Mass spectrometry (LC-MS/MS) identified proteomic biosignatures of breast cancer in proximal fluid

Abstract

We have begun an early phase of biomarker discovery in three clinically important types of breast cancer using a panel of human cell lines: HER2 positive, hormone receptor positive and HER2 negative, and triple negative (HER2-, ER-, PR-). We identified and characterized the most abundant secreted, sloughed, or leaked proteins released into serum free media from these breast cancer cell lines using a combination of protein fractionation methods before LC-MS/MS mass spectrometry analysis. A total of 249 proteins were detected in the proximal fluid of 7 breast cancer cell lines. The expression of a selected group of high abundance and/or breast cancer-specific potential biomarkers including thromobospondin 1, galectin-3 binding protein, cathepsin D, vimentin, zinc-α2-glycoprotein, CD44, and EGFR from the breast cancer cell lines and in their culture media were further validated by Western blot analysis. Interestingly, mass spectrometry identified a cathepsin D protein single-nucleotide polymorphism (SNP) by alanine to valine replacement from the MCF-7 breast cancer cell line. Comparison of each cell line media proteome displayed unique and consistent biosignatures regardless of the individual group classifications, demonstrating the potential for stratification of breast cancer. On the basis of the cell line media proteome, predictive Tree software was able to categorize each cell line as HER2 positive, HER2 negative, and hormone receptor positive and triple negative based on only two proteins, muscle fructose 1,6-bisphosphate aldolase and keratin 19. In addition, the predictive Tree software clearly identified MCF-7 cell line overexpresing the HER2 receptor with the SNP cathepsin D biomarker.

Figure 1

Quantitative comparison of proteins detected by LC-MS/MS in the proximal fluid of seven breast cancer cell lines. In triplicate, MS/MS data of proteins identified in the proximal fluid from each breast cancer cell line was quantitatively determined by spectral counting in Scaffold software. Enolase 1, cathepsin D, CD44,Thrombospondin 1, zinc-α2-glycoprotein, keratin 18, vimentin, galectin-3 binding protein, and EGFR were chosen to represent HER2 negative and hormone receptor positive cell lines T47D and MCF-7, HER2 positive SKBR-3 and MDA-MB-453, triple negative breast cancer MDA-MB-468 and MDA-MB-231, and hormone receptor positive MCF-7 transfected with HER2 (MCF-7HER2).

Figure 2

Western blot comparison of 7 potential biomarkers identified the LC-MS/MS study from whole cell lysates of 7 different breast cancer cell lines. Three known biomarkers, ERα, MUC1, and HER2 were also included in the Western blot study as controls. Minimally three experiments were performed.

Figure 3

Dot blot comparison of 7 LC-MS/MS identified potential biomarkers present in the proximal fluid of 7 different breast cancer cell lines. Serum free media was collected from 7 different breast cancer cell lines as described. Cells were counted and equivalent ratios of media was loaded and analyzed for each breast cancer cell line. Each experiment consisted of three repeats.

Figure 4

Single-nucleotide polymorphism (SNP) identified in cathepsin D by LC-MS/MS. A. MS/MS coverage of cathepsin D in the breast cancer cell lines of SKBR-3 and MCF-7HER2. B. MS/MS spectra of the wild type cathepsin D peptide 55-YSQAVPAVTEGPIPEVLK-72 in SKBR3 (Calc. MH+1898.0270; Exp MH+1898.0046). C. MS/MS spectra of the SNP in cathepsin D peptide 55-YSQVVPAVTEGPIPEVLK-72 in MCF-7HER2 (Calc MH+1926.0582; Exp MH+1926.0621).

Figure 5. Two Principle Component Dimension Analysis

A plot of the first versus second principle component is the projection of the 229 dimensional data in a two dimensional plot. Triplicate replicates of all seven cell lines cluster appropriately, however clusters from each of the two different cell lines in the same group do not cluster together. Each breast cancer cell line is represented by a different symbol in triplicate (see legend).

Figure 6

Tree test predicts that only cathepsin D, muscle fructose 1,6-bisphosphate aldolase and keratin 19 are necessary to predict breast cancer cell line group as hormone receptor positive and HER2 positive (HR+ and HER2+), hormone receptor positive and HER2 negative (HR+ and HER2−), HER2 positive and hormone receptor negative (HER2+ and HR−), and triple negative breast cancer (TNBC). In the right column is a comparison of MS/MS spectral counts across all seven breast cancer cell lines for muscle fructose 1,6-bisphosphate aldolase and keratin 19. In lower right column is a Western blot analysis of fructose 1,6-bisphosphate aldolase, keratin 19 and keratin 8 of breast cancer cell line total extracts. Experiments were performed in triplicate.

Figure 7

Tree Test validation of fructose 1,6 bisphosphate aldolase A and keratin 8 presence in hormone HER2 positive and hormone receptor negative, hormone receptor positive and HER2 negative, triple negative breast cancer tissue. Western blot analysis using fructose 1,6 bisphosphate aldolase A and keratin 8 antibody was conducted on a total of 15 breast cancer tissue samples, five of each type of breast cancer.