Corinne Perreau

Lumican inhibits cell migration through α2β1 integrin.

Lumican, an extracellular matrix protein of the small leucine-rich proteoglycan family, has been shown to impede melanoma progression by inhibiting cell migration. In the present study, we show that lumican targets α2β1 integrin thereby inhibiting cell migration. A375 melanoma cells were transfected with siRNA directed against the α2 integrin subunit. Compared to A375 control cells, the anti-migratory effect of lumican was abrogated on transfected A375 cells. Moreover, lumican inhibited the chemotactic migration of Chinese hamster ovary (CHO) cells stably transfected with α2 integrin subunit (CHO-A2) but not that of wild-type CHO cells (CHO-WT) lacking this subunit. In contrast to CHO-WT cells, we observed in time-lapse microscopy a decrease of CHO-A2 cell migration speed in presence of lumican. Focal adhesion kinase phosphorylated at tyrosine-397 (pFAK) and total FAK were analysed in CHO-WT and CHO-A2 cells. A significant decrease of the ratio pFAK/FAK was shown in presence of recombinant human lumican. Using solid phase assays, a direct binding between lumican and the α2β1 integrin was demonstrated. This interaction did not involve the glycan moiety of lumican and was cation independent. Lumican was also able to bind the activated I domain of the α2 integrin subunit with a K(d)≥200nM. In conclusion, we demonstrated for the first time that the inhibition of cell migration by lumican depends on a direct binding between the core protein of lumican and the α2β1 integrin.

Effect of lumican on the migration of human mesenchymal stem cells and endothelial progenitor cells: involvement of matrix metalloproteinase-14.

Background Increasing number of evidence shows that soluble factors and extracellular matrix (ECM) components provide an optimal microenvironment controlling human bone marrow mesenchymal stem cell (MSC) functions. Successful in vivo administration of stem cells lies in their ability to migrate through ECM barriers and to differentiate along tissue-specific lineages, including endothelium. Lumican, a protein of the small leucine-rich proteoglycan (SLRP) family, was shown to impede cell migration and angiogenesis. The aim of the present study was to analyze the role of lumican in the control of MSC migration and transition to functional endothelial progenitor cell (EPC). Methodology/Principal Findings Lumican inhibited tube-like structures formation on Matrigel® by MSC, but not EPC. Since matrix metalloproteinases (MMPs), in particular MMP-14, play an important role in remodelling of ECM and enhancing cell migration, their expression and activity were investigated in the cells grown on different ECM substrata. Lumican down-regulated the MMP-14 expression and activity in MSC, but not in EPC. Lumican inhibited MSC, but not EPC migration and invasion. The inhibition of MSC migration and invasion by lumican was reversed by MMP-14 overexpression. Conclusion/Significance Altogether, our results suggest that lumican inhibits MSC tube-like structure formation and migration via mechanisms that involve a decrease of MMP-14 expression and activity.

Lumican: A new inhibitor of matrix metalloproteinase‐14 activity

We previously showed that lumican regulates MMP‐14 expression. The aim of this study was to compare the effect of lumican and decorin on MMP‐14 activity. In contrast to decorin, the glycosylated form of lumican was able to significantly decrease MMP‐14 activity in B16F1 melanoma cells. Our results suggest that a direct interaction occurs between lumican and MMP‐14. Lumican behaves as a competitive inhibitor which leads to a complete blocking of the activity of MMP‐14. It binds to the catalytic domain of MMP‐14 with moderate affinity (K D ∼ 275 nM). Lumican may protect collagen against MMP‐14 proteolysis, thus influencing cell–matrix interaction in tumor progression.

Lumican - derived peptides inhibit melanoma cell growth and migration.

Lumican, a small leucine-rich proteoglycan of the extracellular matrix, presents potent anti-tumor properties. Previous works from our group showed that lumican inhibited melanoma cell migration and tumor growth in vitro and in vivo. Melanoma cells adhered to lumican, resulting in a remodeling of their actin cytoskeleton and preventing their migration. In addition, we identified a sequence of 17 amino acids within the lumican core protein, named lumcorin, which was able to inhibit cell chemotaxis and reproduce anti-migratory effect of lumican in vitro. The aim of the present study was to characterize the anti-tumor mechanism of action of lumcorin. Lumcorin significantly decreased the growth in monolayer and in soft agar of two melanoma cell lines – mice B16F1 and human SK-MEL-28 cells – in comparison to controls. Addition of lumcorin to serum free medium significantly inhibited spontaneous motility of these two melanoma cell lines. To characterize the mechanisms involved in the inhibition of cell migration by lumcorin, the status of the phosphorylation/dephosphorylation of proteins was examined. Inhibition of focal adhesion kinase phosphorylation was observed in presence of lumcorin. Since cancer cells have been shown to migrate and to invade by mechanisms that involve matrix metalloproteinases (MMPs), the expression and activity of MMPs were analyzed. Lumcorin induced an accumulation of an intermediate form of MMP-14 (~59kDa), and inhibited MMP-14 activity. Additionally, we identified a short, 10 amino acids peptide within lumcorin sequence, which was able to reproduce its anti-tumor effect on melanoma cells. This peptide may have potential pharmacological applications.

