Reduced Expression of the Hyaluronan and Proteoglycan Link Proteins in Malignant Gliomas

Malignant gliomas have a distinctive ability to infiltrate the brain parenchyma and disrupt the neural extracellular matrix that inhibits motility of axons and normal neural cells. Chondroitin sulfate proteoglycans (CSPGs) are among the major inhibitory components in the neural matrix, but surprisingly, some are up-regulated in gliomas and act as pro-invasive signals. In the normal brain, CSPGs are thought to associate with hyaluronic acid and glycoproteins such as the tenascins and link proteins to form the matrix scaffold. Here, we examined for the first time the expression of link proteins in human brain and malignant gliomas. Our results indicate that HAPLN4 and HAPLN2 are the predominant members of this family in the adult human brain but are strongly reduced in the tumor parenchyma. To test if their absence was related to a pro-invasive gain of function of CSPGs, we expressed HAPLN4 in glioma cells in combination with the CSPG brevican. Surprisingly, HAPLN4 increased glioma cell adhesion and migration and even potentiated the motogenic effect of brevican. Further characterization revealed that HAPLN4 expressed in glioma cells was largely soluble and did not reproduce the strong, hyaluronan-independent association of the native protein to brain subcellular membranes. Taken together, our results suggest that the tumor parenchyma is rich in CSPGs that are not associated to HAPLNs and could instead interact with other extracellular matrix proteins produced by glioma cells. This dissociation may contribute to changes in the matrix scaffold caused by invasive glioma cells.The extracellular matrix (ECM)² of the adult central nervous system lacks most fibrous proteins (collagens, fibronectin, and laminins) that are present in the matrices of other tissues and is formed instead by a scaffold of hyaluronic acid (HA) with associated glycoproteins (1). The major family of HA binding matrix glycoproteins in the central nervous system is formed by the chondroitin sulfate proteoglycans of the lectican family (aggrecan, versican, neurocan, and brevican), the last two expressed almost exclusively in neural tissue (2). These proteoglycans bind both to HA and to cell-surface receptors (3), regulating the cross-linking and compressibility of the matrix scaffold and, therefore, modulating many neural processes including cell motility during development, axonal navigation, and the stabilization of synapses (4). The lecticans have been identified as a major class of molecules that restrict cellular and axonal motility in neural tissue and are a major component of the glial scar that forms after neural injury and prevents axonal regeneration (5).A second family of HA-binding proteins expressed in the central nervous system is formed by small glycoproteins known as HA- and proteoglycan-link proteins (HAPLNs) or, simply, “link proteins.” These glycoproteins bind both to HA and to the lecticans, forming ternary complexes (6, 7). The structure of the link proteins is remarkably similar to the N-terminal region of the lecticans, and the highly homologous HA binding domains from HAPLNs and lecticans are indistinctly known as proteoglycan tandem repeats or link-protein modules.In a striking example of molecular evolution, the genes of the four HAPLNs are located adjacent to the genes of the four lecticans, indicating a common molecular origin by gene duplication (8). Two of the link proteins, HAPLN2 and HAPLN4, have only been detected in neural tissue, and their genes are adjacent to the neural-specific proteoglycans, brevican and neurocan, respectively (8). Both HAPLN2 and HAPLN4, also known as brain-specific link protein (Bral-1) and Bral-2, are up-regulated in the adult central nervous system and match the temporal expression profile of brevican, which is the most abundant CSPG in adult neural tissue (9, 10).Current evidence suggests that the HAPLNs may be key components in the organization of the HA-based matrix scaffold. HAPLN1, the best studied member of the family, increases the affinity of the lecticans for HA (11, 12) and stabilizes lectican-HA matrix aggregates (6, 13). Moreover, the increased expression of lecticans and HAPLNs in the adult central nervous system correlates temporally and spatially with changes in ECM solubility and with appearance of ECM aggregates around subsets of neurons, known as “perineuronal nets.” These changes have been associated with restricted cellular motility and decreased synaptic plasticity (14).The role of the lectican CSPGs as inhibitors of motility in the adult central nervous system contrasts starkly with their pro-invasive role in the highly aggressive brain tumors known as malignant gliomas. These are the most common primary tumors of the brain and are characterized by their extensive and diffuse infiltration of the brain parenchyma (15), which makes them impossible to completely remove and facilitates tumor recurrence even after long term therapies. The invasive ability of gliomas is restricted to neural tissue and is not observed in other tumors that metastasize to the brain, suggesting that glioma invasion may be supported in part by unique mechanisms to remodel the neural microenvironment (16).Two lectican CSPGs, versican and the neural-specific CSPG brevican, are highly up-regulated in gliomas compared with normal brain tissue (3). Although these proteoglycans are thought to inhibit the motility of normal glial cells (17, 18), they instead promote glioma cell adhesion and migration. The underlying molecular mechanisms for this unusual effect are poorly understood, although we and others have demonstrated that these lecticans can activate epidermal growth factor receptor signaling in glioma cells, which leads to an increase of cell-surface adhesion molecules (19). Both brevican and versican can also form adhesive complexes with mesenchymal matrix proteins that are present in the glioma ECM but absent from the normal neural ECM (19, 20).Although the role of CSPGs in brain tumors is starting to become better defined, their HAPLN partners have never been analyzed in human brain or in neuropathologies. Therefore, we still have a highly incomplete picture of the molecular changes that occur in the tumor ECM and of how those changes could affect critical aspects of glioma biology such as invasion of the surrounding tissue.We hypothesized that the gain of function of CSPGs in gliomas could be associated with changes in the levels or molecular associations of specific HAPLNs in the ECM of gliomas. Thus, we studied here the expression and biochemical properties of the HAPLN family in human normal brain and glioma tissue. Our results provide the first biochemical characterization of the brain-specific human HAPLN4 and, in addition, show that both neural-specific link proteins HAPLN2 and HAPLN4, which are abundant in adult brain, are virtually absent from the ECM of malignant gliomas.