Glioma

39 Glioma Anders I. Persson, PhD QiWen Fan, PhD Joanna J. Phillips, PhD William A. Weiss, PhD

Keywords: brain, cancer, glioma, progenitor, tumor, stem cell

I. Introduction II. Nomenclature III. Locations and Symptoms IV. Epidemiology V. Etiology VI. Histopathology VII. Genetic Alterations VIII. Cell of Origin IX. Signaling Pathways X. Pharmacology XI. Future Therapeutic Interventions References

I. Introduction Decades of research on infiltrating gliomas still leave important questions regarding the etiology, cellular origin, and role of different cell types within glial malignancies. Despite surgical resection, radiotherapy, and chemotherapy, the median survival for high-grade glioma remains 1–3 years. Advances in the understanding of the normal

Neurobiology of Disease

development in the central nervous system (CNS) and in the pathogenesis of glioma, specifically focusing on cells of origin and critical signaling pathways, will lead to a better understanding of the biology of these tumors and more effective treatments for patients with glioma. In this chapter, we provide the reader with an overview on 433

Copyright © 2007 by Elsevier (USA). All rights reserved.

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gliomas, including pharmacological treatments and findings regarding cell of origin in relation to cellular heterogeneity in gliomas.

II. Nomenclature Gliomas, including astrocytomas, oligodendrogliomas, mixed oligoastrocytomas, and ependymomas, are classified based on their histological and immunohistological features [1]. The World Health Organization (WHO) classification divides gliomas into grades based on histopathological features of anaplasia, including nuclear atypia, mitotic activity, microvascular proliferation, and/or necrosis. Pilocytic astrocytomas, corresponding to WHO grade I, have clinical, pathological, and molecular features that are distinct from the infiltrating gliomas, which include the diffusely infiltrating astrocytomas, oligodendrogliomas, and mixed oligoastrocytomas. Pilocytic astrocytomas (WHO grade I), diffuse astrocytomas (WHO grade II), oligodendrogliomas (WHO grade II), and oligoastrocytomas (WHO grade II) are all classified as low-grade gliomas. Anaplastic astrocytomas (WHO grade III), anaplastic oligodendrogliomas (WHO grade III), anaplastic oligoastrocytoma (WHO grade III), and glioblastoma multiforme (WHO grade IV) are classified as high-grade gliomas. Glioblastomas themselves can be further classified as primary or secondary. Primary glioblastoma present de novo, whereas secondary glioblastomas arise from a low-grade precursor lesion through a sequential series of genetic events. Oligodendrogliomas differ from astrocytomas in their characteristic morphology and by the observation of a subset of anaplastic tumors (marked by deletion of 1p and 19q) that are responsive to alkylating chemotherapeutic agents. Ependymal tumors and other, less common types of gliomas of different grades are not discussed in this chapter (for a broader review on gliomas, see reference 1).

a comparison between low-grade gliomas and glioblastoma, the low-grade gliomas were more commonly found in supplementary motor area (27.3% vs 10.8%) and within the insula (25% vs 10.8%) [2]. Due to the more aggressive character of high-grade gliomas, signs are similar to low-grade astrocytomas but with a shorter history.

IV. Epidemiology Investigations of the inherited and environmental contributions to glioma have not provided any fundamental insights that can explain the occurrence of this disease [3]. In Western countries, the incidence of brain tumor in adults is approximately 4 to 7 per 100,000 persons per year in males and 3 to 5 per 100,000 in females. Primary brain tumors represent approximately 2% of all cancers in the United States. Pilocytic astrocytoma is the most common glioma in children and usually occurs during the first and second decades of life. All other gliomas are frequently found after 30 years of age (Table 1). Secondary glioblastomas often occur in younger adults (aged 30–45 years), whereas primary glioblastomas are more frequent in older adults (mean age, 55 years). Some studies suggest that brain tumors are more common in Caucasians than in people of African or Asian descent. However, a higher incidence rate of brain tumors in developed, industrial countries might reflect differences in socioeconomic status and diagnostic ascertainment, rather than a true increase in genetic or environmental susceptibility.

V. Etiology Studies of carcinogenic risks have assessed more than 900 exposures classified as carcinogens in humans. For only nine of these exposures have there been reports of possible or weak associations with nervous system tumors in humans. Substances of aromatic nature (petroleum products), metals (lead, arsenic, and mercury) or reactive

III. Locations and Symptoms All low-grade gliomas can be found throughout the CNS, although they occur more frequently in some areas than in others. Depending on the tumor location, signs such as seizures, macrocephaly, headache, increased intracranial pressure, visual deficits, and lack of coordination may be present in patients. In contrast to other low-grade gliomas, pilocytic astrocytomas are not typically localized to cortical areas but are more often found in other areas, including the optic nerve and cerebellum; therefore, seizures are uncommon. All high-grade gliomas develop throughout the CNS, with a preference for the cerebral hemispheres. In

Table 1 Most Common Gliomas as a Function of Age Type Pilocytic astrocytoma Diffuse astrocytoma Oligodendroglioma Mixed oligoastrocytoma Anaplastic astrocytoma Anaplastic oligodendroglioma Anaplastic oligoastrocytoma Secondary glioblastoma multiforme Primary glioblastoma multiforme

