ECM Molecules

The tumor-associated ECM is comprised of adjacent brain-derived and tumor-secreted ECM molecules. The major structural ECM constituents confronted by the tumor cells are members of the proteoglycan, GAG, and glycoprotein superfamilies [20] (Fig. 14.1, Table 14.1).

Proteoglycans

A proteoglycan consists of a core protein having a variable number of GAG side chains [21] (Table 14.1). The ECM proteoglycan superfamily includes the small leucine-rich repeat (LRR) family, the lectican family, the collagen family, and others. Members of these families present in the brain ECM include phosphacan, agrin, versican (also known as PG-M), neurocan, and brain-enriched hyaluronan-binding (BEHAB [22]; also known as brevican), and the vascular basement membrane proteoglycan perlecan. In brain tumors, changes in the expression of several ECM proteoglycans are observed within the tumor-brain adjacent ECM and the tumor vasculature.

Three of the lectican family members are implicated in invasion. BEHAB expression is upregulated in astrocytic tumors in vivo [23], and either protein cleavage [24] or specific isoform expression and upregulation [25] appear to contribute to glioma invasion. Versican is alternatively spliced into 4 different isoforms, and the V2 isoform may be brain-specific. Its expression is decreased in glioma tumor ECM compared to levels seen in normal neuropil, whereas its expression appears to be upregulated in small blood vessels of gliomas in vivo [26]. Its role in glioma cells remains to be deciphered; however, transfection of a dominant-negative versican mutant that inhibits the secretion and binding of endogenous versican inhibited tumor formation, suggesting that versican can play a role in tumor formation [27]. Neurocan, another member of this family can bind to the adhesion molecules neural cell adhesion molecule (NCAM) and neuronglia cell adhesion molecule (NgCAM) (Table 14.2), inhibiting both homophilic cell-cell binding and adhesion [20]. It is present in normal brain and oligodendrogliomas; however, its expression is lost in astrocytic tumors [28].

Phosphacan is a chrondroitin sulfate or keratin sulfate (phosphacan-KS) proteoglycan of nervous

Neural Cell Adhesion Molecule Functions

FIGURE 14.1 Tumor Adjacent External Environment. A schematic representation of the external environment, with an emphasis on the adhesion and ECM molecules involved in glioma invasion. Cell-cell interactions are modulated by the homophilic cell-cell adhesion of cadherins (1) and Ig-superfamily proteins (2), the latter of which can engage in heterophilic cell-cell interactions with members of the RPTP family (3). Both cadherins and RPTP interact with catenins to induce intracellular signaling and cytoskeletal changes (4). The cytoplasmic domain of RPTP can be cleaved to release phosphacan (5), which can interact with the matricellular protein TN-C (6), or the Ig-superfamily molecules (2). Cell-ECM interactions are modulated by CD44, which can bind to its ligand (7), become cleaved (8), or associate with integrin (9), and thereby initiate intracellular signaling, induce cytoskeletal rearrangement, and promote migration and invasion (10). Cell-ECM interactions are also modulated by integrins (11). Ligation of ECM promotes clustering of integrins to focal adhesions (12), which contain actin-associated proteins (13), and which link the integrin to the cytoskeleton (14) thereby triggering activation of FAK and ILK (12) and downstream events involved in cytoskeleton rearrangement and cell migration and invasion (15,10). Association of integrins with uPA/uPAR induces ECM degradation (16). (Adapted from International Journal of Biochemistry and Cell Biology. Vol 36. Bellail, A. C., Hunter, S. B., Brat, D. J., Tan, C., Van Meir, E. G., Microregional extracellular matrix heterogeneity in brain modulates glioma cell invasion. pp 1046-1069, Figures 4 and 5, 2004 with permission from Elsevier) Pax — paxillin, a-act — a-actinin, Ten — tensin, FAK — focal adhesion kinase, ILK — integrin linked kinase, uPA — urokinase plasminogen activator, uPAR — uPA receptor. See Plate 14.1 in Color Plate Section.

tissue, grouped in the "Other" category (Table 14.1). It is the extracellular domain of the receptor-type tyrosine phosphatase adhesion molecule (Table 14.2). Like neurocan, it is present in normal brain and oligodendrogliomas; but expression is lost in astrocytic tumors [28]. It has a high affinity for fibroblast growth factor-2 (FGF2) [21], and like phosphacan, can bind NCAM and NgCAM, suggesting influences on growth and adhesion, respectively. In addition, it can bind to TN-C, and inhibits adhesion of C6 glioma cells to this matricellular protein [29].

Cathepsin Protease Signaling Pathways

figure 14.2 Interactions Between Major Signaling Pathways in Invasion and Subsequent Impact on Proliferation. This is a schematic representation of the interaction between the proteolytic cascade, integrins, ECM molecules, and the matricellular proteins. Pro-cathepsin D is autocatalytically cleaved to generate cathepsin D (1). Cathepsin D and tPA activate procathepsin B (2). Cathepsin B initiates a sequence of events, including the activation of uPA from pro-uPA (3), which then converts plasminogen to plasmin (4). Plasmin reinforces the generation of uPA (5), activates pro-MMPs (6), and degrades ECM (7). This releases latent growth factors (8), permitting their activation by plasmin (9). Growth factors can in turn initiate intracellular signaling by binding to their receptors. They can be inhibited though by binding to the matricellular protein SPARC (10). Membrane-bound MMPs and TIMP2 activate pro-MMP2 to active MMP-2, a step requiring integrins (11). This can be mediated by integrin binding to ECM alone or with other proteins such as PAI-1 (12). The PAI-1 and VN binding to integrin can be modulated by the matricellular protein SPARC (13). The balance of these interactions leads to the modulation of pro-uPA activation (14) and the subsequent ECM degradation of plasmin, as described above. See Plate 14.2 in Color Plate Section.

