenesis are now being reappraised in the hope of constructing a more accurate picture of the origin of gliomas.
Neural Stem Cells and Glial Progenitor Cells
It is now known that there are both neural stem cells and glial progenitor cells in multiple regions of the adult brain. Neural stem cells, which are multipotent and self-renewing, have been isolated from the subventricular zone,7 the lining of the lateral ventricles (Figure 2A), the dentate gyrus,8 within the hippocampus (Figure 2B), and the subcortical white matter9 (Figure 2B). The largest of these germinal regions in humans, the subventricular zone, contains a population of astrocytes that can function as neural stem cells.7 In other adult mammals, glial progenitor cells — self-renewing precursors capable of producing astrocytes and oligodendrocytes — have also been observed throughout the neuraxis,10 including the cortex,11,12 the corpus callosum,13 the periventricular white matter,14 the subventricular zone,15 and the dentate gyrus.8 These stem-cell and progenitor elements, in addition to differentiated adult glia, constitute a substrate for neoplastic transformation.
Figure 2. Germinal Regions of the Adult Human Brain.
Panel A shows the lateral walls of the lateral ventricles, which contain the subventricular zone. Panel B shows the hippocampus, which contains the dentate gyrus and the subcortical white matter. CA1 and CA3 indicate regions with pyramidal cells as their principal neurons.
Properties of Neural Stem Cells
All forms of cancer arise from disturbances of critical cellular functions such as proliferation, apoptosis, and tissue invasion.16 In the adult brain, neural stem cells and progenitor cells (self-renewing precursors capable of producing progeny along either neuronal or glial lineages, but not both) commonly possess features associated with cancer of the central nervous system, including a robust proliferative potential and a diversity of progeny. Neural stem cells are regulated by the same cellular pathways that are active in many brain tumors.17 Consequently, they are capable of exhibiting behavior that is characteristic of gliomas, including high motility, association with blood vessels and white-matter tracts, possession of immature antigenic phenotypes, and activation of "developmental" signaling pathways (Table 2).10,18,19
Table 2. Characteristics Intrinsic to Neural Stem Cells and Gliomas.
Transformation of Neural Stem Cells
Germinal regions such as the subventricular zone have long been proposed as sources of gliomas.20,21 Many gliomas are either periventricular or contiguous with the subventricular zone, and they frequently express the progenitor-cell marker nestin.22,23,24,25,26 It remains unclear, however, whether the expression of nestin is a genetic aberration or a normal property of the precursor cell from which the tumor originated.27 That neural stem cells are potentially susceptible to transformation is suggested by the fact that, in animal models, regions of the brain with a high degree of cellular proliferation — areas with stem-cell populations — are more sensitive to chemical or viral oncogenesis than are areas with a low proportion of proliferating cells.28,29,30 In canine and rodent brains, for example, avian sarcoma viral transformation or systemic exposure to the carcinogen N-ethyl-N-nitrosourea preferentially leads to tumor formation in the proliferative subventricular zone rather than in nonproliferative regions of the brain.29,30,31,32 In one study, intraventricular inoculation with avian sarcoma virus in neonatal canine brains initially led to periventricular glioma microfoci, but as the tumors grew, their continuity with the subventricular zone diminished until, at day 10 after inoculation, they were found in the deep white matter without apparent connection to the subventricular zone.30 This migration of transformed germinal-zone cells may be a mechanism by which human gliomas arise from neural stem cells but then go on to lose any evidence of continuity with these regions.
Molecular Mechanisms
These initial observations suggested that gliomas can originate from neural stem-cell populations, but the genetic alterations necessary for stem-cell and progenitor-cell transformation are only now being uncovered. Recently, progenitor cells activated with Akt and KRas, signal transduction proteins associated with human glioblastomas, were shown to form brain tumors with histologic features of glioblastoma in rodents.33 For that experiment, a retroviral delivery system was designed specifically to infect, in vivo, the progenitor cells of the central nervous system of a transgenic mouse in order to transfer postnatally the Akt and KRas oncogenes exclusively to these cells. When a constitutively active epidermal growth factor receptor (EGFR) was transfected with retrovirus into transgenic Ink4a-Arf-/- mouse neural stem cells, which are deficient in genes that regulate cell-cycle arrest and apoptosis,34 these cells generated high-grade gliomas in vivo once they were transplanted into an adult mouse brain.
