Melanoma is the most aggressive type of cutaneous malignancies. In addition to its role as a regulator of extracellular matrix (ECM) integrity, lumican, a small leucine-rich proteoglycan, also exhibits anti-tumor properties in melanoma. This work focuses on the use of infrared spectral imaging (IRSI) and histopathology (IRSH) to study the effect of lumican-derived peptide (L9Mc) on B16F1 melanoma primary tumor growth. Female C57BL/6 mice were injected with B16F1 cells treated with L9Mc (n = 10) or its scrambled peptide (n = 8), and without peptide (control, n = 9). The melanoma primary tumors were subjected to histological and IR imaging analysis. In addition, immunohistochemical staining was performed using anti-Ki-67 and anti-cleaved caspase-3 antibodies. The IR images were analyzed by common K-means clustering to obtain high-contrast IRSH that allowed identifying different ECM tissue regions from the epidermis to the tumor area, which correlated well with H&E staining. Furthermore, IRSH showed good correlation with immunostaining data obtained with anti-Ki-67 and anti-cleaved caspase-3 antibodies, whereby the L9Mc peptide inhibited cell proliferation and increased strongly apoptosis of B16F1 cells in this mouse model of melanoma primary tumors.
Lumican, a small leucine-rich proteoglycan (SLRP) of the extracellular matrix (ECM), displays anti-tumor properties through its direct interaction with MMP-14. Lumican-derived peptides, such as lumcorin (17 amino acids) or L9M (10 amino acids), are able to inhibit the proteolytic activity of MMP-14 and melanoma progression. This work aimed to visualize the interactions of lumican-derived peptides and MMP-14. Molecular modeling was used to characterize the interactions between lumican-derived peptides, such as lumcorin, L9M, and cyclic L9M (L9Mc, 12 amino acids), and MMP-14. The interaction of L9Mc with MMP-14 was preferential with the MT-Loop domain while lumcorin interacted more with the catalytic site. Key residues in the MMP-14 amino acid sequence were highlighted for the interaction between the inhibitory SLRP-derived peptides and MMP-14. In order to validate the in silico data, MMP-14 activity and migration assays were performed using murine B16F1 and human HT-144 melanoma cells. In contrast to the HT-144 melanoma cell line, L9Mc significantly inhibited the migration of B16F1 cells and the activity of MMP-14 but with less efficacy than lumican and lumcorin. L9Mc significantly inhibited the proliferation of B16F1 but not of HT-144 cells in vitro and primary melanoma tumor growth in vivo. Thus, the site of interaction between the domains of MMP-14 and lumcorin or L9Mc were different, which might explain the differences in the inhibitory effect of MMP-14 activity. Altogether, the biological assays validated the prediction of the in silico study. Possible and feasible improvements include molecular dynamics results.
Small leucine-rich proteoglycans (SLRPs) are major regulators of extracellular matrix assembly and cell signaling. Lumican, a member of the SLRPs family, and its derived peptides possess anti-tumor activity by interacting directly with the catalytic domain of MMP-14 leading to the inhibition of its activity. The aim of this report was to characterize by in silico 3D modeling the structure and the dynamics of four SLRPs including their core protein and their specific polysaccharide chains to assess their capacity to bind to MMP-14 and to regulate its activity. Molecular docking experiments were performed to identify the specific amino acids of MMP-14 interacting with each of the four SLRPs. The inhibition of each SLRP (100nM) on MMP-14 activity and the constants of inhibition (Ki) were evaluated. The impact of the number of glycan chains, structures and dynamics of lumican on the interaction with MMP-14 was assessed by molecular dynamics simulations. Molecular docking analysis showed that all SLRPs bind to MMP-14 through their concave face, but in different regions of the catalytic domain of MMP-14. Each SLRPs inhibited significantly the MMP-14 activity. Finally, molecular dynamics showed the role of glycan chains in interaction with MMP-14 and shielding effect of SLRPs. Altogether, the results demonstrated that each SLRP exhibited inhibition of MMP-14 activity. However, the differential targeting of MMP-14 by the SLRPs was shown to be related not only to the core protein conformation but also to the glycan chain structures and dynamics.
Ovarian cancer remains one of the most fatal cancers due to a lack of robust screening methods of detection at early stages. Extracellular matrix (ECM) mediates interactions between cancer cells and their microenvironment via specific molecules. Lumican, a small leucine-rich proteoglycan (SLRP), maintains ECM integrity and inhibits both melanoma primary tumor development, as well as metastatic spread. The aim of this study was to analyze the effect of lumican on tumor growth of murine ovarian epithelial cancer. C57BL/6 wild type mice (n = 12) and lumican-deficient mice (n = 10) were subcutaneously injected with murine ovarian epithelial carcinoma ID8 cells, and then sacrificed after 18 days. Analysis of tumor volumes demonstrated an inhibitory effect of endogenous lumican on ovarian tumor growth. The ovarian primary tumors were subjected to histological and immunohistochemical staining using anti-lumican, anti-αv integrin, anti-CD31 and anti-cyclin D1 antibodies, and then further examined by label-free infrared spectral imaging (IRSI), second harmonic generation (SHG) and Picrosirius red staining. The IR tissue images allowed for the identification of different ECM tissue regions of the skin and the ovarian tumor. Moreover, IRSI showed a good correlation with αv integrin immunostaining and collagen organization within the tumor. Our results demonstrate that lumican inhibits ovarian cancer growth mainly by altering collagen fibrilogenesis.
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