Astrocytoma adhesion to extracellular matrix: Functional significance of integrin and focal adhesion kinase expression

Evidence is accumulating implicating a role for integrins in the pathogenes is of cancer, a disease in which alterations in cellular growth, differenti ation, and adhesive characteristics are defining features. In the present r eport we studied a panel of 8 human astrocytoma cell lines for their expres sion of integrin subunits by RT-PCR, and of integrin heterodimers by immuno precipitation analyses. The functionality of integrin heterodimers was asse ssed using cell attachment assays to plastic or single matrix substrates. D ownstream effects of integrin activation were studied by western blot analy ses of FAK expression in human astrocytoma cell lines growing on plastic an d on a fibronectin matrix, and in 13 primary human brain tumor specimens of varying histopathological grade. Furthermore, we studied tyrosine phosphor ylation 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 astrocy toma cell lines express various integrin subunits by RT-PCR, and heterodime rs by immunoprecipitation analyses. The beta 1 and alpha v integrin subunit s were expressed by all astrocytoma cell lines. The alpha 3 subunit was exp ressed by all cell lines except SF-188. By immunoprecipitation, the fibrone ctin receptor (alpha 5 beta 1 integrin heterodimer) and the vitronectin rec eptor (alpha v beta 3) were identified in several cell lines. Astrocytoma c ell attachment studies to human matrix proteins suggested that these integr in 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 expres sed in human astrocytoma cell lines, and in several primary human astrocyto ma specimens. When U373 and U87 MG astrocytoma cells bind to a fibronectin matrix, FAK is phosphorylated. GFAP-transfected SF126 human astrocytoma cel ls were shown to overexpress the phosphorylated form of FAK only when these cells were placed on a fibronectin matrix. This result is of interest beca use it suggests that manipulations of the astrocytoma cytoskeleton per se c an bring about potential signaling changes that channel through integrins a nd focal adhesion sites leading to activation of key kinases such as FAK.