Supplemental Material can be found at: http://www.mcponline.org/cgi/content/full/M800493-MCP200 /DC1 Research Author’s Choice Identification of a Putative Protein Profile Associated with Tamoxifen Therapy Resistance in Breast Cancer*□S Arzu Umar‡§, Hyuk Kang¶ʈ, Annemieke M. Timmermans‡, Maxime P. Look‡, Marion E. Meijer-van Gelder‡, Michael A. den Bakker**, Navdeep Jaitly¶‡‡, John W. M. Martens‡, Theo M. Luider§§, John A. Foekens‡, and Ljiljana Pasˇ a-Tolic´¶ -Extracellular matrix metallo .(156 ؍ Tamoxifen resistance is a major cause of death in patients ent patient cohort (n with recurrent breast cancer. Current clinical factors can proteinase inducer levels were higher in therapy-resistant Downloaded from correctly predict therapy response in only half of the tumors and significantly associated with an earlier tumor treated patients. Identification of proteins that are asso- progression following first line tamoxifen treatment (hazard In .(0.002 ؍ ciated with tamoxifen resistance is a first step toward ratio, 1.87; 95% confidence interval, 1.25–2.80; p better response prediction and tailored treatment of pa- summary, comparative proteomics performed on laser tients. In the present study we intended to identify puta- capture microdissection-derived breast tumor cells us- tive protein biomarkers indicative of tamoxifen therapy ing nano-LC-FTICR MS technology revealed a set of www.mcponline.org resistance in breast cancer using nano-LC coupled with putative biomarkers associated with tamoxifen therapy FTICR MS. Comparative proteome analysis was per- resistance in recurrent breast cancer. Molecular & formed on ϳ5,500 pooled tumor cells (corresponding to Cellular Proteomics 8:1278–1294, 2009. ϳ550 ng of protein lysate/analysis) obtained through laser capture microdissection (LCM) from two independently containing both (27 ؍ and n 24 ؍ processed data sets (n Tamoxifen is an antiestrogenic agent that has been widely at Medical Library Erasmus MC on June 8, 2009 tamoxifen therapy-sensitive and therapy-resistant tu- and successfully used in the treatment of breast cancer over mors. Peptides and proteins were identified by matching the past decades (1). Tamoxifen targets and inhibits the es- mass and elution time of newly acquired LC-MS features trogen receptor-␣, which is expressed in ϳ70% of all primary to information in previously generated accurate mass and time tag reference databases. A total of 17,263 unique breast tumors and is known to be important in the develop- peptides were identified that corresponded to 2,556 non- ment and course of the disease. When diagnosed at an early redundant proteins identified with >2 peptides. 1,713 stage, adjuvant systemic tamoxifen therapy can cure ϳ10% overlapping proteins between the two data sets were of the patients (1). In recurrent disease, ϳ50% of patients used for further analysis. Comparative proteome analysis have no benefit from tamoxifen (intrinsic resistance). From the revealed 100 putatively differentially abundant proteins other half of patients who initially respond to therapy with an between tamoxifen-sensitive and tamoxifen-resistant tu- objective response (OR)1 or no change (NC), a majority even- mors. The presence and relative abundance for 47 differ- tually develop progressive disease (PD) due to acquired ta- entially abundant proteins were verified by targeted moxifen resistance (2, 3). With the markers available to date nano-LC-MS/MS in a selection of unpooled, non-micro- we can insufficiently predict therapy response. Therefore, dissected discovery set tumor tissue extracts. ENPP1, EIF3E, and GNB4 were significantly associated with pro- 1 gression-free survival upon tamoxifen treatment for The abbreviations used are: OR, objective response; AMT, accu- recurrent disease. Differential abundance of our top dis- rate mass and time; BRB, biometric research branch; CI, confidence criminating protein, extracellular matrix metalloproteinase interval; CLIC4, chloride intracellular channel protein 4; CR, complete remission; EBP50, ezrin-radixin-moesin-binding phosphoprotein 50; inducer, was validated by tissue microarray in an independ- EIF3E, eukaryotic translation initiation factor 3 subunit 6/E; EMMPRIN, extracellular matrix metalloproteinase inducer; GNB4, guanine nucleotide-binding protein ␤ subunit 4; HR, hazard ratio; IHC, From the ‡Department of Medical Oncology and Cancer Genomics immunohistochemistry; IPI, International Protein Index; LCM, laser Centre, **Department of Pathology, and §§Department of Neurology, capture microdissection; NAP1L1, nucleosome assembly protein Josephine Nefkens Institute, Erasmus Medical Center, 3000 CA 1-like 1; NC, no change; NET, normalized elution time; OCLN, oc- Rotterdam, The Netherlands and ¶Environmental Molecular Sciences cluding; PCA, principal component analysis; PD, progressive disease; Laboratory, Pacific Northwest National Laboratory, Richland, Wash- PR, partial remission; RP, reversed-phase; S100A6, calcyclin; ington 99352 S100A9, calgranulin B; SGPL1, sphingosine-1-phosphate lyase; Author’s Choice—Final version full access. TMA, tissue microarray; TUBB3, ␤-tubulin type 3; UQCRFS1, ubiqui- Received, October 29, 2008, and in revised form, February 24, 2009 nol-cytochrome c reductase iron-sulfur subunit mitochondrial precur- Published, MCP Papers in Press, March 27, 2009, DOI 10.1074/ sor; LTQ, linear trap quadrupole; ENPP1, ectonucleotide phospha- mcp.M800493-MCP200 tase/phosphodiesterase 1. 