In contrast, mature mouse astrocytes expressing the differentiated astrocyte marker glial fibrillary acidic protein (GFAP) were less susceptible to transformation in vivo unless they were converted to an undifferentiated state through retroviral transfection of the platelet-derived growth factor (pdgf) gene35 or through the loss of the Ink4a-Arf locus.34,36 These experiments indicate that the state of differentiation is an important feature of the cell of origin of gliomas, although the influence of the state of differentiation depends on the oncogenes involved. These data suggest that stem cells and progenitor cells in the central nervous system particularly are at risk for malignant transformation, presumably because they have activated the cellular machinery (e.g., promitotic genes, telomerase activity, and antiapoptotic genes) necessary for tumor initiation, progression, or both. Thus, neural stem cells may represent the path of least resistance in tumorigenesis, since they already have the ability to bypass apoptosis and senescence. As a result, neural stem cells may require less than the estimated four to seven mutations needed to effect malignant change in a differentiated cell.16
Stem Cells in Other Neoplasms
The concept of a stem-cell origin for cancer has already been demonstrated in several tumors outside the central nervous system,37,38,39 with the hematopoietic system providing the clearest example.40,41,42 In this system, it is well documented that only a small subgroup of cancer cells from patients with chronic myeloid leukemia and multiple myeloma demonstrate extensive proliferation and tumor initiation. More recently, CD34+CD38– hematopoietic stem cells derived from specimens of human acute myeloblastic leukemia have been identified and enriched for clonogenic capacity. Although these cells represent a fraction of the total population of white cells (0.2 to 100 of 106 white cells), they constitute the only cellular compartment capable of initiating the disease in another host.17 Similar evidence is now being gathered in the field of breast cancer.43
Developmental Patterns
Sonic Hedgehog Pathway
If neural stem cells are the source of initiation for brain cancer, their progression toward a tumorigenic state may be achieved through the operation of abnormal "developmental" programs. As the details regarding the genetics of brain tumors emerge, they point to multiple developmental signaling pathways as critical regulators of tumorigenesis. Although these programs function with the use of normal cellular components, their timing, order, and magnitude are probably abnormal. For example, studies have shown that medulloblastoma, a neuroectodermal tumor of the cerebellum, arises through abnormalities of developmental pathways in a population of progenitor cells.44,45,46 These tumors aberrantly express multiple regulatory genes known to mediate the proliferation of neural stem cells.47
Specifically, dysregulation of the sonic hedgehog (Shh) developmental signaling pathway (Figure 3) leads to the aberrant activation of Shh signaling, which normally regulates the self-renewal of precursors of cerebellar granule cells and the development of their neuronal progeny. The activation of the Shh pathway, in turn, predisposes neural progenitor cells to medulloblastoma formation by mediating the inactivation of the retinoblastoma tumor-suppressor gene (Rb)48 and directly inducing regulators of the cell cycle such as the proto-oncogene N-myc. N-myc, a key transcription factor that mediates the proliferation of neuronal progenitor cells,49,50 is overexpressed in medulloblastomas.51 Thus, activity in the Shh pathway is not only critical for the self-renewal of progenitor cells and the proliferation of their progeny, but it also facilitates the initiation of brain tumors. It is interesting to note that medulloblastoma cells die rapidly when cultured with cyclopamine, an antagonist of the hedgehog family of regulatory pathways,52 suggesting that there is a role for this signaling pathway in tumor maintenance as well as initiation.
Figure 3. Generalized Hedgehog–Gli Signaling Pathway.
When the sonic hedgehog pathway (Shh) binds to the patched (Ptc)-smoothened (Smo) receptor, a cytoskeleton-associated macromolecular complex — including the Gli proteins, suppressor of fused , fused (Fu), protein kinase A (PKA), and other possible components (X, Y, Z) — acts to produce Gli activators. These Gli activators are imported into the nucleus and act on designated target genes. The introduction of cyclopamine inhibits hedgehog signaling by acting on Smo.
Transcription Factors
The hedgehog family of regulatory pathways does not just mediate the proliferation of progenitor cells in the external granular layer of the cerebellum. Hedgehog signaling activates three zinc-finger transcription factors — Gli1, Gli2, and Gli3 — which regulate progenitor cells by promoting cell-cycle entry and DNA replication.53,54 In the adult central nervous system, Gli1 is expressed by neuronal progenitors in germinal regions such as the subventricular zone and the dentate gyrus.55,56 In these regions, the Shh – Gli pathways also play an integral role in the support of germinal niches by maintaining the stem-cell population57 or by facilitating the survival and proliferation of stem-cell progeny.58 It is important to note that Gli is expressed in both low-grade and high-grade gliomas,39 and the Shh – Gli pathway may mediate the initiation and maintenance of these tumors as it does for neural stem cells.39,59 As might be expected, treatment with cyclopamine can inhibit the growth of some glioma cell lines in vitro.39 Thus, the hedgehog family of signaling pathways, key mechanisms of embryonic neural-tube development as well as normal mediators of adult neural stem-cell proliferation, are abnormally activated during gliomagenesis.
Epidermal Growth Factor
The EGF signaling pathway also plays an important part in gliomagenesis and neural stem-cell regulation. Amplification of the EGFR gene is associated with the formation of glioblastomas, and activation of EGFR promotes the growth of both astrocyte precursors60,61 and neural stem cells.62,63 As many as 50 percent of high-grade astrocytomas demonstrate EGFR amplification, and EGFR activation may drive the transformation process in the development of glioblastomas.1,44 In the subventricular zone of the adult rodent, EGF-responsive "C" cells constitute a large population of migratory, rapidly dividing progenitor cells. In response to exposure to EGF in vitro, these C cells give rise to spherical collections of cells (neurospheres) that exhibit properties similar to those of stem cells. In rodents, in vivo, EGF induces the proliferation of C cells, prevents their differentiation, and is associated with tissue infiltration that is analogous to infiltration by high-grade gliomas.18 The human homologue to this cell type has yet to be identified.
PTEN Pathway
Several other genetic pathways are also active in neural stem-cell regulation and gliomagenesis. PTEN, a tumor-suppressor gene often affected in high-grade gliomas and one of the genes most commonly mutated in glioblastomas,64 encodes a phosphatase that regulates the proliferation of neural stem cells.65 PTEN may also play a part in neural stem-cell motility66 and could therefore lead to the extensive infiltration seen in gliomas. Similarly, neurospheres cultured from gliomas and normal neural stem cells express the oncogene bmi-1, a transcriptional repressor necessary for neural stem-cell proliferation in the central nervous system.47,65 Telomerase, which prevents chromosomal shortening and apoptosis after cell division, is also expressed in the majority of high-grade gliomas as well as in progenitor cells found in the subventricular zone,67 although the frequency and timing of telomerase expression remain unknown. Finally, the Wnt signaling pathway, which controls the size of the neural stem-cell population through -catenin activation,68 is mutated in a subgroup of medulloblastomas,69 although its role in gliomas remains unknown.
