Astrocytoma Adhesion to Extracellular Matrix: Functional Significance of Integrin and Focal Adhesion Kinase Expression

Abstract

Evidence is accumulating implicating a role for integrins in the pathogenesis of cancer, a disease in which alterations in cellular growth, differentiation, and adhesive characteristics are defining features. In the present report we studied a panel of 8 human astrocytoma cell lines for their expression of integrin subunits by RT-PCR, and of integrin heterodimers by immunoprecipitation analyses. The functionality of integrin heterodimers was assessed using cell attachment assays to plastic or single matrix substrates. Downstream effects of integrin activation were studied by western blot analyses of FAK expression in human astrocytoma cell lines growing on plastic and on a fibronectin matrix, and in 13 primary human brain tumor specimens of varying histopathological grade. Furthermore, we studied tyrosine phosphorylation of FAK in astrocytoma cells growing on plastic versus fibronectin. Finally, we analyzed the effects of intermediate filament gene transfer on FAK phosphorylation in SF-126 astrocytoma cells. Our data show that astrocytoma cell lines express various integrin subunits by RT-PCR, and heterodimers by immunoprecipitation analyses. The beta1 and alphav integrin subunits were expressed by all astrocytoma cell lines. The alpha3 subunit was expressed by all cell lines except SF-188. By immunoprecipitation, the fibronectin receptor (alpha5beta1 integrin heterodimer) and the vitronectin receptor (alphavbeta3) were identified in several cell lines. Astrocytoma cell attachment studies to human matrix proteins suggested that these integrin heterodimers were functional. Using confocal laser microscopy, we showed that FAK was colocalized to actin stress fibers at sites of focal adhesion complexes. By western blot, FAK was variably but quite ubiquitously expressed in human astrocytoma cell lines, and in several primary human astrocytoma specimens. When U373 and U87 MG astrocytoma cells bind to a fibronectin matrix, FAK is phosphorylated. GFAP-transfected SF-126 human astrocytoma cells were shown to overexpress the phosphorylated form of FAK only when these cells were placed on a fibronectin matrix. This result is of interest because it suggests that manipulations of the astrocytoma cytoskeleton per se can bring about potential signaling changes that channel through integrins and focal adhesion sites leading to activation of key kinases such as FAK.

p300- and Myc-mediated regulation of glioblastoma multiforme cell differentiation

“…Our data show that RNAi-mediated knockdown of p300 significantly increases the migration and invasion of U251 cells in vitro. This finding is consistent with the earlier observations by Rutka et al, who have demonstrated that elimination of GFAP in U251 cells with anti-sense RNA results in the marked decrease in cell adhesion and increase in invasiveness [12], which is associated with increased expression of β1 integrin [62] and CD44, and with redistribution of actin forming actin stress fibers implicated in the regulation of cell motility [63].…”

Epidermal Growth Factor Receptor Variant III–Induced Glioma Invasion Is Mediated through Myristoylated Alanine-Rich Protein Kinase C Substrate Overexpression

“…17). Cells were transfected with MARCKS siRNA, control siRNA, or mock transfected for 72 h and then replated onto the coated 96-well plate.…”

Emerging insights into the molecular and cellular basis of glioblastoma

“…Strikingly, CD44 and RHAMM are both suppressed by p53 (Godar et al 2008;Sohr and Engeland 2008), suggesting that early cellular progression through canonical checkpoints and the ability to migrate/invade are linked, although this possibility remains to be validated in glioma models. Integrins, particularly the avb3 and avb5 heterodimers (Bello et al 2001a), also likely contribute to glioma cell adherence to the ECM in a process that activates cytoskeletal rearrangement through cytoplasmic mediators, including FAK (Rutka et al 1999;Riemenschneider et al 2005) and/or Pyk2 (Lipinski et al 2008). Their putative involvement in glioma pathobiology (D'Abaco and Kaye 2007; Desgrosellier and Cheresh 2010) has prompted the testing of the avb3 and avb5 integrin inhibitor cilengitide in an ongoing phase III CENTRIC clinical trial for newly diagnosed glioblastoma in combination with radiation therapy and temozolomide (Reardon et al 2011a).…”