From: Subject: Malignant astrocytic glioma: genetics, biology, and paths to treatment -- Furnari et al. 21 (21): 2683 -- Genes and Development Date: Mon, 4 Aug 2008 14:55:01 +0200 MIME-Version: 1.0 Content-Type: multipart/related; type="text/html"; boundary="----=_NextPart_000_0000_01C8F642.125CB880" X-MimeOLE: Produced By Microsoft MimeOLE V6.00.2900.5512 This is a multi-part message in MIME format. ------=_NextPart_000_0000_01C8F642.125CB880 Content-Type: text/html; charset="Windows-1252" Content-Transfer-Encoding: quoted-printable Content-Location: http://genesdev.cshlp.org/cgi/content/full/21/21/2683 Malignant astrocytic glioma: genetics, biology, and = paths to treatment -- Furnari et al. 21 (21): 2683 -- Genes and = Development
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REVIEW=20

Malignant astrocytic glioma: genetics, biology, and paths to=20 treatment

Frank B.=20 Furnari1,2,3, Tim=20 Fenton1, Robert M. Bachoo4,=20 Akitake Mukasa1, Jayne M.=20 Stommel5, Alexander Stegh5,=20 William C. Hahn6,7,8, = Keith=20 L. Ligon6,7,9, David N.=20 Louis10, Cameron Brennan11,=20 Lynda Chin5,7,12, = Ronald A.=20 DePinho5,7,8,14,=20 and Webster K.=20 Cavenee1,2,3,13,15=20

1 Ludwig Institute for Cancer Research, University of = California=20 at San Diego, La Jolla, California 92093, USA; 2 Department = of=20 Medicine, University of California at San Diego, La Jolla, California = 92093,=20 USA; 3 Cancer Center University of California at San Diego, = La Jolla,=20 California 92093, USA; 4 Department of Neurology and = Department of=20 Medicine, University of Texas Southwestern Medical School, Dallas, Texas = 75390,=20 USA; 5 Center for Applied Cancer Science of the Belfer = Institute for=20 Innovative Cancer Science, Dana-Farber Cancer Institute, Harvard Medical = School,=20 Boston, Massachussetts 02115, USA; 6 Department of Medicine, = Brigham=20 and Women=92s Hospital, Harvard Medical School, Boston, Massachussetts = 02115, USA;=20 7 Department of Medical Oncology, Dana-Farber Cancer = Institute,=20 Harvard Medical School, Boston, Massachussetts 02115, USA; 8=20 Department of Medicine and Department of Genetics Dana-Farber Cancer = Institute,=20 Harvard Medical School, Boston, Massachussetts 02115, USA; 9=20 Department of Pathology, Brigham and Women=92s Hospital, Harvard Medical = School,=20 Boston, Massachussetts 02115, USA; 10 Department of = Pathology,=20 Massachusetts General Hospital, Harvard Medical School, Boston, = Massachussetts=20 02115, USA; 11 Department of Neurosurgery, Memorial Sloan = Kettering=20 Cancer Institute, New York, New York 10065, USA; 12 = Department of=20 Dermatology, Dana-Farber Cancer Institute, Harvard Medical School, = Boston,=20 Massachussetts 02115, USA; 13 Center for Molecular Genetics,=20 University of California at San Diego, La Jolla, California 92093, USA=20


3D"   =20 Abstract
Top
3D"Abstract
3D"Classification and grading of...
3D"Tumor biological processes and...
3D"Frontiers in glioma research...
Acknowledgments
3D"References =
 
Malignant=20 astrocytic gliomas such as glioblastoma are the most common = and=20 lethal intracranial tumors. These cancers exhibit a = relentless=20 malignant progression characterized by widespread invasion = throughout=20 the brain, resistance to traditional and newer targeted = therapeutic=20 approaches, destruction of normal brain tissue, and certain = death.=20 The recent confluence of advances in stem cell biology, cell=20 signaling, genome and computational science and genetic model = systems=20 have revolutionized our understanding of the mechanisms = underlying=20 the genetics, biology and clinical behavior of glioblastoma. = This=20 progress is fueling new opportunities for understanding the=20 fundamental basis for development of this devastating disease = and=20 also novel therapies that, for the first time, portend = meaningful=20 clinical responses.

[Keywords: Glioma; glioblastoma; neural stem cells; cancer = stem=20 cells; tyrosine kinase inhibitor; genetically engineered models]]

Malignant gliomas are classified and subtyped on the basis of=20 histopathological features and clinical presentation (Fig.=20 1). The most common and biologically aggressive of these = is=20 glioblastoma (GBM), World Health Organization (WHO) grade IV, = and is=20 defined by the hallmark features of uncontrolled cellular=20 proliferation, diffuse infiltration, propensity for necrosis, = robust=20 angiogenesis, intense resistance to apoptosis, and rampant = genomic=20 instability. As reflected in the old moniker "multiforme," = GBM=20 presents with significant intratumoral heterogeneity on the=20 cytopathological, transcriptional, and genomic levels. This=20 complexity, combined with a putative cancer stem cell (CSC)=20 subpopulation and an incomplete atlas of (epi)genetic lesions = driving=20 GBM pathogenesis, has conspired to make this cancer one of = the most=20 difficult to understand and to treat. Despite implementation = of=20 intensive therapeutic strategies and supportive care, the = median=20 survival of GBM has remained at 12 mo over the past = decade.=20



View = larger=20 version (48K):
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  		Figure 1. = Chromosomal and=20 genetic aberrations involved in the genesis of glioblastoma. = Shown=20 are the relationships between survival, pathobiology, and = the=20 molecular lesions that lead to the formation of primary (de = novo)=20 and secondary (progressive) glioblastomas. Although = histologically=20 indistinguishable, these grade IV gliomas occur in different = age=20 groups and present distinct genetic alterations affecting = similar=20 molecular pathways. For example, inactivation of p53 = function occurs=20 due to direct mutation in progressive GBMs or INK4aARF=20 mutation/decrease in expression or MDM2 amplification in de = novo=20 GBMs. Similarly, activation of the PI3K pathway is achieved = by=20 several cooperative mechanisms, including EGFR amplification = and=20 mutation as well as PTEN mutation, although underexpression = of PTEN=20 in the absence of mutation is frequently seen as well. See = the text=20 and Figure = 2 for details on the molecular function of implicated = genes.=20 (OE) Overexpressed; (amp) amplified; (mut) mutated. =

 = ;
In=20 this review, we summarize current basic and translational = challenges=20 and highlight the striking scientific advances that promise = to=20 improve the clinical course of this lethal disease. These = advances=20 include a more comprehensive view of the altered genes and = pathways=20 in glioma and how such alterations drive the hallmark = pathobiological=20 features of the disease, the identification of new molecular = subtypes=20 in GBM, an improved understanding of the cellular origins of = the=20 disease and how CSCs may influence therapeutic responses, = refined=20 model systems for use in research and preclinical = experimental=20 therapeutics, and novel therapeutic strategies for targeting = keystone=20 genetic lesions and their pathways. For reasons of length, we = have=20 not discussed the advances in such important areas as tumor=20 immunology, the blood-brain barrier, and tumor imaging. For = the first=20 time, there is a strong sentiment that meaningful therapeutic = advances will soon flow from this explosion of new molecular = and=20 biological knowledge; the remarkable technological advances = in=20 genomics, proteomics, and model systems; and the systematic = and=20 accurate development of small molecule drugs, therapeutic = antibodies,=20 and the entirely new class of RNA interference (RNAi)-based=20 agents.


3D"   =20 Classification and grading of glioma =
Top
Abstract
3D"Classification and grading of...
3D"Tumor biological processes and...
3D"Frontiers in glioma research...
Acknowledgments
3D"References =
 
The=20 incidence of primary brain tumors worldwide is approximately = seven=20 per 100,000 individuals per year, accounting for 3D~=202% of = primary=20 tumors and 7% of the years of life lost from cancer before = the age of=20 70. The common gliomas affecting the cerebral hemispheres of = adults=20 are termed "diffuse" gliomas due to their propensity to = infiltrate,=20 early and extensively, throughout the brain parenchyma. These = gliomas=20 are classified histologically, immunohistochemically, and/or=20 ultrastructurally as astrocytomas, oligodendrogliomas, or = tumors with=20 morphological features of both astrocytes and = oligodendrocytes,=20 termed oligoastrocytomas. Tumors are then graded on a WHO=20 consensus-derived scale of I to IV according to their degree = of=20 malignancy as judged by various histological features = accompanied by=20 genetic alterations (Fig. = 1;=20 Louis et al. 20073DGo). Grade I tumors are biologically benign and = can be=20 cured if they can be surgically resected; grade II tumors are = low-grade malignancies that may follow long clinical courses, = but=20 early diffuse infiltration of the surrounding brain renders = them=20 incurable by surgery; grade III tumors exhibit increased = anaplasia=20 and proliferation over grade II tumors and are more rapidly = fatal;=20 grade IV tumors exhibit more advanced features of malignancy, = including vascular proliferation and necrosis, and as they = are=20 recalcitrant to radio/chemotherapy they are generally lethal = within=20 12 mo. This review focuses on tumors of the astrocytic = series,=20 emphasizing grade IV GBM.

On the basis of clinical presentation, GBMs have been further=20 subdivided into the primary or secondary GBM subtypes. = Primary=20 GBMs account for the great majority of GBM cases in older = patients,=20 while secondary GBMs are quite rare and tend to occur in = patients=20 below the age of 45 yr. Primary GBM presents in an acute de=20 novo manner with no evidence of a prior symptoms or = antecedent=20 lower grade pathology. In contrast, secondary GBM derives=20 consistently from the progressive transformation of lower = grade=20 astrocytomas, with 3D~=2070% of grade = II gliomas=20 transforming into grade III/IV disease within 5=9610 yr of = diagnosis.=20 Remarkably, despite their distinct clinical histories, = primary and=20 secondary GBMs are morphologically and clinically = indistinguishable=20 as reflected by an equally poor prognosis when adjusted for = patient=20 age. However, although these GBM subtypes achieve a common=20 phenotypic endpoint, recent genomic profiles have revealed = strikingly=20 different transcriptional patterns and recurrent DNA copy = number=20 aberrations between primary and secondary GBM as well as new = disease=20 subclasses within each category (as discussed below; Maher et = al.=20 20063DGo; Phillips et al. 20063DGo). These molecular distinctions make obvious = the need to=20 change the current standardized clinical management of these = truly=20 distinct diseases toward one of rational application of = targeted=20 therapies to appropriate molecular subclasses.

Immunohistochemical markers are important and rapidly evolving=20 tools in the classification and neuropathological diagnosis = of=20 malignant gliomas. Currently, the most clinically useful and = specific=20 of these markers for classification of gliomas are GFAP and = OLIG2.=20 GFAP is universally expressed in astrocytic and ependymal = tumors and=20 only rarely in oligodendroglial lineage tumors. OLIG2, a more = recently discovered stem/progenitor and oligodendroglial = marker, is=20 CNS specific and is universally and abundantly expressed in = all=20 diffuse gliomas, but is rarely expressed at such high levels = in other=20 types of gliomas and CNS malignancies (Ligon et al. 20043DGo;=20 Rousseau et al. 20063DGo). These markers thus serve as effective tools = for=20 unequivocal identification of gliomas and their distinction = from=20 non-CNS tumors while aiding the pathologist in distinction of = different glioma classes.

A recently expanded collection of novel markers has emerged = from=20 numerous avenues of research and holds potential to be = deployed to=20 improve classification and inform the potential clinical = course of=20 glioma patients. Of particular interest are newly discovered = stem and=20 progenitor cell markers that, once clinically validated, may = aid in=20 the differential diagnosis of these tumors as well as = monitoring=20 their responses to therapy. Intensive research efforts are = attempting=20 to uncover agents that may target subpopulations of these = cells with=20 high tumorigenic potential and increased resistance to = current=20 therapies. Along these lines, the cell surface marker, CD133, = and=20 other markers of stem cells, such as Nestin and Musashi, have = been=20 shown to negatively correlate with outcome parameters. These = newly=20 discovered markers suggest that pathologists will soon have = at their=20 disposal highly useful tools for improved clinical diagnosis = and=20 classification of gliomas.

Immunohistochemical markers have also recently been shown to = aid=20 in prediction of the clinical course for certain classes of = tumors.=20 GBMs with intact expression of the PTEN (phosphatase and = tensin=20 homolog deleted on chromosome 10) and EGFRvIII proteins (for = details,=20 see next section) correlated with increased epidermal growth = factor=20 receptor (EGFR) inhibitor response and progression-free = survival=20 compared with those tumors expressing EGFRvIII but lacking = PTEN=20 (Mellinghoff et al. 20053DGo). Also, patients with EGFR protein = expression, mutant=20 or wild-type, have been identified for the sake of targeting = EGFR=20 therapy to the appropriate patient population. Furthermore, a = powerful and widely used molecular marker=97combined loss of = the short=20 arm of chromosome 1 and the long arm of chromosome 19=97is = already=20 widely used in the management of oligodendroglial gliomas, = but its=20 role in the evaluation of astrocytic gliomas such as GBM is = not=20 yet well defined (Reifenberger and Louis 20033DGo;=20 Louis et al. 20073DGo). With the wealth of accumulating profiling = and genomic=20 data, an increase in confidence is merited that useful=20 diagnostic, prognostic, and drug response biomarkers will be=20 incorporated into routine clinical management of GBM in the = near=20 future.


3D"    Tumor=20 biological processes and known underlying genetic alterations in=20 astrocytic gliomas
Top
Abstract
3D"Classification and grading of...
3D"Tumor biological processes=20 and...
3D"Frontiers in glioma research...
Acknowledgments
3D"References =
 
The=20 classical genetic alterations in glioma target pathways = governing=20 cellular proliferation, cellular survival (apoptosis and = necrosis),=20 invasion, and angiogenesis. The following subsections cover = these=20 hallmark biological processes and their links to specific = genetic=20 aberrations and associated signaling pathways (Figs. = 1, 2).= =20



View = larger=20 version (83K):
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  		Figure 2. = Genetic=20 alterations characteristic of astrocytic glioma lead to = aberrant=20 activation of key pathways involved in mitogenic signaling = and cell=20 cycle control. Certain proto-oncogenes (shown in green) such = as EGFR=20 and PIK3CA (p1103D{alpha}=20) are=20 activated by mutation, while other growth-promoting genes = (also=20 green) are commonly overexpressed. Tumor suppressor genes = that are=20 either lost or inactivated by mutation are shown in red. = Knowledge=20 of glioma genetics has driven the development of therapeutic = agents=20 (listed in blue boxes) that specifically target these = pathways=97both=20 those intrinsic to the tumor cells and those that impact on = the=20 surrounding endothelium and extracellular matrix to direct = glioma=20 angiogenesis and invasion. Direct signaling connections, = such as=20 post-translational modification of target proteins, are = shown in=20 solid lines, while dashed lines represent indirect or=20 uncharacterized interactions. The major mitogenic signaling = modules=20 downstream from RTKs (RAS-MAPK and PI3K-mTOR) and the cell = cycle=20 machinery are frequently dysregulated in glioma and are = highlighted=20 (see the text for details). (AKT) Murine thymoma viral = oncogene=20 homolog; (AMPK) AMP-dependent protein kinase; (c-src) = sarcoma=20 (Schmidt-Ruppin A-2) viral oncogene homolog; (ERK) = extracellular=20 signal-regulated kinase; (eIF4E) eukaryotic initiation = factor 1;=20 (4EBP1) eIF4E-binding protein 1; (HDAC) histone deacetylase; = (mdm-2,4) murine double minute 2,4; (MEK) mitogen-activated = protein=20 kinase kinase; (mTOR) mammalian target of rapamycin; = (p90RSK) p90=20 ribosomal protein S6 kinase; (PLC3D{gamma}=20)=20 phospholipase C3D{gamma}=20; (pRb)=20 retinoblastoma protein; (RAF1) v-raf1 murine leukemia viral = oncogene=20 homolog 1; (RAS) rat sarcoma viral oncogene homolog; (REDD1) = regulated in development and DNA damage responses; (RHEB) = Ras=20 homolog enriched in brain; (S6K1) p70 ribosomal protein S6 = kinase 1;=20 (TORC1,2) mTOR complex1,2. =

 = ;

Cell=20 cycle dysregulation and enhanced glioma cell proliferation

Frequent mutations of cell cycle regulatory genes in glioma = have=20 underscored the importance of these genes in cellular proliferation = and senescence. The RB and p53 pathways, which regulate the=20 cell cycle primarily by governing the G1-to-S-phase = transition,=20 are major targets of inactivating mutations in GBM. The = absence=20 of these cell cycle guardians renders tumors particularly = susceptible=20 to inappropriate cell division driven by constitutively = active=20 mitogenic signaling effectors, such as phosphoinositide = 3'-kinase=20 (PI3K) and mitogen-activated protein kinase (MAPK).

The Rb pathway
In = quiescent cells,=20 hypophosphorylated RB blocks proliferation by binding and=20 sequestering the E2F family of transcription factors, which = prevents=20 the transactivation of genes essential for progression = through the=20 cell cycle (Sherr and McCormick 20023DGo). Upon mitogenic stimulation, the activation of the = MAPK=20 cascade leads to the induction of cyclin D1 and its = association=20 with the cyclin-dependent kinases CDK4 and CDK6, as well as = the=20 degradation of the CDK2/cyclin E inhibitor, p27Kip1 = (Albanese=20 et al. 19953DGo;=20 Lavoie et al. 19963DGo;=20 Aktas et al. 19973DGo). These activated CDK complexes in turn = phosphorylate=20 RB, enabling E2F transactivation of its direct = transcriptional=20 targets governing S-phase entry and progression (Weinberg = 19953DGo;=20 Frolov and Dyson 20043DGo).

Gliomas circumvent RB-mediated cell cycle inhibition through = any=20 of several genetic alterations. The Rb1 gene, which maps = to=20 chromosome 13q14, is mutated in 3D~=2025% of = high-grade=20 astrocytomas and the loss of 13q typifies the transition from = low- to=20 intermediate-grade gliomas (James et al. 19883DGo;=20 Henson et al. 19943DGo). Moreover, amplification of the CDK4 = gene on=20 chromosome 12q13-14 accounts for the functional inactivation = of RB in=20 3D~15% high-grade=20 gliomas, and CDK6 is also amplified but at a lower = frequency=20 (Reifenberger et al. 19943DGo; Costello et al. 19973DGo). RB activity is also frequently lost through = the=20 inactivation of a critical negative regulator of both CDK4 = and CDK6,=20 p16Ink4a (Serrano et al. 19933DGo). This gene is one of two transcripts = generated at the=20 CDKN2A locus on chromosome 9p21 (in addition to=20 p14ARF [alternate reading frame p14]; see below), = which is=20 predominantly inactivated by allelic loss or hypermethylation = in=20 50%=9670% of high-grade gliomas and 3D~=2090% of = cultured glioma=20 cell lines (Jen et al. 19943DGo;=20 Schmidt et al. 19943DGo;=20 Merlo et al. 19953DGo;=20 Costello et al. 19963DGo;=20 Fueyo et al. 19963DGo). Consistent with its role as an important glioma = tumor=20 suppressor, p16Ink4a is also a critical inhibitor of = progenitor=20 cell renewal in the subventricular zone of aging mice = (Molofsky=20 et al. 20063DGo). The importance of the inactivation of the RB = pathway=20 in glioma progression is evidenced by the near-universal and=20 mutually exclusive alteration of RB pathway effectors and = inhibitors=20 in both primary and secondary GBM (Schmidt et al. 19943DGo;=20 Ueki et al. 19963DGo). However, numerous in vitro and in vivo assays = have=20 demonstrated that the neutralization of this pathway alone is = insufficient to abrogate cell cycle control to the extent = needed for=20 cellular transformation, suggesting that other important cell = cycle=20 regulation pathways complement its activities in preventing=20 gliomagenesis (Holland et al. 1998a3DGo,=20 b; Rich et al. 20013DGo;=20 Sonoda et al. 20013DGo;=20 Bachoo et al. 20023DGo;=20 Huang et al. 20023DGo; Uhrbom et al. 20023DGo,=20 20053DGo; Xiao et al. 20023DGo).

The p53 pathway
The p53 = tumor=20 suppressor prevents the propagation of cells with unstable = genomes,=20 predominantly by halting the cell cycle in the G1 phase or=20 instigating a program of apoptosis or proliferative arrest = (Vousden=20 and Lu 20023DGo). P53 achieves these ends primarily through = its=20 function as a transcription factor: Upon being post-translationally = modified by various genotoxic and cytotoxic stress-sensing = agents,=20 p53 is stabilized, then binds and transcriptionally regulates = the promoters of >2500 potential effector genes (Hoh et = al.=20 20023DGo;=20 Levine et al. 20063DGo). The best characterized of these effectors = is the=20 transcriptional target CDNK1A; which encodes the protein = for=20 the CDK2 inhibitor p21 (El-Deiry et al. 19933DGo;=20 Harper et al. 19933DGo). Although this gene has not been found to be = genomically=20 altered in gliomas, its expression is frequently abrogated by = p53 functional inactivity as well as by mitogenic signaling=20 through the PI3K and MAPK pathways.

The p53 pathway is nearly invariably altered in sporadic = gliomas:=20 Loss of p53, through either point mutations that prevent DNA=20 binding or loss of chromosome 17p, is a frequent and early = event=20 in the pathological progression of secondary GBM (Louis 19943DGo; Louis and Cavenee 19973DGo). The importance of p53 in gliomagenesis is = also=20 underscored by the increased incidence of gliomas in = Li-Fraumeni=20 syndrome, a familial cancer-predisposition syndrome = associated with=20 germline p53 mutations (Malkin et al. 19903DGo; Srivastava et al. 19903DGo). This genetic linkage has been reinforced by = a=20 glioma-prone condition in mice engineered with a commonly = observed=20 Li-Fraumeni p53 mutation (Olive et al. 20043DGo)=20 as well as in p19ARF-null mice, albeit at a low = frequency=20 (Kamijo et al. 19993DGo).

The finding that a second promoter drives an alternatively = spliced=20 transcript at the CDKN2A locus prompted the discovery of = an=20 additional tumor suppressor gene that is inactivated at this=20 locus (Quelle et al. 19953DGo). The second protein encoded by CDKN2A;=20 p14ARF, was subsequently shown to be an important=20 accessory to p53 activation under conditions of oncogenic = stress due=20 to its neutralization of the p53 ubiquitin ligase, MDM2 = (Kamijo=20 et al. 19983DGo;=20 Pomerantz et al. 19983DGo;=20 Stott et al. 19983DGo;=20 Honda and Yasuda 19993DGo), an oncogene originally discovered amplified = as double=20 minute chromosomes in a spontaneously transformed murine cell = line,=20 and then later found to be a key negative regulator of p53 = during=20 normal development and in tumorigenesis (Fakharzadeh et al. = 19913DGo;=20 Momand et al. 19923DGo;=20 Oliner et al. 19933DGo;=20 Jones et al. 19953DGo;=20 Montes de Oca Luna et al. 19953DGo;=20 Honda et al. 19973DGo;=20 Fang et al. 20003DGo;=20 Honda and Yasuda 20003DGo). Concordantly, the chromosomal region = containing=20 MDM2; 12q14-15, is amplified in 3D~=2010% of primary = GBM, the=20 majority of which contain intact p53 (Reifenberger et = al.=20 19943DGo). The discovery of the MDM2-related = gene,=20 MDM4 (chromosome 1q32), which inhibits p53 = transcription=20 and enhances the ubiquitin ligase activity of MDM2, prompted=20 the finding that the p53 pathway is also inactivated by the=20 amplification of MDM4 in 4% of GBM with neither TP53 = mutation=20 nor MDM2 amplification (Shvarts et al. 19963DGo;=20 Riemenschneider et al. 19993DGo;=20 Gu et al. 20023DGo;=20 Linares et al. 20033DGo). Additionally, the recently discovered tumor = suppressor gene CHD5 (chromodomain helicase = DNA-binding domain=20 5), which maps to chromosome 1p36 and is therefore frequently = hemizygously deleted in those human gliomas that have 1p = loss, has=20 been shown to maintain p53 levels by facilitating expression = of=20 p19Arf (mouse p14Arf ortholog), and = thus=20 presents an additional mechanism for inactivation of this = critical=20 pathway (Bagchi et al. 20073DGo).

Mitogenic signaling = pathways
Many=20 mitogens and their specific membrane receptors are present in = overactive form in gliomas. Proliferation of normal cells = requires=20 activation of mitogenic signaling pathways through diffusible = growth=20 factor binding, cell=96cell adhesion, and/or contact with = extracellular=20 matrix (ECM) components. These signals are transduced = intracellularly=20 by transmembrane receptors that typically activate the PI3K = and MAPK=20 signaling pathways. In contrast, tumor cells acquire genomic=20 alterations that greatly reduce their dependence on exogenous = growth=20 stimulation, enabling their inappropriate cell division, = survival,=20 and motility through the constitutive activation of these = pathways.=20 While gliomas overcome the normal impositions on the control = of=20 mitogenic signaling through multiple mechanisms, activation = of=20 receptor tyrosine kinases (RTKs), discussed in detail below,=20 appears to be the predominant mechanism.

