Genetic Signaling in GBM 805 in any way coordinated, the individual or coordinated contribution of BTSCs to tumor cell proliferation, and how BTSC-specific proteins interact with each other and with chemoactive, antineoplastic agents should be of use not only in expanding our understanding of how brain cancers develop, but also in the design of future neurotherapeutic approaches and multimodal treatment strategies. 4 Gene Expression in the Human Brain Mammalian brain cells have an intrinsically higher index of genetic output and complexity compared to gene transcription profiles in other cells and tissue cell systems (Lukiw et al., 2000; Sutcliffe, 2001; Colangelo et al., 2002; Mattick and Makunin, 2005). Furthermore, complex biological behaviors and functional neurochemical mechanisms that accompany aging and neuropathology, including neuro-oncological change, are probably not controlled by single genes but rather by groups of functionally related genes sometimes referred to as gene families. The use of DNA array technologies is currently capable of interrogating 33,000 genes on a single DNA array, or the levels of all expressed genes in a single brain biopsy sample (Affymetrix Corporation, Santa Clara, CA; Lukiw, 2004; Lukiw et al., 2005; Macdonald et al., 2007). Although some pathogenically related upregulated onco- genic genes thus far identified may share common overlapping functions such as stress response and adaptive processes that support several related aspects of brain inflammation, apoptosis and/or angiogenesis, glioma and GBM may represent some of the most heterogeneous gene expression patterns of any neurological disease known (Kavsan et al., 2006; Idbaih et al., 2007; Juric et al., 2007; Culicchia et al., 2008). 5 Gene Expression in Brain Cancers Molecular–genetic and population-based studies have identified several gene muta- tions that associate with brain tumor development (Tatter, 2005; Nicholas et al., 2006; Van den Bent and Kros, 2007; Juric et al., 2007; Kavsan et al., 2007; Johansson Swartling, 2008). These may be linked to an ordered accumulation of multiple genetic mutations on multiple chromosomes and sequential, temporally mediated pathogenic interactions. For example, in about one third of all cases, the transition from healthy astroglial cells to astrocytoma has been associated with p53 gene mutations at chromosome 17p, and mutations in p53, a tumor suppressor gene, were among the first genetic alterations ever to be identified in astrocytic brain tumors (Tatter, 2005; Nicholas et al., 2006; Van den Bent and Kros, 2007). Deletion or alteration of the p53 gene appears to be present in approximately 25–40% of all GBMs, and p53 immunoreactivity also appears to be associated with tumors that arise in younger patients. Brain cancers also often exhibit loss of heterozygosity (LOH), and LOH at chromosome 10q is the most frequent gene alteration for both 806 W.J. Lukiw and F. Culicchia primary and secondary glioblastomas, ranging from 60 to 90% of all cases. This mutation appears to be specific for GBM, is found rarely in other tumor grades, and is associated with poor survival. LOH at 10q plus 1 or 2 of the additional gene mutations appear to be frequent alterations and are most likely major players in the development of GBM. LOH leads in almost one half of all subsequent cases to anaplastic astrocytoma associated with the retinoblastoma (Rb) gene at chromosome 13q. Involvement of additional mutations at chromosomes 9p and 19q, followed by GBM development associated with chromosome 10 mutations and amplification of the epidermal growth factor receptor gene is further postulated in this highly com- plex pathway of oncogenic development (see, e.g., Tatter, 2005; Nicholas et al., 2006; Van den Bent and Kros, 2007; Johansson Swartling, 2008). The involvement of multiple gene loci and reduced or incomplete penetrance of these gene muta- tions indicate that the resulting altered developmental or oncogenic pathways induce tumors possessing a highly variable phenotype and heterogeneous morphology. Brain cancers are also genetically associated with homeostatic disturbances in the epidermal growth factor