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Proteomic analysis identified N-cadherin, clusterin, and HSP27 as mediators of SPARC (secreted protein, acidic and rich in cysteines) activity in melanoma cells

Authors

  • María Soledad Sosa,

    1. Laboratory of Molecular and Cellular Therapy, Fundación Instituto Leloir-CONICET and Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina
    2. Current address: Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, USA
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    • Both authors contributed equally to this work

  • María Romina Girotti,

    1. Laboratory of Molecular and Cellular Therapy, Fundación Instituto Leloir-CONICET and Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina
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    • Both authors contributed equally to this work

  • Edgardo Salvatierra,

    1. Laboratory of Molecular and Cellular Therapy, Fundación Instituto Leloir-CONICET and Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina
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  • Federico Prada,

    1. Laboratory of Molecular and Cellular Therapy, Fundación Instituto Leloir-CONICET and Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina
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  • Juan Antonio López de Olmo,

    1. Proteomics Unit, Centro Nacional de Biotecnología (CNB), Madrid, Spain
    2. Current address: Proteomics Unit, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain
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  • Silvia Juárez Gallango,

    1. Proteomics Unit, Centro Nacional de Biotecnología (CNB), Madrid, Spain
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  • Juan Pablo Albar,

    1. Proteomics Unit, Centro Nacional de Biotecnología (CNB), Madrid, Spain
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  • Osvaldo Luis Podhajcer,

    1. Laboratory of Molecular and Cellular Therapy, Fundación Instituto Leloir-CONICET and Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina
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  • Andrea Sabina Llera Dr.

    Corresponding author
    1. Laboratory of Molecular and Cellular Therapy, Fundación Instituto Leloir-CONICET and Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina
    • Fundación Instituto Leloir, Av. Patricias Argentinas 435, 1405BWE Buenos Aires, Argentina Fax: +54-11-5238-7501
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Abstract

Secreted protein, acidic and rich in cysteines (SPARC) is a secreted protein associated with increased aggressiveness of different human cancer types. In order to identify downstream mediators of SPARC activity, we performed a 2-DE proteomic analysis of human melanoma cells following antisense-mediated downregulation of SPARC expression. We found 23/504 differential spots, 15 of which were identified by peptide fingerprinting analysis. Three of the differential proteins (N-cadherin (N-CAD), clusterin (CLU), and HSP27) were validated by immunoblotting, confirming decreased levels of N-CAD and CLU and increased amounts of HSP27 in conditioned media of cells with diminished SPARC expression. Furthermore, transient knock down of SPARC expression in melanoma cells following adenoviral-mediated transfer of antisense RNA confirmed these changes. We next developed two different RNAs against SPARC that were able to inhibit in vivo melanoma cell growth. Immunoblotting of the secreted fraction of RNAi-transfected melanoma cells confirmed that downregulation of SPARC expression promoted decreased levels of N-CAD and CLU and increased secretion of HSP27. Transient re-expression of SPARC in SPARC-downregulated cells reverted extracellular N-CAD, CLU, and HSP27 to levels similar to those in the control. These results constitute the first evidence that SPARC, N-CAD, CLU, and HSP27 converge in a unique molecular network in melanoma cells.

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