MAPK
Proliferation signals can be = transduced=20 by the MAPK pathway by both integrins and RTKs. Integrins are = membrane-bound ECM receptors that mediate the interaction = between the=20 ECM and the cytoskeleton. Upon adhesion to ECM, integrins = bind=20 cytoplasmic anchor proteins that coordinate the binding of = integrins=20 to actin filaments, thus creating a focal adhesion complex.=20 Multiple molecules of focal adhesion kinase (FAK) cluster at = these=20 complexes and become activated by cross-phosphorylation, = whereupon=20 FAK activates a signal transduction cascade that leads to=20 extracellular signal-regulated kinase (ERK) phosphorylation = either=20 through activation of Ras by the recruitment of the adaptor=20 protein Grb2 and the Ras guanine nucleotide exchange factor = SOS to=20 phospho-FAK at the plasma membrane, or through Src-dependent=20 phosphorylation of p130Cas (Schlaepfer et al. 19943DGo,=20 19973DGo; Schlaepfer and Hunter 19973DGo). Ras-GTP in turn phosphorylates Raf kinase, which=20 phosphorylates MEK, which phosphorylates ERK, which enters = the=20 nucleus and phosphorylates nuclear transcription factors that = induce=20 the expression of genes promoting cell cycle progression, = such=20 as cyclin D1. RTKs activate the MAPK pathway when activated = by=20 growth factor signaling, mutation, or overexpression. As = discussed=20 in more detail below, RTK activation results in receptor = dimerization=20 and cross-phosphorylation, creating binding sites for adaptor = protein complexes such as Grb2/SOS, which in turn activates=20 Ras. While constitutively activated, mutated forms of Ras are = found in 3D~50% of all human tumors, few Ras mutations have been = found=20 in gliomas. Despite this, high levels of active Ras-GTP are = found in=20 advanced astrocytomas (Guha et al. 19973DGo), suggesting that a more relevant mechanism = for=20 MAPK-dependent mitogenic signaling in GBM is through = inappropriate=20 activation of RTKs and/or integrins.

PI3K/PTEN/AKT
The class I PI3Ks = catalyze the=20 mitogen-stimulated phosphorylation of=20 phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] to = produce PtdIns(3,4,5)P3. This creates docking sites for = a=20 multitude of signaling proteins containing domains capable of = binding either to PtdIns(3,4,5)P3 itself or to the = 5-dephosphorylated product, PtdIns(3,4)P2 (for = reviews,=20 see Vanhaesebroeck et al. 20013DGo;=20 Hawkins et al. 20063DGo). The class IA PI3Ks are heterodimers that = are=20 recruited to activated RTKs and adaptor proteins via their = regulatory=20 subunit, of which there are five isoforms encoded by three = genes:=20 p853D{alpha},=20 p553D{alpha},=20 and p503D{alpha}=20 (PIK3R1); p853Dbeta (PIKR2); and p553D{gamma}=20 = (PIKR3).=20

Since the regulatory subunits appear thus far to be functionally = equivalent, the class IA PI3Ks are currently defined by the=20 catalytic isoform present: p1103D{alpha}=20, p1103Dbeta=20, and = p110, encoded=20 by the PIK3CA, PIK3CB; and PIK3CD genes, = respectively=20 (Hawkins et al. 20063DGo). Evidence for the importance of p1103D{alpha}=20 in = transformation=20 derives from the discovery of a vPIK3CA oncogene in avian=20 sarcoma virus with potent transforming activity in chicken = embryo=20 fibroblasts (CEFs) (Chang et al. 19973DGo). PIK3CA gain-of-function point mutants = have=20 been detected in a variety of cancers, including malignant = gliomas=20 such as GBM, in which the frequency of mutation has been = cited in=20 some studies to be as high as 15% (Samuels et al. 20043DGo;=20 Gallia et al. 20063DGo). Elevated expression of the PIK3D = gene has also=20 been reported in GBM (Knobbe and Reifenberger 20033DGo;=20 S. Kang et al. 20063DGo).

In addition to p85 binding, the p110 subunits can also be = activated=20 by binding to GTP-bound Ras (Rodriguez-Viciana et al. 19943DGo, 19963DGo). Recently, the study of knock-in mice bearing a = p110 point=20 mutant that is unable to bind Ras has revealed that this = interaction=20 is essential both for normal development and for Ras-driven=20 tumorigenesis, as assessed both by transformation of mouse = embryonic=20 fibroblasts (MEFs) by H-Ras and using a mouse model of = K-ras-induced=20 lung adenocarcinomas (Gupta et al. 20073DGo).

The action of class I PI3K enzymes is directly antagonized by = the=20 PtdIns(3,4,5)P3 3-phosphatase encoded by the PTEN gene = located=20 at 10q23.3 (Li et al. 19973DGo;=20 Steck et al. 19973DGo;=20 Maehama and Dixon 19983DGo). PTEN is a major tumor suppressor that is = inactivated=20 in 50% of high-grade gliomas by mutations or epigenetic = mechanisms,=20 each resulting in uncontrolled PI3K signaling in these tumors = (Knobbe and Reifenberger 20033DGo;=20 Ohgaki et al. 20043DGo). In mouse models, brain-specific = inactivation of PTEN=20 caused overgrowth of the mouse brain and aberrant = proliferation of=20 astrocytes both in vivo and in vitro (Fraser et al. 20043DGo). An elegant mouse model of astrocytoma has = been=20 developed in which the Rb family proteins are inactivated by=20 GFAP-directed expression of SV40 T antigen (Xiao et al. = 20023DGo). In this model system, PTEN inactivation was = associated with increased angiogenesis=97a close parallel to = the=20 progression of high-grade disease in humans coincident with = loss of=20 PTEN (Xiao et al. 20023DGo,=20 20053DGo). While regulation of PI3K signaling is = critical to=20 controlling cell growth and survival, a number of recent = studies have=20 pointed to additional levels at which PTEN may act to = suppress=20 transformation and tumor progression. Differentiated and = quiescent=20 cells harbor high levels of nuclear PTEN, which appears to = fulfill=20 important roles in the maintenance of genomic integrity, = through=20 centromere stabilization and promotion of DNA repair (Shen et = al.=20 20073DGo). Importantly, a number of PTEN point = mutations=20 found in familial cancer predisposition syndromes have no = effect on=20 enzyme activity but instead lie within sequences important = for=20 regulating PTEN localization. Analysis of such mutants has = confirmed=20 that aberrant sequestration of PTEN into either the nucleus = or the=20 cytoplasm compromises its tumor suppressor function (Denning = et al.=20 2007; Trotman et al. 20073DGo).

Of the many signaling proteins that are recruited to the = membrane=20 and activated by binding to PtdIns(3,4,5)P3, the=20 phosphoinositide-dependent kinase (PDK1) and Akt/PKB (also = the=20 cellular homolog of a viral oncoprotein), are required for=20 tumorigenesis in PTEN+/=96 mice and for = growth of=20 PTEN=96/=96 embryonic stem (ES) cells as = tumors in nude=20 mice (Stiles et al. 20023DGo;=20 Bayascas et al. 20053DGo;=20 Chen et al. 20063DGo). In response to PI3K activation, PDK1 and = the=20 mammalian target of rapamycin (mTOR, acting in the=20 rapamycin-insensitive TORC2 complex) activate Akt via = phosphorylation=20 of two key residues, T308 and S473, respectively (Mora et al. = 20043DGo;=20 Sarbassov et al. 20053DGo). Assessment of the phosphorylation status of = these=20 residues is often the method of choice for monitoring PI3K = pathway=20 activity in cell lines and primary tumors, including GBM = samples, 85%=20 of which have been reported to display activated Akt (Wang et = al.=20 20043DGo). In addition to aberrant PI3K signaling, = there are a=20 number of other possible mechanisms by which Akt activation = may=20 become dysregulated in GBM. PHLPP (PH domain leucine-rich = repeat=20 protein phosphatase), which dephosphorylates S473, is = expressed at=20 very low levels in certain GBM cell lines, as is CTMP = (C-terminal=20 modulator protein), which binds to Akt and inhibits its=20 phosphorylation (Maira et al. 20013DGo;=20 Knobbe et al. 20043DGo;=20 Gao et al. 20053DGo). PIKE-A, a small GTPase highly expressed in = GBMs and=20 glioma cell lines, binds directly to phosphorylated Akt and = enhances=20 its anti-apoptotic function (Ahn et al. 20043DGo; Knobbe et al. 20053DGo).

Akt phosphorylates many proteins involved in the regulation = of=20 cell growth, proliferation, metabolism, and apoptosis. A = recent study=20 on v-H-ras-induced transformation of MEFs and skin = carcinogenesis=20 indicates that activation of mTOR in the rapamycin-sensitive = TORC1=20 complex via inhibition of the TSC2 tumor suppressor is a key=20 pro-oncogenic function of Akt (Skeen et al. 20063DGo). Since mutant H-ras is seldom seen in = human=20 tumors, it will be important to determine whether = Akt/TSC/TORC1=20 signaling is similarly required downstream from = glioma-relevant=20 perturbations, such as EGFR mutation and = overexpression and/or=20 PTEN loss. Evidence that this may indeed be the case = is=20 provided by the efficacy of PI-103, a small molecule = inhibitor of=20 both p1103D{alpha} and mTOR, which potently blocks the growth of = glioma cell=20 lines and of U87EGFRvIII xenografts following subcutaneous = injection=20 in nude mice, without discernable toxicity to the animals = (Fan et al.=20 2006). The use of TSC2=96/=96 = cells, which=20 display constitutive phosphorylation of the TORC1 substrates = S6K1 and=20 4E-BP1, revealed the existence of a negative feedback loop, = whereby=20 inhibitory phosphorylation of the insulin receptor substrate = (IRS-1)=20 by S6K1 causes a reduction in Akt activation (Harrington et = al.=20 2004; Shah et al. 20043DGo;=20 Riemenschneider et al. 20063DGo;=20 Shah and Hunter 20063DGo). Treatment of glioma cells with = TORC1-specific=20 inhibitors, such as rapamycin, disrupts such feedback = control,=20 resulting in increased Akt activity (Fan et al. 20063DGo). Dual inhibition of PI3K and TORC1 by PI-103 = overcomes=20 these problems and likely explains its increased efficacy. =

In addition, phosphorylation of the FOXO transcription factors = by=20 Akt, which promotes their exclusion from the nucleus, reduces = the=20 expression of a number of important target genes, including = the CDK=20 inhibitors p21WAF1/CIP1 and p27KIP1 (both of which = are also directly targeted by Akt) and the RB family member = p130=20 (Medema et al. 20003DGo;=20 Kops et al. 20023DGo;=20 Seoane et al. 20043DGo). Given the recent data illustrating = context-specific=20 actions of FOXO on various targets in different cell types = and=20 tissues, it may be prudent to validate these FOXO targets=20 specifically in glioma (Paik et al. 20073DGo).

PI3K=96MAPK=96p53=96RB pathway = interactions
While=20 the PI3K, MAPK, p53, and RB pathways are often considered as = distinct=20 entities, there is significant cross-talk among the pathways = that=20 serve to reinforce the inappropriate regulation of any single = pathway=20 perturbation. For example, because p53 enhances PTEN=20 transcription and represses the expression of p1103D{alpha}=20 (Stambolic = et al.=20 20013DGo; Singh et al. 20023DGo), the loss of p53 in cells with = constitutively active=20 RTK signaling can further potentiate PI3K pathway activation. = Therapies aimed at reactivating p53 in GBM may be compromised = by MAPK=20 and PI3K intervention in the activity of p53 and its = effectors. MAPK=20 signaling activates c-myc, which binds the miz-1 = transcriptional=20 repressor to block p21 gene induction (Herold et al. = 20023DGo;=20 Seoane et al. 20023DGo), while Akt impacts on p53 function by = phosphorylation=20 of Mdm2 (Zhou et al. 20013DGo;=20 Shin et al. 20023DGo;=20 Feng et al. 20043DGo)=20 in addition to the direct inhibition of p21 discussed = earlier.=20 Moreover, these pathways can negate each other: p53 can = inhibit=20 activated FOXOs by inducing the expression of the kinase = SGK1, which=20 phosphorylates and exports FOXOs from the nucleus (You et al. = 20043DGo). Conversely, FOXOs can inhibit p53 = transcriptional=20 activity by increasing its association with nuclear export = receptors=20 that translocate it to the cytoplasm (You et al. 20063DGo). The recent finding that Sprouty2, a gene = involved in=20 suppression of Ras signaling during oncogene-induced = senescence, is=20 also a direct transcriptional target of FoxO emphasizes the=20 complexity of cross-talk that exists between the Ras/MAPK and = PI3K=20 pathways (Courtois-Cox et al. 20063DGo;=20 Paik et al. 20073DGo). The complicated interplay among these = critical=20 molecules highlights the need for detailed dissection of the = pathways=20 that are aberrant in each tumor to accurately guide the = choice of=20 combination therapies that can simultaneously target multiple = pathways.

