Cyr61 is a target for heparin in reducing MV3 melanoma cell adhesion and migration via the integrin VLA-4

Summary

The integrin VLA-4 is important for the metastatic dissemination of melanoma cells. We could recently show that heparin can block VLA-4 binding, which contributes, next to blocking P-and L-selectin, to the understanding of antimetastatic activities of heparin. The matricellular ligand Cyr61, secreted by numerous tumours, is responsible for increased tumourigenicity and metastasis. This has been attributed to Cyr61 binding to, and thus activating integrins. However, a VLA-4/Cyr61 axis has not yet been reported. Since Cyr61 possesses heparin binding capabilities, Cyr61 can be supposed as potential target for heparin to indirectly interfere with integrin functions. The present in vitro studies address (i) the existence of a Cyr61/VLA-4 axis and (ii) the functional relevance of heparin interference via Cyr61. The C-terminal module III of Cyr61 could be exposed as nanomolar affine binding site for VLA-4. A shRNA-based knockdown of Cyr61 in MV3 human melanoma cells reduced VLA-4-mediated cell binding to VCAM-1, migration on fibronectin, and integrin signalling functions significantly. Using a biosensor approach we provide insight into heparin interference with this process. The low-molecular-weight heparin tinzaparin, but not the pentasaccharide fondaparinux, binds module IV of Cyr61 with micromolar affinity. But tinzaparin cannot interfere with Cyr61 accumulation onto syndecan-4, indicating different Cyr61 binding sites for heparin and other GAGs. Nonetheless, tinzaparin affects the VLA-4 binding and signalling functions selectively via Cyr61 already at very low concentration most likely by blocking the cellular secreted free Cyr61. This study emphasises Cyr61 as promising, and hitherto not considered target for heparin to selectively influence integrin functions.

Keywords

Cyr61/CCN-1 - heparin - melanoma cell adhesion - migration - VLA-4

^* These authors contributed equally.

