Promotion of Malignant Astrocytoma Cell Migration by Osteopontin Expressed in the Normal Brain: Differences in Integrin Signaling During Cell Adhesion to Osteopontin vs. Vitronectin

Abstract

The extracellular matrix of the normal adult brain lacks expression of most of the adhesive glycoproteins that are known to promote cell attachment, and it has been thought that the malignant invasion of astrocytoma tumor is mediated primarily by remodeling of the matrix by the tumor cells. It has been reported, however, that normal brain neuropil does contain a protein(s) that promotes cell attachment. Therefore, we explored the possibility that the cell attachment protein, osteopontin, is expressed in the normal human brain. Here, we report that osteopontin is expressed in the cortical gray and white matter of normal adult brain, with the levels of osteopontin expression being equivalent to those in malignant astrocytic tumor biopsies as assessed by Western blot analysis. Immunoblotting identified osteopontin polypeptides with relative molecular weights of 60-and 65-kDa in normal brain white matter and in astrocytic tumors, with an additional 70-kDa polypeptide being identified in normal cortical gray matter and in some astrocytic tumors. Recombinant osteopontin was found to promote attachment of U-251MG human malignant astrocytoma cells in a process that was inhibited by anti-integrin monoclonal antibodies anti-␣v␤3 (75%), anti-␣v␤5 (80%), and anti-␣5 (40%). On attachment, integrins ␣v␤5 and ␣v␤3 localized to focal adhesions, and there was an alteration in cell morphology with the formation of lamellae-like processes. The attachment was associated with activation of Rac in a slow and prolonged fashion and rapid activation of Rho. Similarly, integrins ␣v␤5 and ␣v␤3 localized to focal adhesions on attachment of the U-251MG cells to vitronectin, but on this substrate, the cells assumed a spread and flat morphology, and there was rapid activation of both Rac and Rho. Extracts of normal brain white matter were capable of promoting haptotactic migration, and this response was inhibitable by monoclonal antibodies anti-␣v␤3 and anti-␣5. Depletion of the osteopontin in these extracts abrogated the haptotactic response significantly (50%). These data indicate that the cell attachment protein, osteopontin, is expressed in the normal adult brain and that it has the potential to promote malignant astrocytoma cell invasion.

HEF1 is a necessary and specific downstream effector of FAK that promotes the migration of glioblastoma cells

“…FAK overexpression has been shown to promote invasion of A549 lung adenocarcinoma cells and of v-Src-transformed fibroblasts through Matrigel (Sieg et al, 2000;Brabek et al, 2004). Using an invasion assay into detergent-free normal brain homogenate as we have described previously (Ding et al, 2002), we found a twofold increase in invasion of doxycyclinetreated OFAK5 cells as compared to cells administered Figure 1 FAK promotes PDGF-stimulated migration of glioblastoma cells adherent to rec-osteopontin. OFAK5 cells were treated with 2 mg/ml doxycycline or vehicle for 2 days in DMEM-10% FBS, replated onto rec-osteopontin in DMEM-0.4% FBS-1% BSA, and treated with 1 mg/ml doxycycline or vehicle for 2 days.…”

“…The PDGF-stimulated migration of glioblastoma cells on osteopontin and vitronectin is of interest as the PDGF A and B genes, as well as the PDGFr-a and -b genes, are upregulated in glioblastoma tumors (Hermanson et al, 1992), osteopontin and vitronectin are expressed in glioblastoma tumors, and osteopontin is also expressed in the normal brain (Gladson et al, 1995;Ding et al, 2002). We therefore analysed the role of FAK in the migration of glioblastoma cells using previously described clones of U-251MG glioblastoma cells that overexpress wild-type FAK (clone OFAK5) under the regulation of a TET-inducible promoter (Wang et al, 2000).…”

Increased expression of osteopontin gene in atypical teratoid/rhabdoid tumor of the central nervous system

“…18 In a recent study, osteopontin was also found to promote the attachment of malignant astrocytoma cells to become more metastasized and invasive. 19 On the basis of our in vitro and in vivo observations, the high expression of OPN in AT/RT may be associated with tumor cell invasion and dissemination, which may lead to the unique clinical character of the disease and poor outcome of AT/RT patients.…”

Elevation of osteopontin levels in brain tumor cells reduces burden and promotes survival through the inhibition of cell dispersal

“…These observations, combined with low expression levels in benign tumors, suggest that OPN dysregulation is important in cell transformation or that elevated OPN expression contributes to malignant and metastatic character. In the brain OPN is intimately associated with the extracellular matrix (ECM) and its expression is developmentally regulated, suggesting that it may facilitate neuronal or glial cell migration [12,29]. Consistent with its role in the stress response, OPN has been implicated as a possible mediator of CNS inflammatory diseases, as a neuroprotective agent in acute stroke and is up-regulated in models of CNS degenerative disease and epilepsy [6,22,35].…”