Abstract
The spread of cancer cells from a primary tumor to form metastasis at distant sites is a complex multistep process. The cancer cell proteins and plasma membrane proteins in particular involved in this process are poorly defined, and a study of the very early events of the metastatic process using clinical samples or in vitro assays is not feasible. We have used a unique model system consisting of two isogenic human breast cancer cell lines that are equally tumorigenic in mice; but although one gives rise to metastasis, the other disseminates single cells that remain dormant at distant organs. Membrane purification and comparative quantitative LC-MS/MS proteomics identified 13 membrane proteins that were expressed at higher levels and three that were underexpressed in the metastatic compared with the non-metastatic cell line from a total of 1919 identified protein entries. Among the proteins were ecto-5'-nucleotidase (CD73), NDRG1, integrin beta1, CD44, CD74, and major histocompatibility complex class II proteins. The altered expression levels of proteins identified by LC-MS/MS were validated using flow cytometry, Western blotting, and immunocyto- and immunohistochemistry. Analysis of clinical breast cancer biopsies demonstrated a significant correlation between high ecto-5'-nucleotidase and integrin beta1 expression and poor outcome, measured as tumor spread or distant recurrence within a 10-year follow-up. Further the tissue analysis suggested that NDRG1, HLA-DRalpha, HLA-DRbeta, and CD74 were associated with the ER(-)/PR(-) phenotype represented by the two cell lines. The study demonstrates a quantitative and comparative proteomics strategy to identify clinically relevant key molecules in the early events of metastasis, some of which may prove to be potential targets for cancer therapy.
Highlights
The spread of cancer cells from a primary tumor to form metastasis at distant sites is a complex multistep process
Proteomics—To identify proteins, plasma membrane proteins that may influence the ability of breast cancer cells to expand and establish metastasis at distant sites, the isogenic cell line pair M-4A4 and NM-2C5 was used as a model system
Thirteen proteins were found to be quantitatively expressed at higher amounts in the metastatic M-4A4 cells compared with the non-metastatic NM-2C5 cells, whereas three proteins were expressed in lower amounts in M-4A4 compared with NM-2C5 cells, and all of these proteins may be potential markers of cancer cells with the ability to establish metastasis (Table I)
Summary
M-4A4 and NM-2C5 cell lines were cloned from the parental cell line, MDA-MB-435, by serial dilution and screening by orthotopic implantation into the mammary fat pad of athymic mice [13]. Isotope-labeled M-4A4 and unlabeled NM-2C5 cells were combined 1:1 followed by homogenization, fractionation by centrifugation, and Percoll-sucrose density separation of cell contents. The fractions enriched for cell surface proteins and with minimal mitochondrial contamination, as determined by the activities of ␥-glutamyl transpeptidase and succinate dehydrogenase, were either pooled or processed individually for further analysis. The variations included single versus dual isotopic labeling, Percoll-sucrose density separation of crude membranes versus no density separation, and single versus repeated sample analysis to increase the number of proteins identified by LC-MS/MS. Dual isotopic labeling and repeated analysis of a sample with LC-MS/MS (three to four times) increased the percentage of proteins that could be quantified, whereas the use of Percoll-sucrose density separation resulted in enrichment of membrane proteins. The VEMS peptide quantification values (QVs) were based on calculation of QV for each set of the first three peaks in the light and heavy isotopic clusters
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