RTKs
Gliomas may activate=20 receptor-driven pathways by different mechanisms: = overexpression of=20 both ligands and receptors leading to an autocrine loop, = genomic=20 amplification, and/or mutation of the receptor leading to=20 constitutive activation in the absence of ligand. TheEGF and=20 platelet-derived growth factor (PDGF) pathways play important = roles=20 in both CNS development and gliomagenesis, and targeted = therapy=20 against these potentially critical signaling pathways is = currently=20 under vigorous basic and clinical investigation.

EGFR
EGFR gene = amplification occurs=20 in 3D~40% of all=20 GBMs, and the amplified genes are frequently rearranged = (Libermann et=20 al. 19843DGo,=20 19853DGo; Ekstrand et al. 19913DGo;=20 Wong et al. 19923DGo;=20 Louis et al. 20073DGo). An EGFR mutant allele with deletion = of exons=20 2=967 (known variously as EGFRvIII, 3D{Delta}=20EGFR, or = EGFR*) occurs=20 in 20=9630% of all human GBM (and in 50%=9660% of those that = have=20 amplified wild-type EGFR), making it the most common = EGFR=20 mutant (Sugawa et al. 19903DGo;=20 Frederick et al. 20003DGo). EGFRvIII is a highly validated glioma = target as=20 evidenced by the capacity of activated EGFR mutants to = enhance=20 tumorigenic behavior of human GBM cells by reducing apoptosis = and=20 increasing proliferation (Nishikawa et al. 19943DGo;=20 Nagane et al. 19963DGo;=20 Huang et al. 19973DGo;=20 Narita et al. 20023DGo)=20 and to malignantly transform murine Ink4a/Arf-null neural = stem=20 cells (NSCs) or astrocytes in the mouse brain (Holland et al. = 1998a3DGo;=20 Bachoo et al. 20023DGo). Thus, EGFR has been a prime target for = therapeutic=20 intervention in GBM with small molecule kinase inhibitors,=20 antibody-based immunotherapy and immunotoxins (Lorimer et al. = 19953DGo;=20 Mishima et al. 20013DGo;=20 Nagane et al. 20013DGo; Jungbluth et al. 20033DGo), and, more recently, small interfering RNA=20 (siRNA)-directed neutralization of either wild-type EGFR = or=20 the unique junction present in the EGFRvIII allele (Fan and=20 Weiss 20053DGo;=20 C.S. Kang et al. 20063DGo).

Transcriptional profiles of GBM with EGFR overexpression have=20 revealed distinct gene expression profiles that have enabled=20 classification of molecular subgroups among phenotypically=20 undistinguishable tumors (Mischel et al. 20033DGo). Along similar lines, immunohistochemical = studies have=20 demonstrated that GBM could be stratified according to PI3K = pathway=20 activation status and that these activation profiles are = associated=20 with EGFRvIII expression and PTEN loss (Choe et al. = 20033DGo). Such efforts to stratify patients appear to = be=20 important in the optimal deployment of small molecule EGFR = inhibitors=20 as only a small fraction of GBM patients show meaningful = responses to=20 such agents (Rich et al. 20043DGo;=20 Lassman et al. 20053DGo). Thus far, in responsive cases, patients with = coexpression=20 of EGFRvIII (Mellinghoff et al. 20053DGo)=20 or wild-type EGFR (Haas-Kogan et al. 20053DGo), together with PTEN presence or low Akt activation = levels in their GBM cells, exhibited the most favorable = outcomes=20 to EGFR inhibitors. In accordance with findings of multiple=20 activated pathways in GBM, addition of the mTOR inhibitor, = rapamycin,=20 has been shown to enhance the sensitivity of PTEN-deficient=20 tumor cells to the EGFR kinase inhibitor, erlotinib (Fan et = al.=20 2003; Goudar et al. 20053DGo;=20 Wang et al. 20063DGo). Consistent with enhanced apoptosis = resistance by=20 EGFRvIII, activated EGFR has also been shown to confer radio- = and=20 chemo-resistance to GBM cells (Nagane et al. 19983DGo;=20 Chakravarti et al. 20023DGo). These experimental observations and the = capacity of=20 EGFR inhibitors or dominant-negative EGFR-CD533 to sensitize = GBM=20 cells to radiation and chemotherapeutic agents (Nagane et al. = 20013DGo;=20 Stea et al. 20033DGo;=20 Lammering et al. 20043DGo;=20 Sarkaria et al. 20063DGo)=20 predict that disruption of EGFR function at the time of = ionizing=20 radiation and subsequent chemotherapy, instead of at the time = of=20 recurrence, would improve therapeutic outcome (Nyati et al. = 20063DGo). These results, coupled with the recent = identification=20 of EGFR-activating ectodomain mutations in 3D~=2014% of GBMs = that convey=20 sensitivity toward erlotinib (Lee et al. 20063DGo), are beginning to detail tumor molecular = profiles and=20 therapeutic regimens that will best benefit patients with EGF = receptor and downstream pathway genetic lesions.

PDGF receptor (PDGFR)
In addition = to the=20 EGFR signaling axis, PDGFR3D{alpha}=20 and its = ligands,=20 PDGF-A and PDGF-B, are expressed in gliomas, particularly in=20 high-grade tumors, while strong expression of PDGFR3Dbeta occurs = in=20 proliferating endothelial cells in GBM (Hermanson et al. 19923DGo; Plate et al. 19923DGo;=20 Westermark et al. 19953DGo;=20 Di Rocco et al. 19983DGo). PDGF-C and PDGF-D, which require = proteolytic cleavage=20 for activity, are also frequently expressed in glioma cell = lines and=20 in GBM tissues (Lokker et al. 20023DGo). In contrast to EGFR; amplification = or=20 rearrangement of PDGFR3D{alpha}=20 is much = less=20 common, and a relatively rare oncogenic deletion mutation of=20 PDGFR3D{alpha} (loss of exons 8 and 9) has been described = (Clarke and=20 Dirks 20033DGo)=20 that, similar to EGFRvIII, is constitutively active and = enhances=20 tumorigenicity. Given the tumoral coexpression of PDGF and = PDGFR,=20 autocrine and paracrine loops may be the primary means by = which this=20 growth factor axis exerts its effects. Supportive evidence = for a=20 paracrine circuitry initiated by PDGF-B secretion that = enhances=20 glioma angiogenesis has been shown through stimulation of=20 endothelial cells displaying PDGFR3Dbeta=20, in part, to = express=20 VEGF (Guo et al. 20033DGo). Besides glial precursor cells, NSCs in the adult=20 subventricular zone have been shown to express PDGFR3D{alpha}=20 and PDGF = could=20 stimulate these NSCs to form glioma-like lesions in the mouse = (Jackson et al. 20063DGo). Furthermore, mice transgenic for neural = progenitor=20 PDGF-B expression resulted in the formation of = oligodendrogliomas=20 and forced elevation of PDGF-B levels increased overall tumor = incidence (Dai et al. 20013DGo;=20 Shih et al. 20043DGo), suggesting that targeted therapy against = this pathway=20 could have therapeutic potential (Shih and Holland 20063DGo). To this end, an orally active kinase = inhibitor of the=20 2-phenylaminopyrimidine class such as STI571 (imatinib = mesylate,=20 Gleevec) has been shown to be a potent inhibitor of these = oncogenic=20 loops (Kilic et al. 20003DGo;=20 Hagerstrand et al. 20063DGo)=20 and, when combined with hydroxyurea in a phase II study, has = been=20 shown to achieve durable anti-tumor activity in some patients = with=20 recurrent GBM (Reardon et al. 20053DGo); in contrast, when used alone, imatinib has=20 demonstrated minimal activity in malignant glioma (see below; = Table = 1; Wen=20 et al. 20063DGo).


View = this=20 table:
[in=20 this window]
[in a new window]

  		Table 1. = Inhibitors being=20 used in clinical trials and their targets =

 = ;
RTK coactivation and cooperation
One = additional=20 potential explanation for the failure of EGFR and PDGFR = inhibitors to=20 elicit significant clinical outcomes is that additional RTKs = may=20 cooperate to provide a signaling threshold that prevents the=20 inhibition of mitogenic and survival signals through the = inactivation=20 of any single RTK. This hypothesis is supported by recent = work that=20 demonstrates that multiple RTKs in addition to EGFR and PDGFR = are=20 activated simultaneously in primary GBM patient samples = (Stommel et=20 al. 20073DGo), and oncogenic signaling, survival, and=20 anchorage-independent growth were not fully abrogated until = cell=20 lines with endogenous coactivation of RTKs were treated with=20 pharmacological agents or siRNAs targeting at least three = different=20 receptors. Importantly, these effects were observed = irrespective of=20 PTEN status, indicating that the presence of this = tumor=20 suppressor may not be a critical determinant of therapeutic = success=20 as long as upstream signaling effectors are sufficiently = inhibited.=20 The discovery of receptor coactivation or cooperation = suggests that=20 tumor RTK profiling may be an important step in the = development of a=20 personalized GBM therapeutic regimen. Another study (Huang et = al.=20 2007) showed that glioma cells engineered to = overexpress=20 EGFRvIII to levels observed in GBM caused increased c-MET=20 phosphorylation that was dependent on the kinase activity and = levels=20 of this mutant EGFR. The cross-talk between the receptors = could be=20 targeted with specific inhibitors to both, resulting in = enhanced=20 cytotoxicity of EGFRvIII-expressing cells compared with = either=20 compound alone. It appears that the initially disappointing = clinical=20 trials using RTK-targeted agents in GBM should be reanalyzed = with=20 respect to the RTK activation profiles of the responders and=20 nonresponders, and that future trials could take RTK coactivation=20 into account when selecting combination inhibitor regimens. =


Apoptosis

A hallmark feature of malignant glioma cells is an intense = resistance=20 to death-inducing stimuli such as radiotherapy and = chemotherapy.=20 This biological property has been linked to genetic = alterations=20 of key regulatory molecules involved in mitogenic signaling,=20 most prominently RTKs and the PI3K=96PTEN=96Akt signaling = axis, as=20 well as regulatory and effector molecules residing in = classical cell=20 death networks of both extrinsic (death receptor-mediated) = and=20 intrinsic (mitochondria-dependent) apoptosis signaling pathways. =

The "death receptors" are cell surface molecules that, upon=20 binding their cognate ligands, recruit adapter molecules to=20 provide a molecular scaffold for the autoproteolytic = processing=20 and activation of caspases (for review, see Lavrik et al. 20053DGo). The most important death receptor systems = include=20 TNFR1 (DR1/CD120a), TRAILR1 (DR4/APO-2), TRAILR2 = (DR5/KILLER/TRICK2),=20 and CD95 (DR2/Fas/APO-1). Several lines of evidence support = important=20 roles of these death receptors in glioma pathogenesis. First, = various=20 human glioma cell lines and primary glioma-derived cell = cultures are=20 sensitive to death ligand-mediated apoptosis in vitro and in=20 xenograft model systems in vivo (Weller et al. 19943DGo;=20 Roth et al. 19973DGo; Shinoura et al. 19983DGo;=20 Nagane et al. 20003DGo;=20 Maleniak et al. 20013DGo; Rohn et al. 20013DGo). Second, expression levels of these death = receptors=20 and in particular of their corresponding (antagonistic) decoy = receptors may correlate with susceptibility of glioma cells = to death=20 ligand-induced apoptosis. A prominent example is the decoy = receptor=20 for CD95 ligand (CD95L), soluble decoy receptor 3 (DcR3). It = is=20 expressed on malignant glioma cell lines, and its expression = pattern=20 correlates with the grade of malignancy in human glioma = specimens=20 (Roth et al. 20013DGo). Interestingly, infiltration of = CD4+ and=20 CD8+ T cells and microglia/macrophages was = significantly=20 decreased in DcR3-driven xenografts, suggesting that glioma = cells may=20 escape CD95L-dependent immune-cytotoxic attack by expressing = a decoy=20 receptor that neutralizes CD95L by preventing its interaction = with=20 the receptor (Roth et al. 20013DGo).