receptor (EGFR), MDM2, platelet-derived growth factor- alpha (PDGFα) and PTEN genes. The EGFR gene is involved in the control of cell proliferation and EGFR overexpression and mutant EGFR expression occurs in approximately 50% of patients with GBM (Nicholas et al., 2006; Voelzke et al., 2008). In fact multiple genetic mutations in EGFR are apparent, including overex- pression of the receptor as well as rearrangements that result in truncated isoforms (Voelzke et al., 2008). However, all the clinically relevant EGFR mutations appear to generate a similar phenotype resulting in increased EGFR activity. Amplification or overexpression of MDM2 constitutes an alternative mechanism to escape from p53-regulated control of cell growth by binding to p53 and blunting its activity. Overexpression of MDM2 is the second most common gene mutation in GBMs and is observed in 10–15% of patients. Some studies show that this mutation asso- ciates with a poor prognosis. The PDGF gene acts as a major mitogen for glial cells by binding to the PDGF receptor (PDGFR) and amplification or overexpres- sion of PDGFR is typical (60%) in the pathway leading to secondary glioblastomas. PTEN (also known as MMAC and TEP1) encodes a tyrosine phosphatase located at chromosome 10q23.3 that functions as a cellular phosphatase, turning off signaling pathways, and is consistent with possible tumor-suppression activities. When phos- phatase activity is lost because of genetic mutation, signaling pathways can become activated constitutively, resulting in aberrant proliferation. PTEN mutations have been found in as many as 30% of all glioblastoma cases studied (Koul, 2008; Cheng et al., 2009). Current DNA array technologies and statistical and comparative bioinformat- ics analysis enable a comprehensive examination of the expression of all genes associated with brain health and disease. Genomewide gene expression patterns of neoplastic brain cells in their various developmental stages provide a fascinat- ing reflection of the physiological and pathological status of those pathogenic brain cells. Robust gene expression analyses have been applied to whole tumor cells and some recurrent themes, besides such variables as patient age, sex, affected lobe and disease onset, duration, and other clinical parameters, are emerging for specific pathology-related genes. Interestingly, the progression from low-grade glioma to Genetic Signaling in GBM 807 high-grade GBM may be associated with distinct molecular–genetic changes that vary according to WHO grade (MacDonald et al., 2007; Juric et al., 2007). Several excellent reviews of DNA array analysis of glioma and GBM have recently appeared and the material in t hem is not reiterated here (Boudreau et al., 2005; Belda-Iniesta et al., 2006; Kavsan et al., 2005; Tso et al., 2006; Faury et al., 2007; Idbaih et al., 2007; Juric et al., 2007; MacDonald et al., 2007; Johansson Swartling, 2008). Rather we focus on some current observations on increases in the expression of glioma and GBM of several altered markers involved in brain cell contact, cell cycle, cell death, and vascular proliferation markers at the level of gene expression in virtually all brain tumors examined: beta-amyloid precursor protein (βAPP), the apoptosis effector caspase-3, a cell–cell contact neuronal-enriched protein pentraxin-2, and the angiogenesis promoting vascular endothelial growth factor (VEGF). 5.1 Beta-Amyloid Precursor Protein (βAPP) Beta amyloid precursor protein (βAPP), a brain-abundant trans-membrane glyco- protein “sensor” implicated in neuronal–glial intercellular contact and progressive apoptotic and necrotic brain cell death appears to be part of a pathogenic gene family that associates with glial cell proliferation in glioma, GBM, and neurodegeneration (Colangelo et al., 2002; Lukiw, 2004; Fuso et al., 2007; Culicchia et al., 2008). In fact most of the original studies on the role of βAPP in neurobiology and neu- rodegeneration were first performed in transformed human glioblastoma cell lines (Lahiri et al., 1997; Paris et al., 2005; Fuso et al., 2007; Culicchia et al., 2008). Cancer-affected glial cells are characterized by highly unusual