Heterogeneity of Gliomas
The neural stem-cell model for the origin of gliomas sheds light on their heterogeneity. In several solid tumors outside the central nervous system, only a small fraction of the total population of cancer cells appears to be clonogenic. Examples of such tumors include those in the lung and ovary, from which only 1 in 1000 to 1 in 5000 cells form colonies in agar.70 Recently, self-renewing, multipotent cells expressing the CD133 cell-surface marker were isolated from human gliomas and transplanted into adult mouse brains, where they recapitulated the parent tumor's histology.71,72 These findings suggest that gliomas, too, are initiated and maintained by a small fraction of cancerous neural stem cells.
There is ample evidence of a far greater level of heterogeneity among gliomas than that allowed for in the current classification system. Since the expression of normal differentiation markers within these tumors varies, at least some of this heterogeneity probably arises from the anomalous differentiation of tumor cells.17 For a single glioma, karyotype analysis reveals substantial differences from region to region; however, a pattern of shared chromosomal abnormality, and hence clonal origin, is conserved.73,74 Although it is uncommon for a single glioma to exhibit the entire range of cell types generated by a neural stem cell, gliomas are often composed of multiple cell types, such as astrocytes and oligodendrocytes.75
The genetic and cellular diversity within each glioma may be due to secondary changes within tumor subclones, but it is possible that the origin is a multipotent tumor cell. The cause of mixed-cell gliomas is not likely to be the independent transformation of two differentiated cells but, rather, the transformation of a single, bipotential progenitor cell.76 For example, an oligoastrocytoma may originate from a bipotential adult oligodendrocyte-type-2 astrocyte progenitor cell, which normally generates both astrocytic and oligodendrocytic progeny in vitro.77 The transformation of such a population of progenitor cells, which are already capable of self-renewal through asymmetric cell division, could generate astrocytomas, oligodendrogliomas, or oligoastrocytomas.78
It is important to note that the loss of heterozygosity on chromosomes 1p and 19q in both the astrocytic and oligodendrocytic components in mixed oligoastrocytomas has been reported,79 suggesting that there is a common cell of origin. In vivo gene transfer of the polyomavirus middle T antigen, an oncogene that activates multiple signal-transduction pathways involved in gliomagenesis, into differentiated, GFAP-positive astrocytes has been shown to result in the production of both oligodendrogliomas and astrocytomas.80 Thus, the cell of origin of gliomas not only may be capable of producing multiple cell types, including oligodendrocytes and astrocytes, but also may resemble an astrocyte in terms of phenotype. It remains unknown whether such a multipotent astrocyte is related to the astrocytic neural stem cells lining the subventricular zone of the adult human lateral ventricles, as recently described (Figure 4).7
Figure 4. Adult Human Subventricular Zone.
The cells of the subventricular zone, labeled with the astrocyte marker GFAP (shown in green), line the lateral walls of the lateral ventricles. This is the largest known region of adult neural stem cells in the human brain; it is composed of the deep subcortical white matter (A), a periventricular ribbon of astrocytes that can function as neural stem cells (B), a dense layer of astrocytic processes (C), and the ependymal lining (D). Throughout adult life, astrocytes from the subventricular zone exhibit a unique capacity for multipotency and self-renewal in vitro. (From Sanai et al.7)
Cellular Environment
With regard to mixed-cell gliomas, there is also the question of how the cellular environment directs gliomagenesis. Since gliomas can contain nonneoplastic astrocytes and endothelial cells within their stroma, a potentially important factor in the formation of gliomas may be the germinal niche re-created by the coexistence of neoplastic and nonneoplastic cells of the central nervous system. This relationship is highly evocative of the environmental interplay known to exist between adult neural stem cells and the supporting cells of the subventricular zone and dentate gyrus.58
Blocked Differentiation
Although the presence of multipotent progenitor cells can explain the heterogeneity of brain tumors, the diversity of their progeny may be limited by the blocked-differentiation phenomenon that was first observed in human leukemias.81 This process entails an accrual of immature, self-renewing progenitor cells in which cell proliferation and maturation are uncoupled. The transformed progenitor cells divide rapidly, but their progeny are incapable of complete differentiation (Figure 5).82 Thus, the tumor phenotype may be defined by the direction and degree of differentiation of the transformed progenitor population. For example, astrocytomas may arise from glial progenitors whose progeny can differentiate only along astrocytic lines. More complex phenomena, such as the transformation of an oligodendroglioma to a glioblastoma, may be explained by a secondary wave of accelerated growth and arrest of maturation among a subgroup of progenitor cells. This progression of a glioma from low grade to high grade is common and may be driven by various combinations of mutations in genes that regulate growth, differentiation, senescence, or all three and that are propagated, possibly in parallel, by continued cell division throughout the tumor-cell population.
Figure 5. Maturation-Arrest Theory.
