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: non-tumorigenic 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. Prot. Res. 5, 899-906 2006). The search files produced from 5 analyses (3 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 non-classical secretion (SecretomeIP, 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 and 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.

S100B and S100A6 Differentially Modulate Cell Survival by Interacting with Distinct RAGE (Receptor for Advanced Glycation End Products) Immunoglobulin Domains

“…S100B is actively secreted by cells, and previous studies have shown the effect of extracellular S100B on cellular functions. S100A6 was found in the extracellular medium of breast cancer cells (13), but to our knowledge, no data are available on the active secretion of S100A6 by cells and on the extracellular effect of the protein on cellular functions. To determine whether S100A6 was actively released from brain-related cells, we used human U87-MG glioblastoma cells, which endogenously express S100A6 ( Fig.…”

“…Similarly S100A12 is found in synovial fluid of patients suffering from rheumatoid arthritis and also triggers neuritogenesis of hippocampal neurons when added extracellularly at submicromolar to micromolar concentrations (11,12). Finally S100A6 was found in the extracellular medium of breast cancer cells (13). S100B is mainly expressed in the brain and primarily in astrocytes of the human cortex and hippocampus but is also present in certain populations of oligodendrocytes and neurons (14 -18).…”

Global secretome analysis identifies novel mediators of bone metastasis

“…The first breast cancer metastasis secretome study profiled the secretomes of the MCF10A metastasis progression series, finding proteins such as alpha-1-antichymotrypsin and galectin-3-binding protein to be upregulated in conditioned media from the metastatic variants [48]. More recently, many secretome analyses of cell lines representing several different cancer types have been reported [8,[30][31][32][33][34][35][36][37][38][39][40].…”

Optimization and establishment of RNA interference-mediated knockdown of the progestagen-associated endometrial protein gene in human metastatic melanoma cell lines

“…To observe changes in the secreted protein profiles of melanoma samples following PAEP-silencing, mass spectral analysis was conducted on a Thermo Fisher Orbitrap mass spectrometer (Thermo Fisher, Waltham, MA, USA) with an Agilent 1200 Series Nanoflow LC (Agilent Technologies, Santa Clara, CA, USA) for sample introduction, as previously described (13)(14)(15). Prior to analysis, secreted proteins isolated from PAEP-knockdown and non-targeting knockdown 624.38-Mel cells were digested with trypsin in the presence of a reducing agent.…”