The 67 kDa laminin receptor increases tumor aggressiveness by remodeling laminin-1.
The association between expression of the 67 kDa laminin receptor (67LR) and tumor aggressiveness has been convincingly demonstrated although the exact function of this molecule in the metastatic process has remained unclear. In this study, we tested whether the laminin-1, upon interaction with 67LR, promotes tumor cell aggressiveness; the investigation was based on: (i) the previous demonstration that soluble 67LR, as well as a 20-amino-acid peptide corresponding to the 67LR laminin binding site, changes the conformation of laminin upon interaction with this adhesion molecule and (ii) the known relevance of microenvironment remodeling by the tumor, leading to structural modification of extracellular matrix components in tumor progression. MDAMB231 breast carcinoma cells plated on peptide G-treated laminin-1 exhibited a polygonal array of actin filament bundles compared with cells seeded on native laminin-1 which presented the actin bundles organized as multiple cables parallel to margins. Furthermore, in cells seeded on peptide G-treated laminin-1, 67LR was distinct from the alpha6 integrin subunit in filopodia protrusions in addition to colocalizing with this integrin in focal adhesion plaques as it occurs when cells are plated on native laminin-1. In addition to differences in tumor cell adhesion and migration found in cells exposed to peptide G-treated vs native laminin-1, breast carcinoma cells seeded on modified laminin-1 showed a 6-fold increase in invasion capability compared with cells seeded on unmodified laminin-1. Alterations in actin organization as well as adhesion, migration and especially invasion observed in MDAMB231 cells in the presence of peptide G-treated laminin-1 were even found in MDAMB231 cells that, after selection for 67LR high expression, were seeded on native laminin-1. As the 67LR shedding is proportional to its expression level, these findings indicate a role for 67LR in changing laminin structure. Expression analysis of 97 genes encoding proteins that mediate cell matrix interactions, revealed significant differences between cells exposed to modified vs unmodified laminin-1 in 19 genes, 17 of which--including those encoding alpha3 integrin, extracellular matrix protein 1, proteolytic enzymes (such as MT1-MMP, stromelysin-3 and cathepsin L) and their inhibitors--were up-modulated in cells treated with modified laminin-1. Zymogram analysis clearly indicated a significant increase in the activity of the gelatinolytic enzyme MMP-2 in the culture supernatant from cells exposed to modified laminin-1, without an increase in mRNA abundance as observed in microarray analysis. Invasiveness of tumor cells conditioned by modified laminin-1, evaluated as the capability to cross Matrigel basement, was significantly more inhibited by MMPinhibitor TIMP-2 than invasiveness induced by native laminin-1. Taken together, our findings indicate that the role of 67LR in tumor aggressiveness rests in its ability to modify laminin-1 thereby activating proteolytic enzymes that promote tumor cell invasion through extracellular matrix degradation.
Berno
V
V
Molecular Targeting Unit, Department of Experimental Oncology, Istituto Nazionale Tumori, Via Venezian 1 20133 Milan, Italy.
Porrini
D
D
Castiglioni
F
F
Campiglio
M
M
Casalini
P
P
Pupa
S M
SM
Balsari
A
A
Ménard
S
S
Tagliabue
E
E
D001665:Binding Sites; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology*; D002448:Cell Adhesion; D002465:Cell Movement; D005109:Extracellular Matrix / Q000378:metabolism; D005260:Female; D020869:Gene Expression Profiling; D006801:Humans; D007797:Laminin / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D009361:Neoplasm Invasiveness; D017474:Receptors, Laminin / Q000502:physiology*
D007797:Laminin; D017474:Receptors, Laminin; C121346:laminin 1