This hypothesis predicts the transformation of progenitor cells and an ensuing accrual of immature, self-renewing progenitor cells in which cellular proliferation and maturation are uncoupled. Panel A shows the normal neural stem-cell production of progenitor cells, which subsequently generates the three differentiated cell types of the central nervous system (neurons, astrocytes, and oligodendrocytes). The formation of an astrocytoma (Panel B) may follow the neoplastic transformation of a glial progenitor cell, whose abnormal progeny then phenotypically resemble an astrocyte. Subsequent malignant degeneration of the astrocytoma may generate a glioblastoma (Panel C) after a subpopulation of the transformed glial progenitor cells accumulate additional mutations, leading to accelerated growth of the glioblastoma and complete arrest of maturation. The blue arrows indicate cell differentiation.
Tumor initiation from a genetically altered progenitor cell, however, would be expected to yield mutant progeny. For example, gliomas removed from children exhibit self-renewal and multipotency in vitro, but tumor-derived progenitors generate phenotypically abnormal progeny that recapitulate the properties of their tumor of origin on the basis of immunohistochemistry, degree of differentiation, and types of cells produced.47 A similar phenomenon has been observed in gliomas removed from adults; when multipotent, self-renewing cells were isolated, their capacity for self-renewal increased according to tumor grade, and these multipotent cells had an abnormal karyotype identical to that of the tumor of origin. In addition, the progeny of these cells expressed the unique phenotype and genotype of the tumor of origin.83 Although this scenario suggests that the cell of origin for gliomas is a stem cell, it also indicates that this cell of origin has undergone a transformation event that limits the normal differentiation of its progeny.
Migration of Glioma Cells
The migratory capacity of malignant gliomas represents a significant challenge to any therapeutic strategy. Cancer cells often disseminate far from primary tumors, although many of the growths that arise in these sites do not manifest as disease.84 In the injured adult brain, nestin-positive cells migrate to the site of injury from the subventricular zone, indicating that progenitor cells retain the ability to travel through mature parenchyma.85,86,87,88 This capacity for migration is an important feature shared by gliomas and neural stem cells. Consequently, insight into the biology of progenitor-cell motility in the central nervous system may lead to a better understanding of the invasion of brain tumors, because the mechanisms underlying the latter may represent a reactivation of the former.
In adult mice exposed to EGF, precursors of the subventricular zone are found, aberrantly, in the overlying parenchyma, outside their usual stem-cell niche.11 Also, when neural stem cells are injected into an adult rodent brain, they travel to sites adjacent to tumor cells that themselves had migrated through the parenchyma of the central nervous system.89 As is true of gliomas, neural stem cells and progenitor cells have a predilection for white-matter tracts and blood-vessel basement membranes.18,19 These findings suggest that neural stem cells and tumor cells share a common substrate for motility. At least two novel mechanisms of cellular migration exist in adult germinal regions of the mammalian brain.90,91 The function of each mechanism is to translocate many cells rapidly across vast territories of mature parenchyma. It will be exciting to see whether the same molecular mechanisms underlying this movement also account for the astounding distances negotiated by malignant glioma cells in the human brain.
Conclusions
Malignant gliomas are the most common primary brain tumors, and glioblastomas are among the deadliest of human cancers. Since gliomas were first recognized and treated in the mid-19th century, we have accrued a tremendous amount of data on this disease but have enjoyed little improvement in its survivability. In the light of the significant advances that have been made in the treatment of many other cancers during the same period of time, this inadequate progress has inspired a reevaluation of the gliomagenesis theory. In the wake of the information explosion that has occurred in the field of neural stem-cell biology, a new path is now emerging to link neuro-oncology with developmental neurobiology. The adult human brain, for many years thought to be a static, fully differentiated organ, is now considered to be a surprisingly dynamic environment driven by multiple neuronal and glial progenitor-cell populations. Future investigations of human neural stem cells and their potential for malignancy will be indispensable to our continued struggle with brain cancer, since identifying the true source of human gliomas may lead to better therapeutic targeting, the identification of new markers for the progression of gliomas, earlier cancer detection, and the development of new therapeutic agents.
Source Information
From the Department of Neurological Surgery, Brain Tumor Research Center, and the Developmental Stem Cell Biology Program, University of California, San Francisco.
Address reprint requests to Dr. Sanai at the Department of Neurological Surgery, University of California, San Francisco, 505 Parnassus Ave., M-779, Campus Box 0112, San Francisco, CA 94143, or at sanain@neurosurg.ucsf.edu.
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Figure 1. Grades I to IV (World Health Organization) Astrocytic Tumors.
Panels A and B show circumscribed astrocytomas. Pilocytic astrocytomas (Panel A) are typically indolent, have a limited invasive capacity, and rarely undergo anaplastic progression. These tumors may have microvascular hyperplasia and cellular pleomorphism despite their designation as grade I tumors. Pleomorphic xanthoastrocytomas (Panel B) are also relatively circumscribed and, despite their distinct, conspicuous cellular pleomorphism, tend to be low-grade (grade II) tumors with limited capacity for brain invasion. Panels C through F show diffuse-type astrocytomas, which have the capacity for dispersion into the surrounding brain and a high frequency of anaplastic progression. A grade II astrocytoma (Panel C) is well-differentiated, with mild-to-moderate nuclear pleomorphism. A grade III astrocytoma (Panel D) has a high rate of cell proliferation, as indicated by the mitotic figures. These tumors commonly have a moderate degree of cellular pleomorphism and more heterogeneous cellularity. Glioblastoma multiforme, grade IV, is the most aggressive glial tumor and has the distinctive features of palisading or geographic necrosis (Panel E) and conspicuous microvascular hyperplasia (Panel F) in addition to marked cellular pleomorphism. (Tissue samples, stained with hematoxylin and eosin, courtesy of Dr. Scott R. VandenBerg.)