The TRAIL death receptor system in particular has gained = considerable=20 interest as a specific inducer of cancer cell apoptosis as = its=20 expression has been positively correlated with survival of = patients=20 with primary GBM (Kuijlen et al. 20063DGo). In this regard, loco-regional = administration of TRAIL=20 inhibited growth of human glioma cell xenografts (Roth et al. = 19993DGo)=20 and acted synergistically with chemotherapeutic drugs (Nagane = et al.=20 20003DGo; Rohn et al. 20013DGo), in part through up-regulation of TRAIL-R2 = and Bak=20 protein and down-regulation of the caspase-8-specific = inhibitor=20 cFLIPs (LeBlanc et al. 20023DGo;=20 Arizono et al. 20033DGo;=20 J.H. Song et al. 20033DGo). In addition, peptides derived from the = second=20 mitochondria-derived activator of caspases (Smac), a potent=20 antagonist of members of the IAP family of caspase = inhibitors, acted=20 synergistically with TRAIL to induce tumor cell apoptosis in = vitro=20 and in vivo without demonstrable neurotoxicity (Fulda et al. = 20023DGo). Mechanistically, these peptides abrogate = IAP-binding=20 activity and, consequently inhibition of effector caspase-9,=20 caspase-3, and caspase-7 activity downstream from = mitochondrial=20 membrane disintegration, underscoring the importance of=20 post-mitochondrial caspase activation for apoptosis = propagation in=20 glioma cell lines and its validity as a therapeutic target = (Fulda et=20 al. 2002).

The role of the Bcl-2 family in gliomagenesis has also been=20 extensively studied. On the mechanistic level, classical=20 anti-apoptotic Bcl-2 family members (BAK, BAD, BID, BAX,=20 BCL-XL, MCL-1) modulate apoptosis signaling by = preserving=20 mitochondrial membrane integrity and the release of = cytochrome c,=20 which effects the caspase cascade and the apoptotic program = (for=20 review, see Green and Kroemer 20043DGo). On the clinical level, there is a correlation = between=20 tumor grade and expression of several anti-apoptotic Bcl-2 = proteins=20 (BCL-2 and MCL-1) (Weller et al. 19953DGo;=20 Krajewski et al. 19973DGo), and in general, this Bcl-2 "rheostat" is = shifted=20 toward an anti-apoptotic balance during the transition from = initial=20 to recurrent GBM (Strik et al. 19993DGo). Additionally, Bcl-xL is up-regulated = by=20 overexpression of EGFRvIII in glioma cells and this = up-regulation=20 confers resistance to the chemotherapeutic agent cisplatin = (Nagane=20 et al. 19983DGo). In addition to their classical roles, Bcl2 family = members may contribute to gliomagenesis through enhancement = of=20 migration and invasion by altering the expression of a set of = metaloproteinases and their inhibitors (Wick et al. 19983DGo, 20013DGo,=20 20043DGo). Due to their central role and importance in = apoptosis=20 signaling, neutralization of anti-apoptotic Bcl-2 proteins by = antisense technology (Julien et al. 20003DGo), small molecules that block BcL2 = interactions with=20 other families (Fesik 20053DGo), or by viral-mediated delivery of select = proapoptotic=20 members (Naumann et al. 20033DGo), may represent promising future avenues of = therapeutic=20 intervention.


Necrosis

While highly resistant to therapeutic apoptotic stimuli, GBM = tumor=20 cells exhibit the paradoxical propensity for extensive = cellular=20 necrosis. Indeed, necrosis is the most prominent form of = spontaneous=20 cell death in GBM, presented as foci of micronecrosis = surrounded by=20 broad hypercellular zones contiguous with normal tissue or by = parenchymal infiltrates (Raza et al. 20023DGo;=20 Brat and Van Meir 20043DGo). While limited blood supply and anoxia due = to a=20 microthrombotic process has been identified as an important = cause of=20 necrosis, the molecular basis for this necrotic phenotype,=20 particularly in the context of intense apoptotic therapy = resistance,=20 has recently come into focus with the discovery and = characterization=20 of the Bcl2-like 12 (Bcl2L12) protein.

Bcl2L12 has been shown to be a potent inhibitor of = post-mitochondrial=20 apoptosis signal transduction that is significantly = overexpressed=20 in primary GBMs (Stegh et al. 20073DGo). Bcl2L12 is a proline-rich protein = characterized by a=20 C-terminal 14-amino-acid sequence with significant homology = with the=20 BH (Bcl-2 Homology) 2 domain found in several members of the = Bcl-2=20 protein family (Scorilas et al. 20013DGo). Enforced expression of Bcl2L12 in primary = cortical=20 astrocytes inhibited apoptosis, and its RNAi-mediated = knockdown=20 sensitizes human glioma cell lines to drug-induced apoptosis=20 and reduces tumor formation in an orthotopic transplant model = in vivo (Stegh et al. 20073DGo). The anti-apoptotic actions of Bcl2L12 = relate=20 significantly to its capacity to neutralize effector caspase = activity=20 downstream from mitochondrial dysfunction and apoptosome = activity,=20 likely through specific interaction with effector caspase-7 = (Stegh et=20 al. 20073DGo). These activities of Bcl2L12 are highly = relevant to=20 the necrotic process in the light of studies showing that = suppression=20 of caspase activity downstream from mitochondria redirects = the death=20 program from apoptosis to necrosis (for review, see Nicotera = and=20 Melino 20043DGo), indicating that post-mitochondrial caspase = activation=20 acts as a molecular switch between apoptotic and necrotic = cell death=20 paradigms (for review, see Nicotera and Melino 20043DGo).

In support of this model, germline deletion of = post-mitochondrial=20 apoptosis signaling components, such as the caspase activator = Apaf-1, or blockage of effector caspase maturation by = pan-specific=20 caspase inhibitors results in decreased apoptosis, yet causes = an increase in necrosis (for review, see Nicotera and Melino=20 20043DGo). Mechanistically, oxidative phosphorylation and = consequently=20 intracellular ATP levels decrease due to extensive cytochrome = c=20 release and mitochondrial dysfunction, rendering cells unable = to=20 maintain ion homeostasis and provoking cellular edema, dissolution = of=20 organelles, and plasma membranes (for review, see Nicotera = and Melino=20 20043DGo). That apoptosis and necrosis signaling pathways = are=20 interconnected is evidenced by the ability of enforced Bcl2L12=20 expression to provoke necrotic cell morphology, as evidenced = by=20 substantial plasma membrane disintegration and enhanced nuclear = and=20 subcellular organelle swelling in apoptosis-primed astrocytes = (Stegh=20 et al. 20073DGo). Therefore, up-regulation of Bcl2L12 as a = novel=20 regulator of the apoptosis/necrosis balance in glial cells = may=20 represent an important event in malignant glioma pathogenesis. =


Angiogenesis

GBMs are among the most highly vascular of all solid tumors.=20 Microvascular hyperplasia, the defining histopathological = phenotype=20 of both primary and secondary GBM, consists of proliferating=20 endothelial cells that emerge from normal parent microvessels = as tufted microaggregates (glomeruloid bodies) accompanied by = stromal elements, including pericytes and basal lamina = (Stiver=20 et al. 20043DGo). Microvascular density, a measure of microvascular = proliferation, is an independent prognostic factor for adult=20 gliomas (Leon et al. 19963DGo;=20 Birlik et al. 20063DGo). The idea that angiogenesis is rate limiting = for tumor=20 growth, and therefore a rational therapeutic target, is = strongly=20 supported by animal studies that have shown that angiogenesis = is=20 vital for macroscopic solid tumor growth (Folkman 20073DGo).

One common feature in the transition from low-grade or = anaplastic=20 astrocytomas to secondary GBM is a dramatic increase in = microvascular=20 proliferation. An equivalently robust microvasculature = proliferation=20 phenotype is observed in primary GBM. Since there are marked=20 genomic differences between primary and secondary GBM (Maher = et=20 al. 20063DGo), it is likely that different genetic programs = converge=20 on a final common angiogenesis pathway involving HIF and=20 non-HIF-dependent downstream effectors that include positive = (VEGF,=20 PDGF, bFGF,IL-8, SDF-1) and negative (thrombospondin1,=20 thrombospondin2, endostatin, tumstatin, interferons) = regulators of=20 this process (Nyberg et al. 20053DGo). A comprehensive understanding of the molecular = mechanisms=20 driving angiogenesis in GBM will be necessary for the = rational=20 development and deployment of anti-angiogenesis therapies.=20 Increasingly, it is becoming evident that tumor-associated=20 angiogenesis is not simply a physiological adaptation to = hypoxia as a=20 result of an increasing tumor cell mass. Rather it appears to = be=20 the result of critical genetic mutations that activate a=20 transcriptional program for angiogenesis with local tumor = oxygen=20 status further modifying this response. The relative = contributions of=20 these two mechanisms are not yet fully defined, but it is = likely=20 that both may operate to different extents in different = tumors=20 or even in different regions of the same tumor. Recently, a=20 number of experimental studies have shown that key=20 glioma-relevant mutations=97including those in the PTEN, = EGFR;=20 and CMYC genes=97may act as an "angiogenic switch" by=20 stabilizing HIF-13D{alpha}=20 or one of = its=20 downstream targets, VEGF (Watnick et al. 20033DGo;=20 Blum et al. 20053DGo;=20 Phung et al. 20063DGo;=20 Shchors et al. 20063DGo). The distinction between microvascular = proliferation=20 being an adaptive response to hypoxia or it being an = epiphenomenon=20 of critical genetic mutations that also activate a cascade of = proangiogenesis pathways has clinical and therapeutic=20 importance.

Another issue is the functional consequences of tumor = angiogenesis,=20 with respect to tissue perfusion (Vogel et al. 20043DGo). Tumor microvessels are highly tortuous with = sluggish=20 flow and diminished gradient for oxygen delivery and = increasing=20 susceptibility to thrombosis and microhemorrhages (Kaur et = al. 20043DGo). Thus, the GBM microvasculature = proliferation may=20 provide little support in oxygen/nutrient delivery but rather = paradoxically contribute to further exacerbating a metabolic = mismatch=20 between the "supply and demand," leading to progressive = hypoxia and=20 eventually necrosis. This scenario is supported by the recent = experience with anti-angiogenesis drugs, where their limited = clinical=20 benefit seems to be the result of "pruning" immature vessel = growth=20 and allowing "normalization" of the pre-existing vasculature = (see=20 below; Horsman and Siemann 20063DGo). In addition to the poor vascular architecture, = endothelial=20 cells associated with the tumor vasculature fail to form = tight=20 junctions and have few associated pericytes or astrocytic = foot=20 processes leaving the integrity of the BBB compromised, = resulting=20 in increased interstitial edema. Interstitial edema may = further=20 compromise regional blood flow and exacerbate tumor hypoxia=20 leading to areas of necrosis. In addition to these maladapted = biophysical properties of GBM microvasculature, specific = genetic=20 mutations in GBM likely contribute to compromised tumor=20 bioenergetics, specifically the shift in energy reduction = from=20 oxidative phosphorylation to glycolysis (Elstrom et al. = 20043DGo;=20 Fantin et al. 20063DGo). These interrelated mechanisms lead to a = level of=20 metabolic demand that may exceed the ability of the = cerebrovascular=20 system to maintain adequate blood flow to prevent hypoxia and = necrosis. The histological evidence of thrombosis and = degenerating=20 vessels with microhemorrhages are a common feature of GBM and = likely reflect these biological processes.

Anti-angiogenesis = therapies
The=20 hypothesis that interruption of blood supply to the tumor = will lead=20 to regression or dormancy of the tumor has led to the = development of=20 several drugs that target multiple steps in angiogenesis (Table = 1; Fig. = 2).=20 Currently three approaches are in advanced stages of clinical = testing=20 that aim to target VEGF/VEGFR signaling pathways: (1) = monoclonal=20 antibodies directed against VEGF or its receptor(s) (Winkler = et al.=20 20043DGo; Vredenburgh et al. 20073DGo), (2) small molecule inhibitors of VEGFR-2 tyrosine = kinase activity (Batchelor et al. 20073DGo), and (3) soluble decoy receptors created = from VEFGR1=20 receptor that selectively inhibits VEGF (Folkman 20073DGo). A fourth approach targeting 3D{alpha}=20V3Dbeta=203 and 3D{alpha}=20V3Dbeta=205 = integrin=20 receptors on endothelial cells (Nabors et al. 20073DGo)=20 is also in early clinical trials as an anti-angiogenesis = therapy=20 in GBM.

