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. 2020 Mar 27;12(4):801.
doi: 10.3390/cancers12040801.

Extracellular Protease ADAMTS1 Is Required at Early Stages of Human Uveal Melanoma Development by Inducing Stemness and Endothelial-Like Features on Tumor Cells

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Free PMC article

Extracellular Protease ADAMTS1 Is Required at Early Stages of Human Uveal Melanoma Development by Inducing Stemness and Endothelial-Like Features on Tumor Cells

Carlos Peris-Torres et al. Cancers (Basel). .
Free PMC article

Abstract

Extracellular matrix remodeling within the tumor microenvironment has been recognized as a relevant dynamic framework during tumor growth. However, research on proteases that trigger this remodeling keeps revealing a wide range of actions including both pro- and anti-tumorigenic. The extracellular protease ADAMTS1 exemplifies this dual role. In this work, we first confirmed a positive correlation of ADAMTS1 with endothelial-like phenotype of human melanoma cells together with the finding of associated signatures, including key genes such as endothelial CDH5. Using a CRISPR-Cas9 approach, we observed that the inhibition of ADAMTS1 in an aggressive uveal melanoma model compromised its endothelial-like properties, and more importantly, caused a robust blockade on the progression of tumor xenografts. Although vasculature emerged affected in ADAMTS1-deficient tumors, the most relevant action implied the downregulation of endothelial CDH5 in tumor cells, in association with stemness markers. Indeed, melanoma sphere assays also revealed a deficient commitment to form spheres in the absence of ADAMTS1, directly correlating with stemness markers and, remarkably, also with CDH5. Finally, taking advantage of advanced bioinformatics tools and available public data of uveal melanomas, we disclosed new prognosis factors, including endothelial elements and ADAMTS proteases. Our findings support the key role of ADAMTS proteases for uveal melanoma development since earlier stages, modulating the complex crosstalk between extracellular matrix and the induction of stemness and endothelial-like features. To our knowledge, this is the first report that supports the development of therapeutic targets on the extracellular matrix to overcome uveal melanoma.

