keratan-sulfate and Melanoma

Mammals express a wide variety of glycans that include N-glycans, O-glycans, proteoglycans, glycolipids, etc. Glycan expression can modulate the cellular functions, and hence is strongly involved in the onset and progression of numerous diseases. Here, we report the relevance of the ectopic expression of keratan sulfate (KS) glycan chains in human malignant melanomas. Using a human melanoma cell line, we found that the KS enhanced the invasiveness of the cells but caused no change in the growth rate of the cells. The phosphorylation of paxillin, a focal adhesion-associated adaptor protein, was strong at the region where KS was expressed in the melanoma tissues, indicating that KS stimulated the phosphorylation of paxillin. We also observed that KS enhanced the adhesion of melanoma cells and this was accompanied by a greatly increased level of phosphorylation of paxillin. These data suggest that the expression of KS contributes to the development of malignant phenotypes such as strong cell adhesion and the invasiveness of melanoma cells.

Topics: Cell Line, Tumor; Glycolipids; Humans; Keratan Sulfate; Melanoma; Melanoma, Cutaneous Malignant; Paxillin; Proteoglycans; Skin Neoplasms

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.

Topics: Cell Line, Tumor; Cell Movement; Chondroitin Sulfate Proteoglycans; HT29 Cells; Humans; Keratan Sulfate; Lumican; Matrix Metalloproteinase 14; Melanoma; Snail Family Transcription Factors; Transcription Factors

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.

Topics: Cell Movement; Cell Proliferation; Chondroitin Sulfate Proteoglycans; Enzyme Activation; Focal Adhesion Protein-Tyrosine Kinases; Gene Expression Regulation, Neoplastic; Humans; Keratan Sulfate; Lumican; Matrix Metalloproteinase 14; Melanoma; Melanoma, Experimental; Peptide Fragments; Peptides; Phosphorylation

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.

Topics: Animals; Cell Line, Tumor; Cell Movement; CHO Cells; Chondroitin Sulfate Proteoglycans; Cricetinae; Cricetulus; Focal Adhesion Protein-Tyrosine Kinases; Humans; Integrin alpha2; Integrin alpha2beta1; Keratan Sulfate; Lumican; Melanoma; Phosphorylation; Protein Binding

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.

Topics: Actins; Blotting, Western; Cell Adhesion; Cell Line, Tumor; Cell Movement; Chondroitin Sulfate Proteoglycans; Collagen Type I; Cytoskeleton; Extracellular Matrix; Fibronectins; Focal Adhesion Protein-Tyrosine Kinases; Humans; Immunohistochemistry; Integrin beta1; Keratan Sulfate; Lumican; Melanoma; Microscopy, Confocal; Phosphorylation; Reverse Transcriptase Polymerase Chain Reaction; Vinculin

We previously showed that lumican decreases melanoma progression. The aim of the present study was to determine the active sequence of the lumican core protein responsible for the inhibition of melanoma cell migration. Using different recombinant and synthetic peptides derived from lumican, we localized an active site in the leucine-rich repeat 9 domain of the lumican core protein. We propose the name lumcorin (fragment of lumican core protein) for the active peptide derived from this site. Lumcorin was able to inhibit melanoma cell migration in vitro.

Topics: Apoptosis; Catalytic Domain; Cell Line, Tumor; Cell Movement; Cell Survival; Chondroitin Sulfate Proteoglycans; Humans; Keratan Sulfate; Lumican; Melanoma; Peptide Fragments

Lumican, a small leucine-rich proteoglycan (SLRP), has attracted attention as a molecule of the extracellular matrix possibly involved in signalling pathways affecting cancer cell behaviour. The remodelling of the actin cytoskeleton, induced in response to external stimuli, is crucial for cell motility and intracellular signal transduction. The main goal of this study was to examine the effects of recombinant lumican on actin organization, the state of actin polymerization, actin isoform expression, and their sub-cellular distribution in the A375 human melanoma cell line.. Fluorescence and confocal microscopy were used to observe actin cytoskeletal organization and the sub-cellular distribution of cytoplasmic beta- and gamma-actins. The ability of actin to inhibit DNaseI activity was used to quantify actin. Western blotting and real-time PCR were used to determine the expression levels of the actin isoforms.. A375 cells grown on lumican coatings changed in morphology and presented rearranged actin filament organization: from filaments evenly spread throughout the whole cell body to their condensed sub-membrane localization. In the presence of lumican, both actin isoforms were concentrated under the cellular membrane. A statistically significant increase in the total, filamentous, and monomeric actin pools was observed in A375 cells grown on lumican.. Novel biological effects of lumican, an extracellular matrix SLRP, on the actin pool and organization are identified, which may extend our understanding of the mechanism underlying the inhibitory effect of lumican on the migration of melanoma cells.

Topics: Actins; Apoptosis; Blotting, Western; Cell Survival; Chondroitin Sulfate Proteoglycans; Collagen; Cytoskeleton; Cytosol; Fibronectins; Humans; Isomerism; Keratan Sulfate; Lumican; Melanoma; Microscopy, Confocal; Microscopy, Fluorescence; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Staurosporine; Tetrazolium Salts; Thiazoles

Lumican is a small leucine-rich proteoglycan (SLRP) present in the dermal extracellular matrix. Previous data from our laboratory demonstrated that lumican decreases melanoma progression in vivo. Here, we show that melanoma cell migration is decreased by lumican and that this effect is due to an enhanced cell adhesion. The adhesion of A375 human melanoma cells on lumican was dose-dependent and required Mg2+ and Mn2+ divalent cations. Using a panel of monoclonal antibodies directed against integrin subunits, we showed that A375 cells can bind to recombinant lumican through beta1 type integrins. Moreover, the use of rhodocetin, an inhibitor of alpha2 integrin, suggested that this particular subunit might also be involved in the interaction with lumican. The increased beta1 integrin-mediated adhesion of melanoma cells to lumican might explain, at least in part, the anti-invasive effect of this SLRP.

