title: Metastasis-related plasma membrane proteins of human breast cancer cells identified by comparative quantitative mass spectrometry.
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.
authors.affiliation: Medical Biotechnology Center, Institute of Medical Biology, University of Southern Denmark, Winsloewparken 25.3, 5000 Odense C, Denmark.
authors.forename: Rikke
authors.initials: R
authors.lastname: Leth-Larsen
authors.forename: Rikke
authors.initials: R
authors.lastname: Lund
authors.forename: Helle V
authors.initials: HV
authors.lastname: Hansen
authors.forename: Anne-Vibeke
authors.initials: AV
authors.lastname: Laenkholm
authors.forename: David
authors.initials: D
authors.lastname: Tarin
authors.forename: Ole N
authors.initials: ON
authors.lastname: Jensen
authors.forename: Henrik J
authors.initials: HJ
authors.lastname: Ditzel