TY - JOUR

T1 - Metastasis-related plasma membrane proteins of human breast cancer cells identified by comparative quantitative mass spectrometry

AU - Leth-Larsen, Rikke

AU - Lund, Rikke

AU - Hansen, Helle V

AU - Laenkholm, Anne-Vibeke

AU - Tarin, David

AU - Jensen, Ole N

AU - Ditzel, Henrik J

PY - 2009/6

Y1 - 2009/6

N2 - 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.

AB - 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.

KW - Animals

KW - Base Sequence

KW - Blotting, Western

KW - Breast Neoplasms/metabolism

KW - Cell Line, Tumor

KW - Chromatography, Liquid

KW - DNA Primers

KW - Female

KW - Flow Cytometry

KW - Humans

KW - Immunohistochemistry

KW - Membrane Proteins/metabolism

KW - Mice

KW - Neoplasm Metastasis

KW - Neoplasm Proteins/metabolism

KW - Polymerase Chain Reaction

KW - Tandem Mass Spectrometry/methods

U2 - 10.1074/mcp.M800061-MCP200

DO - 10.1074/mcp.M800061-MCP200

M3 - Journal article

C2 - 19321434

VL - 8

SP - 1436

EP - 1449

JO - Molecular and Cellular Proteomics

JF - Molecular and Cellular Proteomics

SN - 1535-9476

IS - 6

ER -