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Secreted proteins (the secretome) have high potential use as sources of biomarkers of cancer. An optimized method for isolating the secreted proteome of an isogenic series of breast cancer cell lines varying from the non-tumorigenic and non-metastatic MCF10A to the metastatic MCF10CA cl. d has been developed. Cells are grown in serum free media which is harvested and partially fractionated using a tC2 SepPak cartridge. The fractions are then digested and analyzed using LC-MS/MS (peptide sequence based analyses). Repeatable protein profiles were obtained from 3 separate preparations of each cell line, and from duplicate analyses of the same line. The combined data from all fractions and analyses of each cell line were searched against the NCBI reference sequence database using a Mascot search engine. There were significant changes observed between the secreted proteomes of the cell lines. The most significant changes occurred with alpha-1-antichymotrypsin and galectin-3-binding protein which were highly secreted from the MCF10 DCIS.com and MCF10A cl. d, but undetected in MCF10A and MCF10AT cell lines. These proteins were also secreted from unrelated metastatic cell lines in our earlier research (Mbeunkui, et al., J. Prot. Res. 2006, 5, 899-906). Other proteins showing increasing abundance in the more aggressive cell lines included alpha-1-antitrypsin, cathepsin D and quiescin Q6. The S100 proteins, associated with aggressive cancers, showed variable changes in abundance. Secretion of proteins often believed to be strictly cytosolic, such as annexin A2, was observed. These proteins are all predicted to undergo non-classical secretion (SecretomeIP, Center for Biological Sequence Analysis). The results were validated for five selected proteins by western blot analysis. The comparison of the secretome of an isogenic series of cell lines reveals proteins related to cancer aggressiveness/progression, and proteins which are potentially highly specific markers for the early detection of cancer.

This research was partially supported by grants from the National Cancer Institute (5R21CA116070 to LKP) and the Susan G. Komen Breast Cancer Foundation (BCTR0402317 to LAS).

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA