DIGE analysis reveals molecular mediators of SPARC activity in tumor progression

MARÍA ROMINA GIROTTI; MARISOL FERNÁNDEZ; JUAN PABLO ALBAR; JUAN ANTONIO LÓPEZ; EMILIO CAMAFEITA; OSVALDO PODHAJCER; ANDREA S. LLERA

Pilar

Congreso; First Annual Iberoamerican Proteomics Congress; 2007

Latin American Human Proteome Organization

SPARC is a secreted glycoprotein involved in tissue remodelling, endothelial cell migration, morphogenesis, angiogenesis and increased aggressiveness of different human cancer types. Although SPARC expression is upregulated in many types of cancer, little is known about the molecular mechanisms affected by SPARC during tumor growth. Previous studies of our laboratory showed that stable transfection of tumor cells with antisense SPARC DNA abolished tumorigenicity in an in vivo melanoma murine model through still unclear molecular mechanisms. We have now developed a stable cell clone of human melanoma cells (L2F6) in which SPARC expression was downregulated by the use of a RNAi. SPARC downregulation in L2F6 cells abolished tumor growth in a murine immunodeficient in vivo model. In order to identify putative secreted proteins that may mediate SPARC biological function, we performed a differential proteomic analysis of conditioned media of L2F6 cell clone and the control cell line LBLAST. For this purpose we applied the novel technology of DIGE (DIfferential Gel Electrophoresis). Image analysis of DIGE bidimensional spot maps using DeCyder software and statistical analysis by unpaired T-test rendered 98 differentially expressed proteins that were identify by using MALDI TOF-TOF technology. A predominant subset of differential proteins belongs to the familiy of proteases, which have been extensively associated with tumor invasion and metastasis. Interestingly, unsupervised multivariate analysis was also able to identify a set of spots that could distinguish the two different treatments used in the study. These results define a set of proteins potentially related to SPARC role in tumor progression. Functional and biological validation of selected proteins confirmed the differences observed and further work is in progress to determine their biological relevance to SPARC effects.