**3. Migration vs. proliferation**

acid and N-acetylgalactosamine linked via a β-glycosidic bond and is polymerized into chains through the activity of chondroitin synthase and polymerizing factor [15]. Variability within CSPG derives from variation within the core protein of CSPGs as well as the number and

112 Neurooncology - Newer Developments

**Figure 3. Chondroitin sulfate proteoglycan structure**. (A) Binding of glycosaminoglycan side chains to core protein through serine residues (Xyl, xylose; Gal, galactose). Sulfate groups added at R. (B) CSPG core protein with N-terminal hyaluronan binding domain and a C-terminal tenascin-binding domain. Central domain binds GAG side chains. (C)

Structure of Lecticans (Reproduced with permission from Miyata et al. [14]).

One ofthe characteristics of malignant gliomas that makes them universally fatal is their ability to infiltrate normal brain parenchyma. This diffuse spread makes surgical cure impossible and makes treatment with radiation and chemotherapy difficult and inefficient. The rapid proliferation of cells in malignant gliomas changes the tumor microenvironment, which becomes hypoxic, acidic, and devoid of glucose and other nutrients. Tumor cells must adapt to these changes to survive and thus change from a proliferative to a migratory phenotype in order to reach a more favorable environment. The mechanism by which tumor cells transi‐ tion to this migratory state and the factors which trigger this process are therefore important to understand as it serves as an excellent target for therapy. This complex metabolic change, sometimes called the "epithelial-mesenchymal transition," is poorly understood, however, and involves multiple signal transduction pathways with many molecules needed to effect the changes in gene expression needed to bring about this transition.

One ofthe key molecules involved with sensing stress in a cell's microenvironment is the AMPactivated serine/threonine protein kinase (AMPK) [37]. AMPK is activated by a high AMP/ATP ratio and other conditions of metabolic stress and causes cells to conserve energy, thus regulating their cellular homeostasis in response to environmental cues. AMPK is activated in response to environmental stress through phosphorylation by three known protein kinases: liver kinase-B1 (LKB1) and the two calmodulin-dependent protein kinases, CaMKKα and CaMKKβ, that phosphorylate AMPK in response to high intracellular calcium levels [38]. Once activated, AMPK exerts its effects on cellular metabolism through many downstream molecules, one of which is cyclooxygenase-2 (COX2), whose inhibition by AMPK leads to more aggressive tumor growth and invasion [39]. Overexpression of COX2 has been seen in many types of cancer including colon, breast, and lung [40].

LKB1, a tumor suppressor gene, is constitutively active and is the primary AMPK kinase. Its phosphorylation of AMPK sets off a cascade that results in liberation of intracellular ATP and conservation of energy through regulation of biosynthetic pathways [41]. Mutations in LKB1 are found in Peutz-Jeghers syndrome [42] as well as melanoma [43], lung [44], and pancreat‐ ic cancers [45]. LKB1 signaling pathways also are involved in cell migration by virtue of their control of cytoskeletal proteins involved in cell polarization and migration. LKB1 deficiency leads to alterations in cell polarity and impaired migration of neural progenitor cells *in vivo* [46], while LKB1 activation is known to inhibit cell proliferation and can affect cellular polarity, which is essential for cell migration [47]. The latter effect is thought to be mediated, in part, through phosphorylation of MAP/microtubule affinity regulating kinase-3 (MARK-3), which regulates phosphorylation of microtubule-associated proteins [48] and phosphorylation of myosin light chain-2 directly by AMPK [49].

While the activation of AMPK leads to energy conservation in nutrient-poor environments, the mammalian target of rapamycin complex-1 (mTORC1) is a serine/threonine kinase, which promotes cell growth and proliferation. Inhibition of apoptosis [50] by mTOR overactivity has been observed in several types of cancers [51]. The balance between AMPK and mTOR is maintained in part by the tuberous sclerosis complex-2 gene (TSC-2), which is activated by AMPK and which, in turn, inactivates mTOR [50] (**Figure 4**). In addition, AMPK directly phosphorylates Raptor, a scaffold protein in the mTOR1 complex, resulting in direct inactivation of mTORC1 [52]. Mammalian target of rapamycin complex-2 (mTORC2) is considered resistant to rapamycin and is not sensitive to nutrients in the cellular microenvironment. It activates PKC-α and AKT to regulate the structure of the actin cytoskeleton [53].

makes treatment with radiation and chemotherapy difficult and inefficient. The rapid proliferation of cells in malignant gliomas changes the tumor microenvironment, which becomes hypoxic, acidic, and devoid of glucose and other nutrients. Tumor cells must adapt to these changes to survive and thus change from a proliferative to a migratory phenotype in order to reach a more favorable environment. The mechanism by which tumor cells transi‐ tion to this migratory state and the factors which trigger this process are therefore important to understand as it serves as an excellent target for therapy. This complex metabolic change, sometimes called the "epithelial-mesenchymal transition," is poorly understood, however, and involves multiple signal transduction pathways with many molecules needed to effect the

One ofthe key molecules involved with sensing stress in a cell's microenvironment is the AMPactivated serine/threonine protein kinase (AMPK) [37]. AMPK is activated by a high AMP/ATP ratio and other conditions of metabolic stress and causes cells to conserve energy, thus regulating their cellular homeostasis in response to environmental cues. AMPK is activated in response to environmental stress through phosphorylation by three known protein kinases: liver kinase-B1 (LKB1) and the two calmodulin-dependent protein kinases, CaMKKα and CaMKKβ, that phosphorylate AMPK in response to high intracellular calcium levels [38]. Once activated, AMPK exerts its effects on cellular metabolism through many downstream molecules, one of which is cyclooxygenase-2 (COX2), whose inhibition by AMPK leads to more aggressive tumor growth and invasion [39]. Overexpression of COX2 has been seen in many

