Identification of differentially secreted biomarkers using LC-MS/MS in isogenic cell lines representing a progression of breast cancer

Abstract

Proteins secreted (the secretome) from cancer cells are potentially useful as biomarkers of the disease. Using LC-MS/MS, the secreted proteomes from a series of isogenic breast cancer cell lines varying in aggressiveness were analyzed by mass spectrometry: nontumorigenic MCF10A, premalignant/tumorigenic MCF10AT, tumorigenic/locally invasive MCF10 DCIS.com, and tumorigenic/metastatic MCF 10CA cl. D. Proteomes were obtained from conditioned serum-free media, partially fractionated using a small reverse phase C2 column, and digested with trypsin for analysis by LC-MS/MS, using a method previously shown to give highly enriched secreted proteomes (Mbeunkui et al. J. Proteome Res. 2006, 5, 899-906). The search files produced from five analyses (three separate preparations) were combined for database searching (Mascot) which produced a list of over 250 proteins from each cell line. The aim was to discover highly secreted proteins which changed significantly in abundance corresponding with aggressiveness. The most apparent changes were observed for alpha-1-antichymotrypsin and galectin-3-binding protein which were highly secreted proteins from MCF10 DCIS.com and MCF10CA cl. D, yet undetected in the MCF10A and MCF10AT cell lines. Other proteins showing increasing abundance in the more aggressive cell lines included alpha-1-antitrypsin, cathepsin D, and lysyl oxidase. The S100 proteins, often associated with metastasis, showed variable changes in abundance. While the cytosolic proteins were low (e.g., actin and tubulin), there was significant secretion of proteins often associated with the cytoplasm. These proteins were all predicted as products of nonclassical secretion (SecretomeP, Center for Biological Sequence Analysis). The LC-MS/MS results were verified for five selected proteins by western blot analysis, and the relevance of other significant proteins is discussed. Comparisons with two other aggressive breast cancer cell lines are included. The protein with consistent association with aggressiveness in all lines, and in unrelated cancer cells, was the galectin-3-binding protein which has been associated with breast, prostate, and colon cancer earlier, supporting the approach and findings. This analysis of an isogenic series of cell lines suggests the potential usefulness of the secretome for identifying prospective markers for the early detection and aggressiveness/progression of cancer.

Figure 1

The profile of the secreted proteome changes as the cells become progressively more aggressive. Conditioned serum free media from the cell lines was concentrated and the total protein estimated. Eight micrograms of total protein was resolved by SDS-PAGE and immunoblotted for LGAL3BP (galectin-3-binding protein), AACT (alpha-1-chymoantitrypsin), AAT (alpha-1-antitrypsin), SPARC (osteonectin) and MSLN (mesothelin). NS represents a non-specific band used to show equal loading.

Figure 2

The profile of the secreted proteome remains unchanged even in the absence of any serum in the growth medium for 16–18 hours. Conditioned media (containing 5% FBS (CM) as well as serum free (SF) media) from the cell lines was concentrated eight-fold. Albumin was depleted from the serum-containing samples. Eight micrograms of total protein was resolved by SDS-PAGE and immunoblotted for galectin-3-binding protein (LGALS3BP). 10A corresponds to MCF10A cells and 10CA to MCF10CA cl. D cells.

Scheme 1

View of the overall strategy for secreted proteome comparisons by LC-MS/MS with Mascot searching and verification by western blotting.