Grade

Average Age in Years

I II II II III III III IV IV

<20 30–40 35–45 35–45 ∼40 ∼50 40–50 <45 ∼55

Glioma dipolar substances (vinyl chloride) have all been associated with glioma risk (for lead: odds ratio 2:1, 95% confidence interval [CI] 1.1–4.0) [3]. Because these families of molecules alter developmental processes, it is perhaps not surprising that the incidence of glial tumors and primitive neuroectodermal tumors was increased in children with parents working in agriculture (odds ratio 1:3, 95% CI 1.0–1.8). The same was true for children with male parents working as electricians (odds ratio 1:1, 95% CI 0.9–1.5), drivers (odds ratio 1:3, 95% CI 1.0–1.7), or mechanics (odds ratio 1:5, 95% CI 1.0–2.3). Children with female parents in the textile industry had a significantly higher incidence of these brain tumors (odds ratio 1:7, 95% CI 1.1–2.7) [4]. Parents working in chemical industry were at higher risk of having children with astroglial tumors (father’s odds ratio 2:1, 95% CI 1.1–3.9 and mother’s odds ratio 3:3, 95% CI 1.4–7.7). Physicians and firefighters represent other occupations linked to higher glioma risk (physician’s odds ratio 4:7, 95% CI 0.5–42.7, and firefighter’s odds ratio 2:7, 95% CI 0.3–26.1). Diets high in N-nitroso compounds, which are present in cured meat, cooked ham, processed pork, and fried bacon, were associated with elevated glioma risk (relative risk [RR] of 1.48, 95% CI 1.20–1.83) in adults [3]. An inverse association has been noted for frequent intake of fruits, fresh vegetables, and vitamin C. The developmental aspect is important because several studies found that high consumption of meats by women during pregnancy was associated with elevated risk of astrocytic gliomas (RR of 1.68, 95% CI 1.30–2.17) in children. Ingestion of meats may lead to increased oxidative stress, which has been implicated as a contributing factor in tumorigenesis. Leukemia patients who received therapeutic intracranial irradiation had an increased risk of brain tumors. This risk was most evident in children younger than 5 or 6 years who were treated for acute lymphoblastic leukemia. Some of these children developed glioma as early as 7–9 years after gamma-irradiation. The number of brain tumors among patients who had received cranial radiation was nearly 27 times greater than expected, whereas no such tumors were seen after chemotherapy. The developing brain contains a significant number of proliferating progenitor cells. Studies in rodents have shown that g-irradiation causes acute loss of neural progenitors [5]. The neural progenitors reappeared after 1 week but may be more susceptible to transformation into tumorigenic cells due to damage to deoxyribonucleic acid (DNA). Registers of the 80,160 survivors of the Hiroshima and Nagasaki atomic bombings also demonstrated a dose-related increase in CNS tumors (RR per sievert dose 1.2, 95% CI 0.6–2.1). Risk of glioma development was also increased but not statistically significant (RR per sievert dose 0.6, 95% CI 0.2–2.0). No clear correlation has been found between exposure to electromagnetic fields or cellular phones and CNS tumors.

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VI. Histopathology A. Low-Grade Gliomas Pilocytic astrocytomas are slow-growing, generally wellcircumscribed tumors. Histologically, these tumors are of low to moderate cellularity and show a biphasic architecture, with compact areas composed of bipolar (piloid) tumor cells alternating with loose, microcystic areas composed of multipolar tumor cells. Other common features include Rosenthal fibers, eosinophilic granular bodies, and low mitotic activity (Fig. 1A). The bipolar tumor cells exhibit strong immunoreactivity for glial fibrillary acidic protein (GFAP). Diffuse astrocytomas exhibit marked cellular differentiation and are characterized by slow growth. These tumors infiltrate neighboring brain structures and over time can undergo malignant progression. Histologically, tumors demonstrate a moderate increase in cellularity, mild to moderate nuclear atypia, and little mitotic activity. Tumors are composed of well-differentiated neoplastic astrocytes that can have a fibrillary, gemistocytic, or protoplasmic appearance. In biopsy samples, the presence of bare neoplastic nuclei in a moderately hypercellular background often suggests the diagnosis (see Fig. 1B). The most common variant, fibrillary astrocytoma, is composed of multipolar neoplastic GFAP-positive astrocytes with scant cytoplasm and fine cell processes that form a fiber-rich glial matrix. Tumor cells have few mitoses and show neither vascular proliferation nor necrosis. Oligodendrogliomas are well-differentiated, diffusely infiltrating tumors composed of monomorphic neoplastic cells with round, relatively uniform nuclei. Typically the tumors have well-delineated borders, may appear gelatinous, and frequently contain calcifications. A characteristic artifact of major diagnostic relevance is the perinuclear cytoplasmic clearing seen on standard tissue preparation (see Fig. 1C). The isomorphic nuclei reveal a speckled, dense chromatin pattern and are slightly larger than the nuclei of oligodendrocytes. The proliferative index is generally low, and a dense network of branching capillaries often appears in the background. By far the most common mixed gliomas are the oligoastrocytomas. The diagnosis of oligoastrocytoma requires the identification of two distinct populations of neoplastic cells types that resemble the neoplastic cells in oligodendroglioma and diffuse astrocytoma. Both biphasic and intermingled variants are recognized. Overall, oligoastrocytomas are moderately cellular and exhibit no or low mitotic activity.