Clinical studies, in which anti-angiogenesis drugs have been = used=20 as "single" agents to treat GBM, have shown little efficacy. = This may=20 reflect the fact that these drugs have no direct effect on = the=20 pre-existing stable microvasculature that may be co-opted to = support=20 tumor growth especially at the infiltrating tumor edge. = Recent data,=20 however, suggest that anti-angiogenesis drugs may be more = effective=20 when combined with cytotoxic therapy (Table=20 1). Recently a single-arm phase II study of bevacizumab=20 (Avastin; Genetech, Inc.) (Vredenburgh et al. 20073DGo), a recombinant, humanized monoclonal = antibody=20 targeting VEGF, plus irinotecan (CPT-11) in patients with = recurrent=20 high-grade gliomas reported dramatic rates (63%) of = radiographic=20 response and a near doubling of 6 mo and median progression = free=20 survival (PFS) in the patients with GBM (30% and 20 wk, = compared with=20 historical controls of 15% and 9 wk). The therapeutic = benefits in the=20 setting of combination therapy (radiation and/or conventional = chemotherapy) could be attributed to (1) improved drug = delivery=20 because of improved vascular flow, (2) improved drug = penetration into=20 the tumor because of reduced interstitial pressure, and/or = (3)=20 improved radiation/chemotherapy response as a result of = reducing=20 tumor hypoxia. Hypoxia is well known to create radiation=20 resistance and reduce efficacy of chemotherapies (Semenza = 20033DGo). Overall, the early clinical data for the=20 anti-angiogenic drugs when used in combination with radiation = or=20 conventional chemotherapies is encouraging. The possibility = that=20 anti-angiogenic drugs may enhance intratumoral concentration = of=20 conventional chemotherapeutics raises the intriguing = possibility that=20 these drugs may improve the efficacy profile of some of the = currently=20 available drugs. A possible mechanism for such synergy could = be=20 enhanced drug delivery, although off-target drug effects = and/or=20 poorly understood pharmacological mechanisms remain = possibilities.=20 The full benefit of anti-angiogenesis will derive from an = improved=20 understanding of the molecular basis of tumor angiogenesis = process,=20 how tumor cell metabolism drives angiogenesis versus = cooptation of=20 normal brain microvascular networks, and definition of those=20 patients that are likely to benefit from various types of=20 anti-angiogenic therapies operating on different levels of = the=20 process.


Tumor cell invasion

Infiltration throughout the brain is prominent feature of low- = and=20 high-grade malignant glioma (Lefranc et al. 20053DGo)=20 and is the principal basis for the lack of surgical cure. In=20 >90% of cases, the recurrent tumor develops immediately=20 adjacent to the resection margin or within several = centimeters of=20 the resection cavity. Invasion by glioma cells into regions = of=20 normal brain is driven by a multifactorial process involving = cell=20 interactions with the ECM and with adjacent cells, as well as = accompanying biochemical processes supportive of proteolytic=20 degradation of ECM and active cell movement. These processes = bear a=20 striking resemblance to the robust inherent migration = potential of=20 glial cells during embryogenesis (Hatten 19993DGo).

The most frequent route of invasion of glial tumor cells is = along=20 white matter tracts and basement membranes of blood vessels. = Whether=20 this route offers a path of least resistance or there are = biochemical=20 substrates that mediate adhesion and promote migration, or = both, is=20 unclear. Invasion and migration of glial tumors differs from = other=20 tumors where local spread is very limited and dissemination = occurs=20 hematogenously or via the lymphatic system. In fact, glioma = cells=20 lack the ability to penetrate the basement membrane of blood = vessels=20 (Bernstein and Woodard 19953DGo), and cells gaining access to the blood through a = disrupted=20 blood vessel within the tumor are unable to establish robust=20 tumor growth outside the CNS. The molecular basis for this = curious=20 inability of glioma cells to metastasize outside of the CNS = is=20 not known and warrants further investigation.

Several genes involved in glioma invasiveness have been = identified=20 and include members of the family of metalloproteases (MMP) = and=20 their endogenous tissue inhibitors (TIMPs). Expression of = MMP-2 and,=20 to a lesser extent, MMP-9 correlate with invasiveness, = proliferation=20 and prognosis in astrocytomas (M. Wang et al. 20033DGo). Other non-MMP proteases, including urokinase-type=20 plasminogen activator (uPA) (Landau et al. 19943DGo;=20 Yamamoto et al. 1994a3DGo, b3DGo)=20 and cysteine proteases (e.g., cathepsin B) (McCormick 19933DGo), are elevated in high-grade malignant = gliomas (for=20 review, see Uhm et al. 19973DGo). Despite these findings, the role of proteases = in=20 glioma invasion remains unclear since low-grade astrocytomas=20 infiltrate diffusely throughout the brain, despite relatively = normal levels of the proteases.

Integrins, especially 3D{alpha}=20V3Dbeta=203 complexes, = are elevated=20 in GBM and appear to be relevant to processes of glioma = invasion and=20 angiogenesis (Kanamori et al. 20043DGo). Several studies have also reported potential = novel=20 glioma invasion genes. Invasion inhibitory protein 45 = (IIp45),=20 a potential tumor suppressor gene on chromosome 1p36,=20 is frequently down-regulated in GBMs. Its product inhibits = invasion=20 through the binding of IGFBP2 (S.W. Song et al. 20033DGo). In contrast, IGFBP2 promotes invasion in = GBM by=20 up-regulating a panel of genes involved in invasion, one of = which is=20 MMP-2 (H. Wang et al. 20033DGo). Other proteins are overexpressed in invasive = areas of=20 GBM, such as angiopoietin-2, which in addition to its involvement = in=20 angiogenesis also plays a role in inducing tumor cell infiltration = by=20 activating MMP-2 (Hu et al. 20033DGo). Ephrin receptors and their ligands, the = ephrins,=20 mediate neurodevelopmental processes such as axon guidance = and cell=20 migration and in glioma have been shown to regulate migration = and=20 invasion. Compared with low-grade astrocytoma or normal = brain, GBMs,=20 in particular the migratory tumor cells, overexpress EphB2 = (Hu et al.=20 2003). Intriguingly, EphA2 overexpression has been = linked to=20 poor survival in GBM (Liu et al. 20063DGo).

Other novel invasion- and migration-associated genes have been=20 identified using oligonucleotide microarray technology = (Demuth=20 and Berens 20043DGo;=20 Tatenhorst et al. 20043DGo)=20 on RNA isolated by laser-captured microdissection of cryostat = sections from human glioma biopsy tumor cores and invasive = edges.=20 These genes include P311; a 68-amino-acid polypeptide = that has=20 been described in embryonic neuronal migration (Studler et = al. 19933DGo); death-associated protein 3 (DAP3), = which has=20 been shown to confer protection from Fas-induced, ionizing=20 radiation-induced, and streptonigrin-induced cell death = (Kissil et=20 al. 19993DGo); and FN14; which encodes a cell = surface=20 receptor for the tumor necrosis factor superfamily member = named=20 TWEAK, all of which have functionally been shown to modulate = glioma=20 cell migration and apoptosis (Taylor et al. 20003DGo;=20 Mariani et al. 20013DGo;=20 Wiley and Winkles 20033DGo).

Since migrating glioma cells show increased levels of = phosphorylated=20 Akt, PI3K inhibitors have been tested experimentally on these = cells, resulting in a decrease in migration and an increase = in=20 apoptosis sensitivity (Joy et al. 20033DGo). In conjunction with this, PTEN = mutation has=20 been implicated in an invasive phenotype, not only as = contributing to=20 deregulated PI3K signaling but also in its ability to = stabilize=20 E-cadherin and modulate cell matrix adhesion complexes = (Kotelevets et=20 al. 20013DGo). These findings highlight the multitude of = ways that=20 gliomagenic lesions effect a broad spectrum of the tumor = phenotypes=20 ranging from aberrant cell proliferation to invasion and = resistance=20 to apoptosis.


3D"   =20 Frontiers in glioma research and therapy =
Top
Abstract
3D"Classification and grading of...
3D"Tumor biological processes and...
3D"Frontiers in glioma = research...
Acknowledgments
3D"References=20
 

Genomic = profiles of=20 GBM

Copy number = analysis
Comparative=20 genomic hybridization (CGH) analysis of astrocytic tumors has = revealed numerous recurrent copy number alterations (CNAs), = pointing=20 to the existence of many additional oncogenes or tumor = suppressor=20 genes beyond the handful of classical GBM mutation targets = described=20 in the previous sections. Conventional and array-based CGH = (aCGH)=20 profiling have cataloged the multitude of recurrent CNAs, = including=20 gains/amplifications of 1p34-36, 1q32, 3q26-28, 5q, 7q31, = 8q24, 11q,=20 12q13, 13q, 15p15, 17q22-25, 19q, 20p, and 20q and = losses/deletions=20 of 3q25-26, 4q, 6q26-27, 9p, 10p, 10q, 11p, 11q, 12q22, 13q, = 14q13,=20 14q23-31, 15q13-21, 17p11-13, 18q22-23, 19q, and 22q = (Reifenberger et=20 al. 19943DGo; Nishizaki et al. 20003DGo;=20 Hui et al. 20013DGo;=20 Burton et al. 20023DGo; Nutt et al. 20033DGo;=20 Misra et al. 20053DGo;=20 Nigro et al. 20053DGo;=20 Phillips et al. 20063DGo). These high-resolution studies also revealed new=20 molecular markers of glioma that complement and extend = histological=20 (astrocytoma, oligodendroglioma, and GBM) (Kotliarov et al.=20 20063DGo)=20 and tumor grade classifiers (Nishizaki et al. 20003DGo). This is particularly evident for primary = and=20 secondary GBMs, which are histopathologically = indistinguishable yet=20 show dramatically different patterns with the majority of = recurrent=20 CNAs being unique, rather than overlapping between the two = entities.=20 Analysis of these patterns using an unsupervised = classification=20 algorithm, termed genomic nonnegative matrix factorization = (gNMF),=20 showed that primary and secondary GBMs segregate distinctly = into=20 two classes, and that secondary GBM can be further stratified = into two subgroups with different times to progression from=20 low-grade to secondary GBM (Maher et al. 20063DGo). Some of the recurrent genomic alterations = have been=20 shown to be prognostic=97loss of 6q or 10q or gain of 19q is = associated=20 with shorter survival, while loss of 19q tracks with = long-term=20 survival (>3 yr) (Burton et al. 20023DGo). Current efforts are now directed toward = identifying=20 the clinically relevant genes residing in these = loci=97efforts strongly=20 motivated by the discovery of molecular signatures of drug = response=20 in the clinic (Haas-Kogan et al. 20053DGo;=20 Hegi et al. 20053DGo;=20 Mellinghoff et al. 20053DGo).

Transcriptional = profiling
Gene=20 expression profiling has proven to be a highly effective = method to=20 obtain global signatures reflecting the biological state of = the tumor=20 and underlying pathogenic mechanisms and providing markers = for use in=20 diagnosis and clinical management. Initial applications of=20 transcriptional profiling to GBM confirmed that defined gene=20 signatures could be used to classify different histological = grades=20 (Rickman et al. 20013DGo;=20 Godard et al. 20033DGo; van den Boom et al. 20033DGo). Indeed, among nonclassical lesions, = classification by=20 gene expression signatures more accurately predicted survival = than=20 standard pathological evaluation (Nutt et al. 20033DGo). More recently, even among histologically = indistinguishable=20 GBMs, expression profiling was able to classify GBM into = subgroups=20 with different overall survival. Although further validation=20 studies are needed to confirm that these signatures can be = used=20 prospectively, these studies suggest that gene expression = profiling=20 represents a useful approach in classifying categorize GBM = (Liang=20 et al. 20053DGo).