Table 1. Cell Types and Associated Tumors of the Central Nervous System.
The Cell of Origin of Gliomas
The identification of the cellular origin of gliomas presents an opportunity for improving our understanding of this disease. Although the neoplastic transformation of fully differentiated glia is widely assumed to be the mechanism of gliomagenesis, this hypothesis has never been adequately tested. Furthermore, the concept of dedifferentiation of mature glia is questionable and fails to explain adequately the origin of some gliomas, such as the mixed oligoastrocytoma. The astrocytoma, another glioma variant, does possess some morphologic characteristics of mature astrocytes, but the origin of a neoplasm is not necessarily reflected in the appearance of its most common cellular component: brain tumors of apparently comparable histologic structure can exhibit vastly different behaviors. Adult glia were once thought to be the only dividing cells in the postnatal brain, making them the only brain cells susceptible to transformation. We now know that this is not the case, because other proliferative populations of cells have since been discovered in the adult human brain, as discussed below. For this reason, the classic theories regarding gliomagenesis are now being reappraised in the hope of constructing a more accurate picture of the origin of gliomas.
Neural Stem Cells and Glial Progenitor Cells
It is now known that there are both neural stem cells and glial progenitor cells in multiple regions of the adult brain. Neural stem cells, which are multipotent and self-renewing, have been isolated from the subventricular zone,7 the lining of the lateral ventricles (Figure 2A), the dentate gyrus,8 within the hippocampus (Figure 2B), and the subcortical white matter9 (Figure 2B). The largest of these germinal regions in humans, the subventricular zone, contains a population of astrocytes that can function as neural stem cells.7 In other adult mammals, glial progenitor cells — self-renewing precursors capable of producing astrocytes and oligodendrocytes — have also been observed throughout the neuraxis,10 including the cortex,11,12 the corpus callosum,13 the periventricular white matter,14 the subventricular zone,15 and the dentate gyrus.8 These stem-cell and progenitor elements, in addition to differentiated adult glia, constitute a substrate for neoplastic transformation.
Figure 2. Germinal Regions of the Adult Human Brain.
Panel A shows the lateral walls of the lateral ventricles, which contain the subventricular zone. Panel B shows the hippocampus, which contains the dentate gyrus and the subcortical white matter. CA1 and CA3 indicate regions with pyramidal cells as their principal neurons.
Properties of Neural Stem Cells
All forms of cancer arise from disturbances of critical cellular functions such as proliferation, apoptosis, and tissue invasion.16 In the adult brain, neural stem cells and progenitor cells (self-renewing precursors capable of producing progeny along either neuronal or glial lineages, but not both) commonly possess features associated with cancer of the central nervous system, including a robust proliferative potential and a diversity of progeny. Neural stem cells are regulated by the same cellular pathways that are active in many brain tumors.17 Consequently, they are capable of exhibiting behavior that is characteristic of gliomas, including high motility, association with blood vessels and white-matter tracts, possession of immature antigenic phenotypes, and activation of "developmental" signaling pathways (Table 2).10,18,19
Table 2. Characteristics Intrinsic to Neural Stem Cells and Gliomas.
Transformation of Neural Stem Cells
Germinal regions such as the subventricular zone have long been proposed as sources of gliomas.20,21 Many gliomas are either periventricular or contiguous with the subventricular zone, and they frequently express the progenitor-cell marker nestin.22,23,24,25,26 It remains unclear, however, whether the expression of nestin is a genetic aberration or a normal property of the precursor cell from which the tumor originated.27 That neural stem cells are potentially susceptible to transformation is suggested by the fact that, in animal models, regions of the brain with a high degree of cellular proliferation — areas with stem-cell populations — are more sensitive to chemical or viral oncogenesis than are areas with a low proportion of proliferating cells.28,29,30 In canine and rodent brains, for example, avian sarcoma viral transformation or systemic exposure to the carcinogen N-ethyl-N-nitrosourea preferentially leads to tumor formation in the proliferative subventricular zone rather than in nonproliferative regions of the brain.29,30,31,32 In one study, intraventricular inoculation with avian sarcoma virus in neonatal canine brains initially led to periventricular glioma microfoci, but as the tumors grew, their continuity with the subventricular zone diminished until, at day 10 after inoculation, they were found in the deep white matter without apparent connection to the subventricular zone.30 This migration of transformed germinal-zone cells may be a mechanism by which human gliomas arise from neural stem cells but then go on to lose any evidence of continuity with these regions.
Molecular Mechanisms
These initial observations suggested that gliomas can originate from neural stem-cell populations, but the genetic alterations necessary for stem-cell and progenitor-cell transformation are only now being uncovered. Recently, progenitor cells activated with Akt and KRas, signal transduction proteins associated with human glioblastomas, were shown to form brain tumors with histologic features of glioblastoma in rodents.33 For that experiment, a retroviral delivery system was designed specifically to infect, in vivo, the progenitor cells of the central nervous system of a transgenic mouse in order to transfer postnatally the Akt and KRas oncogenes exclusively to these cells. When a constitutively active epidermal growth factor receptor (EGFR) was transfected with retrovirus into transgenic Ink4a-Arf-/- mouse neural stem cells, which are deficient in genes that regulate cell-cycle arrest and apoptosis,34 these cells generated high-grade gliomas in vivo once they were transplanted into an adult mouse brain.
