Comprehensive Proteomic Analysis of Breast Cancer Cell Membranes Reveals Unique Proteins with Potential Roles in Clinical Cancer

Abstract

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 proteinbinding partners that elucidate potential functionality in cancer.

S100A14: Novel Modulator of Terminal Differentiation in Esophageal Cancer

“…Previously, S100A14 was identified as a plasma membrane-associated protein in breast cancer cell lines and exhibited an increased expression in breast cancer tissues versus matched normal tissues. Accordingly, S100A14 exhibited different patterns of subcellular distribution, typified by plasma membrane localization in breast cancer tissues but cytosolic expression in nontumor breast epithelial cells (35). Previous studies showed that some members of the S100 family of proteins exhibit calcium-dependent translocation (36,37), and the translocation of S100A14 is regulated in a calcium-dependent manner through interaction with nucleobindin, which has strong association with Ga proteins (38).…”

CD70 (TNFSF7) is expressed at high prevalence in renal cell carcinomas and is rapidly internalised on antibody binding

“…Preparation of membrane fractions and mass spectrometry on a 20 cm 6% Laemlli one-dimensional gel (BioRad, Hemel Hempstead, UK) and 0.5 mm slices were subjected to trypsinolysis and prepared for MALDI-TOF mass spectrometry (Voyager STR, Applied Biosystems, Framingham, MA, USA) as previously described (Adam et al, 2003). Selected peptide masses for CD70 (M þ H) ¼ 1217.6 and 1142.6 were characterised further by MS/MS using a tandem quadrupole TOF mass spectrometer equipped with a nanospray ion source (Micromass UK Ltd, Manchester, UK).…”

“…To compile a list of candidate RCC cell-surface proteins, we used a previously described proteomics approach adapted for breast cancer cell lines (Adam et al, 2003) using plasma membrane preparations from the A498, SW839 and CAKI-2 RCC-derived cell lines. As described previously (Adam et al, 2003), potential targets were analysed further using real-time quantitative RT -PCR and immunohistochemistry to quantify expression in normal tissues and clinical RCC samples.…”

“…To compile a list of candidate RCC cell-surface proteins, we used a previously described proteomics approach adapted for breast cancer cell lines (Adam et al, 2003) using plasma membrane preparations from the A498, SW839 and CAKI-2 RCC-derived cell lines. As described previously (Adam et al, 2003), potential targets were analysed further using real-time quantitative RT -PCR and immunohistochemistry to quantify expression in normal tissues and clinical RCC samples. We found CD70, a type II transmembrane cell-surface protein of the tumour necrosis factor (TNF) receptor family (Goodwin et al, 1993;Bowman et al, 1994;Hintzen et al, 1994), to be expressed at high levels in RCC, with expression retained in metastatic RCC tissues.…”

RT in situ PCR detection of MART-1 and TRP-2 mRNA in formalin-fixed, paraffin-embedded tissues of melanoma and nevi

“…13,14 The oligonucleotide primer sequences (Invitrogen, Carlsbad, CA, USA) were derived from a previous paper 15 as shown in Table 1. For RT-PCR of MART-1 mRNA, the forward primer, 5 0 -CACGGCCACTCTTACAC CAC-3 0 , and the reverse primer, 5 0 -GGAGCATTGG GAACCACAGG-3 0 , yielded a product of 254 bp.…”