B. High-Grade Gliomas Relative to the diffuse astrocytoma, anaplastic astrocytomas show increased cell density, increased nuclear atypia, and significant mitotic activity (see Fig. 1D). Many tumor cells express GFAP. Anaplastic oligodendrogliomas

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Figure 1 Common glial neoplasms of the central nervous system (hemotoxylin & eosin stain). A, Pilocytic astrocytomas often have a biphasic appearance with loose cystic areas and compact fascicular areas. Eosinophilic granular bodies are a common finding (arrow). B, Diffuse astrocytomas, WHO grade II, insidiously infiltrate brain tissue and typically exhibit mild cytologic atypia with mild nuclear pleomorphism and hyperchromasia (arrow). C, Oligodendroglioma, WHO grade II, is composed of neoplastic cells with round, relatively uniform nuclei and a fine capillary network. D, Anaplastic astrocytomas, WHO grade III, demonstrate increased cellularity, increased cytologic atypia, and significant mitotic activity (arrows). E, Glioblastoma, WHO grade IV, often exhibits prominent cytologic atypia, mitotic activity, and well-developed microvascular proliferation (arrow). F, Anaplastic oligodendroglioma, WHO grade III, demonstrates increased cytologic atypia, increased cellularity, and high mitotic activity. Microvascular proliferation (arrow) and necrosis may also be present.

retain certain histological features of oligodendroglioma but also demonstrate anaplastic features such as increased cellularity, nuclear atypia, increased mitotic activity, and sometimes microvascular proliferation and necrosis (see Fig. 1E). Anaplastic mixed oligoastrocytomas also show histological features of malignancy including increased cellularity, nuclear atypia, cellular pleomorphism, and high mitotic activity. The anaplastic features can be present in the oligodendroglioma-like components, astrocytoma-like components, or both. Glioblastoma is a densely cellular malignant neoplasm composed of poorly differentiated neoplastic astrocytes. The neoplastic cells exhibit prominent cytologic and nuclear atypia and high mitotic activity. The presence of welldeveloped microvascular proliferation, necrosis, or both is essential for the diagnosis (see Fig. 1F). Necrosis within a glioblastoma can consist of large areas of ischemic necrosis or multiple, small foci of pseudopalisading necrosis. Tumors can show extensive cellular heterogeneity, including multinucleated cells; undifferentiated, round tumor cells; bipolar fusiform cells; and more differentiated, neoplastic, GFAPpositive astrocytic-like cells. If the tumor is dominated by multinucleated cells, it is considered giant cell glioblastoma. Vascular proliferation is often found in necrotic areas and the peripheral zone

of the tumor. GFAP staining is typical but may be patchy because more astrocytic-like cells express GFAP whereas small, highly proliferative and undifferentiated tumor cells are negative.

VII. Genetic Alterations Cancer development is characterized by the accumulation of mutations in distinct molecular pathways that regulate fundamental aspects of proliferation and homeostasis. Gain of function is acquired in genes that positively regulate proliferation; loss of function mutations are acquired in genes that negatively regulate proliferation (Table 2). The genetic alterations of gliomas involve three main signaling pathways: RB1 (retinoblastoma 1 gene) dysregulation, p53 pathway abnormalities, and aberrant signaling through receptor tyrosine kinases and downstream components (reviewed in [6]). In low-grade gliomas, two genetic alterations are frequently observed: p53 inactivation (associated with astrocytomas) and the loss of 1p and 19q chromosome arms in oligodendrogliomas. High-grade gliomas show deletion of CDKN2A (p16 INK4A /p14ARF ) on chromosome 9p21, inactivation of RB1 on chromosome 13q, and amplification of CDK4 on chromosome

Glioma

437 Table 2 Common Genetic and Epigenetic Alterations in Glioma

Gene Tumor suppressor genes TP53

Typical Alteration

Protein Functions

Locations of Common Alterations

Mutation

Regulator of apoptosis, cell cycle progression, DNA repair

Rb1 CDKN2A

Mutation hypermethylation Homozygous deletion Hypermethylation

Cell cycle regulation Inhibitor of CDK4/6

P14ARF

Homozygous deletion Hypermethylation Mutation, hypermethylation

Inhibitor of MDM2

PTEN

Oncogenes EGFR PDGFR

Amplification and overexpression Genomic rearrangement Amplification and overexpression

CDK4

Amplification and overexpression

MDM2

Amplification and overexpression

Diffuse astrocytomas, anaplastic astrocytomas, glioblastomas (secondary > primary) Glioblastomas Glioblastomas, anaplastic astrocytomas, anaplastic oligodendrogliomas Glioblastomas

Lipid phosphatase, negative regulator of PI3K

Anaplastic astrocytomas, anaplastic oligodendrogliomas, glioblastomas

Tyrosine kinase growth factor receptor Tyrosine kinase growth factor receptor Cyclin-dependent kinase, promoter of G1/S phase progression Inhibitor of p53 function

Diffuse and anaplastic astrocytomas, glioblastomas Glioblastomas

12q. Primary glioblastoma show frequent amplification of EGFR, inactivation of PTEN, deletion of 10q, and loss of CDKN2A. Secondary glioblastoma progresses over time from low- to high-grade tumor and usually shows mutations in p53 and RB and amplification of CDK4. Some of the most common genetic and epigenetic changes in gliomas are presented in Table 39.2. These genes are downstream effectors of growth factor signaling and are discussed later in this chapter (Fig. 2). Epigenetic changes that alter acetylation of histones and methylation of CpG islands within DNA sequences influence gene expression and contribute to malignant progression in glioma. Dividing glioma cells depend on correct acetylation of core histone proteins during replication, when cells have a looser chromatin structure. DNA is packed by histones into nucleosomes, which are composed of 147 base pairs of DNA and core histone proteins. The histone acetyltransferases and histone deacetylases regulate acetylation and deacetylation of the conserved lysine residues present in the amino terminal tails of the core histones. Acetylation of the core histones has been shown to weaken the histone-DNA interactions and consequently increase DNA accessibility. However, pretreatment with histone deacetylase inhibitors of cultured glioblastoma cells increased the efficiency of anticancer drugs that target DNA [7]. Several tumor suppressor genes in gliomas are hypermethylated (see Table 2). DNA methylation involves transfer of a methyl group to cytosine in a CpG dinucleotide by DNA methyltransferases that create or maintain methylation patterns. Methylation of CpG islands located in the 5 promoter region of the genes occurs frequently in glioblastomas, is also observed in mouse models for glioma, and