In contrast, mature mouse astrocytes expressing the differentiated astrocyte marker glial fibrillary acidic protein (GFAP) were less susceptible to transformation in vivo unless they were converted to an undifferentiated state through retroviral transfection of the platelet-derived growth factor (pdgf) gene35 or through the loss of the Ink4a-Arf locus.34,36 These experiments indicate that the state of differentiation is an important feature of the cell of origin of gliomas, although the influence of the state of differentiation depends on the oncogenes involved. These data suggest that stem cells and progenitor cells in the central nervous system particularly are at risk for malignant transformation, presumably because they have activated the cellular machinery (e.g., promitotic genes, telomerase activity, and antiapoptotic genes) necessary for tumor initiation, progression, or both. Thus, neural stem cells may represent the path of least resistance in tumorigenesis, since they already have the ability to bypass apoptosis and senescence. As a result, neural stem cells may require less than the estimated four to seven mutations needed to effect malignant change in a differentiated cell.16
Stem Cells in Other Neoplasms
The concept of a stem-cell origin for cancer has already been demonstrated in several tumors outside the central nervous system,37,38,39 with the hematopoietic system providing the clearest example.40,41,42 In this system, it is well documented that only a small subgroup of cancer cells from patients with chronic myeloid leukemia and multiple myeloma demonstrate extensive proliferation and tumor initiation. More recently, CD34+CD38– hematopoietic stem cells derived from specimens of human acute myeloblastic leukemia have been identified and enriched for clonogenic capacity. Although these cells represent a fraction of the total population of white cells (0.2 to 100 of 106 white cells), they constitute the only cellular compartment capable of initiating the disease in another host.17 Similar evidence is now being gathered in the field of breast cancer.43
Developmental Patterns
Sonic Hedgehog Pathway
If neural stem cells are the source of initiation for brain cancer, their progression toward a tumorigenic state may be achieved through the operation of abnormal "developmental" programs. As the details regarding the genetics of brain tumors emerge, they point to multiple developmental signaling pathways as critical regulators of tumorigenesis. Although these programs function with the use of normal cellular components, their timing, order, and magnitude are probably abnormal. For example, studies have shown that medulloblastoma, a neuroectodermal tumor of the cerebellum, arises through abnormalities of developmental pathways in a population of progenitor cells.44,45,46 These tumors aberrantly express multiple regulatory genes known to mediate the proliferation of neural stem cells.47
Specifically, dysregulation of the sonic hedgehog (Shh) developmental signaling pathway (Figure 3) leads to the aberrant activation of Shh signaling, which normally regulates the self-renewal of precursors of cerebellar granule cells and the development of their neuronal progeny. The activation of the Shh pathway, in turn, predisposes neural progenitor cells to medulloblastoma formation by mediating the inactivation of the retinoblastoma tumor-suppressor gene (Rb)48 and directly inducing regulators of the cell cycle such as the proto-oncogene N-myc. N-myc, a key transcription factor that mediates the proliferation of neuronal progenitor cells,49,50 is overexpressed in medulloblastomas.51 Thus, activity in the Shh pathway is not only critical for the self-renewal of progenitor cells and the proliferation of their progeny, but it also facilitates the initiation of brain tumors. It is interesting to note that medulloblastoma cells die rapidly when cultured with cyclopamine, an antagonist of the hedgehog family of regulatory pathways,52 suggesting that there is a role for this signaling pathway in tumor maintenance as well as initiation.
Figure 3. Generalized Hedgehog–Gli Signaling Pathway.
When the sonic hedgehog pathway (Shh) binds to the patched (Ptc)-smoothened (Smo) receptor, a cytoskeleton-associated macromolecular complex — including the Gli proteins, suppressor of fused , fused (Fu), protein kinase A (PKA), and other possible components (X, Y, Z) — acts to produce Gli activators. These Gli activators are imported into the nucleus and act on designated target genes. The introduction of cyclopamine inhibits hedgehog signaling by acting on Smo.
Transcription Factors
The hedgehog family of regulatory pathways does not just mediate the proliferation of progenitor cells in the external granular layer of the cerebellum. Hedgehog signaling activates three zinc-finger transcription factors — Gli1, Gli2, and Gli3 — which regulate progenitor cells by promoting cell-cycle entry and DNA replication.53,54 In the adult central nervous system, Gli1 is expressed by neuronal progenitors in germinal regions such as the subventricular zone and the dentate gyrus.55,56 In these regions, the Shh – Gli pathways also play an integral role in the support of germinal niches by maintaining the stem-cell population57 or by facilitating the survival and proliferation of stem-cell progeny.58 It is important to note that Gli is expressed in both low-grade and high-grade gliomas,39 and the Shh – Gli pathway may mediate the initiation and maintenance of these tumors as it does for neural stem cells.39,59 As might be expected, treatment with cyclopamine can inhibit the growth of some glioma cell lines in vitro.39 Thus, the hedgehog family of signaling pathways, key mechanisms of embryonic neural-tube development as well as normal mediators of adult neural stem-cell proliferation, are abnormally activated during gliomagenesis.
