DIGE Profiling of Melanoma Secretome Identified Molecular Mediators of SPARC Activity

MARIA ROMINA GIROTTI; MARISOL FERNÁNDEZ; EMILIO CAMAFEITA; JUAN ANTONIO LOPEZ; JUAN PABLO ALBAR; OSVALDO LUIS PODHAJCER; ANDREA LLERA

Pamplona, España

Congreso; Joint Congress of the Spanish Society and the Latin American Human Proteome Organization; 2009

SEPROT - LAHUPO

SPARC is a secreted glycoprotein related to tumor progression and metastasisand overexpressed in different tumors. Its role has been expanded to include tissueremodelling, endothelial cell migration, morphogenesis, angiogenesis and increasedaggressiveness of different human cancer types. However, little is known about themolecular mechanisms affected by SPARC during tumor growth. We have showed thatstable transfection of tumor cells with antisense SPARC DNA abolished tumorigenicityin an in vivo melanoma murine model through still unclear molecular mechanisms.In order to identify putative secreted proteins that may mediate SPARC biologicalfunction, we performed a proteomic analysis of conditioned media of a stable cell cloneof human melanoma cells (L2F6) in which SPARC expression was downregulated bythe use of a RNAi versus the control cell line LBLAST. For this purpose we appliedDIGE (DIfferential Gel Electrophoresis). After analyzing 2D-gel images with DeCydersoftware, we obtained 98 differentially expressed proteins that were identify by usingMALDI TOF-TOF technology. Ontological studies shown that the predominant groupof differential proteins belongs to the family of proteases, proteins which have beenextensively associated with tumor progression. Functional and biological validationconfirmed the differences observed by DIGE. Thus, our results define a set of proteinspotentially related to SPARC role in tumor progression, many of them not previouslyassociated with SPARC. Further work is in progress to elucidate the molecularinteractions between SPARC and this set of novel proteins.