and diverse mor- phology that often correlates to the grade of the neoplasm and display noncontact inhibited cells, lack of cell–cell adhesion and connectivity, and a highly varied range of cellular morphology (Fig. 1) (Hyun Huang et al., 2007; Caltagarone et al., 2007; Culicchia et al., 2008). Bizarre glial cell morphology in malignant gliomas and GBM have been correlated with the depletion of cytoskeletal-matrix actin-bundling proteins and alterations in integrin-mediated communication between the extracellu- lar matrix and the actin cytoskeleton (Venezia et al., 2007; Caltagarone et al., 2007; Young-Pearse et al., 2007). Although the abundant cytoskeletal protein β-actin itself is not upregulated, β-actin-associated cytoskeletal proteins and integrins that further drive glial cell division and proliferation processes during the cell cycle have been implicated in brain cancer development. Interestingly βAPP structural orientation within the membrane, βAPP trafficking, and intracellular signaling functions are associated with β-actin-associated proteins and β-actin-mediated cellular shape. It is difficult to rationalize whether βAPP upregulation in glioma and GBM is either a consequence of, or contributory factor to altered microfilament of microtubule cytoarchitecture, or increased cellular proliferation, or both (Venezia et al., 2007; Caltagarone et al., 2007; Young-Pearse et al., 2007; Culicchia et al., 2008). Although βAPP is known to be variably upregulated in chronic neurodegenera- tive disease, depending on the stage of the disease, the observation of upregulated proinflammatory and amyloidogenic neural degenerative markers in glioma and 808 W.J. Lukiw and F. Culicchia GBM is a relatively recent one (Lukiw, 2004; Lukiw et al., 2005; Fuso et al., 2007; Sin et al., 2008; Culicchia et al., 2008). Excessive βAPP-mediated signaling is thought to be responsible, in part, for driving neural inflammation, glial cell growth and expansion, and brain cell degeneration events such as apoptosis (Melhorn et al., 2000; Radde et al., 2007; Venezia et al., 2007; Herber et al., 2007). Ischemic brain damage is also known to induce inflammatory cytokine and βAPP over-expression that further induce widespread brain cell death via apoptosis or necrosis (Pluta, 2002; Bates et al., 2002). Chronic gliosis is, in addition, associated with altered processing of βAPP in vivo, and thus may trigger pathological changes associated with aberrant interneural communication between brain cells, thus contributing to progressive alterations in glial cell morphology (Pluta, 2002; Bates et al., 2002; Young-Pearse et al., 2007). Increased upregulation of βAPP expression in glioma and GBM further suggests that unscheduled proliferative events of brain cells are accompanied by the signifi- cant elevation of integral transmembrane receptors that are pathogenic markers for neurodegeneration. βAPP appears to be part of a poorly understood cell-contact signaling pathway whose disruption induces cell-cycle s ignaling, mitotic abnormal- ities, and glial cell expansion (Paris et al., 2005; Venezia et al., 2007; Young-Pearse et al., 2007; Fuso et al., 2007; Lukiw et al., 2008). Increased βAPP expression has long been associated with gliosis, the localized expansion of astrocyte populations, and the production of dense fibrous networks of neuroglia in the area of a pathogenic lesion. Similar gliosis-related increases in the expression of βAPP in glioma and GBM and in the neurodegenerating brain tissues support some underlying com- monality in disorganized interneural signaling, aberrant cytoarchitecture and neural cell shape characteristic of both neurological conditions. Interestingly, Alzheimer’s disease and GBM have similar age-specific incidence rates and accumulation of senile plaque deposits consisting of amyloid beta (Aβ) peptides derived from the secretase cleavage of βAPP holoprotein. About one third of all GBM cases exhibit age-related plaque scores indicative or suggestive of AD; and progressive neurode- generative pathology is present in about half of all cases of GBM (Nelson, 2002; Lukiw et al., 2008). 