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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: 7.25). 04/2009; 8(6):1436-49. DOI: 10.1074/mcp.M800061-MCP200
Source: PubMed

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.

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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.
    Glycobiology 08/2013; DOI:10.1093/glycob/cwt060 · 3.14 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: Glycoproteomics has emerged as a prime area of interest within the field of proteomics because glycoproteins have been shown to function as biomarkers for disease and as promising therapeutic targets. A significant challenge in the study of glycoproteins is the fact that they are expressed in relatively low abundance in cells. In response, various enrichment methods have been developed to improve the detection of glycoproteins. One such method involves their capture via oxidation of their glycan chains and covalent attachment with hydrazide resins which, when catalyzed by PNGase F, release N-linked glycans and convert the glycosite Asn to Asp; this conversion is identifiable with LC/ESI-MS/MS as a corresponding increase of 0.984 Da in molecular weight. The present study builds on this body of work, providing evidence of three additional strategies that improve glycoprotein identification: (1) use of a high resolution mass spectrometer-the Q Exactive MS-which delivers 2-3 times more glycoprotein identifications than a low resolution MS; (2) optimization of instrument settings and database search parameters to reduce misidentification of N-linked glycopeptides to ~1 percent; and (3) labeling glycopeptides with (18)O during PNGase F treatment to locate N-linked glycosites within peptides containing multiple N-linked sequons.
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    • "Then, modulation of cell functions can occur through activation of some member of the adenosine receptors family (Fredholm et al., 2011). Expression of CD73 has recently been associated with a prometastatic phenotype in melanoma and breast cancer (Lee et al., 2003; Sadej et al., 2006; Leth-Larsen et al., 2009; Stagg et al., 2010). Further, it has been demonstrated that tumor-derived CD73 activity is involved in the mechanism of tumor immune escape (Wang et al., 2008; Stagg et al., 2011) and in the proliferation of glioma cells (Bavaresco et al., 2008). "
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