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Originally published In Press as doi:10.1074/mcp.M500178-MCP200 on October 8, 2005.
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Molecular & Cellular Proteomics 5:157-171, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

Biomarker Discovery from Pancreatic Cancer Secretome Using a Differential Proteomic Approach*,S

Mads Grønborg{ddagger},§, Troels Zakarias Kristiansen{ddagger},§, Akiko Iwahori{ddagger}, Rubens Chang, Raghunath Reddy{ddagger}, Norihiro Sato, Henrik Molina{ddagger},§, Ole Nørregaard Jensen§, Ralph H. Hruban,||, Michael G. Goggins,**, Anirban Maitra{ddagger},¶,||,{ddagger}{ddagger} and Akhilesh Pandey{ddagger},¶,||,§§

From the {ddagger} McKusick-Nathans Institute of Genetic Medicine and the Department of Biological Chemistry, The Johns Hopkins University, Baltimore, Maryland 21205, the § Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark, and the Department of Pathology and The Sol Goldman Pancreatic Cancer Research Center and the || Department of Oncology, The Johns Hopkins University, Baltimore, Maryland 21287

Quantitative proteomics can be used as a screening tool for identification of differentially expressed proteins as potential biomarkers for cancers. Candidate biomarkers from such studies can subsequently be tested using other techniques for use in early detection of cancers. Here we demonstrate the use of stable isotope labeling with amino acids in cell culture (SILAC) method to compare the secreted proteins (secretome) from pancreatic cancer-derived cells with that from non-neoplastic pancreatic ductal cells. We identified 145 differentially secreted proteins (>1.5-fold change), several of which were previously reported as either up-regulated (e.g. cathepsin D, macrophage colony stimulation factor, and fibronectin receptor) or down-regulated (e.g. profilin 1 and IGFBP-7) proteins in pancreatic cancer, confirming the validity of our approach. In addition, we identified several proteins that have not been correlated previously with pancreatic cancer including perlecan (HSPG2), CD9 antigen, fibronectin receptor (integrin ß1), and a novel cytokine designated as predicted osteoblast protein (FAM3C). The differential expression of a subset of these novel proteins was validated by Western blot analysis. In addition, overexpression of several proteins not described previously to be elevated in human pancreatic cancer (CD9, perlecan, SDF4, apoE, and fibronectin receptor) was confirmed by immunohistochemical labeling using pancreatic cancer tissue microarrays suggesting that these could be further pursued as potential biomarkers. Lastly the protein expression data from SILAC were compared with mRNA expression data obtained using gene expression microarrays for the two cell lines (Panc1 and human pancreatic duct epithelial), and a correlation coefficient (r) of 0.28 was obtained, confirming previously reported poor associations between RNA and protein expression studies.

§§ Supported by a Beckman Young Investigator Award, a Sidney Kimmel Scholar Award, and National Institutes of Health Grant U54 RR020839. To whom correspondence should be addressed. Tel.: 410-502-6662; Fax: 410-502-7544; E-mail: pandey{at}jhmi.edu


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