1278 Molecular & Cellular Proteomics 8.6 This paper is available on line at http://www.mcponline.org Tamoxifen Resistance Protein Profile in Breast Cancer identification of new biomarkers that can more effectively identifying peptides in subsequent quantitative LC-MS anal- predict response to treatment and that can potentially func- yses. Substituting routine LC-MS/MS analyses (shotgun ap- tion as drug targets is a major focus of research. proach) with LC-FTICR MS analyses (AMT tag approach) The search for new biomarkers has been enhanced by the significantly increases overall throughput and sensitivity while introduction of microarray technology. Gene expression stud- reducing sample requirements. Additionally quantitative in- ies have resulted in a whole spectrum of profiles for e.g. tensity information related to the abundance of the protein molecular subtypes, prognosis, and therapy prediction in can be discerned from these MS analyses (24). In the present breast cancer (4–10). Corresponding studies at the protein study, we used the same strategy to analyze eight pools of level are lagging behind because of immature technology. tumor cells in duplicate or triplicate (resulting in 19 samples) However, protein-level information is crucial for the functional derived from 51 fresh frozen primary invasive breast carcino- understanding and the ultimate translation of molecular mas that appeared to be either sensitive or resistant to ta- knowledge into clinical practice, and proteomics technologies moxifen treatment after recurrence. This work resulted in the continue to progress at a rapid pace. identification of a putative protein profile associated with ta- Proteomics studies reported so far have mainly been per- moxifen therapy resistance. In addition, the top discriminating Downloaded from formed with breast cancer cell lines using either two-dimen- protein of the putative profile, extracellular matrix metallopro- sional gel electrophoresis (11–14) or LC-MS for protein sep- teinase inducer (EMMPRIN), was validated in an independent aration (15–17). However, it is known that the proteomic patient cohort and was significantly associated with resist- makeup of a cultured cell is rather different from that of a ance to tamoxifen therapy and shorter time to progression tumor cell surrounded by its native microenvironment (18). upon tamoxifen treatment in recurrent breast cancer. Furthermore cell lines lack the required follow-up information www.mcponline.org for answering important clinical questions. In addition, tumor EXPERIMENTAL PROCEDURES tissues in general and breast cancer tissues in particular are Patients and Tumor Tissues—For the discovery phase of the study, very heterogeneous in the sense that they harbor many dif- 51 different fresh frozen primary breast cancer tissues from our liquid ferent cell types, such as stroma, normal epithelium, and N2 tissue bank were used. Primary tumors were selected from pa- tumor cells. LCM technology has emerged as an ideal tool for tients that did not receive any systemic adjuvant hormonal therapy and were treated with the antiestrogen tamoxifen as first line therapy at Medical Library Erasmus MC on June 8, 2009 selectively extracting cells of interest from their natural envi- upon detection of recurrent breast cancer. Furthermore tumors were ronment (19) and has therefore been an important step for- selected on the basis of positive estrogen receptor-␣ expression as ward in the context of genomics and proteomics cancer bi- assessed by ligand binding assay or enzyme-linked immunosorbent omarker discovery research. LCM-derived breast cancer assay (Ն10 fmol/mg of cytosolic protein). Tumor tissues were divided tumor cells have been used for comparative proteomics anal- into two classes based on the type of response to tamoxifen therapy. 24 tumors were sensitive to tamoxifen therapy, showing either com- yses in the past using both two-dimensional gel electrophore- plete remission (CR) or partial remission (PR), and were assigned as sis (20, 21) and LC-MS (22). This has resulted in the identifi-