Metastasis-related Plasma Membrane Proteins of Human Breast Cancer Cells Identified by Comparative Quantitative Mass Spectrometry

Medical Biotechnology Center, Institute of Medical Biology, University of Southern Denmark, Winsloewparken 25.3, 5000 Odense C, Denmark.
Molecular &amp Cellular Proteomics (Impact Factor: 6.56). 04/2009; 8(6):1436-49. DOI: 10.1074/mcp.M800061-MCP200
Source: PubMed


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.

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Available from: Henrik J Ditzel, Sep 23, 2014
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    • "In the case of biomarkers, there is a necessity of quantification of the expression levels of polypeptides . Several strategies have been used in the field of breast cancer biomarkers including Stable Isotope Labelling by Amino Acid in Cell Culture (SILAC) [13] [14] [15] [16] [17] [18], 2D-Fluorescence Difference in Gel Electrophoresis (2D-DIGE) [19] [20] [21], and Isobaric Tag for Relative and Absolute Quantitation (iTRAQ) [22] [23] [24] [25], among others. There are very few reports of quantitative proteomics applied to several breast cancer cell lines using iTRAQ and tandem mass spectrometry. "
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    ABSTRACT: Breast cancer is the principal cancer in women worldwide. Although there are serum tumor markers such as CEA and HER2, they are detected in advanced stages of the disease and used as progression and recurrence markers. Therefore, there is a necessity for the identification of new markers that might lead to an early detection and also provide evidence of an effective treatment. The aim of this work was to determine the differential protein expression profiles of four breast cancer cell lines in comparison to a normal control cell line by iTRAQ labelling and tandem mass spectrometry, in order to identify putative biomarkers of the disease. We identified 1,020 iTRAQ-labelled polypeptides with at least one peptide identified with more than 95% in confidence. Overexpressed polypeptides in all cancer cell lines were 78, whilst the subexpressed were 128. We categorised them with PANTHER program into biological processes, being the metabolic pathways the most affected. We detected six groups of proteins with the STRING program involved in DNA topology, glycolysis, translation initiation, splicing, pentose pathway, and proteasome degradation. The main subexpressed protein network included mitochondrial proteins involved in oxidative phosphorylation. We propose BAG6, DDX39, ANXA8 and COX4 as putative biomarkers in breast cancer. We report a set of differentially expressed proteins in the MCF7 and T47D (Luminal A), MDA-MB-231 (Claudin low) and SK-BR-3 (HER2(+)) breast cancer cell lines that have not been previously reported in breast cancer disease. From these proteins, we propose BAG6, DDX39, ANXA8 and COX4 as putative biomarkers in breast cancer. On the other hand, we propose sets of unique polypeptides in each breast cancer cell line that can be useful in the classification of different subtypes of breast cancer. Copyright © 2015. Published by Elsevier B.V.
    Journal of proteomics 04/2015; 124. DOI:10.1016/j.jprot.2015.04.018 · 3.89 Impact Factor
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    • "Interestingly, FN is implicated in epithelial-to-mesenchymal transition (EMT) [16] and both the β1 integrin and FN are required in the formation of lamellipodia, filopodia and invadopodia [17, 18]; potentially supporting their role in ECM remodelling and subsequent tumour cell invasion [19]. Indeed, the β1 integrin has been reported to be more highly expressed in primary tumours with LN metastases [20] and with poor survival outcomes [21]. This fits in well with our findings that suggest an increase in β1 integrin immunoreactivity with increasing invasiveness. "
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    ABSTRACT: HCP and ASK are co-first authors. Cancer metastasis is the main contributor to breast cancer fatalities as women with the metastatic disease have poorer survival outcomes than women with localised breast cancers. There is an urgent need to develop appropriate prognostic methods to stratify patients based on the propensities of their cancers to metastasise. The insulin-like growth factor (IGF)-I: IGF binding protein (IGFBP):vitronectin complexes have been shown to stimulate changes in gene expression favouring increased breast cancer cell survival and a migratory phenotype. We therefore investigated the prognostic potential of these IGF- and extracellular matrix (ECM) interaction-induced proteins in the early identification of breast cancers with a propensity to metastasise using patient-derived tissue microarrays.
    BMC Cancer 08/2014; 14(1):627. DOI:10.1186/1471-2407-14-627 · 3.36 Impact Factor
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    • "There are a number of other proteomics studies of the breast cancer cell lines, but most consider only lines from malignant tumors, or cover many fewer cell lines than described here (Kulasingam and Diamandis 2007; Leth-Larsen et al. 2009; Whelan et al. 2009; Bateman et al. 2010; Drake et al. 2012; Geiger et al. 2012). An exception is a study by Boersema et al. (2013), who quantified the N-linked glycoproteins found in conditioned medium from four nonmalignant and seven malignant breast cancer cell lines. "
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    ABSTRACT: Breast cancer cell lines express fewer transmemebrane and secreted glycoproteins than non-malignant ones. The objective of these experiments was to characterize the changes in expression of several hundred glycoproteins quantitatively. Secreted and cell surface glycoproteins were isolated using a glycoprotein capture protocol, then identified by tandem mass spectrometry. Glycoproteins expressed by a group of cell lines originating from malignant tumors of the breast were compared to those expressed by a non-malignant set. The average number of spectral counts (proportional to relative protein abundance) and the total number of glycopeptides in the malignant samples were reduced to about 2/3 of the level in the non-malignant samples. Most glycoproteins were expressed at a different level in the malignant samples, with nearly as many increasing as decreasing. The glycoproteins with reduced expression accounted for a larger change in spectral counts, and hence for the net loss of spectral counts in the malignant lines. Similar results were found when the glycoproteins were studied via identified glycosylation sites only, or through identified sites together with nonglycopeptides. The overall reduction is largely due to the loss of integrins, laminins and other proteins that form or interact with the basement membrane.
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