SPARC Promotes Cathepsin B-Mediated Melanoma Invasiveness through a Collagen I/α2β1 Integrin Axis  María R. Girotti, Marisol Fernández, Juan A. López, Emilio Camafeita, Elmer A. Fernández, Juan P. Albar, Lorena G. Benedetti, María P. Valacco, Rolf A. Brekken, Osvaldo L. Podhajcer, Andrea S. Llera  Journal of Investigative Dermatology  Volume 131, Issue 12, Pages 2438-2447 (December 2011) DOI: 10.1038/jid.2011.239 Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Validation of SPARC-induced changes in extracellular proteins in melanoma cells. (a–c) Immunoblotting and immunostaining confirms that SPARC (secreted protein acidic and rich in cysteine) downregulation decreases extracellular proteases and epithelial to mesenchymal transition (EMT)-related proteins. Different conditioned media from control LBLAST (LB) and SPARC-deficient L2F6 (L2) cells were labeled a, b, or c. The optical density of bands were normalized to SYPRO Ruby-stained loading control intensities and expressed either as single relative percentages or the mean±SEM (n=3; *P<0.05). Small L2 experimental tumors, contrary to LB tumors, show an absence of positive vimentin or cathepsin B staining. Bar=100μm. Inset: × 2 enlarged image. Arrows indicate tumor cells. (d) A lower cathepsin B enzymatic activity in L2 cells correlates with decreased cathepsin B levels (**P<0.01). (e) The addition of SPARC reverts cathepsin B deficiency in L2 cells. FAM3C/ILEI, family with sequence similarity 3, member C/interleukin-like EMT-inducer. Journal of Investigative Dermatology 2011 131, 2438-2447DOI: (10.1038/jid.2011.239) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 SPARC-dependent cathepsin B effect on melanoma cell invasion. (a) Matrigel-invading LBLAST (LB) and L2F6 (L2) cells (blue nuclei)±SPARC (secreted protein acidic and rich in cysteine) (SP) and/or the cathepsin B inhibitor CA-074. Bar=100μm. (b) Invading cells (% of control LB cells)±CA-074 or anti-SPARC antibodies (Anti-SP). Error bars=mean±SEM (n=3). ***P<0.001 with respect to LB; ###P<0.001 with respect to L2; <<<P<0.001 with respect to LB-SP; +++P<0.001 with respect to L2-SP. SPARC mRNA (c), protein (d), and extracellular cathepsin B (e) in clone A3, which expresses SPARC short hairpin RNA, with respect to A375 wild-type cells (AW) and control (empty vector) cells (AE). (f) Invading cells (% of control AE cells), ±CA-074. Error bars=mean±SEM (n=3). ***P<0.001 with respect to AE. Journal of Investigative Dermatology 2011 131, 2438-2447DOI: (10.1038/jid.2011.239) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Effects of collagen I on LBLAST (LB) and L2F6 (L2) cell invasion. (a) Real-time PCR for collagen I α2 chain in LB and L2 cells. *P<0.05, ***P<0.001 with respect to LB. (b) Invasiveness after plating on collagen I or fibronectin with or without SPARC (secreted protein acidic and rich in cysteine) and/or CA-074. Error bars=mean±SEM (n=3). ***P<0.001 with respect to LB in plastic, collagen, or fibronectin; ##P<0.01 with respect to L2; ^^^P<0.001 with respect to L2-SP. (c) A representative immunoblot of secreted procathepsin B after treatment with SPARC and/or collagen I. Numbers represent normalized band intensities. (d) Invasiveness after treatment with anti-α2β1 integrin or control (MOPC) antibodies, in the presence of SPARC, collagen I, and/or CA-074. Error bars=mean±SEM (n=3). ***P<0.001 with respect to its corresponding MOPC control. Journal of Investigative Dermatology 2011 131, 2438-2447DOI: (10.1038/jid.2011.239) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Transforming growth factor (TGF)-β1 effects on melanoma cell invasion. Anti-TGF-β1 antibodies (a) or TGF-β1-receptor inhibitor SB-431542 (b) partially inhibited the SPARC-dependent invasiveness of LBLAST (LB) and L2F6 (L2) cells preincubated with SPARC, but it did not alter the collagen I effect. (c) Anti-SPARC (secreted protein acidic and rich in cysteine) antibodies supersede the anti-TGF-β1 antibody effect on SPARC-dependent LB and -L2 cell invasiveness. (d) TGF-β1 induces L2 cell invasion; however, this effect is completely abolished by anti-SPARC antibodies. Error bars=mean±SEM (n=3). *P<0.05 with respect to LB-MOPC or LB-DMSO; **P<0.01; ***P<0.001 with respect to LB-MOPC; #P<0.05 with respect to L2-SPARC-MOPC or L2-SPARC-DMSO; ###P<0.001 with respect to L2-SPARC-MOPC; ^P<0.05 with respect to the corresponding cell line plated on collagen I and MOPC-treated; +++P<0.001 with respect to L2-MOPC. COLA, collagen. Journal of Investigative Dermatology 2011 131, 2438-2447DOI: (10.1038/jid.2011.239) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 N-cadherin effect on transendothelial migration of melanoma cells. (a) Flow cytometry analysis of E-cadherin (E-CAD) and N-cadherin (N-CAD) in LBLAST (LB) or L2F6 (L2) cells in the presence or absence of SPARC (secreted protein acidic and rich in cysteine) (SP). Cell populations expressing high (+) and low (-) levels of cadherins are indicated. (b) Representative images of a transmigration assay. Blue nuclei represent total cells, and the red cytoplasm corresponds to migrated cells. Top right, a human dermal microvascular endothelial cell cell monolayer is shown. A-CAM, anti-N-cadherin antibody. Bar=100μm. (c) Cells (% of control) that transmigrated in the presence or absence of SPARC, CA-074, anti-N-cadherin (anti-N-CAD), anti-TGF-β1, anti-α2β1 integrin (α2β1-INT), and/or control (MOPC) antibodies. Error bar=mean±SEM (n=3). ***P<0.001 with respect to LB; ###P<0.001 with respect to L2; ^^P<0.01 with respect to L2-SP. FL1-H, height of fluorescence intensity. Journal of Investigative Dermatology 2011 131, 2438-2447DOI: (10.1038/jid.2011.239) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Proposed model for the SPARC tumor progression effect. SPARC (secreted protein acidic and rich in cysteine) functions as a driver of a cathepsin B-mediated proinvasive axis that involves transforming growth factor (TGF)-β1 and collagen type I/α2β1 integrin (α2 int) as mediators. Engagement of integrins by SPARC-derived collagen I might affect trafficking of endocytic compartments, forcing accumulation of procathepsin B in pericellular locations from where it could be secreted in local favorable conditions. SPARC also promotes the E- to N-cadherin shift that enhances transendothelial migration of melanoma cells through a mechanism not linked to TGF-β1 and collagen type I/integrin α2β1. This cadherin switch favors the establishment of N-cadherin homotypic contacts with fibroblasts and endothelial cells, enabling malignant cells to enter the bloodstream, traveling away from the primary tumor and disseminating to establish new metastatic foci. Journal of Investigative Dermatology 2011 131, 2438-2447DOI: (10.1038/jid.2011.239) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions