Plasma membrane proteins that are exposed on the cell surface have important biological functions, such as signaling into and out of the cells, ion transport, and cell-cell and cell-matrix interactions. The expression level of many of the plasma membrane proteins involved in these key functions is altered on cancer cells, and these proteins may also be subject to post-translational modification, such as altered phosphorylation and glycosylation. Additional protein alterations on cancer cells confer metastatic capacities, and some of these cell surface proteins have already been successfully targeted by protein drugs, such as human antibodies, that have enhanced survival of several groups of cancer patients. The combination of novel analytical approaches and subcellular fractionation procedures has made it possible to study the plasma membrane proteome in more detail, which will elucidate cancer biology, particularly metastasis, and guide future development of novel drug targets. The technical advances in plasma membrane proteomics and the consequent biological revelations will be discussed herein. Many of the advances have been made using cancer cell lines, but because the main goal of this research is to improve individualized treatment and increase cancer patient survival, further development is crucial to direct analysis of clinically relevant patient samples. These efforts include optimized specimen handling and preparation as well as improved proteomics platforms.

Recent studies have shown that Abs that target the cell-surface enzyme CD73 (ecto-5′-nucleotidase) reduce growth of primary tumors and metastasis in syngenic mice by inhibiting the catalytic activity of CD73, and thus increasing the activity of cytotoxic T lymphocytes. In this article, we report another anticancer mechanism of anti-CD73 Abs and show that an anti-CD73 mAb (AD2) inhibits metastasis formation by a mechanism independent of CD73 catalytic activity and inhibition of primary tumor growth. This mechanism involves clustering and internalization of CD73, but does not require cross-linking of CD73, because both whole IgG anti-CD73 AD2 mAb and Fab′ fragments thereof exhibited this effect. Ex vivo treatment of different breast cancer cell lines with anti-CD73 AD2 mAb before i.v. injection into mice inhibited extravasation/colonization of circulating tumor cells and significantly reduced metastasis development. This effect was also observed when the cancer cell-surface expression of CD73 was significantly reduced by small interfering RNA knockdown. The antimetastatic activity is epitope specific, as another Ab that efficiently binds CD73-expressing live cancer cells did not lead to CD73 internalization and metastasis inhibition. Furthermore, anti-CD73 AD2 mAb inhibited development of metastasis in a spontaneous animal model of human metastatic breast cancer. Our study shows that some anti-CD73 mAbs cause cell-surface clustering of CD73 followed by internalization, thus inhibiting the ability of circulating tumor cells to extravasate and colonize, leading to inhibition of metastasis. Ab-based CD73 cancer therapy should include a combination of Abs that target the catalytic activity of CD73, as well as those with the characteristics described in this article.

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 ␤1, 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 ␤1 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-DR␣, HLA-DR␤, and CD74 were associated with the ER

Cell surface membrane proteins are involved in central processes such as cell signaling, cell-cell interactions, ion and solute transport, and they seem to play a pivotal role in several steps of the metastatic process of cancer cells. The low abundance and hydrophobic nature of cell surface membrane proteins complicate their purification and identification by MS. We used two isogenic cell lines with opposite metastatic capabilities in nude mice to optimize cell surface membrane protein purification and to identify potential novel markers of metastatic cancer. The cell surface membrane proteins were isolated by centrifugation/ultracentrifugation steps, followed by membrane separation using a Percoll/sucrose density gradient. The gradient fractions containing the cell surface membrane proteins were identified by enzymatic assays. Stable isotope labeling of the proteome of the metastatic cell line by SILAC followed by mass spectrometry analysis enabled identification and quantification of proteins that were differentially expressed in the two cell lines. Dual stable isotopic labels ((13)C-arginine and (13)C-lysine) instead of a single label ((13)C-arginine) increased the percentage of proteins that could be quantified from 40 to 93%. Repeated LC-MS/MS analyses (3-4 times) of each sample increased the number of identified proteins by 60%. The use of Percoll/sucrose density separation allowed subfractionation of membranes leading to enrichment of membrane proteins (66%) and reduction from 33% to only 16% of protein from other membranous organelles such as endoplasmatic reticulum, Golgi, and mitochondria. In total, our optimized methods resulted in 1919 protein identifications (corresponding to 826 at similarity level 80% (SL80); 1145 (509 at SL80) were identified by two or more peptides of which 622 (300 at SL80) were membrane proteins. The quantitative proteomic analysis identified 16 cell surface proteins as potential markers of the ability of breast cancer cells to form distant metastases.