5.2 Caspase-3 Whether brain cell death in the malignant neoplasms, triggered by hypoxia, lack of nutritive support or other pathogenic factors, is driven by apoptosis or necrosis is not well understood. In fact both neural-destructive processes may be operating simultaneously. Apoptosis and necrosis appear to lie at either end of a spectrum of functional brain cell dysfunction and progressive cell loss spanning from pro- grammed cell death (apoptosis; internucleosomal DNA fragmentation) at one end to induced and premature cell death (necrosis; randomized DNA fragmentation) of brain cells at the other. Cysteine–aspartic acid protease-3 (caspase-3), a key mem- ber of a family of 11 human cysteine proteases, plays key essential effector roles in Genetic Signaling in GBM 809 both apoptosis and necrosis and in neuroinflammatory aspects of neurodegeneration and brain tumor growth. There is evidence for both caspase-3 upregulation (Ray et al., 2002; Lukiw et al., 2009) and caspase-3 downregulation in human brain tumors (Stegh et al., 2008). Nonhomeostatic levels of caspase-3 indicate alterations in the cellular balance of both pro-apoptotic and anti-apoptotic signals (Takuma et al., 2004; Lefranc et al., 2007). The increased expression of the pro-apoptotic Bax protein, upregulation of calpain and caspase-3, and occurrence of internucleo- somal DNA fragmentation indicate that one mechanism of cell death in malignant brain tumors is apoptosis (Ray et al., 2002; Lukiw et al., 2009). These results may be explained by the fact that the apoptotic process only approaches the stage of caspase-3 activation, followed by a subsequent variable activation of the apoptotic cascade and “programmed” cell death mechanism, resulting in apoptotic blockage and an accumulation of brain cell mass. 5.3 Pentraxin-2 (NP2; NPTX2) Pentraxins are a family of pentameric calcium-dependent ligand-binding proteins bearing a highly distinctive structure similar to that of the ring-shaped lectins (Emsley et al., 1994). Pentraxins represent a novel neuronal uptake pathway that functions during intercellular and extracellular signaling, synapse formation and clustering, remodeling, and cell–cell contact (Gerrow and El-Husseini, 2007). “Short” pentraxins include the inflammation-related serum amyloid P component (SAP) and C reactive protein (CRP) and the “long” pentraxins include PTX3 (a cytokine-modulated molecule) and several prominent brain-enriched pentrax- ins such as neuronal pentraxin-2 (NP2; NPTX2). Interestingly, NPTX2, normally expressed in the CNS, is a member of a family of proteins related to CRP and other acute-phase inflammatory mediators, and has been found to be correlated with glioma and GBM edema, the swelling of soft tissues as the result of loss of brain water balance and excessive water accumulation. Increased NPTX2 are in turn strongly associated with poorer survival rates in tumors with the high- est levels of edema (Hsu and Perin, 1995; Goodman et al., 1996). Several gene expression studies have shown NPTX2 to be consistently and significantly upreg- ulated in glioma and GBM (Carlson et al., 2007; Pope et al., 2008; unpublished observations). It is important to note that the NPTX2 upregulation associated with angiogenic- and edema-related signaling is often coregulated with the simultaneous upregulation of vascular endothelial growth factor (VEGF) and the proliferation of neovascularization. 5.4 Vascular Endothelial Growth Factor (VEGF) Angiogenesis, the proliferation of vascular growth that provides nutritive sup- port to the expanding tumor cell mass, is one of the hallmarks of all cancers. 