Focal adhesions and associated proteins in medullary thyroid carcinoma cells.
BACKGROUND
In medullary thyroid carcinoma (MTC), mutations in the RET protooncogene lead to oncogenic transformation. RET activation in other cell types has been shown to cause phosphorylation of the focal adhesion-associated proteins focal adhesion kinase (FAK), paxillin, and p130(CAS). We hypothesized that adhesion-dependent signaling might be deranged in MTC cells.


METHODS
Indirect immunofluorescence was used to label beta(1) integrin, FAK, paxillin, and p130CAS. Rhodamine-labeled phalloidin was used to visualize actin microfilaments. Phosphorylated protein was detected by immunoprecipitation followed by Western blotting for phosphotyrosine. MTC cell invasiveness was quantified using a modified Boyden chamber assay.


RESULTS
Clustering of beta(1) integrin, FAK, paxillin, and p130(CAS) into focal adhesions were not detected in MTC cells under any conditions, although clustering was seen as expected in control HeLa cells. Despite this failure, FAK, paxillin and p130(CAS) were all found to be phosphorylated. Actin microfilaments were generally not seen although in a few cells, small, poorly formed microfilaments could be detected. MTC cells invaded poorly as compared with highly invasive cell lines. However a clear difference was noted between invasiveness on growth factor depleted Matrigel and regular Matrigel.


CONCLUSIONS
In MTC cells, focal adhesions are not seen in response to interaction with extracellular matrix. Consistent with this failure, actin microfilaments are absent or poorly formed and invasion is weak. Despite the absence of focal adhesions, focal adhesion proteins remain phosphorylated, even though in normal cells their signaling activity is dependent on focal adhesion formation. Deranged adhesion-dependent signaling may contribute to MTC pathogenesis.
Kim
Lawrence T
LT
Surgical Service, Central Arkansas Veterans Healthcare System and University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.
Fleming
Jason B
JB
Lopez-Guzman
Christie
C
Nwariaku
Fiemu
F
D008841:Actin Cytoskeleton / Q000473:pathology; D000199:Actins / Q000032:analysis; D015153:Blotting, Western; D018276:Carcinoma, Medullary / Q000737:chemistry* / Q000473:pathology*; D050719:Crk-Associated Substrate Protein; D003598:Cytoskeletal Proteins / Q000032:analysis; D019084:Fluorescent Antibody Technique, Indirect; D051416:Focal Adhesion Protein-Tyrosine Kinases; D022001:Focal Adhesions / Q000473:pathology*; D007163:Immunosorbent Techniques; D019012:Integrin beta1 / Q000032:analysis; D051419:Paxillin; D010750:Phosphoproteins / Q000032:analysis; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000032:analysis; D011506:Proteins; D050717:Retinoblastoma-Like Protein p130; D015398:Signal Transduction; D013964:Thyroid Neoplasms / Q000737:chemistry* / Q000473:pathology*; D014407:Tumor Cells, Cultured
D000199:Actins; D050719:Crk-Associated Substrate Protein; D003598:Cytoskeletal Proteins; D019012:Integrin beta1; D051419:Paxillin; D010750:Phosphoproteins; D011506:Proteins; D050717:Retinoblastoma-Like Protein p130; D011505:Protein-Tyrosine Kinases; D051416:Focal Adhesion Protein-Tyrosine Kinases