Topics: Animals; Cell Adhesion; Cell Line, Tumor; Cell Movement; Chondroitin Sulfate Proteoglycans; Dose-Response Relationship, Drug; Humans; Integrin beta1; Keratan Sulfate; Lumican; Melanoma; Mice

The family of small leucine-rich proteoglycans (SLRPs), which includes decorin, lumican, biglycan and fibromodulin, constitutes an abundant component of the skin extracellular matrix. We previously demonstrated that human lumican inhibits melanoma growth and progression in a mouse experimental model, by regulating cell migration, proliferation and apoptosis.. The aim of this study was to investigate the expression of lumican and decorin in human malignant melanoma and adjacent peritumoral tissue, to understand better their role in the control of growth and invasion of human melanoma.. Expression of both proteoglycans was studied by immunohistochemistry using specific antibodies in 34 malignant melanomas, 12 Hutchinson's melanotic freckles and 4 cutaneous metastatic melanomas.. We showed that lumican and decorin are located in the dermis and in the peritumoral stroma of malignant melanoma, but are not found in melanoma cells or dense tumour tissue. In the healthy dermis, distant from the tumour, the increasing ratio of lumican to decorin was inversely correlated with the proliferation of the tumour cells (P = 0.035). The comparison of the level of expression of lumican protein in superficial vs. nodular subtypes of malignant melanomas showed a decrease of lumican but not decorin in the peritumoral stroma of nodular subtypes. In the peritumoral stroma, the level of expression of lumican but not decorin decreased significantly (P = 0.016) with increasing Clark levels. In addition, immunocytochemical and reverse transcription PCR analyses of malignant melanoma cell lines (A-375, HT-144) and of MRC-5 and dermal fibroblasts from healthy donors in vitro confirmed that dermal fibroblasts are responsible for lumican and decorin synthesis in skin. CONCLUSIONS. Lumican may regulate vertical progression of human malignant melanoma, but further study is necessary to clarify the antitumour mechanism and the downstream signal transduction pathways involved.

Topics: Adult; Aged; Aged, 80 and over; Chondroitin Sulfate Proteoglycans; Decorin; Extracellular Matrix Proteins; Female; Humans; Immunohistochemistry; Keratan Sulfate; Lumican; Male; Melanoma; Middle Aged; Neoplasm Proteins; Proteoglycans; Reverse Transcriptase Polymerase Chain Reaction; Skin Neoplasms; Stromal Cells

Melanoma is a frequent and therapy-resistant human disease. Malignant melanocytes modulate their microenvironment in order to penetrate the dermal/epidermal junction and eventually invade the dermis. The small leucine-rich proteoglycans (SLRPs) constitute important constituents of the dermis extracellular matrix (ECM), participating in both the structural and the functional organization of the skin. The role of a keratan sulphate SLRP lumican, has recently been investigated in the growth and metastasis of several cancers. In this study, the expression of lumican was studied in two human melanoma cell lines (WM9, M5) as well as in normal neonatal human melanocytes (HEMN) using real time PCR, western blotting with antibodies against the protein core and keratan sulfate, and treatments with specific enzymes. Both human metastatic melanoma cell lines were found to express lumican mRNA and effectively secrete lumican in a proteoglycan form, characterized to be substituted mostly with keratan sulfate chains. Lumican mRNA was not detected in normal melanocytes. This is the first time that the synthesis and secretion of lumican in human melanoma cell lines is reported. The role of this proteoglycan in the development and progression of malignant melanoma has to be further investigated.

Topics: Cell Line, Tumor; Cells, Cultured; Chondroitin Sulfate Proteoglycans; Humans; Keratan Sulfate; Lumican; Melanocytes; Melanoma; RNA, Messenger

Lumican is a member of the small leucine-rich proteoglycan (SLRP) family. It contributes to the organisation of the collagen network and plays an important role in cell migration and tissue repair. The present study aimed to determine the influence of lumican expression on adhesion, anchorage-dependent and -independent growth, migration, in vitro invasion and in vivo melanoma growth. For that purpose, B16F1 mouse melanoma cells were stably transfected with an expression plasmid containing the complete lumican cDNA. Lumican expression by tumor cells did not change the proliferative activity of mouse melanoma cells in monolayer culture and did not influence either cell adhesion to extracellular matrix gel or type I collagen or cell spreading on these substrates. In contrast, lumican-transfected cells were characterized by a strong reduction of their anchorage-independent proliferation in agarose gel and capacity to invade extracellular matrix gel. After subcutaneous injections of transfected B16F1 cells in syngenic mice, lumican expression significantly decreased subcutaneous tumor formation in vivo, with a concomitant decrease of cyclin D1 expression. Lumican induced and/or increased the apoptosis of B16F1 cells. The results suggest that lumican is involved in the control of melanoma growth and invasion and may be considered, like decorin, as an anti-tumor factor from the extracellular matrix.

Topics: Animals; Apoptosis; Cell Adhesion; Cell Division; Cell Line, Tumor; Chondroitin Sulfate Proteoglycans; Cyclin D1; Disease Progression; Female; Humans; Keratan Sulfate; Lumican; Melanoma; Mice; Mice, Inbred C57BL; Neoplasm Invasiveness; Neoplasm Transplantation; Transfection