Glioblastomas Glioblastomas

Ligand Growth factor receptor Plasma membrane p

PLC- γ

p

JAK-STAT

RAS

PI3K PTEN Cell growth

MAPK

mTOR

Akt

Apoptosis Nucleus

P16/Ink4A

p27 CyclinD

CDK4/6 CyclinE

CDK2

pRb p21

E2F

P19/Arf

MDM2

p53

Cell cycle progression

Figure 2 Interactions between RTK pathways, the cell cycle, and apoptosis. RTKs are activated in gliomas by growth factors, such as EGF and PDGF, that in turn signal through PI3-K/Akt, RAS/MAPK, PLC, and JAK-STAT pathways. Ras and PI3-K engage the cell cycle machinery and apoptosis at different levels. Most identified genetic alterations result in either abnormal activation of signal transduction pathways downstream of growth factor receptors or disruption of cell cycle arrest pathways.

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results in gene silencing [8]. Epigenetic silencing of the O6-methylguanine-DNA methyltransferase (MGMT) gene by promoter methylation has been associated with longer overall survival in glioblastoma patients treated with radiation and the alkylating agent temozolomide [9]. Epigenetic changes in gliomas have also been observed for the tumor suppressor genes PTEN, Rb1, p14ARF  p15INK4B , and p16 INK4A [10].

VIII. Cell of Origin Similar to normal stem cells, tumor stem cells are rare cells that also have the ability to perpetuate themselves through self-renewal and to differentiate into the various cell types that drive tumorigenesis. The clear identification of stem cells in hematopoietic malignancies has led investigators to attempt comparable isolation of tissue-specific stem and progenitor cells in solid tumors, including breast, prostate, pancreatic, and brain tumors. These cells presumably comprise a small minority of the cells in different cancers and are hypothesized to be tumor-initiating cells. Gliomas have previously been thought to arise from dedifferentiation of a mature brain cell in response to genetic alterations. An alternative hypothesis suggested that these tumors arise from transformation of a resident immature brain cell. Since the 1990s, it has been commonly accepted that the adult mammalian hippocampus and subventricular zone (SVZ) contain neural stem cells (NSCs) [11]. This was a rediscovery based on original findings in the 1960s. The adult human brain has since been shown to contain multipotent NSCs with extensive capacity for self-renewal and the ability to differentiate into neurons and glia. It was recently shown that radial glia give rise to adult NSCs in the SVZ. These NSCs express nestin and GFAP, two proteins that

Figure 3 Cellular heterogeneity in primary cultures of human glioblastoma. The cultures present populations of cells with varying morphology and expression of markers. Some elongated cells (long white arrows) that are positive for nestin (green) also express GFAP (red). Small round cells (short white arrows) are often negative for these markers but express high levels of Sox2 (blue).

are found in subpopulations of tumorigenic cells isolated from human gliomas (Fig. 3). A subpopulation of the nestinpositive NSCs and isolated glioma cells also express the cell surface marker CD133. The CD133 negative cells comprise the majority of the tumor and therefore largely comprise the characteristic cells on which diagnostic criteria are based (Fig. 4A). Both CD133 immunoreactive SVZ NSCs and their progenitors may be the targets for transformation, leading to glioma (see Fig. 4B). The longevity of the NSCs targets them for the accumulation of genetic mutations. Chromosome loss occurs frequently in SVZ NSCs as well as neurons and glia. Several recent studies have identified a subpopulation of cells in brain tumors that express immunohistochemical markers often found on NSCs [12]. Do NSCs or immature glial precursor cells represent the cellular origin of gliomas? The cancer stem cell hypothesis suggests that not all tumor cells have the same ability to proliferate and maintain the growth of the tumor. In

Figure 4 A, Only a few percent of tumor cells (nuclear counterstain in blue) isolated from glioblastomas express the CD133 cell surface protein (green). B, It has been hypothesized that the progeny of transformed SVZ CD133-positive NCSs (red arrows) migrate away from the ventricles and form gliomas. C, Recent studies suggest that only CD133-positive cancerous stem cells (gray) are able to maintain tumor growth. These cells generate more differentiated tumor cells (green) with limited self-renewal capacity. Therefore future treatments should target both populations of cells. It is most important to target the CD133-positive cancerous stem cells that can give rise to new tumors.

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fact, gliomas are heterogenous and are comprised of cells expressing phenotypes of more than one neural lineage (see Fig. 3). Neoplastic cells in gliomas that express the cell surface marker CD133 have been identified as cancerous stem cells due to their increased self-renewal capacity, ability to form tumor spheres in culture, and ability to generate cellular phenotypes expressing both neuronal and glial markers [12,13]. Even more important, separation of CD133-positive and CD133-negative cells demonstrated that only the CD133-positive subpopulation supported tumor growth when xenotransplanted into mice [14]. Moreover, signalling pathways that mediate self-renewal and proliferation of NSCs have also been shown to induce brain tumors in mice. Using viruses that infected only subpopulations of nestin- or GFAP-positive cells, researchers have shown that expression of platelet-derived growth factor (PDGF), epidermal growth factor receptor (EGFR), or the downstream targets K-Ras or Akt can induce glioma in mice. Recent data suggest that chronic PDGF receptor activation in glial progenitors gives rise to oligodendroglioma, whereas chronic activation of Ras and Akt gives rise to astrocytoma.