810 W.J. Lukiw and F. Culicchia Vascular endothelial growth factor stands out as a key mediator of tumor-associated angiogenesis among a complex signaling system involving pro- and antiangiogenic factors (Chamberlain, 2008; Grothey et al., 2008; Pope et al., 2008). The upregula- tion of VEGF, originally described as a vascular permeability factor in brain tumors, has often been proposed to be the major cause of both vasogenic edema in gliomas and neovascularization (NV) (Bruce et al., 1987; Buie and Valgus, 2008; Norden et al., 2008). A consistent observation in brain cancer biology is that malignant gliomas invariably express vast amounts of VEGF, now regarded as an important pathogenic marker of angiogenesis and NV, essential for the proliferation and the survival at any cost for malignant glioma cells. NV is orchestrated by the coordinate induction of a family of growth-factor genes and most prominently by VEGF which also possesses endothelial cell-specific mitogenic effects that closely correlate with NV during embryonic development and normal systemic physiology, fetal anemia, in retinal NV, in models of hypoxic ischemia, and in malignant tumors. These com- bined observations are suggestive of VEGF’s key role in vascular proliferation in growth, health, and disease. Hypoxia is thought to be one crucial physiological stimulus for VEGF upregulation that precedes NV, and low cellular oxygen tension rapidly induces a number of transient genetic signals through which this is accom- plished (Larrivee and Karsan, 2000; Hasan and Jayson, 2001; Giles, 2001; L. Lukiw et al., 2003; Norden et al., 2008). The multiple roles of VEGF in brain tumor development and proliferation and anti-VEGF-based therapies have been recently examined in the last year in several excellent reviews and interested readers are encouraged to refer to these thoughtful works and the published papers referenced within (Brandsma et al., 2008; Chamberlain, 2008; Grothey and Ellis, 2008; Pope et al., 2008; Reardon et al., 2008; Wong and Brem, 2008). 6 Specific Alterations in the Expression of Brain-Enriched Genes Neurological disorders including glioblastoma involve a highly complex patho- genesis with multiple etiological factors, and this is reflected in the expression of brain genes in this disease. Several glioma and GBM tumor cell lines have been immortalized and “standardized brain tumor cell cultures” are available to oncol- ogy researchers through government-funded sources such as the American Type Tissue Collection (ATCC, Bethesda MD). Commonly used human brain cell cul- tures include glioma cell line HTB-138 (Hs683) and glioblastoma tumor cell lines CRL-2020 (DBTRG-05MG), CRL-1690 (T98G), CRL-2365 (M059K), and CRL- 2366 (M059J). The majority of these standardized neoplastic, immortalized cell lines develop as a mixture of floating and adherent cells growing as heterogeneous clusters of neuroblastic cells with multiple, short, fine cell processes (neurites) that often aggregate, forming clumps, detach from solid surface, and float within the cell culture medium (Fig. 1). Total DNA, RNA, and protein can be effectively and effi- ciently isolated from these archived cell lines and are subsequently used for gene Genetic Signaling in GBM 811 expression analysis and downstream molecular–genetic investigations. Recent stud- ies in these cell lines have indicated increases in the integral membrane β-amyloid precursor protein (βAPP) as a proinflammatory, neurodegenerative, and proliferative pathogenic marker (Culicchia et al., 2008). Indeed, from the perspective of dys- regulated pathogenic gene expression, glioma and glioblastoma multiforme display significant upregulation of disease markers such as βAPP and caspase-3 with fea- tures of rapid-onset, progressive, glial cell proliferating, degenerative brain disease. The known disease-related functions of these inflammatory and neurodegenera- tive markers may further contribute to the pathogenic phenotype and unscheduled misregulated propagation of glial cells in the brain. The important point is that brain tumors consist of a spectrum of tumors of varying differentiation, malignancy grades, and gene expression profiles. Despite the fact that all tumors have an initially invasive phenotype, early genetic events appear to differ between astrocytic and oligodendroglial tumors, and this may form, in part, the molecular genetic basis for variation in brain cancer cell composition that complicates more effective therapies. Knowledge of malignant glioma genetics has already affected clinical management of these tumors, and researchers and clinicians can only hope that further knowl- edge of the evolution of the molecular pathology of malignant gliomas will result in novel therapies that employ multiple, multimodal treatment strategies. 