Keywords: ADAMTS; cancer stem cell; endothelial-like phenotype; extracellular matrix; vasculogenic mimicry.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Endothelial-like properties of melanoma cells and correlation with ADAMTS1 expression. (a) Representative images of 3D Matrigel-based assay of human melanoma cell lines, 24 h after seeding. 20,000 cells/well were cultured for MUM-2B, SK-MEL-147, C8161, MUM-2C and SK-MEL-103; and 30,000 cells/well for SK-MEL-28, G-361 and A-375 (white scale bar = 500 µm); (b) Graph representing mRNA fold change expression of ADAMTS1 in human melanoma cell lines. Values are relative to MUM-2B (n = 21 for MUM-2B, n = 17 for MUM-2C, n = 11 for A-375, n = 5 for C8161 and SK-MEL-28, n = 6 for G-361, n = 15 for SK-MEL-103 and n = 9 for SK-MEL-147). EL+ and EL− phenotypes are indicated; (c) Graph representing mRNA fold change expression of ADAMTS1 in human melanoma cell lines, according to their EL+ or EL− phenotype (values are based in same data that Figure 1C); (d) Heatmap showing differential gene expression between EL+ (including HUVECs) and EL− cell lines. Only significant differently expressed genes are depicted (47 upregulated and 420 downregulated, FDR < 0.05). Gene Expression Omnibus (GEO) ID samples are listed and color coded in Table S2; (e) Representation of top ten GO Biological Processes after enrichment analysis using significantly upregulated genes in EL+ cells. Red line determined the limit of significance: -log (0.05). (****, p < 0.0001; ***, p < 0.001; and **, p < 0.01).
Figure 2
Figure 2
ADAMTS1 inhibition affects in vitro endothelial-like phenotypic properties and endothelial-related signature. (a) Western blot analysis of conditioned media and cell lysates of ADAMTS1 in MUM-2B WT and ATS1-KO cells. Black arrows point full-length (FL) ADAMTS1. Red Ponceau staining and Actin were used as loading controls for conditioned media and cell lysates, respectively (uncut blots including a densitometry analysis are shown in Figure S1c); (b) Representative images (original and WimTube filtered) of Matrigel assay for MUM-2B WT and ATS1-KO cells, 24 h after seeding 20,000 cells/well. Scatter plots represent the parameters resulting of WimTube analysis: total tubes, total branching points and total loops (n = 12 for all groups, white scale bar = 500 µm); (c) Graphs representing mRNA fold change expression of CDH5, ENG, EPHA2, KDR, LAMC2, TEK and TIE1 in MUM-2B WT and ATS1-KO cells (n = 3–5 for WT, n = 3–6 for ATS1-KO1 and n = 2–4 for ATS1-KO2); (d) Graph representing mRNA fold change expression of CDH5 in C8161 WT and ATS1-KO cells (n = 4 for all groups). (****, p < 0.0001; ***, p < 0.001; **, p < 0.01; and *, p < 0.05. WT cells were used as control for statistical analyses).
Figure 3
Figure 3
ADAMTS1 inhibition blocks tumorigenesis and alters tumor vasculature. (a) Table indicating the number of mice that developed tumors and the total number of injected mice of every experimental group (in parenthesis); (b) Graph representing final tumor weight of different experimental groups, according to panel A; (c) Representative pictures of tumors from SwN-Matrigel and NSG groups (black scale bar = 1 cm); (d) Graphs representing tumor evolution for each experimental group. (e) Representative images of EMCN immunofluorescence analysis of tumor sections from WT and ATS1-KO NSG xenografts (white scale bar = 100 µm); (f) Graphs representing tumor vasculature quantification of NSG xenografts: vessel density, vessel perimeter and percentage area (n = 5 for WT; n = 7 for ATS1-KO1; n = 6 for ATS1-KO2). (***, p < 0.001 and **, p < 0.01. Tumors generated with WT cells were used as control for statistical analyses).
Figure 4
Figure 4
ADAMTS1 inhibition compromises the stemness capacities and endothelial-like phenotype in tumor xenografts. (a–c) Graphs representing mRNA fold change expression of NANOG, POU5F1, PROM1 and SOX2 (a), ADAMTS1 (b) and CDH5 (c), in MUM-2B WT 2D cultured cells and NSG xenografts (n = 4–6 for cells and n = 3–4 for NSG); (d-e) Graphs representing mRNA fold change expression of NANOG, POU5F1, PROM1 and SOX2 (d), and CDH5 (e) in NSG xenografts generated with WT and ATS1-KO cells (n = 3 for WT, n = 5 for ATS1-KO1 and n = 4–6 for ATS1-KO2); (f) Representative images of IF analysis of WT and ATS1-KO NSG xenografts, at low and high magnification. For low magnification, columns from left to right: CDH5, EMCN, DAPI and merge; (g) Representative images of IHC staining of CDH5 and NANOG in consecutive sections of WT and ATS1-KO NSG xenografts; (h) Representative images of IHC co-staining of CDH5 and PAS, in WT and ATS1-KO NSG xenografts. (****, p < 0.0001; **, p < 0.01; and *, p < 0.05. White scale bar = 50 µm).
Figure 5
Figure 5
Inhibition of ADAMTS1 compromises melanoma sphere formation. (a) Graphs representing mRNA fold-change expression of NANOG, POU5F1, PROM1, ADAMTS1 and CDH5 in MUM-2B WT 2D culture, primary and secondary melanoma spheres (n = 4 for 2D culture, except for ADAMTS1 which is 12; n = 2–5 for primary spheres, and n = 4–6 for secondary spheres). 2D cultured cells were used as control for statistical analyses; (b) Violin plots representing volume of primary (n = 49 for WT, n = 62 for ATS1-KO1, and n = 58 for ATS1-KO2) and secondary (n = 46 for WT, n = 34 for ATS1-KO1 and n = 21 for ATS1-KO2) melanoma spheres. WT spheres volume was used as control for statistical analyses; (c) Representative images of WT and ATS1-KO primary melanoma spheres grown in CSC medium; (d) Graphs representing mRNA fold-change expression of NANOG and CDH5 in 2D culture (n = 4–6 for ATS1-KO1 and n = 2–4 for ATS1-KO2) and primary melanoma spheres (n = 2 for ATS1-KO1 and n = 4–7 for ATS1-KO2). 2D cultured conditions were used as control for statistical analyses; (e) Schematic protocol for melanoma spheres formation, using CSC medium and ADAMTS1+ CM; (f) Violin plots representing volume of WT and ATS1-KO primary melanoma spheres grown in CSC medium (n = 49 for WT, n = 62 for ATS1-KO1, and n = 58 for ATS1-KO2) or in ADAMTS1+ CM (n = 44 for WT, n = 48 for ATS1-KO1, and n = 49 for ATS1-KO2); (g) Representative images of WT and ATS1-KO primary melanoma spheres in ADAMTS1+ CM; (h) Violin plots representing WT and ATS1-KO1 primary melanoma spheres, grown in control CSC (n = 27 for WT, and n = 44 for ATS1-KO1) or rhATS1+ CSC medium (n = 35 for WT, and n = 28 for ATS1-KO1); (i) Representative images of WT and ATS1-KO1 primary melanoma spheres grown in control and rhATS1+ CSC medium. (****, p < 0.0001; ***, p < 0.001; **, p < 0.01; *, p < 0.05. Violin plots indicate the median of every experimental group. White scale bar = 100 µm).
Figure 6
Figure 6
Identification of EL and ECM molecules as poor prognosis factors in TCGA Uveal Melanoma Project (TCGA-UVM). (a) Kaplan–Meier survival curves for low and high gene expression levels of EL markers CDH5 and KDR; (b) Scatter plot representing Pearson correlation analysis between gene expression levels of CDH5 and endothelial-related KDR and TIE1; (c) Representation of top ten GO Biological Processes after enrichment analysis using genes that positively correlated (q-value < 0.05) with CDH5. Red line determined the limit of significance: -log (0.05); (d) Box graph representing ADAMTS1 expression among different clinical stages of human uveal melanoma, from stage IIA to IV (n = 4 for IIA, n = 32 for IIB, n = 27 for IIIA, n = 10 for IIIB, n = 3 for IIIC and n = 4 for IV); (e) Kaplan–Meier survival curves for low and high gene expression levels of extracellular proteases ADAMTS4, ADAMTS5, ADAMTS9, ADAMTS12, ADAMTS2 and ADAMTS14; (f) Scatter plot representing Pearson correlation analysis between gene expression levels of CDH5 and extracellular proteases ADAMTS4, ADAMTS9, ADAMTS12 and ADAMTS2. (Survival probability is depicted in correlation analysis plots (panels B and F) with light and dark red dots, representing low and high survival probability, respectively. r = Pearson correlation coefficient).

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