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In order to identify potential markers of renal cancer, the plasma membrane protein content of renal cell carcinoma (RCC)-derived cell lines was annotated using a proteomics process. One unusual protein identified at high levels in A498 and 786-O cells was CD70 (TNFSF7), a type II transmembrane receptor normally expressed on a subset of B, T and NK cells, where it plays a costimulatory role in immune cell activation. Immunohistochemical analysis of CD70 expression in multiple carcinoma types demonstrated strong CD70 staining in RCC tissues. Metastatic tissues from eight of 11 patients with clear cell RCC were positive for CD70 expression. Immunocytochemical analysis demonstrated that binding of an anti-CD70 antibody to CD70 endogenously expressed on the surface of A498 and 786-O cell lines resulted in the rapid internalisation of the antibody-receptor complex. Coincubation of the internalising anti-CD70 antibody with a saporin-conjugated secondary antibody before addition to A498 cells resulted in 50% cell killing. These data indicate that CD70 represents a potential target antigen for toxin-conjugated therapeutic antibody treatment of RCC.

Comparative two-dimensional proteome analysis was used to identify proteins differentially expressed in multiple clinical normal and breast cancer tissues. One protein, the expression of which was elevated in invasive ductal and lobular breast carcinomas when compared with normal breast tissue, was arylamine N-acetyltransferase-1 (NAT-1), a Phase II drug-metabolizing enzyme. NAT-1 overexpression in clinical breast cancers was confirmed at the mRNA level and immunohistochemical analysis of NAT-1 in 108 breast cancer donors demonstrated a strong association of NAT-1 staining with estrogen receptor-positive tumors. Analysis of the effect of active NAT-1 overexpression in a normal luminal epithelial-derived cell line demonstrated enhanced growth properties and etoposide resistance relative to control cells. Thus, NAT-1 may not only play a role in the development of cancers through enhanced mutagenesis but may also contribute to the resistance of some cancers to cytotoxic drugs.

B-cell-specific plasma-membrane proteins are potential targets for either small molecule or antibody-based therapies. We have sought to annotate proteins expressed at the cell surface membrane in patients with chronic lymphocytic leukemia (CLL) using plasma-membrane-based proteomic analysis to identify previously uncharacterized and potentially B-cell-specific proteins. Proteins from plasma-membrane fractions were separated on one-dimensional gels and trypsinized fractions subjected to high-throughput MALDI-TOF mass spectrometry. Using this method, many known B-cell surface antigens were detected, but also known proteins not previously described in this disease or in this cellular compartment, including cell surface receptors, membrane-associated enzymes and secreted proteins, and completely unknown proteins. To validate the method, we show that BLK, a B-cell-specific kinase, is located in the CLL-plasma-membrane fraction. We also describe two novel proteins (MIG2B and B-cell novel protein #1, BCNP1), which are expressed preferentially in B cells. MIG2B is in a highly conserved and defined gene family containing two plasma-membrane-binding ezrin/radixin/moesin domains and a pleckstrin homology domain; the Caenorhabditis elegans homolog (UNC-112) is a membrane-associated protein that colocalizes with integrin at cell-matrix adhesion complexes. BCNP1 is a completely unknown protein with three predicted transmembrane domains, with three alternatively spliced final exons. Proteomic analysis may thus define new potential therapeutic targets.

hAG-2 and hAG-3 are recently discovered human homologues of the secreted Xenopus laevis proteins XAG-1/2 (AGR-1/2) that are expressed in the cement gland, an ectodermal organ in the head associated with anteroposterior fate determination during early development. Although the roles of hAG-2 and hAG-3 in mammalian cells are unknown, both proteins share a high degree of protein sequence homology and lie adjacent to one another on chromosome 7p21. hAG-2 mRNA expression has previously been demonstrated in oestrogen receptor (ER)-positive cell lines. In this study, we have used real-time quantitative RT - PCR analysis and immunohistochemistry on tissue microarrays to demonstrate concordant expression of hAG-2 and hAG-3 mRNA and protein in breast tumour tissues. Tumour expression of both genes correlated with OR (hAG2, P=0.0002; hAG-3, P=0.0012), and inversely correlated with epidermal growth factor receptor (EGFR) (P=0.003). Yeast two-hybrid cloning identified metastasis-associated GPI-anchored C4.4a protein and extracellular alpha-dystroglycan (DAG-1) as binding partners for both hAG-2 and hAG-3, which if replicated in clinical oncology would demonstrate a potential role in tumour metastasis through the regulation of receptor adhesion and functioning. hAG-2 and hAG-3 may therefore serve as useful molecular markers and/or potential therapeutic targets for hormone-responsive breast tumours.

Proteins associated with cancer cell plasma membranes are rich in known drug and antibody targets as well as other proteins known to play key roles in the abnormal signal transduction processes required for carcinogenesis. We describe here a proteomics process that comprehensively annotates the protein content of breast tumor cell membranes and defines the clinical relevance of such proteins. Tumor-derived cell lines were used to ensure an enrichment for cancer cell-specific plasma membrane proteins because it is difficult to purify cancer cells and then obtain good membrane preparations from clinical material. Multiple cell lines with different molecular pathologies were used to represent the clinical heterogeneity of breast cancer. Peptide tandem mass spectra were searched against a comprehensive data base containing known and conceptual proteins derived from many public data bases including the draft human genome sequences. This plasma membrane-enriched proteome analysis created a data base of more than 500 breast cancer cell line proteins, 27% of which were of unknown function. The value of our approach is demonstrated by further detailed analyses of three previously uncharacterized proteins whose clinical relevance has been defined by their unique cancer expression profiles and the identification of protein-binding partners that elucidate potential functionality in cancer.