• References

• 1 Chen C-C, Lau LF. Functions and mechanisms of action of CCN matricellular proteins. Int J Biochem Cell Biol 2009; 41: 771-783.
• 2 Walsh CT, Stupack D, Brown JH. G protein-coupled receptors go extracellular: RhoA integrates the integrins. Mol Interv 2008; 8: 165-173.
• 3 Dhar A, Ray A. The CCN family proteins in carcinogenesis. Exp Oncol 2010; 32: 2-9.
• 4 Leask A, Abraham DJ. All in the CCN family: essential matricellular signaling modulators emerge from the bunker. J Cell Sci 2006; 119: 4803-4810.
• 5 Sun Z-J, Wang Y, Cai Z. et al. Involvement of Cyr61 in growth, migration, and metastasis of prostate cancer cells. Br J Cancer 2008; 99: 1656-1667.
• 6 Tsai M-S, Bogart DF, Castañeda JM. et al. Cyr61 promotes breast tumorigenesis and cancer progression. Oncogene 2002; 21: 8178-8185.
• 7 Xie D, Yin D, Tong X. et al. Cyr61 is overexpressed in gliomas and involved in integrin-linked kinase-mediated Akt and beta-catenin-TCF/Lef signaling pathways. Cancer Res 2004; 64: 1987-1996.
• 8 Haque I, Mehta S, Majumder M. et al. Cyr61/CCN1 signaling is critical for epithelial-mesenchymal transition and stemness and promotes pancreatic carcinogenesis. Mol Cancer 2011; 10: 8
• 9 Babic AM, Chen CC, Lau LF. Fisp12/mouse connective tissue growth factor mediates endothelial cell adhesion and migration through integrin alphavbeta3, promotes endothelial cell survival, and induces angiogenesis in vivo. Mol Cell Biol 1999; 19: 2958-2966.
• 10 Menendez JA, Vellon L, Mehmi I. et al. A novel CYR61-triggered „CYR61-alphavbeta3 integrin loop“ regulates breast cancer cell survival and chemosensitivity through activation of ERK1/ERK2 MAPK signaling pathway. Oncogene 2005; 24: 761-779.
• 11 Menéndez JA, Mehmi I, Griggs DW. et al. The angiogenic factor CYR61 in breast cancer: molecular pathology and therapeutic perspectives. Endocr. Relat. Cancer 2003; 10: 141-152.
• 12 Jandova J, Beyer TE, Meuillet EJ, Watts GS. The matrix protein CCN1/CYR61 is required for α(V) β(5) -mediated cancer cell migration. Cell Biochem Funct 2012; 30: 687-695.
• 13 Leu S-J, Liu Y, Chen N. et al. Identification of a novel integrin alpha 6 beta 1 binding site in the angiogenic inducer CCN1 (CYR61). J Biol Chem 2003; 278: 33801-33808.
• 14 Chen N, Chen CC, Lau LF. Adhesion of human skin fibroblasts to Cyr61 is mediated through integrin alpha 6beta 1 and cell surface heparan sulfate proteoglycans. J Biol Chem 2000; 275: 24953-24961.
• 15 Schober JM, Lau LF, Ugarova TP. et al. Identification of a novel integrin alphaMbeta2 binding site in CCN1 (CYR61), a matricellular protein expressed in healing wounds and atherosclerotic lesions. J Biol Chem 2003; 278: 25808-25815.
• 16 Lin B-R, Chang C-C, Chen L-R. et al. Cysteine-rich 61 (CCN1) enhances chemotactic migration, transendothelial cell migration, and intravasation by concomitantly up-regulating chemokine receptor 1 and 2. Mol Cancer Res 2007; 5: 1111-1123.
• 17 Okahara H, Yagita H, Miyake K. et al. Involvement of very late activation antigen 4 (VLA-4) and vascular cell adhesion molecule 1 (VCAM-1) in tumor necrosis factor alpha enhancement of experimental metastasis. Cancer Res 1994; 54: 3233-3236.
• 18 Garofalo A, Chirivi RG, Foglieni C. et al. Involvement of the very late antigen 4 integrin on melanoma in interleukin 1-augmented experimental metastases. Cancer Res 1995; 55: 414-419.
• 19 Schlesinger M, Schmitz P, Zeisig R. et al. The inhibition of the integrin VLA-4 in MV3 melanoma cell binding by non-anticoagulant heparin derivatives. Thromb Res 2012; 129: 603-610.
• 20 Schlesinger M, Simonis D, Schmitz P. et al. Binding between heparin and the integrin VLA-4. Thromb Haemost 2009; 102: 816-822.
• 21 Borsig L. Antimetastatic activities of heparins and modified heparins. Experimental evidence. Thromb Res 2010; 125: 66-71.
• 22 Akl EA, Gunukula S, Barba M. et al. Parenteral anticoagulation in patients with cancer who have no therapeutic or prophylactic indication for anticoagulation. Cochrane Database Syst Rev 2011; 1: CD006652
• 23 Ludwig RJ, Boehme B, Podda M. et al. Endothelial P-selectin as a target of heparin action in experimental melanoma lung metastasis. Cancer Res 2004; 64: 2743-2750.
• 24 Koenig A, Norgard-Sumnicht K, Linhardt R. et al. Differential interactions of heparin and heparan sulfate glycosaminoglycans with the selectins. Implications for the use of unfractionated and low molecular weight heparins as therapeutic agents. J Clin Invest 1998; 101: 877-889.