7 Micro RNAs (miRNAs): Specific miRNA and mRNA Alterations in Human Brain Cancer Micro RNAs (miRNAs) are small RNA polymerase II and III transcribed, noncoding RNA molecules that play important posttranscriptional regulatory roles by recogniz- ing and binding to the 3 untranslated region (3 UTR) of mature messenger RNAs (mRNAs). By doing so, miRNAs repress translation and expression of their partic- ular mRNA targets (Mattick and Makunin, 2005; Cao et al., 2006; Lukiw, 2007; Lukiw and Pogue, 2007; Cho, 2007; Amaral et al., 2008; Dogini et al., 2008; Zeng 2009). Transcription of protein-encoding genes and miRNAs by RNA polymerase II and III and their interrelated functions in the modulation of gene expression sug- gests the possibility of some coordinated mode of interaction, possibly through miRNA interaction with specific transcription factors (Mattick and Makunin, 2005; Hobert, 2008; Lukiw et al., 2008; Makeyev and Maniatis, 2008; Williams et al., 2008; Amaral et al., 2008). Interestingly, small signaling molecules such as miRNA may transfer epigenetic information not only within cells but also between cells and organ systems as part of a dynamic RNA-mediated interplay between the environ- ment and the genome (Zhao et al., 2006; Hill et al., 2009; Mattick et al., 2009). Such novel genetic mechanisms may explain, in part, cancer invasiveness and metasta- sis throughout cells, organs, and tissue systems (Louis, 2006; Hyun Hwang et al., 2008). To date about 911 miRNAs in the human brain have been identified (Lukiw, 2007; Lukiw and Pogue, 2007). The miRNA-mediated regulation of messenger RNA (mRNA) complexity in the human central nervous system is evolving as a 812 W.J. Lukiw and F. Culicchia critical and determining factor in regulating CNS-specific gene expression during development, plasticity, aging, and disease (Hobert, 2008; Makeyev and Maniatis, 2008; Williams et al., 2008). Several excellent recent reviews on miRNA specia- tion in the CNS and specific examples in brain tumors have recently appeared in the literature and the authors would encourage interested researchers, clinicians, and medical and graduate students to read them over (Mattick and Makunin, 2005; Ciafrè et al., 2005; Zhang et al., 2007; Mourelatos, 2008; Nicoloso and Calin, 2008; Papagiannakopoulos and Kosik, 2008; Silber et al., 2008; Hobert, 2008;Makeyev and Maniatis, 2008; Williams et al., 2008; Zeng, 2009, Lukiw et al., 2009). Current studies indicate that specific miRNAs may function at multiple hierar- chical levels in gene regulatory networks, from targeting hundreds of effector genes to controlling the levels of global regulators of transcription and alternative pre- mRNA splicing (Cao et al., 2006; Makeyev and Maniatis, 2008; Silber et al., 2008). An expanding number of miRNAs have been reported to be altered in abundance in glioma and GBM, and largely because of their disease-related expression and selection of specific mRNA targets in the brain, these miRNAs are strongly impli- cated as important regulatory controls in neoplastic onset and evolution. In general, abrogation of global miRNA-mediated mRNA processing and homeostatic control is associated with accelerated cellular transformation and tumorigenesis, and some specific examples are given below (Lukiw, 2004; Pogue and Lukiw, 2004; Lukiw and Bazan, 2006; Kumar et al., 2007; Lukiw and Bazan, 2008; Lukiw, 2009; Zeng, 2009). Several decreased or increased miRNA species implicated in miRNA-mediated brain cell tumor growth, oncogenesis, apoptosis, and survival (sometimes referred to as oncomirs) are miRNA-124 and miRNA-137 (Ciafrè et al., 2005; Cho, 2007; Silber et al., 2008; Papagiannakopoulos and Kosik, 2008; unpublished observa- tions). In one recent study the expression levels of miRNA-124 and miRNA-137 were found to be significantly decreased in anaplastic astrocytoma (WHO grade III) and GBM (WHO grade IV) relative to nonneoplastic control tissue (Silber et al., 2008; Papagiannakopoulos and Kosik, 2008). Interestingly, when miRNA- 124 was introduced into nonneuronal mammalian cells a preferential reduction in the amounts of nonneuronal