LKB1, a tumor suppressor gene, is constitutively active and is the primary AMPK kinase. Its phosphorylation of AMPK sets off a cascade that results in liberation of intracellular ATP and conservation of energy through regulation of biosynthetic pathways [41]. Mutations in LKB1 are found in Peutz-Jeghers syndrome [42] as well as melanoma [43], lung [44], and pancreat‐ ic cancers [45]. LKB1 signaling pathways also are involved in cell migration by virtue of their control of cytoskeletal proteins involved in cell polarization and migration. LKB1 deficiency leads to alterations in cell polarity and impaired migration of neural progenitor cells *in vivo* [46], while LKB1 activation is known to inhibit cell proliferation and can affect cellular polarity, which is essential for cell migration [47]. The latter effect is thought to be mediated, in part, through phosphorylation of MAP/microtubule affinity regulating kinase-3 (MARK-3), which regulates phosphorylation of microtubule-associated proteins [48] and phosphorylation of

While the activation of AMPK leads to energy conservation in nutrient-poor environments, the mammalian target of rapamycin complex-1 (mTORC1) is a serine/threonine kinase, which promotes cell growth and proliferation. Inhibition of apoptosis [50] by mTOR overactivity has been observed in several types of cancers [51]. The balance between AMPK and mTOR is maintained in part by the tuberous sclerosis complex-2 gene (TSC-2), which is activated by AMPK and which, in turn, inactivates mTOR [50] (**Figure 4**). In addition, AMPK directly phosphorylates Raptor, a scaffold protein in the mTOR1 complex, resulting in direct inactivation of mTORC1 [52]. Mammalian target of rapamycin complex-2 (mTORC2) is considered resistant to rapamycin and is not sensitive to nutrients in the cellular

changes in gene expression needed to bring about this transition.

114 Neurooncology - Newer Developments

types of cancer including colon, breast, and lung [40].

myosin light chain-2 directly by AMPK [49].

**Figure 4. The AMPK/mTOR system**. AMPK, AMP-activated serine/threonine protein kinase; CAMKK, Ca(2+)/calmo‐ dulin-dependent protein kinase kinase; COX2, cyclooxygenase-2; LKB1, liver kinase B1, mTOR, mammalian target of rapamycin; TSC2, tuberous sclerosis-2.

In addition to intracellular energy levels, hypoxia and acidity are triggers for cells to regu‐ late their gene expression to adapt to a hostile environment. In tumors, hypoxia occurs due to rapid cell proliferation and inadequate blood supply from aberrant blood vessels. It leads to resistance to radiation and chemotherapy and is associated with a more aggressive disease and a poorer outcome. Oxygen homeostasis is mediated by the hypoxia-inducible factor (HIF) family of basic helix-loop-helix transcription factors, which consist of heterodimer of a constitutively expressed beta-subunit and an alpha-subunit which, when translated, is only stabilized under hypoxic conditions and is degraded once hypoxia has been corrected [54–56]. HIF-1 induces expression of dozens of target genes involved in the regulation of angiogene‐ sis, cellular metabolism, and cell migration by binding to hypoxia-responsive elements (HREs) in their promoters. HIF-1α directly activates transcription of vascular endothelial growth factor (VEGF) [57, 58], which is the major regulator of angiogenesis and directs new blood vessel growth into hypoxic areas. HIF-2α knockdown leads to reduced levels of VEGF and poorly vascularized, highly necrotic tumors in neuroblastoma [59]. In order to adapt to hypoxia, cells switch from aerobic to anaerobic metabolism, and this shift is regulated, in part, by HIF-1. Glycolytic enzymes such as pyruvate kinase M2, phosphoglycerate kinase, and aldolase are induced by HIF-1 [60, 61] as are the glucose transporters, GLUT-1 and GLUT-3 [62]. Additionally, pyruvate dehydrogenase kinase-1 is activated, reducing mitochondrial oxygen consumption by preventing pyruvate from entering the citric acid cycle [63]. Finally,

HIF-1 is essential in the epithelial-mesenchymal transition by directly regulating the expres‐ sion of Twist, which is essential for cancer metastasis [64]. Twist is a basic helix-loop-helix transcription factor whose expression is regulated through a number of signal transduction pathways including Akt, Ras, and Wnt and whose expression correlates with higher tumor grade [65]. It inhibits the E-cadherin-mediated adhesion between cells, which enables tumor cells to adopt a more motile phenotype [66]. Twist also serves as a survival factor by inhibit‐ ing p53-induced apoptosis by counteracting the effects of c-MYC in neuroblastoma [67]. HIF-1 regulates expression of a number of adhesion molecules, such as alpha- and beta-integrins and E-cadherin [68–70], matrix metalloproteinase-2 and -9 [71, 72] as well as a number of chemo‐ kines and their receptors including c-Met and CXCR4 [73–75], suggesting how hypoxia may play a role in triggering cell migration and digestion of the ECM.

Much remains to be elucidated regarding the molecular cascades through which cells transition to a migratory phenotype. Rapid proliferation creates a toxic microenvironmentthat, when sensed by the cell, sends a signal through the AMPK-mTORC axis or by HIF-1 and others to effect the changes in transcription needed to bring about the transformation to a migrato‐ ry phenotype so the cell may escape to a more favorable environment. As they leave the main tumor mass and move into the brain parenchyma which limits and inhibits their migration, glioma cells remodel their environment by secreting degradative enzymes and novel ECM components which attempt to recapitulate the more permissive, primitive structure of the developing brain.