IX. Signaling Pathways A. Receptor Tyrosine Kinase Signaling in Glioma Primary glioblastomas frequently amplify and/or overexpress oncogenes such as EGFR and PDGFR. In addition, mutations that commonly occur in glioma impact the p53, RB1, and phosphatidylinositol 3-kinase (PI3-K) signaling pathways. Mutations such as amplification of EGFR, gain of function in PIK3CA, or loss of PTEN all activate the PI3-K/Akt signaling pathway, downstream targets of which include mTOR and the FOXO proteins. The PDGFR is also frequently overexpressed, resulting in activation of many of the same pathways as EGFR and the constitutively activated EGFRvIII (also known as EGFR or del2-7EGFR). Therefore EGFR, EGFRvIII, PDGFR, and the RAS/mitogen-activated protein kinase (MAPK) and PI3-K/mTOR pathways present attractive targets for glioblastoma therapy (see Fig. 2) [15]. 1. PI3-K, Akt and PTEN Signaling Activation of PI3-K can occur from numerous agonists and receptors, including PDGF, EGFR, fibroblast growth factor receptor (FGFR), insulin-like growth factor-1 (IGFR1), vascular endothelial growth factor receptor (VEGFR), interleukin (IL) receptors, interferon (IFN) receptors, integrin receptors, and the Ras pathway. The PI3-Ks can be grouped into three classes based on substrate preference and sequence homology. The substrate preference of the Class

I PI3Ks is the lipid phosphatidylinositol 4,5-bisphosphate PIP2 , which is phosphorylated by PI3-K to phosphatidylinositol 3,4,5 trisphosphate PIP3 . Other targets of Class I PI3-Ks include Rac, p70s6k , and certain isoforms of protein kinase C (PKC). Class I PI3-Ks are heterodimers of approximately 200 kDa, composed of a regulatory subunit (p55 or p85) and a catalytic subunit (p110). The regulatory subunit, in particular p85, contains several different regions including Src homology domains (SH3 domain and two SH2 domains). The p110 subunit has   , and  isoforms; each contains a kinase domain and a Ras interaction site. In addition, the  , and  isoforms have an interaction site for the p85 subunit, which stabilizes p110. Class 1 PI3-Ks can be further subdivided into A and B groups. The class 1A p110 isoforms  , and  are able to active PDK-1 and AKT. Class 1B PI3-Ks contain the  isoform of p110 and cannot interact with Akt. Class II PI3-Ks are all 170-210kDa monomeric proteins that preferentially phosphorylate PtdIns and PtdIns(4)P. Class III PI3-Ks only phosphorylate PtdIns. The best evidence supporting a role for class I PI3-Ks in glioma comes from recent studies in which this family of proteins was sequenced in a number of patients with glioma. Approximately 5–25% of patients showed activating mutation in p110a, with no mutations observed in other p110 molecules. AKT (PKB) is a 56-kDa serine/threonine protein kinase with three human isoforms (AKT1-3) and two phosphorylation sites. Once AKT is associated with the membrane and bound to PIP3 , a conformational change occurs that allows phosphorylation at the threonine 308-position of the catalytic domain by protein-dependent kinase-1 (PDK-1). A second phosphorylation event is then mediated by PDK2. PDK-2 phosphorylates the serine 473-position of the hydrophobic carboxy-terminal tail. Phosphorylation of both sites is required for full activation of AKT. Activated AKT phosphorylates numerous downstream effectors, including glycogen synthase kinase (GSK)-3 and GSK-3, mTOR, IkB, the pro-apoptotic molecules caspase 3 and 9, and the forkhead transcription factors FOXO1, FOXO3, and FOXO4, among others. One signal responsible for cell survival is mediated through BAD, a proapoptotic member of the Bcl-2 family. Akt phosphorylates BAD on serine 136, creating a binding site for the adaptor protein 14-3-3. Once BAD is bound by 14-3-3, it is unable to heterodimerize with and inhibit the pro-survival proteins Bcl-2 or Bcl-XL . Akt is also involved modifying cell cycle function and enhanced cell proliferation. Akt phosphorylates and inhibits the CDK inhibitors p21WAF 1/CIP1 and p27 KIP1 . Phosphorylation of mdm2 by Akt promotes the degradation of p53 and induction of E2F transcriptional activity, leading to enhanced cell cycle activity at the G1 /S interface. In addition, Akt is able to modify GSK3-mediated phosphorylation and degradation of cyclin D1. PTEN mutation and methylation are frequent in highgrade astrocytomas and are responsible for the abnor-