mRNAs, including those encoding protein required for cell proliferation or neural stem cell function was observed, and promotion of a neuronal-like mRNA profile (Conaco et al., 2006; Makeyev et al., 2007). Conversely, a depletion of miRNA-124 from primary neurons accumulated a number of nonneuronal mRNA targets, suggesting that miRNA-124 ensures that progenitor genes are posttranscriptionally inhibited in neurons (Makeyev et al., 2007; Cao et al., 2007). Such evidence suggests the roles of miRNAs are in con- trolling cell fate and the proliferating capacity of brain cells. That miRNA-124 and miRNA-137 induce differentiation of adult neural stem cells, oligodendroglioma- derived stem cells, and human GBM-derived stem cells and induce cell cycle arrest in GBM suggests that targeted delivery of these highly soluble and mobile small RNAs to glioma and GBM cells may provide an efficacious and novel therapeutic treatment strategy for containing the growth of cancerous brain cells (Silber et al., 2008; unpublished observations). Genetic Signaling in GBM 813 Another miRNA species implicated in cell tumor growth, oncogenesis, apoptosis, and survival is miRNA-221 (Ciafrè et al., 2005; Gillies and Lorimer, 2007; Medina et al., 2008; Lukiw et al., 2009). Support for the pathogenic role of miRNA-221 in tumor growth comes from the recent observations that upregulated miRNA-221 targets the cell growth suppressive cyclin-dependent kinase inhibitors p27 and p57, thus linking a cell-cycle checkpoint at S phase initiation with growth factors that trigger cell proliferation (Li et al., 1999; le Sage et al., 2007; Mellai et al., 2008; Medina et al., 2008; Lukiw et al., 2009). Other recent work reported a selective upregulation of miRNA-221 and downregulation of a miRNA-221 messenger RNA target encoding the survivin-1 homologue BIRC1, a neuronal inhibitor of apopto- sis protein and brain cell marker for neural degeneration (Lukiw et al., 2009). In these later studies the expression of BIRC5 (survivin-1) and caspase-3 was found to be significantly upregulated, particularly in the more advanced stages of GBM. It is important to note that paracrine signaling between adjacent brain cells may contribute to significant positive feedback regulation and the progressive intercellu- lar proliferation of pathogenic signaling in both degenerating brain cells and brain tumors (Zhao et al., 2006; Culicchia et al., 2008). Indeed, tumor invasion occurs not only through dysfunction of the adhesive properties of tumor cells but also in their pathogenic secretion of small lysosomal proteolytic enzymes such as cathepsin-L (Levicar et al., 2002, 2003). Use of online accessible miRNA–mRNA database searches, other miRNA-221-targeted components of apoptotic signaling in glioma and GBM, and interactions with the Bcl-2 protein family of apoptosis include anti- apoptotic protein Bcl-2-binding component 3 and other Bcl-2-modifying factors (Sanger mirBase version 10.1; http://microrna.sanger.ac.uk/cgi-bin), hence miRNA- 221 may further modulate apoptotic signaling via quenching or augmentation of the expression of a number of alternate antiapoptotic mRNA targets, such as additional Bcl-2-modifing factors. Again the small size and high solubility of specific brain- enriched miRNA species suggests that they may perform ancillary intracellular and extracellular signaling roles involved in the spreading and propagation of tumor cell growth and associated metastatic events (Lukiw, 2007; Lukiw and Pogue, 2007; Felicetti et al., 2008; Mattick et al., 2009). More recently, miRNA-125b has been shown to be upregulated in interleukin-6 (IL-6)-stressed normal human astrocytes (NHA), a treatment known to induce astrogliosis, and in vitro, anti-miRNA-125b added exogenously to IL-6-stressed NHA cultures attenuated both glial cell prolif- eration and increased the expression of the cyclin-dependent kinase inhibitor 2A (CDKN2A), a miRNA-125b target, and negative regulator of cell growth (Pogue et al., 2010). 