Gliomas are the most common intracranial malignant tumors in humans, and high-grade Gliomas in particular pose a unique challenge due to their propensity for proliferation and tissue invasion (Ref.1). The invasion of neoplastic cells into healthy brain tissue is a pathologic hallmark of Gliomas and contributes to the failure of current therapeutic modalities (surgery, radiation and chemotherapy). Transformed glial cells share the common attributes of the invasion process, including cell adhesion to ECM (Extracellular Matrix) components, cell locomotion, and the ability to remodel extracellular space. However, Glioma cells have the ability to invade as single cells through the unique environment of the normal CNS (Central Nervous System). The brain parenchyma has a unique composition, mainly Hyaluronan and is devoid of rigid protein barriers composed of Collagen, Fibronectin and Laminin. The Integrins and the Hyaluronan receptor, CD44 (CD44 Antigen) are specific adhesion receptors active in Glioma-ECM adhesion. The glycosaminoglycan Hyaluronan, a universal component of ECM, is the major component of the ECM within the brain and facilitates primary brain tumor invasion and migration through its two cellular receptors, CD44 and RHAMM (Hyaluronan-Mediated Motility Receptor). These adhesion molecules play a major role in Glioma cell-matrix interactions because the neoplastic cells use these receptors to adhere to and migrate along the components of the brain ECM (Ref.2).
During brain development, the ECM modulates the migration of glial and neuronal precursor cells, guides axonal growth cones, synapse formation and cell proliferation. In normal reparative processes, as well as in primary brain neoplasia, the ECM is present in increased amounts and undergoes remodeling. A critical factor in the process of invasion is the interaction of neoplastic cells or Glioma cells with the ECM (Ref.1). This process is realized via specific receptors expressed on their surface. There are many cell surface receptors involved in Glioma cell adhesion, migration and invasion. The Integrins, as a class of adhesion molecules, and the Hyaluronan receptors, CD44 and RHAMM play major roles in Glioma cell-matrix adhesion. Integrins are expressed as cell surface heterodimers consisting of Alpha and Beta-subunits. Integrins regulate many aspects of cell behavior including survival, proliferation, migration, differentiation, cytoskeletal rearrangement and gene transcription. They achieve their effects by two means: first, they provide a physical transmembrane link between the ECM and the cytoskeleton; second, they transduce bi-directional signals across the cell membrane. Adhesion to the substrate also requires the presence of divalent cations such as Ca2+ (Calcium ions) and Mg2+ (Magnesium ions). Each Integrin recognizes specific ligands, which are either molecules of the ECM (like Vtn (Vitronectin)) or other cell surface counter-receptors of the family of GPI-anchored membrane proteins that lack transmembrane and cytoplasmic domains, like UPAR (Urokinase Plasminogen Activator Surface Receptor). Glioma cells activate zymogens like Plg (Plasminogen) and inactive serine proteases like proUPA. UPAR is composed of three homologous domains (D1, D2, and D3) and is extensively glycosylated and attached to the cell membrane by a GPI (Glycosyl Phosphatidylinositol) anchor. In a mutually interdependent process, UPA, the active form of proUPA converts Plg to the active serine-protease Plasmin, whereas, Plasmin as a proteolytic enzyme enhance the ability of UPAR to localize the proteolytic activity of UPA (via UPA-UPAR Signaling) on the cell surface which is extremely important for the invasive ability of tumor cells. Receptor-bound UPA is inhibited by PAI1 (Plasminogen Activator Inhibitor-1). PAI1 acts as an important biologic regulator of this cascade, which promotes the internalization of UPA bound UPAR. This internalization promotes binding of receptor-bound UPA with Alpha/Beta-Integrins. The simultaneous recognition of Vtn by UPAR, co-localize Integrins to these receptors to form adhesion structures and potentially activate the Plasmin/MMP (Matrix Metalloproteinase) proteolytic cascade by converting Plg to Plasmin. This leads to ECM degradation. Consecutive clustering Integrins, trigger multiple second messenger events, such as the activation of FAK, which appears to be the most important second messenger. The phosphorylation of FAK (Focal Adhesion Kinase) activates a cascade of kinases involving MAPK (Mitogen-Activated Protein Kinase), PI3K (Phosphatidylinositde-3 Kinase); cytoskeletal modulators like Rho, Ras; which ultimately leads to the rearrangement of the cytoskeleton and cell locomotion/invasion (Ref.2 & 3).
Extracellular proteolytic enzymes are critical for the invasive properties of malignant neoplasms. This requirement is self-evident for most neoplasms, such as carcinomas and sarcomas, which must break down rigid protein barriers that include Collagen, Elastin and Laminin in order to invade adjacent structures and metastasize. Plasmin as a broad-specificity protease degrades several ECM components, such as Fibronectin, Laminin and Collagen. In addition, UPA triggers a proteolytic cascade that involves the activation of MMPs, which are responsible for ECM degradation. Plasmin activates several Matrix Metalloproteinases such as MMP1, MMP2, MMP3 and MMP9 from the inactive forms or proMMPs. These MMPs have a “hemopexin” domain, which contains a binding site for tissue inhibitors of MMPs or TIMPs (Tissue Inhibitor of Metalloproteinases). Plasmin cleaves TIMPs to inhibits their function and promote tissue invasion (Ref.2 & 4). In human Glioblastoma Multiforme (GBM), MMP2 is expressed most intensely by Glioma cells, whereas MMP9 is expressed by proliferating endothelial cells, suggesting that MMP2 is more important in the invasive properties of neoplastic cells, while MMP9 may regulate Angiogenic remodeling. Further CD44 exhibits Hyaluronan-independent role in cell migration where it acts as a cell surface anchor for MMP9. CD44 is implicated in a various range of physiological and pathological processes including cell-matrix interactions, cell migration, regulation of tumor growth and metastasis. CD44 is also essential for Hyaluronan uptake and transportation into lysosomes for degradation. The gene encoding the CD44 proteins contains 20 exons of which up to 10 variant exons encoding a portion of the ectodomain are alternatively spliced in various combinations, thereby generating numerous CD44 splice variant isoforms. The standard form of CD44 is abbreviated “sCD44” while its multiple variant isoforms are abbreviated as “vCD44”. sCD44 is expressed in a wide variety of cells including Gliomas, however its expression in the brain is restricted to the white matter. In Glioblastomas, several species of NM_000610
mRNA are expressed and encode standard CD44 (Ref.2 & 5).
More recently, a proteolytic cleavage of the extracellular portion of CD44 is known to occur in Gliomas but not in normal brain. In several tumor cell lines including Glioma, the cleavage leads to the release of a soluble fragment and also a membrane-bound cleavage product. The soluble and the membrane-bound fragments of CD44 promote tumor cell migration, mostly detected in low-grade (WHO (World Health Organization) Grade-II) and high-grade (WHO Grades, III and IV) Gliomas. This cleavage also occurs in Pilocytic Astrocytoma (WHO Grade-I). Plasmin also activates PAR1 (Protease Activated Receptor-1) and latent Growth Factors to enhance cell invasion and proliferation leading to Gloima metastasis and manifestation of brain tumors (Ref.5 & 6). This potential link between cellular anchoring to ECM by Integrins and co-localization of protease receptors appear to have an important role in tumor progression, but not all Integrins will be of equal importance. In addition, the implication of Integrins in the neovascularization of tumors is essential because understanding the mechanisms of tumor cell invasion is critical as it plays a central role in Glioma progression and failure of current treatment due to tumor recurrence from micro-disseminated disease (Ref.2 & 7).