Lumican Inhibits SNAIL-Induced Melanoma Cell Migration Specifically by Blocking MMP-14 Activity

Lumican, a small leucine rich proteoglycan, inhibits MMP-14 activity and melanoma cell migration in vitro and in vivo. Snail triggers epithelial-mesenchymal transitions endowing epithelial cells with migratory and invasive properties during tumor progression. The aim of this work was to investigate lumican effects on MMP-14 activity and migration of Snail overexpressing B16F1 (Snail-B16F1) melanoma cells and HT-29 colon adenocarcinoma cells. Lumican inhibits the Snail induced MMP-14 activity in B16F1 but not in HT-29 cells. In Snail-B16F1 cells, lumican inhibits migration, growth, and melanoma primary tumor development. A lumican-based strategy targeting Snail-induced MMP-14 activity might be useful for melanoma treatment.

Lumican effectively regulates the estrogen receptors-associated functional properties of breast cancer cells, expression of matrix effectors and epithelial-to-mesenchymal transition

Lumican is a small leucine-rich proteoglycan that has been shown to contribute in several physiological processes, but also to exert anticancer activity. On the other hand, it has been recently shown that knockdown of the estrogen receptor α (ERα) in low invasive MCF-7 (ERα+) breast cancer cells and the suppression of ERβ in highly aggressive MDA-MB-231 (ERβ+) cells significantly alter the functional properties of breast cancer cells and the gene expression profile of matrix macromolecules related to cancer progression and cell morphology. In this report, we evaluated the effects of lumican in respect to the ERs-associated breast cancer cell behaviour, before and after suppression of ERs, using scanning electron and confocal microscopies, qPCR and functional assays. Our data pinpointed that lumican significantly attenuated cell functional properties, including proliferation, migration and invasion. Furthermore, it modified cell morphology, inducing cell-cell junctions, evoked EMT/MET reprogramming and suppressed the expression of major matrix effectors (matrix metalloproteinases and EGFR) implicated in breast cancer progression. The effects of lumican were found to be related to the type of breast cancer cells and the ERα/β type. These data support the anticancer activity of lumican and open a new area for the pharmacological targeting of the invasive breast cancer.

Involvement of lysine 1047 in type I collagen-mediated activation of polymorphonuclear neutrophils

Oxidative functions of polymorphonuclear neutrophils (PMNs), which play a deciding role in the phagocytosis process, are stimulated by extracellular matrix proteins such as type I collagen. Previous studies have demonstrated the involvement of a DGGRYY sequence located within the α1 chain C‐terminal telopeptide in type I collagen‐induced PMN activation, but so far the mechanism has not been completely elucidated. We have recently demonstrated that collagen carbamylation (i.e. post‐translational binding of cyanate to lysine ε‐NH2 groups) impairs PMN oxidative functions, suggesting the potential involvement of lysine residues in this process. The present study was devoted to the identification of lysine residues involved in the collagen‐induced activation of PMNs. The inhibition of PMN activation by collagen in the presence of 6‐amino‐hexanoic acid, a structural analogue of lysine residues, confirmed the involvement of specific lysine residues. Modification of lysine residues by carbamylation demonstrated that only one residue, located within the α1CB6 collagen peptide, was involved in this mechanism. A recombinant α1CB6 peptide, designed for the substitution of lysine 1047 by glycine, exhibited decreased activity, demonstrating that the lysine residue at position 1047 within the collagen molecule played a significant role in the mechanism of activation. These results help to understand in more detail the collagen‐mediated PMN activation mechanism and confirm the prominent involvement of lysine residues in interactions between extracellular matrix proteins and inflammatory cells.

Regulation of the Inflammatory Response by the α3 Chain of Type IV Collagen

Our initial observation (Monboisse et al, 1987; Monboisse et al, 1990) that type I collagen activates polymorphonuclear leukocytes (PMN) prompted the testing of the activating potential of type IV collagen. It was noted, however, that type IV collagen isolated from bovine lens capsule did not activate PMN but rather prevented their stimulation by N-formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe), phorbol myristate acetate (PMA) or type I collagen (3). This observation led to the present study (4) which demonstrates that the inhibitory effect of lens capsule type IV collagen resides in the non-collagenous (NCl) domain of the α3 chain and specifically in the region comprising residues 185–203 of the NCl domain of both the human and bovine molecules. Synthetic peptides from the same region of the NCl domains of the α1, α2, α4 and α5 chains did not possess the inhibitory effect seen with the α3 chain. The sequence S-N-S (residues 189–191) is unique to the peptide of the α3 chain and substitution of either serine with alanine abolishes the inhibition. Type IV collagen isolated from the mouse EHS tumor, a molecule that lacks the α3 chain, did not prevent PMN activation but instead stimulated the secretion of elastase and type IV collagenase. Incubation of PMN with intact lens capsule type IV collagen or a peptide comprising residues 185–203 of the α3(IV) chain resulted in a 3-fold increase of intracellular cAMP, whereas, Ca++ levels remained unchanged. Incubating PMN with forskolin or with dibutyryl-cAMP resulted in the inhibition of both O2- production and degranulation by PMN, thus mimicking the effects of type IV collagen and the α3(IV) 185–203 peptide. Pertussis toxin abolished the inhibitory effect of type IV collagen and its peptide.