Lumican core protein inhibits melanoma cell migration via alterations of focal adhesion complexes.
Lumican is a small leucine-rich proteoglycan (SLRP) of the extracellular matrix (ECM) with anti-tumor activity. We recently demonstrated that lumican inhibits the migration of melanoma cells and identified beta1 integrin as mediator of this effect [M.F. D'Onofrio, S. Brézillon, T. Baranek, C. Perreau, P.J. Roughley, F.X. Maquart, Y. Wegrowski, Identification of beta1 integrin as mediator of melanoma cell adhesion to lumican, Biochem. Biophys. Res. Commun. 365 (2008) 266-272]. The aim of the present work was to study beta1 integrin, focal adhesion complexes, actin distribution and expression in the presence of lumican substratum in comparison to type I collagen or fibronectin substrata in A375 human melanoma cells. The protein distribution was investigated by immunocytochemistry and confocal microscopy. In parallel, their expression was evaluated by Western immunoblotting and Real-time Reverse Transcription-PCR analyses. The interaction of melanoma cells with the lumican substratum resulted in heterogeneous distribution of beta1 integrin on cell membrane after 24h of seeding. Concomitantly, a reorganization of actin stress fibers and a significant decrease in vinculin immunostaining at focal adhesion complexes were observed. No alteration of the expression was detected at protein and mRNA levels. However, a cytosolic accumulation of vinculin focal adhesion protein was observed on lumican substratum by confocal microscopy. Moreover, vinculin expression was significantly increased in cytosolic fractions in comparison to cells seeded on type I collagen or fibronectin substrata. Our results suggest that lumican induces an alteration of the link between actin filaments and beta1 integrin, characterized by a cytosolic accumulation of vinculin focal adhesion protein, which could lead to a destabilization of focal adhesion complexes. In addition, focal adhesion kinase phosphorylated at tyrosine-397 (pFAK) was significantly decreased. Therefore, the cytoskeleton remodeling and the decreased pFAK phosphorylation induced by lumican in melanoma cells might explain, at least in part, the anti-invasive effect of this SLRP.
Brézillon
Stéphane
S
Laboratoire de Biochimie Médicale et Biologie Moléculaire, CNRS UMR 6237, Faculté de Médecine, Université de Reims-Champagne-Ardenne, 51095 Reims Cedex, France. stephane.brezillon@univ-reims.fr
Radwanska
Agata
A
Zeltz
Cedric
C
Malkowski
Andrzej
A
Ploton
Dominique
D
Bobichon
Hélène
H
Perreau
Corinne
C
Malicka-Blaszkiewicz
Maria
M
Maquart
François-Xavier
FX
Wegrowski
Yanusz
Y
D000199:Actins / Q000378:metabolism; D015153:Blotting, Western; D002448:Cell Adhesion / Q000502:physiology; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000502:physiology*; D011508:Chondroitin Sulfate Proteoglycans / Q000378:metabolism*; D024042:Collagen Type I / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism*; D005109:Extracellular Matrix / Q000378:metabolism*; D005353:Fibronectins / Q000378:metabolism; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D019012:Integrin beta1 / Q000378:metabolism; D007632:Keratan Sulfate / Q000378:metabolism*; D000071234:Lumican; D008545:Melanoma / Q000378:metabolism / Q000473:pathology*; D018613:Microscopy, Confocal; D010766:Phosphorylation; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016596:Vinculin / Q000378:metabolism
D000199:Actins; D011508:Chondroitin Sulfate Proteoglycans; D024042:Collagen Type I; D005353:Fibronectins; D019012:Integrin beta1; C000604306:LUM protein, human; D000071234:Lumican; D016596:Vinculin; D007632:Keratan Sulfate; D051416:Focal Adhesion Protein-Tyrosine Kinases