Tamoxifen-resistance is a major cause of death in patients with recurrent breast cancer. Current clinical parameters can correctly predict therapy response in only half of the treated patients. Identification of proteins that associate with tamoxifen-resistance is a first step towards better response prediction and tailored treatment of patients. In the present study we aimed to identify putative protein biomarkers indicative of tamoxifen therapy-resistance in breast cancer, using nanoLC-FTICR MS. Comparative proteome analysis was performed on ~5,500 pooled tumor cells obtained through laser capture microdissection from two independently processed data sets (n=24 and n=27) of tamoxifen therapy-sensitive and -resistant tumors. Peptide and protein identifications were acquired by matching mass and elution time features to information in previously generated accurate mass and time tag reference data bases. A total of 17,263 unique peptides were identified that corresponded to 2,556 non-redundant proteins identified with >=2 peptides. From this total, 1,713 proteins overlapped between the two data sets, which were used for further statistical analysis. Comparative proteome analysis of the two data sets combined revealed 100 putatively differentially abundant proteins (p<0.05) between tamoxifen-sensitive and -resistant tumors. The presence and abundance of 47 of these proteins was verified by targeted nanoLC-MS/MS in the same, individual, non-microdissected tumor tissue extracts. ENPP1, EIF3E, and GNB4 significantly associated with progression-free survival upon tamoxifen treatment (p=0.005, p=0.03 and p= 0.04, respectively). Differential abundance of our top discriminating protein, EMMPRIN, was validated by tissue microarray in an independent patient cohort (n=156). EMMPRIN was not only higher expressed in PD tumors, it also significantly associated with shorter time to progression upon tamoxifen treatment (p=0.002). In summary, quantitative comparative proteomics was performed on LCM-derived breast tumor cells using ultra-sensitive nanoLC-FTICR technology; this resulted in the identification of putative biomarkers associating with tamoxifen therapy-resistance in recurrent breast cancer.

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Keywords *Drug Resistance, Neoplasm, Amino Acid Sequence, Breast Neoplasms/*drug therapy/metabolism, Breast cancer, Chromatography, Liquid, Female, Humans, Immunohistochemistry, Molecular Sequence Data, Neoplasm Proteins/chemistry/*metabolism, Peptide Mapping, Tamoxifen/*therapeutic use, Tandem Mass Spectrometry, Trypsin/chemistry, laser capture microdissection, protein profile, tamoxifen resistance, tissue proteomics
Persistent URL hdl.handle.net/1765/22152
Citation
Umar, A., Martens, J.W.M., Foekens, J.A., Paša-Tolić, L., Kang, H., Timmermans, A.M., … den Bakker, M.A.. (2009). Identification of a putative protein-profile associating with tamoxifen therapy-resistance in breast cancer. Molecular and Cellular Proteomics, 8(6), 1278–1294. Retrieved from http://hdl.handle.net/1765/22152