title: Proteomic analysis identified N-cadherin, clusterin, and HSP27 as mediators of SPARC (secreted protein, acidic and rich in cysteines) activity in melanoma cells.
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.
authors.affiliation: Laboratory of Molecular and Cellular Therapy, Fundación Instituto Leloir-CONICET and Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina.
authors.forename: María Soledad
authors.initials: MS
authors.lastname: Sosa
authors.forename: María Romina
authors.initials: MR
authors.lastname: Girotti
authors.forename: Edgardo
authors.initials: E
authors.lastname: Salvatierra
authors.forename: Federico
authors.initials: F
authors.lastname: Prada
authors.forename: Juan Antonio López
authors.initials: JA
authors.lastname: de Olmo
authors.forename: Silvia Juárez
authors.initials: SJ
authors.lastname: Gallango
authors.forename: Juan Pablo
authors.initials: JP
authors.lastname: Albar
authors.forename: Osvaldo Luis
authors.initials: OL
authors.lastname: Podhajcer
authors.forename: Andrea Sabina
authors.initials: AS
authors.lastname: Llera
mesh_terms: D015820:Cadherins / Q000032:analysis / Q000378:metabolism*; D045744:Cell Line, Tumor; D051152:Clusterin / Q000032:analysis / Q000378:metabolism*; D055551:HSP27 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D008545:Melanoma / Q000378:metabolism*; D018832:Molecular Chaperones; D009363:Neoplasm Proteins / Q000032:analysis / Q000378:metabolism*; D015676:Osteonectin / Q000096:biosynthesis / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D014407:Tumor Cells, Cultured
chemical_list: D015820:Cadherins; D051152:Clusterin; D055551:HSP27 Heat-Shock Proteins; C057725:HSPB1 protein, human; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D009363:Neoplasm Proteins; D015676:Osteonectin