figure 14.2 Interactions Between Major Signaling Pathways in Invasion and Subsequent Impact on Proliferation. This is a schematic representation of the interaction between the proteolytic cascade, integrins, ECM molecules, and the matricellular proteins. Pro-cathepsin D is autocatalytically cleaved to generate cathepsin D (1). Cathepsin D and tPA activate procathepsin B (2). Cathepsin B initiates a sequence of events, including the activation of uPA from pro-uPA (3), which then converts plasminogen to plasmin (4). Plasmin reinforces the generation of uPA (5), activates pro-MMPs (6), and degrades ECM (7). This releases latent growth factors (8), permitting their activation by plasmin (9). Growth factors can in turn initiate intracellular signaling by binding to their receptors. They can be inhibited though by binding to the matricellular protein SPARC (10). Membrane-bound MMPs and TIMP2 activate pro-MMP2 to active MMP-2, a step requiring integrins (11). This can be mediated by integrin binding to ECM alone or with other proteins such as PAI-1 (12). The PAI-1 and VN binding to integrin can be modulated by the matricellular protein SPARC (13). The balance of these interactions leads to the modulation of pro-uPA activation (14) and the subsequent ECM degradation of plasmin, as described above. See Plate 14.2 in Color Plate Section.

Glycosaminoglycans (GAGs)

The GAG side-chains of the proteoglycans are the most abundant heteropolysaccharides in the body (Table 14.1). These side chains include chrondroitin sulfate, heparin sulfate, keratin sulfate, and dermatan sulfates. GAGs provide structural integrity to cells and provide passageways between cells, allowing for cell migration. The content of GAGs increases in gliomas, mainly due to increases in heparin sulfate and dermatan sulfate [30]. Hyaluronan is a major GAG constituent of brain ECM often found non-covalently attached to other proteins including members of the lectican proteoglycan family (Table 14.1). In normal adult brain, hyaluronan constitutes 36 and 54 per cent of grey and white matter GAG, respectively. In brain tumors, the percentage of hyaluronan is highest in astrocytoma (56 per cent), decreasing to 32 per cent in anaplastic astrocytomas, and 29 per cent in glioblastoma [20]. Hyaluronan can induce intracellular signaling by binding to its cell surface proteoglycan receptor, the adhesion molecule CD44 (Table 14.2, Fig. 14.1; see CD44 below). This GAG can therefore contribute structurally to the ECM, as well as serve as a regulatory signal. The binding of hyaluronan to its receptor is enhanced by the ECM glycoprotein collagen 1 [31], demonstrating the ability of other ECM components to influence hyaluronan-induced intracellular signaling. Its role in invasion has been substantiated by perturbation of hyaluronan-CD44 interactions using either small hyaluronan oligosac-charides that compete for endogenous hyaluronan polymer interactions, or by overexpression of soluble hyaluronan-binding proteins, which leads to inhibition of anchorage-dependent growth and decreased invasion [32]. These experiments suggest that

FIGURE 14.3 Interactions Between Integrins, Matricellular and Extracellular (ECM) Proteins. The matricellular proteins have a significant impact on integrin signaling. They can either bind ECM proteins (1) or integrins (2) to disrupt integrin-ECM interactions (3), and thereby influence adhesion, motility and signaling. Colored circles to the left indicate the matricellular proteins that can bind to the same ECM protein (4). The ECM proteins can often bind to more than one integrin (5). The grey and black circles connected by the black lines depict the major fi, a, and av integrins involved in glioma adhesion and invasion (6). Integrins can bind to more than one ECM. Integrins can also directly bind to matricellular proteins, the colored circles depicted on the right. Therefore, there are many potential interactions that govern the state of adhesion, and a change to just one component, the integrin, ECM, or matricellular protein could significantly impact the state of adhesion and consequently migration and invasion. OPN — osteopontin, TSP — thrombospondin, TN — Tenascin C, CN — collagen, LN — laminin, FN — fibronectin, and VN — vitronectin. See Plate 14.3 in Color Plate Section.

FIGURE 14.3 Interactions Between Integrins, Matricellular and Extracellular (ECM) Proteins. The matricellular proteins have a significant impact on integrin signaling. They can either bind ECM proteins (1) or integrins (2) to disrupt integrin-ECM interactions (3), and thereby influence adhesion, motility and signaling. Colored circles to the left indicate the matricellular proteins that can bind to the same ECM protein (4). The ECM proteins can often bind to more than one integrin (5). The grey and black circles connected by the black lines depict the major fi, a, and av integrins involved in glioma adhesion and invasion (6). Integrins can bind to more than one ECM. Integrins can also directly bind to matricellular proteins, the colored circles depicted on the right. Therefore, there are many potential interactions that govern the state of adhesion, and a change to just one component, the integrin, ECM, or matricellular protein could significantly impact the state of adhesion and consequently migration and invasion. OPN — osteopontin, TSP — thrombospondin, TN — Tenascin C, CN — collagen, LN — laminin, FN — fibronectin, and VN — vitronectin. See Plate 14.3 in Color Plate Section.

molecular targets TABLE 14.1 ECM Molecules

Proteoglycan/GAG Superfamily

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