• 25 Jun J-I, Lau LF. Taking aim at the extracellular matrix: CCN proteins as emerging therapeutic targets. Nat Rev Drug Discov 2011; 10: 945-963.
• 26 Walsh CT, Radeff-Huang J, Matteo R. et al. Thrombin receptor and RhoA mediate cell proliferation through integrins and cysteine-rich protein 61. FASEB J 2008; 22: 4011-4021.
• 27 Naggi A, Casu B, Perez M. et al. Modulation of the heparanase-inhibiting activity of heparin through selective desulfation, graded N-acetylation, and glycol splitting. J Biol Chem 2005; 280: 12103-12113.
• 28 Schütze N, Kunzi-Rapp K, Wagemanns R. et al. Expression, purification, and functional testing of recombinant CYR61/CCN1. Protein Expr Purif 2005; 42: 219-225.
• 29 Kaplan RS, Pedersen PL. Determination of microgram quantities of protein in the presence of milligram levels of lipid with amido black 10B. Anal Biochem 1985; 150: 97-104.
• 30 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-685.
• 31 Fritzsche J, Alban S, Ludwig RJ. et al. The influence of various structural parameters of semisynthetic sulfated polysaccharides on the P-selectin inhibitory capacity. Biochem Pharmacol 2006; 72: 474-485.
• 32 Liang C-C, Park AY, Guan J-L. In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc 2007; 2: 329-333.
• 33 Fritzsche J, Simonis D, Bendas G. Melanoma cell adhesion can be blocked by heparin in vitro: suggestion of VLA-4 as a novel target for antimetastatic approaches. Thromb Haemost 2008; 100: 1166-1175.
• 34 Singh P, Carraher C, Schwarzbauer JE. Assembly of fibronectin extracellular matrix. Annu Rev Cell Dev Biol 2010; 26: 397-419.
• 35 Leiss M, Beckmann K, Girós A. et al. The role of integrin binding sites in fibronectin matrix assembly in vivo. Curr Opin Cell Biol 2008; 20: 502-507.
• 36 Chen N, Leu S-J, Todorovic V. et al. Identification of a novel integrin alphavbeta3 binding site in CCN1 (CYR61) critical for pro-angiogenic activities in vascular endothelial cells. J Biol Chem 2004; 279: 44166-44176.
• 37 Grzeszkiewicz TM, Kirschling DJ, Chen N. et al. CYR61 stimulates human skin fibroblast migration through Integrin alpha vbeta 5 and enhances mitogenesis through integrin alpha vbeta 3, independent of its carboxyl-terminal domain. J Biol Chem 2001; 276: 21943-21950.
• 38 Jedsadayanmata A, Chen CC, Kireeva ML. et al. Activation-dependent adhesion of human platelets to Cyr61 and Fisp12/mouse connective tissue growth factor is mediated through integrin alpha(IIb)beta(3). J Biol Chem 1999; 274: 24321-24327.
• 39 Todorovic V, Chen C-C, Hay N. et al. The matrix protein CCN1 (CYR61) induces apoptosis in fibroblasts. J Cell Biol 2005; 171: 559-568.
• 40 Hostettler N, Naggi A, Torri G. et al. P-selectin- and heparanase-dependent antimetastatic activity of non-anticoagulant heparins. FASEB J 2007; 21: 3562-3572.
• 41 Vlodavsky I, Mohsen M, Lider O. et al. Inhibition of tumor metastasis by heparanase inhibiting species of heparin. Invasion Metastasis 1994; 14: 290-302.
• 42 Sciumbata T, Caretto P, Pirovano P. et al. Treatment with modified heparins inhibits experimental metastasis formation and leads, in some animals, to long-term survival. Invasion Metastasis 1996; 16: 132-143.
• 43 Zhang C, Liu Y, Gao Y. et al. Modified heparins inhibit integrin alpha(IIb)beta(3) mediated adhesion of melanoma cells to platelets in vitro and in vivo. Int J Cancer 2009; 125: 2058-2065.
• 44 Bendas G, Borsig L. Cancer cell adhesion and metastasis: selectins, integrins, and the inhibitory potential of heparins. Int J Cell Biol 2012; 2012: 676-731.
• 45 Chong HC, Tan CK, Huang R-L. et al. Matricellular proteins: a sticky affair with cancers. J Oncol 2012; 2012: 351-389.
• 46 Niers TMH, Brüggemann LW, Klerk CPW. et al. Differential effects of anticoagulants on tumor development of mouse cancer cell lines B16, K1735 and CT26 in lung. Clin Exp Metastasis 2009; 26: 171-178.
• 47 Mousa SA, Linhardt R, Francis JL. et al. Anti-metastatic effect of a non-anticoagulant low-molecular-weight heparin versus the standard low-molecular-weight heparin, enoxaparin. Thromb Haemost 2006; 96: 816-821.
• 48 Ludwig RJ, Alban S, Bistrian R. et al. The ability of different forms of heparins to suppress P-selectin function in vitro correlates to their inhibitory capacity on bloodborne metastasis in vivo. Thromb Haemost 2006; 95: 535-540.