A proteomic approach to study the molecular mechanism of the protumoral protein SPARC.

ANDREA S. LLERA; MARIA ROMINA GIROTTI; MARÍA SOLEDAD SOSA; EDGARDO SALVATIERRA; MARISOL FERNÁNDEZ; SILVIA JUÁREZ; EMILIO CAMAFEITA; JUAN PABLO ALBAR; OSVALDO PODHAJCER

Pilar, Buenos Aires

Congreso; First Annual Iberoamerican Proteomics Congress; 2007

Latin American Human Proteome Organization

SPARC is a glycoprotein from the extracellular matrix that elicits changes in cell shape and proliferation. SPARC is overexpressed in different tumors, in association with tumor progression. We have showed that downregulation of SPARC expression by antisense and RNAi techniques in human melanoma cells abolished tumorigenicity in an in vivo immunodeficient murine model, through still not clear molecular mechanisms. In the pursuit of molecular mediators of SPARC activity that may explain its role in tumor malignization, we have performed a proteomic analysis of proteins secreted by MEL-LES human melanoma cells with antisense- or RNAi-mediated downregulated SPARC expression. We have used differential bidimensional electrophoresis to compare conditioned media from human melanoma MEL-LES cells with antisense-mediated downregulation of SPARC expression (clone L-1D) and its control cell line L-CMV. We have also studied the secretome of a stable MEL-LES-derived cell clone (L2F6), in which SPARC expression was downregulated by the use of a RNAi, and compared it to its control cell line LBLAST. Overall, around 12% of detected spots in conditioned medium were significantly up- or down-regulated by changes in expression levels of SPARC. After identification of differential spots by peptide mass fingerprinting or MS/MS, a selected group of these proteins was chosen for validation by immunoblotting, real time PCR and other techniques. Differences in these proteins were confirmed not only in the aforementioned cells but also using transient (i.e. adenoviral) methods of SPARC down-regulation on MEL-LES cells. Furthermore, restoration of SPARC expression levels using SPARC-expressing adenovirus also reverted levels of differential proteins to those in the control. Most interestingly, several of the validated proteins are well-known mediators of tumor progression but were not previously related to SPARC effects on malignization. Our results constitute the first evidence that SPARC and these proteins may participate in a single molecular network that leads to tumor progression.