8 Therapeutic Strategies for the Clinical Management of Glioma and GBM Current treatment strategies for GBM are multimodal and typically involve sur- gical resection followed by radiation therapy and chemotherapy. Upon tumor recurrence repeat resection or stereotactic radiosurgery followed by additional 814 W.J. Lukiw and F. Culicchia radiotherapy and chemotherapy may improve outcome in certain cases; several new strategies have been developed to optimize designer therapies for GBM (Sathornsumetee and Rich, 2008; Sathornsumetee and Reardon, 2009; Tentori and Graziani, 2009). The most commonly used chemotherapeutic drug for GBM, temo- zolomide (TMZ), typically administered both during and after radiotherapy, is a potent DNA methylating agent that generates a wide s pectrum of random methyl adducts in the genome. The antitumor activity of TMZ and related alkylating agents has been mainly attributed to the production of O(6)-methylguanine as a potent cytotoxic and antimitotic (Tentori and Graziani, 2009). TMZ also pro- motes autophagic cell death, a caspase-independent process characterized by the accumulation of cytoplasmic autophagic vacuoles and accompanied by extensive degradation of polyribosomes, the endoplasmic reticulum, and the Golgi apparatus that precedes the destruction of the nucleus (Lefranc et al., 2007). As brain neo- plasms are generally associated with altered βAPP, pentraxin, caspase-3, VEGF expression, and the kinases that modify these effector molecules, co-ordinated inhibition of these oncogenic markers might be an effective therapeutic strat- egy. These kinds of treatment approaches have recently been reviewed (Anderson et al., 2008; Lakka and Rao, 2008; Hide et al., 2008; Norden et al., 2008). Anti- inflammatory, anti-βAPP, and antiamyloid pharmacologic strategies directed at neurodegenerative processes may also have some therapeutic value in the treat- ment of glioma- or glioblastoma-affected brain cells (Nelson, 2002; Lukiw and Bazan, 2006; Lukiw and Bazan, 2008; Tschape and Hartmann, 2008; Culicchia et al., 2008). Unfortunately, chemotherapeutic drug resistance occurs relatively often and effective drug delivery to cancer targets remains an accessory concern affecting the clinical response in brain cancer patients. Because malignant gliomas are highly vascularized tumors that produce VEGF, a key mediator of angiogenesis, and given the fact that angiogenesis is essential for the proliferation and survival of malignant glioma cells, angiogenesis antagonists such as angiostatin, endostatin, and vaso- statin may provide yet another specifically targeted, therapeutic strategy. Recent studies have investigated the use of bevacizumab—a humanized monoclonal anti- body against VEGF—for patients with recurrent malignant glioma, however, the results have been inconsistent, and larger, randomized clinical trials are needed to determine the magnitude of the benefit (Buie and Valgus, 2008; Norden et al., 2008). Moreover, angiogenesis antagonists have numerous unwanted side effects in interfering with normal wound healing, bleeding, and blood clotting, and are associated with heart, immune, and reproductive dysfunction (Norden et al., 2008). Interestingly, gamma- and beta-secretases that act on βAPP processing appear to play an essential role during angiogenesis, and inhibitors of these secretases may constitute a novel evolving class of antiangiogenic and antitumoral compounds (Paris et al., 2005). Just as for angiogenesis antagonists, toxic metal-based anticancer drugs (MBADs), including cisplatin, carboplatin, and oxaliplatin, and other arsenic-, cadmium-, copper-, gallium-, lanthanum-, platinum-, ruthenium-, or titanium- containing antitumor drug complexes have adverse effects on physiological systems . gliosis-related increases in the expression of βAPP in glioma and GBM and in the neurodegenerating brain tissues support some underlying com- monality in disorganized interneural signaling, aberrant. protein (CRP) and the “long” pentraxins include PTX3 (a cytokine-modulated molecule) and several prominent brain-enriched pentrax- ins such as neuronal pentraxin-2 (NP2; NPTX2). Interestingly,. resulting in apoptotic blockage and an accumulation of brain cell mass. 5.3 Pentraxin-2 (NP2; NPTX2) Pentraxins are a family of pentameric calcium-dependent ligand-binding proteins bearing a