In addition, given the observed intratumoral heterogeneity in = GBM,=20 these studies have provided important biological insights = into the=20 pathogenesis of GBM. When histologically distinct lesions = from the=20 same patient were compared, the gene signatures from these = lesions=20 more closely resembled each other than lesions from other = patients,=20 suggesting that they arise from a common precursor and share = a common=20 molecular life history (Liang et al. 20053DGo). Such analyses have implicated angiogenesis, = immune=20 cell infiltration, and extracellular remodeling as drivers of = differences between tumor subtypes (Godard et al. 20033DGo;=20 Liang et al. 20053DGo). In a few cases, these studies have facilitated = the=20 identification of specific genes that predict survival, such = as=20 FABP7, DLL; and ASPM (Liang et al. 20053DGo;=20 Horvath et al. 20063DGo;=20 Phillips et al. 20063DGo)=20 and permit one to predict the clinical response to EGFR = kinase=20 inhibitors (Haas-Kogan et al. 20053DGo;=20 Mellinghoff et al. 20053DGo). These studies suggest that further = characterization,=20 validation, and application of this technology will provide = improved=20 metrics for prognostication and choice of therapy. =


Current status of targeted therapy in GBM

While surgery remains the primary intervention, several = early-phase=20 clinical trials of targeted therapies in high-grade glioma = have=20 been completed or are underway either singly or in = combination=20 with standard chemotherapy and/or radiation therapy (for a = detailed=20 review, see Sathornsumetee et al. 20073DGo=20 and references therein). A listing of such agents is = presented in Table = 1=20 (compiled from http://www.clinicaltrials.gov/). The=20 compendium of GBM agents reflects the prevalence of = alterations in=20 EGFR; such as the EGFRvIII deletion mutation = (Scott et=20 al. 20073DGo)=20 and PDGF signaling and modulators of PI3K signaling, as well = as the=20 prominence of biological processes such as angiogenesis and = invasion.=20 Clinical outcomes in these trials are often difficult to = interpret=20 and are best considered in the context of standard therapy. = The=20 mainstay of initial treatment for GBM has changed little over = the last 25 yr and is based primarily on external beam = radiation=20 delivered conformally to the tumor volume, now commonly = determined=20 by both MRI contrast-enhancement and surrounding T2 signal=20 hyperintensity (Walker et al. 19783DGo,=20 19803DGo). In conjunction with surgery and medical = management,=20 radiation therapy doubles median survival to 12 mo and = extends 2-yr=20 survival to 10%, with little added benefit from conventional=20 chemotherapies (Shapiro et al. 19893DGo; Fine et al. 19933DGo;=20 DeAngelis et al. 19983DGo;=20 Stewart 20023DGo). More recently, a notable randomized = prospective study=20 demonstrated the first clear survival benefit for = chemotherapy in the=20 treatment of GBM: The oral alkylating agent temozolomide = (TMZ),=20 given concurrently with radiation and continued thereafter, = was=20 found to extend median survival to 15 mo and 2-yr survival to = 26% (Stupp et al. 20053DGo). Supporting the concept of molecularly informed=20 clinical management, further analysis revealed that the = survival=20 benefit was largely restricted to those patients whose tumors = showed epigenetic silencing of the DNA repair gene MGMT:=20 Median survival was extended to nearly 2 yr when MGMT = was=20 methylated, whereas little benefit was seen in patients with = tumors=20 expressing MGMT (Hegi et al. 20053DGo). It is tempting to speculate that MGMT-mediated = DNA=20 repair may itself be considered a potentially valuable therapeutic=20 target for the 50% of patients that express the MGMT gene=20 (Hegi et al. 20063DGo).

Clinical trials of single-agent-targeted therapies typically=20 recruit from the molecularly heterogeneous group of patients=20 who have tumor relapse following radiation and other = therapies.=20 The difficulties in interpreting outcomes, whether = radiographic=20 response to treatment or overall survival, are well = documented=20 (Grant et al. 19973DGo). Several studies have established the validity = of 6-mo=20 progression-free survival (PFS6), determined radiographically, = as a=20 meaningful endpoint in defining response to treatment: A PFS6 = of 15%=20 or less has been estimated as a benchmark for inactive = therapy (Wong=20 et al. 19993DGo;=20 Ballman et al. 20073DGo). In comparison, TMZ given at first = recurrence in GBM=20 yields a PFS6 of 21% (Yung et al. 20003DGo). In this challenging patient group, the = initial=20 results of the EGFR inhibitors erlotinib and gefitinib in=20 single-agent trials have shown little activity overall, although = a=20 modest response may be seen in the subset of patients with = intact=20 PTEN (Prados et al. 20033DGo;=20 Rich et al. 20043DGo;=20 Mellinghoff et al. 20053DGo). Interpretation may be complicated by variable = EGFR=20 inhibition in tumors treated by erlotinib and gefitinib, as = measured=20 by abundance of phosphorylated EGFR, Akt, and Erk (Lassman et = al.=20 20053DGo). Phase II single-agent trials for inhibitors = of PDGFR=20 (imatinib), RAS (tipifarnib), and mTor (temsirolimus) have = shown=20 minimal activity overall in GBM as well, although further = analysis of=20 tumor material and clinical parameters from sporadic = responders may=20 indicate prognostic features (Chang et al. 20053DGo;=20 Galanis et al. 20053DGo;=20 Cloughesy et al. 20063DGo;=20 Wen et al. 20063DGo;=20 Franceschi et al. 20073DGo). VEGF/R inhibitors and multitarget tyrosine = kinase=20 inhibitors with anti-VEGFR potency are theoretically = attractive=20 agents for attacking GBM, particularly in combination with = therapies=20 that have direct tumor cytotoxicity. A recent trial of = AZD2171, a=20 multikinase inhibitor with pan-VEGFR, c-Kit, and PDGFR = selectivity,=20 demonstrated primary effects of tumor vasculature = "normalization":=20 namely, a reduction in contrast-enhancing tumor volume and=20 surrounding edema that Batchelor et al. (2007)3DGo were able to link to vessel pruning and = reconstitution=20 of the blood-brain barrier through analysis of perfusion MRI = and=20 model systems. It remains to be seen whether these potent=20 effects on tumor vasculature may be vividly improving the=20 patient=92s MRI without impacting progression of the = underlying tumor.=20 Nonetheless, VEGF inhibition is likely to have a useful role = in=20 combination therapy (Vredenburgh et al. 20073DGo). While targeted inhibitors have shown little = durable=20 effect as monotherapies, their specificity and generally = modest side=20 effect profiles facilitate combination together and with = conventional=20 cytotoxic agents. Table = 1=20 lists currently active clinical trials investigating combined = therapies. Although there is reason to believe that certain=20 combinations will be effective in certain patients, the added = complexity presents a challenge to clinical trial design, = patient=20 stratification and logistics.


Evidence for glioma origins

There is growing evidence that only a minor population of cells = in=20 solid tumors, including primary brain tumors (GBM, medulloblastoma, = and ependymoma), are capable of forming a tumor when = orthotopically=20 transplanted into an immunocompromised mouse (Singh et al. 20043DGo). The concept of the brain CSC (Reya et al. = 20013DGo)=20 is based on the observation that only a small fraction of = primary=20 leukemic (AML) cells are capable of initiating and sustaining = clonogenic growth and inducing leukemia in immunocompromised = mice=20 (Lapidot et al. 19943DGo;=20 Bonnet and Dick 19973DGo). Importantly, these leukemic subclones = shared the same=20 cell surface markers (CD43+, CD38=96) = as "normal"=20 human hematopoietic stem cells (HSCs), while the progeny of = these=20 leukemic clones, the blast cells, often expressed more = differentiated=20 lymphoid or myeloid lineage markers and were not capable of = producing=20 leukemic disease. At present it is unclear whether the CSC = derives=20 from a normal stem cell compartment or from a more = differentiated=20 progenitor that dedifferentiates into a stem cell-like state = is not=20 yet clear. The identification of the "cell of origin" remains = an area=20 of active research for both hematological malignancies = (Passegue et=20 al. 20033DGo)=20 and solid tumors (Al-Hajj et al. 20033DGo;=20 Singh et al. 20043DGo;=20 Sanai et al. 20053DGo;=20 Taylor et al. 20053DGo;=20 Patrawala et al. 20063DGo;=20 Li et al. 20073DGo; O=92Brien et al. 20073DGo;=20 Prince et al. 20073DGo).

The CSC hypothesis was independently proposed for GBM (Singh = et=20 al. 20033DGo)=20 and pediatric gliomas (Hemmati et al. 20033DGo). There were two critical findings from these = studies.=20 First, only a minor population of cells identified in cell = cultures,=20 from a variety of primary CNS tumors (including GBM,=20 medulloblastoma, ganglioglioma, ependymoma, and pilocytic=20 astrocytomas) was able to self-renew and form clonogenic=20 neurospheres. These self-renewing brain tumor cells were = identified=20 (Singh et al. 20033DGo)=20 by the expression of the cell surface marker, = CD133+=20 (1%=9635% of total population). In contrast, the = CD133=96=20 population failed to proliferate and remained as an adherent=20 monolayer and expressed mature lineage-specific markers. = Second,=20 CD133+ tumor neurospheres under NSC culture = conditions=20 expressed the stem cell marker Nestin and, upon exposure to = serum,=20 differentiated into a mixed population of neurons = (Tuj1+),=20 astrocytes (GFAP+), and oligodendrocytes = (PDGFR+), which mirrored the mixed cell types = found in=20 the original patient=92s tumor. These observations provide = support for=20 a hierarchical CSC hypothesis, suggesting that only = CD133+=20 brain tumor cells can self-renew and undergo lineage-specific = differentiation.

Subsequently, substantial enrichment of the tumor-forming = ability=20 of FACS-sorted CD133+ cells (as few as 100 implanted = cells=20 were able to produce orthotopic tumors) following in vitro=20 expansion of these cells was reported (Singh et al. 20043DGo). In contrast, CD133=96 cells = failed to form=20 tumors, even following injection of a much larger cell = innoculum=20 (105 per injection). The orthotopic tumors = mirrored the=20 original tumor heterogeneity, with CD133+ cells = forming a=20 minor fraction and the CD133=96 cells failing to = form tumors=20 on serial transplantation. These data suggest that loss of = CD133=20 expression reflects an "irreversible" loss of cellular = ability to=20 propagate a tumor. Whether CD133+ cells are only = important=20 for tumor initiation and are less critical for tumor = progression will=20 require a genetic strategy, similar to that used to monitor = skin stem=20 cells in vivo using a doxycyline-inducible H2B-eGFP reporter = tag that=20 enabled selection of CD133+ cells over time = (Tumbar et al.=20 20043DGo).

There is now substantial evidence for the enrichment of in vivo=20 cancer-forming ability of CD133+-expressing cells for = GBM=20 (Singh et al. 20043DGo;=20 Bao et al. 2006a3DGo;=20 Piccirillo et al. 20063DGo)=20 and more recently in colon cancer (O=92Brien et al. 20073DGo;=20 Ricci-Vitiani et al. 20073DGo). There are, however, a number of reports that = suggest=20 a less clear distinction between the ability of CD133+ = and=20 CD133=96 cells to form orthotopic tumors (Bao et = al. 2006b3DGo;=20 Sakariassen et al. 20063DGo;=20 Beier et al. 20073DGo;=20 Zheng et al. 20073DGo). For example, it has been reported recently = (Beier et=20 al. 2007) that CD133=96 cells isolated from = primary GBM=20 tumors were equally capable of forming orthotopic tumors as = the=20 CD133+ subpopulation, while under the same = conditions,=20 none of the secondary GBM tumors (zero of seven) produced = viable=20 neurosphere cultures. They also reported that for four out of = 11=20 primary GBM tumors, CD133=96 cells grew as an = adherent=20 monolayer yet were able to produce orthotopic tumors. = Similarly,=20 CD133=96 primary GBM tumor cells, maintained as an = adherent=20 monolayer by addition of serum to stem cell culture media, = were also=20 able to produce highly infiltrative orthotopic tumors = (Sakariassen et=20 al. 20063DGo). These data suggest that even brief ex vivo=20 manipulations may alter the molecular and phenotypic = properties of=20 freshly isolated tumor cells, may complicate the conclusions = that can=20 be drawn from these sorts of experiments, and point at the = need for=20 studies using directly isolated tumor cells from fresh = specimens=20 and immediate implantation into immunocompromised mice. While = the GBM-stem cell idea is in its infancy and many questions=20 remain, its potential for our understanding of tumor = development=20 and therapy design and selection is exciting indeed. =


Genetically engineered models of glioma

There is little debate of the importance of murine models in=20 advancing our understanding of the complex biology of = gliomas.=20 Various types of in vivo model systems have been developed = and=20 utilized, including traditional orthotopic xenotransplants = with=20 established human glioma cell lines and, more recently, with=20 primary human glioma cells enriched for surface expression of = CD133 (Singh et al. 20043DGo). There is great interest in the further = development of=20 the CD133 primary tumor model system as this appears to be = superior=20 in recapitulating well the diffuse infiltrative nature of the = primary=20 human disease. Whether the CD133 primary tumor system will = prove to=20 be a more accurate biological model or be more predictive in = drug=20 testing than xenotransplant models with established cell = lines is an=20 area of significant current investigation.