Epidermal Growth Factor
The EGF signaling pathway also plays an important part in gliomagenesis and neural stem-cell regulation. Amplification of the EGFR gene is associated with the formation of glioblastomas, and activation of EGFR promotes the growth of both astrocyte precursors60,61 and neural stem cells.62,63 As many as 50 percent of high-grade astrocytomas demonstrate EGFR amplification, and EGFR activation may drive the transformation process in the development of glioblastomas.1,44 In the subventricular zone of the adult rodent, EGF-responsive "C" cells constitute a large population of migratory, rapidly dividing progenitor cells. In response to exposure to EGF in vitro, these C cells give rise to spherical collections of cells (neurospheres) that exhibit properties similar to those of stem cells. In rodents, in vivo, EGF induces the proliferation of C cells, prevents their differentiation, and is associated with tissue infiltration that is analogous to infiltration by high-grade gliomas.18 The human homologue to this cell type has yet to be identified.
PTEN Pathway
Several other genetic pathways are also active in neural stem-cell regulation and gliomagenesis. PTEN, a tumor-suppressor gene often affected in high-grade gliomas and one of the genes most commonly mutated in glioblastomas,64 encodes a phosphatase that regulates the proliferation of neural stem cells.65 PTEN may also play a part in neural stem-cell motility66 and could therefore lead to the extensive infiltration seen in gliomas. Similarly, neurospheres cultured from gliomas and normal neural stem cells express the oncogene bmi-1, a transcriptional repressor necessary for neural stem-cell proliferation in the central nervous system.47,65 Telomerase, which prevents chromosomal shortening and apoptosis after cell division, is also expressed in the majority of high-grade gliomas as well as in progenitor cells found in the subventricular zone,67 although the frequency and timing of telomerase expression remain unknown. Finally, the Wnt signaling pathway, which controls the size of the neural stem-cell population through -catenin activation,68 is mutated in a subgroup of medulloblastomas,69 although its role in gliomas remains unknown.
Heterogeneity of Gliomas
The neural stem-cell model for the origin of gliomas sheds light on their heterogeneity. In several solid tumors outside the central nervous system, only a small fraction of the total population of cancer cells appears to be clonogenic. Examples of such tumors include those in the lung and ovary, from which only 1 in 1000 to 1 in 5000 cells form colonies in agar.70 Recently, self-renewing, multipotent cells expressing the CD133 cell-surface marker were isolated from human gliomas and transplanted into adult mouse brains, where they recapitulated the parent tumor's histology.71,72 These findings suggest that gliomas, too, are initiated and maintained by a small fraction of cancerous neural stem cells.
There is ample evidence of a far greater level of heterogeneity among gliomas than that allowed for in the current classification system. Since the expression of normal differentiation markers within these tumors varies, at least some of this heterogeneity probably arises from the anomalous differentiation of tumor cells.17 For a single glioma, karyotype analysis reveals substantial differences from region to region; however, a pattern of shared chromosomal abnormality, and hence clonal origin, is conserved.73,74 Although it is uncommon for a single glioma to exhibit the entire range of cell types generated by a neural stem cell, gliomas are often composed of multiple cell types, such as astrocytes and oligodendrocytes.75
The genetic and cellular diversity within each glioma may be due to secondary changes within tumor subclones, but it is possible that the origin is a multipotent tumor cell. The cause of mixed-cell gliomas is not likely to be the independent transformation of two differentiated cells but, rather, the transformation of a single, bipotential progenitor cell.76 For example, an oligoastrocytoma may originate from a bipotential adult oligodendrocyte-type-2 astrocyte progenitor cell, which normally generates both astrocytic and oligodendrocytic progeny in vitro.77 The transformation of such a population of progenitor cells, which are already capable of self-renewal through asymmetric cell division, could generate astrocytomas, oligodendrogliomas, or oligoastrocytomas.78
It is important to note that the loss of heterozygosity on chromosomes 1p and 19q in both the astrocytic and oligodendrocytic components in mixed oligoastrocytomas has been reported,79 suggesting that there is a common cell of origin. In vivo gene transfer of the polyomavirus middle T antigen, an oncogene that activates multiple signal-transduction pathways involved in gliomagenesis, into differentiated, GFAP-positive astrocytes has been shown to result in the production of both oligodendrogliomas and astrocytomas.80 Thus, the cell of origin of gliomas not only may be capable of producing multiple cell types, including oligodendrocytes and astrocytes, but also may resemble an astrocyte in terms of phenotype. It remains unknown whether such a multipotent astrocyte is related to the astrocytic neural stem cells lining the subventricular zone of the adult human lateral ventricles, as recently described (Figure 4).7
Figure 4. Adult Human Subventricular Zone.
The cells of the subventricular zone, labeled with the astrocyte marker GFAP (shown in green), line the lateral walls of the lateral ventricles. This is the largest known region of adult neural stem cells in the human brain; it is composed of the deep subcortical white matter (A), a periventricular ribbon of astrocytes that can function as neural stem cells (B), a dense layer of astrocytic processes (C), and the ependymal lining (D). Throughout adult life, astrocytes from the subventricular zone exhibit a unique capacity for multipotency and self-renewal in vitro. (From Sanai et al.7)
Cellular Environment
With regard to mixed-cell gliomas, there is also the question of how the cellular environment directs gliomagenesis. Since gliomas can contain nonneoplastic astrocytes and endothelial cells within their stroma, a potentially important factor in the formation of gliomas may be the germinal niche re-created by the coexistence of neoplastic and nonneoplastic cells of the central nervous system. This relationship is highly evocative of the environmental interplay known to exist between adult neural stem cells and the supporting cells of the subventricular zone and dentate gyrus.58
Blocked Differentiation
Although the presence of multipotent progenitor cells can explain the heterogeneity of brain tumors, the diversity of their progeny may be limited by the blocked-differentiation phenomenon that was first observed in human leukemias.81 This process entails an accrual of immature, self-renewing progenitor cells in which cell proliferation and maturation are uncoupled. The transformed progenitor cells divide rapidly, but their progeny are incapable of complete differentiation (Figure 5).82 Thus, the tumor phenotype may be defined by the direction and degree of differentiation of the transformed progenitor population. For example, astrocytomas may arise from glial progenitors whose progeny can differentiate only along astrocytic lines. More complex phenomena, such as the transformation of an oligodendroglioma to a glioblastoma, may be explained by a secondary wave of accelerated growth and arrest of maturation among a subgroup of progenitor cells. This progression of a glioma from low grade to high grade is common and may be driven by various combinations of mutations in genes that regulate growth, differentiation, senescence, or all three and that are propagated, possibly in parallel, by continued cell division throughout the tumor-cell population.