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mally high levels of activity in the PI3-K/Akt signaling pathway. The PTEN gene, which consists of nine exons, is located on chromosome 10q23.3 and encodes a 403-amino acid cytoplasmic protein that contains two domains in the N -terminus, a region with lipid phosphatase activity that dephosphorylates PIP3 to PIP2 , and a region that interacts with cellular cytoskeleton. PTEN can dephosphorylate tyrosine-, serine-, and threonine-phosphorylated peptides, although no physiologic protein substrates have been identified. In normal cells with a functioning PTEN gene, PIP3 and activated Akt are maintained at low levels. In tumors and PTEN-deficient tumor cell lines, basal levels of PIP3 and phospho-Akt [16] are high, and multiple proteins are activated to promote cellular survival. 2. mTOR Signaling mTOR (also known as FRAP, RAFT, or RAPT) is a recently characterized signal transduction mediator that is intimately linked with the PI3-K/Akt signaling pathway and regulates protein synthesis and cell growth. mTOR is activated in response to growth factor signals through the PI3-K/Akt pathway. Stimulation of PI3-K leads to activation of Akt, with subsequent phosphorylation of mTOR on ser-2448. In addition, phospho-Akt is able to phosphorylate the Tsc1/Tsc2 complex on the Thr-1462 of Tsc2 that is inhibitory to mTOR [16]. Recent studies demonstrate that inhibitors of mTOR activate Akt signaling, suggesting that the clinical efficacy of mTOR inhibitors may be augmented by concurrent blockade of Akt. 3. RAS/RAF/MEK/ERK Signaling The small guanosine-triphosphate (GTP)–binding proteins include several important mediators of the growth factor receptor pathway, including RAS, RHO, RAC, and CDC42 family members. These proteins promote cell cycle progression, invasion, motility, and angiogenesis. Unlike many systemic cancers, the infiltrating gliomas typically do not express mutant RAS but may exhibit increased levels of all three major RAS proteins (N-RAS, H-RAS, and Ki-RAS), resulting in increased levels of MAPK [17,18].

B. Hypoxic Regulation of Angiogenesis in Glioblastoma When grade III astrocytomas progress to grade IV glioblastomas, tumor biology changes dramatically. Actively proliferating tumor cells outgrow their existing blood supply and progressively become hypoxic and necrotic. A key transcription factor mediating such responses is hypoxia inducible factor (HIF-1), a heterodimer

that consists of a HIF-1 subunit and a constitutively expressed HIF-1. In low oxygen conditions, HIF-1 binds to hypoxia-response elements (HREs), thereby inducing the expression of numerous hypoxia-responsive genes involved in tumor angiogenesis and invasion, cell survival, and glucose metabolism. Angiogenesis is a complex and tightly controlled physiological process that involves growth and maintenance of blood vessels within tissues and organs. A delicate equilibrium exists between opposing proangiogenic (e.g., vascular endothelial growth factor [VEGF], transforming growth factor-b [TGF-], PDGF, IL-8, and FGF) and antiangiogenic factors (e.g., thrombospondin TSP-1, endostatin, and angiostatin). Glioblastomas are among the most highly vascularized tumors with elevated levels of numerous proangiogenic factors. Hypoxia is one of the most potent stimulators of VEGF expression (Fig. 5). In addition to hypoxic regulation, VEGF expression and glioma angiogenesis are also affected by the genetic status of the tumor cells. EGFR activation leads to an up-regulation of VEGF by increasing the levels of the hypoxia inducible factor (HIF1-) via a PI3-K dependent pathway that is distinct from signals induced by hypoxia. Mutation or loss of PTEN function is also characteristic of these tumors and activates the PI3-K pathway.

C. Mechanisms of Glioma Cell Migration and Invasion Cellular migration is dependent on adhesion to the surrounding matrix. Extracellular matrix (ECM) proteins (fibronectin, laminin, collagen, vitronectin, and tenascin) in the brain are localized to the perivascular space. These proteins interact with adhesion molecules such as integrins and cadherins. Altered levels of the ECM proteins in the tumor cells and surrounding tissue contribute to tumor cell migration. One of the most important hallmarks of malignant gliomas is their invasive behavior. Glioma migration and invasion are influenced by overexpression of matrix metalloproteinases (MMPs) in glioma. Invasion of tumor cells is regulated by several proteins. Rho proteins are proteins that cycle between GTP- and GDP-bound states and therefore have a multitude of functions, including effects on the migratory behavior of glioma cells. Focal adhesion kinase (FAK) and proline-rich tyrosine kinase (PYK2) are nonreceptor tyrosine kinases. Inhibition or overexpression of these proteins has been shown to alter migration of glioma cells. Some studies suggest that radiotherapy reduces glioma invasion in xenografts and cultures. However, clinical trials to date have not demonstrated reduced invasion of tumor cells in patients.

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CBP/p300 HIF-1 α HIF-1 β

Hypoxia

HRE

Transcription activation

Release of VEGF from glioma

HRE-targeted genes

Host blood vessel

VEGF

VEGFR

Signal transduction (PKC, PLC, PLP, MAPK, Akt)

Release of proteases, endothelial cell proliferation Migration and tube formation

Tumor angiogenesis Figure 5 HIF- and VEGF-mediated regulation of glioma angiogenesis. Hypoxia is one of the most potent stimulators of VEGF expression and acts in part through transcription factors of the HIF family. VEGF acts to bind to either the homodimers or heterodimers of the VEGFR and subsequently activates intrinsic tyrosine kinase in the intracellular domain followed by autophosphorylization of the receptor. The tyrosine-phosphorylated receptor is reorganized by cytoplasmatic signaling molecules. The activated receptor signals through the transduction cascade and promotes cellular responses. Activated endothelial cells release proteases, proliferate, and migrate towards the glioma.

X. Pharmacology The standard of care for gliomas includes surgery followed by radiation and chemotherapy. Chemotherapy has typically used nonspecific cytotoxic approaches that generally act via damaging DNA. Alkylating agents such as temozolomide and nitrosoureas have been more commonly used in recent research trials. The purpose of this section is to highlight therapies that target signaling pathways discussed earlier in this review; this section is not intended to be a comprehensive review of all targets and therapeutics currently under development.

they have a set of defined molecular lesions and signaling pathway disruptions that present clear targets. However, the ability of drugs to cross the blood-brain barrier (BBB) and the low incidence of glioma in relation to more common adult neoplasms present relative barriers to glioma-specific drug development. The initial studies of targeted molecular therapies for malignant gliomas have focused on the inhibition of tyrosine kinases such as EGFR, PDGFR, and the mTOR, mainly due to availability of agents that target these pathways.

