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.