Astrocytoma adhesion to extracellular matrix: functional significance of integrin and focal adhesion kinase expression.
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.
Arthur and Sonia Labatt Brain Tumor Research Center, Division of Neurosurgery, The Hospital for Sick Children, The University of Toronto, Ontario, Canada.
J T
JT
Rutka
M
M
Muller
S L
SL
Hubbard
J
J
Forsdike
P B
PB
Dirks
S
S
Jung
A
A
Tsugu
S
S
Ivanchuk
P
P
Costello
S
S
Mondal
C
C
Ackerley
L E
LE
Becker
D000328:Adult; D001254:Astrocytoma; D015153:Blotting, Western; D002448:Cell Adhesion / Q000502:physiology; D015815:Cell Adhesion Molecules / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D002648:Child; D003599:Cytoskeleton / Q000378:metabolism; D017931:DNA Primers; D019281:Dimerization; D016326:Extracellular Matrix Proteins / Q000378:metabolism*; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D005904:Glial Fibrillary Acidic Protein / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D016023:Integrins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000201:enzymology; D014443:Tyrosine / Q000378:metabolism
D015815:Cell Adhesion Molecules; D017931:DNA Primers; D016326:Extracellular Matrix Proteins; D005904:Glial Fibrillary Acidic Protein; D016023:Integrins; D012333:RNA, Messenger; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human