B. Receptor Tyrosine Kinase Inhibitors A. Targeted Molecular Therapy Responses to pharmacological treatment vary considerably even within a classified type of glioma. This is not surprising because these tumors are found in different areas of the CNS, contain high cellular heterogeneity, and also show variations in genetic alterations. Glioblastomas are highly suitable for targeted molecular therapy because

The EGFR is an attractive therapeutic target in glioblastoma. The EGFR gene is amplified in 40–50% and overexpressed in more than 60% of glioblastomas. Approximately 40% of tumors with EGFR amplification have gene rearrangements in EGFR that result in the constitutive activation. Overactivity of the EGFR pathway results in cell proliferation; increased tumor invasiveness, motility, and angiogenesis; inhibition of apoptosis; and

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is associated with resistance to treatment with radiation therapy and chemotherapy. Several small-molecule tyrosine kinase inhibitors of the EGFR are being evaluated in malignant gliomas [19]. Gefitinib (ZD1839, Iressa) and erlotinib (OSI-774, Tarceva) are small molecule inhibitors of the EGFR. The use of gefitinib alone in patients with glioblastoma has been disappointing: the 6-month median progression-free survival (PFS) was 13.2% [19]. Erlotinib showed slightly encouraging efficacy in a phase II trial. The PFS for glioblastoma was 12 weeks, with all patients progressing at 24 weeks [19]. PDGFR is also an attractive target for therapy because PDGFR signaling promotes glioblastoma proliferation and survival. Imatinib is an ATP competitive inhibitor of Abl that also has a high level of activity against PDGFR. Imatinib has already dramatically impacted the survival of chronic myeloid leukemia patients. Imatinib also inhibits the growth of glioblastoma xenografts in vivo; however, clinical trials with this agent have not documented activity against glioma.

C. Combination Therapy An obvious issue that could explain the failure of EGFR inhibitors in patients relates to the ability of these inhibitors to shut down signaling through PI3-K. If PTEN is lost, or if PI3-K shows gain-of-function mutations, then inhibition of EGFR will not lead to inhibition of PI3-K. We have found in our laboratory that gefitinib and LY294002 showed combinatorial efficacy on growth of xenografts derived from PTEN-mutant U87MG glioblastoma cells transduced with EGFRvIII. This experiment demonstrates that biologically based therapies targeting EGFR and PI3-K effectively cooperate in the preclinical treatment of gliomas [20] and argues that a similar approach should be applied in patients with glioma. The PI3-Ks are lipid kinases. The basic chemical understanding of inhibitors of lipid kinases lags about a decade behind the basic chemical understanding of protein kinases structure and function. Existing inhibitors of the PI3-Ks, wortmannin and LY294002, have demonstrated activity in preclinical testing but are not applicable to the clinical arena because of unfavorable pharmacological characteristics, including problems with instability and insolubility. Another concern is that the first-generation PI3-K inhibitors have such a broad inhibitory spectrum against the entire class of PI3-Ks, as well as more distant PI3-K-like kinases (e.g., ATM). This lack of specificity translates into a poor therapeutic index and unacceptable clinical toxicity. In order to translate our preclinical observations to test inhibitors of PI3-K in combination with inhibitors of EGFR, new drugs targeting PI3-K need to be developed. The isoformselective inhibitors of p110 show particular promise in this

regard [16] because these should effectively block specific isoforms of PI3-K active in tumors while sparing those isoforms critical to growth and development of normal cells.

D. Inhibition of the RAS/MAPK Pathway Constitutive Ras activation in glioblastoma arises primary from EGFR and PDGFR signaling, suggesting that patients whose tumors overexpress these receptors may derive benefit. The synthetic farnesyltransferase inhibitors SCH66336 and R115777 demonstrate promising results in preclinical models, including glioblastoma cell lines, although it is unclear whether these agents actually impact Ras. In early clinical trials, these drugs have been effective in some patients with a variety of cancers, particularly when used in combination with conventional cytotoxic agents.

E. mTOR Inhibitors Whereas direct PI3-K inhibitors are generally toxic, there are several well-tolerated inhibitors of mTOR, including sirolimus (Rapamycin), temsirolimus (CCI-779), everolimus (RAD001), and AP23573. These mTOR inhibitors inhibit glioblastoma cell proliferation in culture and intracerebral xenografts and are undergoing clinical testing [19]. However, if these drugs block mTOR and feedback to activate Akt, then these drugs will likely be effective only in combination with PI3-K or Akt inhibitors, neither of which are currently available for use in patients.