In recent years, important advances have been made in the = construction=20 of genetically engineered mouse (GEM) models harboring=20 glioma-relevant mutations or combinations of mutations. In = several=20 cases, such GEMs predictably develop gliomas with many of the = features of the human disease (Table = 2;=20 Weissenberger et al. 19973DGo;=20 Uhrbom et al. 19983DGo;=20 Kamijo et al. 19993DGo;=20 Holland et al. 20003DGo;=20 Reilly et al. 20003DGo;=20 Dai et al. 20013DGo;=20 Ding et al. 20013DGo,=20 2003; Rich et al. 20013DGo;=20 Sonoda et al. 20013DGo;=20 Bachoo et al. 20023DGo;=20 Uhrbom et al. 20023DGo;=20 Xiao et al. 20023DGo;=20 Weiss et al. 20033DGo;=20 Holmen and Williams 20053DGo;=20 Zhu et al. 20053DGo;=20 Charest et al. 20063DGo;=20 Tchougounova et al. 20073DGo). Given the experimentally tractable nature of the=20 mouse, these glioma-prone GEM models are beginning to shed = light=20 on a number of key issues such as, for example, the glioma = cell=20 of origin (Zhu et al. 20053DGo), the ordering of mutations and whether such = events=20 underlie various glioma subtypes (Hu et al. 20053DGo), the cooperative and epistatic relationship = of such=20 mutations, and the complex heterotypic interactions between = the=20 evolving tumor cell and the host microenvironment, among = other=20 issues central to the problem of gliomagenesis. With further=20 refinement, there is now increasing evidence that these GEM = model=20 systems will provide an additional vantage with which to test = the=20 timing, dosing, and combination of drugs in the pipeline and=20 assist in the development of drug response biomarkers (Momota = et=20 al. 20053DGo;=20 Xiao et al. 20053DGo).


View = this=20 table:
[in=20 this window]
[in a new window]

  		Table 2. Mouse = and human=20 models of gliomagenesis based on genetic alterations found = in=20 astrocytic glioma =

 = ;
Each=20 of the GEM models in Table = 2=20 offers distinct advantages and limitations for certain types = of=20 experimental inquiry. In particular, these models are ideal = for=20 investigation of biological mechanisms underlying = tumorigenesis and=20 for the functional validation of candidate genes identified = through=20 large-scale genomic analysis of tumor specimens. The need for = accurate models is perhaps most acute in preclinical testing, = where=20 experimental data often determine the fate of a drug in = development.=20 Although additional study is needed, it is widely anticipated = that=20 refined GEM models of glioma should enable the identification = of=20 tumor maintenance genes and the testing of agents targeting = such=20 mission critical lesions, thereby identifying key targets, = the best=20 agent, and the right patient population (i.e., genotype) (for = review,=20 see Sharpless and Depinho 20063DGo). Thus, GEM models may allow for culling of = ineffective=20 drugs and improved clinical trials design for those entering = phase=20 I/II clinical trials. In addition, the availability of = refined GEM=20 models that evolve through stages may help define the tumor = grade=20 where an agent or combination of agents may be most = effective.=20

While current efforts are focused on the development of GEMs=20 harboring signature mutations in human glioma, there remains = a=20 great utility for models engineered with nonstereotypical = lesions=20 that yet capture aspects of human disease behavior and = appearance,=20 including invasion, angiogenesis, necrosis, and tumor=96ECM=20 interactions. Novel therapies developed to block these = biological=20 pathways could be tested in such a model. Similarly, a model = that=20 recapitulates the genetics but lacks several of the clinical = features=20 of the tumor can be valuable. For example, a tumor driven by = PDGF=20 (Dai et al. 20013DGo)=20 could be used to study the downstream targets and the = biological=20 consequences of neutralization of the pathway. Finally, = inducible and=20 conditional models are gaining popularity as ideal systems = for the=20 somatic activation of genes in specific cell populations and = for the=20 assessment of genetic lesions in tumor progression and=20 maintenance.

The recently developed glioma-prone GEM models have been notable = for recapitulating most of the cardinal histological features = of the human disease. That said, a fully accurate genocopy = and=20 phenocopy of the human disease has yet to be developed in = which=20 the most common mutations are engineered, genome instability = is=20 rampant, and orthologous acquired events are documented.=20 Nevertheless, current models have provided important lessons=20 for understanding the nature of gliomas: (1) Loss of a single = tumor suppressor gene or overexpression of an oncogene is=20 insufficient to induce high-grade gliomas with high = penetrance; (2)=20 modifying mutations are important in gliomagenesis; (3)=20 cell-of-origin and the mutations or set of mutations in such = cells=20 plays a significant role in transformation; (4) dysregulating = various family members of a pathway or regulatory machinery = may=20 have similar biological consequences; and (5) the mutation or = combination of mutations has stark effects on a given state = of=20 differentiation.

Thus, while further refinement is needed, these GEM models have=20 afforded opportunities to better understand many enigmatic = aspects=20 of human glioma development and therapy. Given the wealth of=20 new data anticipated from The Cancer Genome Atlas (TCGA) = (Hanauer=20 et al. 20073DGo), for which GBM is one of the select cancer types = to be=20 analyzed, a key challenge will be to assign the plethora of = newly=20 discovered cancer-associated genetic alterations with cancer=20 relevance. Here, mouse models can serve two key roles: First, = they=20 can be used in comparative oncogenomics to identify = loci/genes that=20 are commonly targeted in cancer development across evolution, = and=20 second, they can serve as relevant model systems to validate = genes as=20 well as determine whether new genes cooperate (or not) with=20 specifically engineered mutations=97ultimately allowing for = the=20 placement of genetic lesions into certain pathways and the = testing of=20 drugs targeting these activities.


Future directions

The progress and depth of understanding of the biology and = genetics=20 of glioma, together with truly manipulable experimental = models,=20 now offer very real opportunities for the development of = effective=20 targeted therapy. Despite significant gaps in our = understanding,=20 a wealth of information now exists about the clinical and = biological=20 behavior of the tumors, the genetic pathways involved in=20 gliomagenesis, and the nature and role of signature = alterations in=20 these pathways. The challenge now is to integrate all of this = knowledge in an interdisciplinary way to fully understand = this=20 disease and how its signature heterogeneity contributes to = its=20 intractability. For example, the relatively poor response of = GBM=20 patients to EGFR inhibitors, together with emerging data = showing that=20 those who do respond have specific genetic combinations,=20 suggests that a pathway targeting approach requires a more = thorough=20 understanding. Moreover, the fact that even those patients = who do=20 respond to these therapies eventually progress suggests that = the=20 evolution of therapeutic resistance is a hallmark feature in = their=20 effectiveness. This raises critical questions as to which = genetic=20 alterations should be targeted as drivers of tumor = maintenance, which=20 should be ignored because they are initially needed for tumor = establishment, and which drive the glioma stem cell niche, = thus=20 providing a reservoir from which such therapeutic resistant = cells can=20 emerge (Bao et al. 2006a3DGo). These studies, along with new data that = will emerge=20 from the TCGA initiative, will likely transform our = understanding of=20 genetics underlying GBM.

To fully understand the relevance of this niche in driving = therapeutic=20 resistance (Bao et al. 2006a3DGo), many critical questions remain to be = answered,=20 including whether CD133+ cells are equivalent to = the=20 actively proliferating tumor cells seen on routine histological=20 analysis or represent a quiescent population that is = activated=20 by ex vivo manipulations. It is also not yet clear whether = there=20 is a prognostic correlation between CD133+ and patient=20 outcome, and if CD133+ cells are selectively = spared by=20 radiation and chemotherapeutic drugs. Finally, it is not = clear=20 whether de novo CD133+ cells are preferentially = found in=20 the neurovascular niche, as was recently proposed based on in = vitro=20 studies (Calabrese et al. 20073DGo).

Beyond the stem cell issue is the emerging data noted above=20 regarding RTK coactivation that provides a rational = explanation=20 for the feeble ability of RTK inhibitor monotherapy to effect = durable clinical responses in GBM patients, in that the = inhibition=20 of a single RTK is insufficient to block signaling through = critical=20 growth and survival pathways (Huang et al. 20073DGo;=20 Stommel et al. 20073DGo). This suggests that RTK profiling will be = necessary to=20 rationally determine an appropriate combination of inhibitors = that=20 will achieve a significant clinical outcome. Thus, a systematic = study=20 of combination RTK therapies in cancers harboring specific = RTK=20 coexpression patterns represents an important next step in = the design=20 of new clinical trials, and the secondary analysis of such = tumor=20 samples will yield valuable insight into mechanisms of = response and=20 resistance. Because FDA-approved RTK inhibitors already exist = and=20 additional novel drugs are under development, this treatment = paradigm=20 may be implemented in a relatively timely fashion for GBM and = other=20 cancers that are currently highly refractory to virtually all = existing therapies.

Our ability to isolate and culture neural and CSCs, astrocytes = and=20 oligodendrocytes and the creation of faithful models of this = disease=20 coupled to enormous advances in genomic characterization of = gliomas=20 and exquisite functional validation of causative mutations = offer the=20 very real prospect of rapid and thorough preclinical testing = of=20 compounds and other agents to directly answer these = questions. By=20 identifying the weaknesses of the tumor, useful treatments = for=20 patients with these devastating diseases will become a = reality.=20


3D"   =20 Acknowledgments
Top
Abstract
3D"Classification and grading of...
3D"Tumor biological processes and...
3D"Frontiers in glioma research...
3D"Acknowledgments
3D"References =
 
This work=20 was supported in part by Scholar Awards for cancer research = from the=20 Kimmel Foundation and the V Foundation (to F.B.F.), grants = CA95616=20 (to W.K.C., R.M.B., F.B.F., L.C., and R.A.D.) and CA099041 = (to L.C.)=20 from the National Cancer Institute, and a Fellow Award from = the=20 National Foundation for Cancer Research (to W.K.C.). R.A.D. = is an=20 American Cancer Society Research Professor and an Ellison = Medical=20 Foundation Scholar and is supported by the Robert A. and = Renee E.=20 Belfer Foundation Institute for Innovative Cancer = Science.=20


3D"   =20 Footnotes
 
14 Corresponding authors. =

E-MAIL ron_depinho{at}dfci.harvard.edu ; FAX (617) 632-6069.

15 E-MAIL wcavenee{at}ucsd.edu ; FAX (858) 534-7750.

Article is online at http://www.g= enesdev.org/cgi/doi/10.1101/gad.1596707=20


3D"   =20 References
Top
Abstract
3D"Classification and grading of...
3D"Tumor biological processes and...
3D"Frontiers in glioma research...
Acknowledgments
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P. Y.=20 Wen and S. Kesari
Malignant Gliomas in = Adults
N.=20 Engl. J. Med., July 31, 2008; 359(5): 492 - 507.
[Full = Text]=20 [PDF] =
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N. de=20 la Iglesia, G. Konopka, K.-L. Lim, C. L. Nutt, J. F. Bromberg, D. = A.=20 Frank, P. S. Mischel, D. N. Louis, and A. = Bonni
Deregulation of=20 a STAT3-Interleukin 8 Signaling Pathway Promotes Human = Glioblastoma Cell=20 Proliferation and Invasiveness
J. Neurosci.,=20 June 4, 2008; 28(23): 5870 - 5878.
[Abstra= ct]=20 [Full=20 Text] [PDF] =
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S. E.=20 Kendall, J. Najbauer, H. F. Johnston, M. Z. Metz, S. Li, M. = Bowers, E.=20 Garcia, S. U. Kim, M. E. Barish, K. S. Aboody, et=20 al.
Neural Stem Cell Targeting of Glioma Is = Dependent on=20 Phosphoinositide 3-Kinase Signaling
Stem Cells,=20 June 1, 2008; 26(6): 1575 - 1586.
[Abstract]=20 [F= ull=20 Text] [PDF]=20
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N. de=20 la Iglesia, G. Konopka, S. V. Puram, J. A. Chan, R. M. Bachoo, M. = J. You,=20 D. E. Levy, R. A. DePinho, and A. Bonni
Identification = of a=20 PTEN-regulated STAT3 brain tumor suppressor = pathway
Genes=20 & Dev., February 15, 2008; 22(4): 449 - 462.
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