Figure 5. Maturation-Arrest Theory.
This hypothesis predicts the transformation of progenitor cells and an ensuing accrual of immature, self-renewing progenitor cells in which cellular proliferation and maturation are uncoupled. Panel A shows the normal neural stem-cell production of progenitor cells, which subsequently generates the three differentiated cell types of the central nervous system (neurons, astrocytes, and oligodendrocytes). The formation of an astrocytoma (Panel B) may follow the neoplastic transformation of a glial progenitor cell, whose abnormal progeny then phenotypically resemble an astrocyte. Subsequent malignant degeneration of the astrocytoma may generate a glioblastoma (Panel C) after a subpopulation of the transformed glial progenitor cells accumulate additional mutations, leading to accelerated growth of the glioblastoma and complete arrest of maturation. The blue arrows indicate cell differentiation.
Tumor initiation from a genetically altered progenitor cell, however, would be expected to yield mutant progeny. For example, gliomas removed from children exhibit self-renewal and multipotency in vitro, but tumor-derived progenitors generate phenotypically abnormal progeny that recapitulate the properties of their tumor of origin on the basis of immunohistochemistry, degree of differentiation, and types of cells produced.47 A similar phenomenon has been observed in gliomas removed from adults; when multipotent, self-renewing cells were isolated, their capacity for self-renewal increased according to tumor grade, and these multipotent cells had an abnormal karyotype identical to that of the tumor of origin. In addition, the progeny of these cells expressed the unique phenotype and genotype of the tumor of origin.83 Although this scenario suggests that the cell of origin for gliomas is a stem cell, it also indicates that this cell of origin has undergone a transformation event that limits the normal differentiation of its progeny.
Migration of Glioma Cells
The migratory capacity of malignant gliomas represents a significant challenge to any therapeutic strategy. Cancer cells often disseminate far from primary tumors, although many of the growths that arise in these sites do not manifest as disease.84 In the injured adult brain, nestin-positive cells migrate to the site of injury from the subventricular zone, indicating that progenitor cells retain the ability to travel through mature parenchyma.85,86,87,88 This capacity for migration is an important feature shared by gliomas and neural stem cells. Consequently, insight into the biology of progenitor-cell motility in the central nervous system may lead to a better understanding of the invasion of brain tumors, because the mechanisms underlying the latter may represent a reactivation of the former.
In adult mice exposed to EGF, precursors of the subventricular zone are found, aberrantly, in the overlying parenchyma, outside their usual stem-cell niche.11 Also, when neural stem cells are injected into an adult rodent brain, they travel to sites adjacent to tumor cells that themselves had migrated through the parenchyma of the central nervous system.89 As is true of gliomas, neural stem cells and progenitor cells have a predilection for white-matter tracts and blood-vessel basement membranes.18,19 These findings suggest that neural stem cells and tumor cells share a common substrate for motility. At least two novel mechanisms of cellular migration exist in adult germinal regions of the mammalian brain.90,91 The function of each mechanism is to translocate many cells rapidly across vast territories of mature parenchyma. It will be exciting to see whether the same molecular mechanisms underlying this movement also account for the astounding distances negotiated by malignant glioma cells in the human brain.
Conclusions
Malignant gliomas are the most common primary brain tumors, and glioblastomas are among the deadliest of human cancers. Since gliomas were first recognized and treated in the mid-19th century, we have accrued a tremendous amount of data on this disease but have enjoyed little improvement in its survivability. In the light of the significant advances that have been made in the treatment of many other cancers during the same period of time, this inadequate progress has inspired a reevaluation of the gliomagenesis theory. In the wake of the information explosion that has occurred in the field of neural stem-cell biology, a new path is now emerging to link neuro-oncology with developmental neurobiology. The adult human brain, for many years thought to be a static, fully differentiated organ, is now considered to be a surprisingly dynamic environment driven by multiple neuronal and glial progenitor-cell populations. Future investigations of human neural stem cells and their potential for malignancy will be indispensable to our continued struggle with brain cancer, since identifying the true source of human gliomas may lead to better therapeutic targeting, the identification of new markers for the progression of gliomas, earlier cancer detection, and the development of new therapeutic agents.
Source Information
From the Department of Neurological Surgery, Brain Tumor Research Center, and the Developmental Stem Cell Biology Program, University of California, San Francisco.
Address reprint requests to Dr. Sanai at the Department of Neurological Surgery, University of California, San Francisco, 505 Parnassus Ave., M-779, Campus Box 0112, San Francisco, CA 94143, or at sanain@neurosurg.ucsf.edu.
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