XI. Future Therapeutic Interventions Due to the heterogeneity of gliomas, successful future treatment will most likely include multimodal therapy. For example, recent studies have shown that treatment of glioblastoma patients using radiotherapy in combination with temozolomide increases the median survival by a few months compared with radiotherapy alone. Furthermore, the benefit from temozolomide in glioblastoma patients has been positively correlated with epigenetic silencing of the MGMT promoter through methylation. Maximal potentiation by the MGMT inhibitor O6 -benzylguanine requires complete and prolonged suppression of MGMT. This supports the use of O6 -benzylguanine as a pretreatment to achieve full benefit of alkylating agents, particularly temozolomide, in the chemotherapy of gliomas. The role of drug delivery in the treatment of CNS neoplasms is a crucial consideration in the development of any agent. The delivery of all substances into the brain is tightly regulated by the BBB. Lipophilic substances can penetrate the BBB whereas large or charged

Glioma molecules cannot. Alternative approaches involve the use of intracranial catheters (termed convection-enhanced delivery), specialized vehicles (e.g., liposomes) or stem cells, or compounds to improve BBB penetration. Another approach relies on the ability of monoclonal antibodies to find targets on tumor cells. As important components of the immune system, antibodies can potentially initiate immune responses, including complementmediated or antibody-dependent cytotoxicity. Antibodies may also block protein-protein interactions, including growth-factor and integrin-ligand binding. Internalizing antibodies have been labelled with radioisotopes or toxins to kill tumor cells. Thus far, antibodies directed against the EGFR have shown promising results in preclinical and clinical studies, although delivery of antibodies across the BBB remains an active issue. In other clinical trials, ligands specific for cell surface receptors on glioma cells have been fused to toxins. The advantage of this approach is that ligands are usually very specific and can be used in low concentrations, reducing unwanted side effects. NSCs have been shown to target glioma cells that are transplanted into murine brains. In these experiments, NSCs were distributed around tumor cells and reduced tumor growth. Due to the infiltrative nature of gliomas, the migration of NSCs might represent an alternative approach to target tumor cells dispersed throughout the brain. In most studies, NSCs have been used as vectors expressing interleukins or enzymes that are know to reduce tumor growth. However, even endogenous NSCs have been shown to “home in” on tumor cells and reduce tumor growth. This migratory capacity of NSCs is not restricted to tumors. In a similar way, NSCs also migrate toward injured and ischemic parts of the brain. The NSCs seem to migrate toward a gradient of stromal cell–derived factor-1 (SDF-1) because they express the receptor chemokine receptor 4 (CXCR4), and the effect is inhibited using an antagonist against this receptor. The understanding of the cellular heterogeneity of gliomas further suggest that future interventions will be based on combined effects from different therapies. Although some of the treatments mentioned here reduce tumor growth, the majority of glioma patients develop recurrent and lethal tumors. These observations suggest that our current therapeutic approaches do not affect a substantial reservoir of neoplastic tumor cells and that this reservoir is sufficient to reestablish the malignancy. Both NSCs and glial progenitors are found in brain areas including the SVZ, hippocampus, and subcortical white matter. Glial progenitors that are unable to generate neuronal progeny have been isolated throughout the neuraxis. Abnormal activation of the sonic hedgehog (SHH), WNT, NOTCH-1, and EGFR pathways in NSCs and glial progenitors may initiate hyperplasia by increasing proliferation, altering differentiation, or changing the proportion of cells undergoing asymmetrical division. Alternatively, differentiated adult glia may undergo neoplastic

443 transformation in response to carcinogens through reactivation or overexpression of these pathways. Glioblastoma is a multiforme tumor and is comprised of diverse cell types. The stem cell hypothesis suggests that this diversity results, in part, from the differentiation of a relatively homogeneous progenitor population. If tumor stem cells represent the ideal therapeutic target in glioma, then preclinical and clinical interventions should be tested on this population specifically, in addition to, or instead of the entire tumor as a whole (see Fig. 4C). Existing data suggest that SHH signaling plays a significant role in initiating and sustaining medulloblastoma growth. Gli, a transcription factor family activated by SHH, is expressed in both low- and high-grade gliomas, suggesting a possible role in this cancer. While SHH is involved in generation of the ventral telencephalic cells during neural development, the sequential signaling of WNT and fibroblast growth factor specifies cells of dorsal telencephalic character. Overexpression of human Dkk-1, a gene encoding a WNT antagonist, sensitizes glioblastoma cells to apoptosis following alkylation damage to DNA, suggesting a protective role for the WNT pathway in glioblastoma cells. Glioblastoma progenitor cells may be isolated based on excluding Hoechst dye (side-population). The more tumorigenic cells in the side-population express high levels of both -catenin, a transcription factor downstream of WNT, and Notch-1. Oncogenic Ras also activates Notch1 signaling and is necessary to maintain the neoplastic phenotype of fibroblasts. Notch-1 therefore represents a potential therapeutic target for various cancers including gliomas. In the SVZ of the adult rodent, EGF-responsive, transit-amplifying progenitors constitute a large population of migratory, rapidly dividing cells. Stable transfection of the EGFRvIII variant into neural progenitor cells resulted in highly migratory and fast-dividing cells that infiltrated different brain regions when transplanted into the rodent brain. These and other pathways shared between NSCs and glioma cells represent potential therapeutic targets. Research should develop markers to identify glioma stem cells and other subpopulations of tumor cells and to assess the impact of preclinical and clinical interventions on this population specifically, rather than on the entire tumor as a whole (see Fig. 4C). Identification of differentially expressed targets for signaling pathways or cell-surface antigens on cancerous stem cells and the more differentiated tumor cells in gliomas may enable the development of therapies that not only inhibit tumor growth but also reduce the recurrence of tumor formation.

Acknowledgments We thank Eric Burton for his critical review of the manuscript. A.I.P. was supported by grants from the Medical Faculty of Göteborg University, Hjärnfonden, the

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Swedish Society of Medicine, the Swedish Society for Medical Research, the Lars Hiertas Minne, the Sahlgrenska University Hospital, and the Assar Gabrielsson and Edit Jacobsson Foundations. W.A.W acknowledges research support from the Goldhirsh, Sandler, and Waxman Foundations; Thrasher and Burroughs Wellcome Funds; and the Brain Tumor Society.

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