Journal of Cancer 2012, 3 269 Ivyspring International Publisher Journal of Cancer 2012; 3: 269-284. doi: 10.7150/jca.4592 Research Paper Identification of Potential Glycoprotein Biomarkers in Estrogen Receptor Positive (ER+) and Negative (ER-) Human Breast Cancer Tissues by LC-LTQ/FT-ICR Mass Spectrometry Suzan M. Semaan1, Xu Wang1, Alan G. Marshall1,2, Qing-Xiang Amy Sang1 1. Department of Chemistry and Biochemistry and Institute of Molecular Biophysics; 2. Center for Interdisciplinary Magnetic Resonance, National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida 32306-4390, USA. Corresponding author: Tel: +1-850-644-8683. Fax: +1-850-644-8281. Email: [email protected]. Room 3501, Chemical Sciences Labor- atory Building, 102 Varsity Way, Florida State University, Tallahassee, Florida 32306-4390, U.S.A. © Ivyspring International Publisher. This is an open-access article distributed under the terms of the Creative Commons License (http://creativecommons.org/ licenses/by-nc-nd/3.0/). Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited. Received: 2012.05.14; Accepted: 2012.06.20; Published: 2012.06.21 Abstract Breast cancer is the second most fatal cancer in American women. To increase the life ex- pectancy of patients with breast cancer new diagnostic and prognostic biomarkers and drug targets must be identified. A change in the glycosylation on a glycoprotein often causes a change in the function of that glycoprotein; such a phenomenon is correlated with cancerous transformation. Thus, glycoproteins in human breast cancer estrogen receptor positive (ER+) tissues and those in the more advanced stage of breast cancer, estrogen receptor negative (ER-) tissues, were compared. Glycoproteins showing differences in glycosylation were examined by 2-dimensional gel electrophoresis with double staining (glyco- and total protein staining) and identified by reversed-phase nano-liquid chromatography coupled with a hybrid linear quadrupole ion trap/ Fourier transform ion cyclotron resonance mass spectrometer. Among the identified glycosylated proteins are alpha 1 acid glycoprotein, alpha-1-antitrypsin, calmodulin, and superoxide dismutase mitochondrial precursor that were further verified by Western blotting for both ER+ and ER- human breast tissues. Results show the presence of a possible glycosylation difference in alpha-1-antitrypsin, a potential tumor-derived biomarker for breast cancer progression, which was expressed highest in the ER- samples. Key words: Two-dimensional gel electrophoresis; Estrogen receptor positive and negative breast cancer; Biomarkers; Alpha-1-antitrypsin; Fourier transform ion cyclotron resonance mass spec- trometry; Proteomics Introduction Breast cancer, if cancers of the skins are exclud- carcinomas will be diagnosed in 2012 [1]. Based on ed, is the most frequently diagnosed cancer in Amer- proteomic and genomic studies, prognostic biomarker ican women and ranks immediately after lung cancer candidates identified from breast cancer include in mortality. According to the American Cancer Soci- growth factor receptors, steroid receptors, p53, Ki-67, ety Breast Cancer Facts and Figures, approximately cyclins, BRCA1 and BRCA2, urokinase plasminogen 39,510 women (i.e., 14.4% of those diagnosed) will die activator, p21, and pro- and anti-apoptotic factors [2]. from breast cancer in the USA. In 2012, estimated new The Food and Drug Administration has approved breast cancer occurrences will reach 226,870, not in- biomarkers CA 15.3, human epidermal growth factor cluding in situ carcinoma. About 63,300 in situ breast receptor 2 (Her-2/neu), and CA27-29. However, the http://www.jcancer.org Journal of Cancer 2012, 3 270 combination of several biomarkers may produce the Furthermore, those cancer-related peptide epitopes best prognosis [3]. have been exploited as immunogens for cancer [32]. Breast cancer development and progression are Therefore, identification of glycoproteins in the ER+ regulated by estrogen and its receptor (ER) that is one and ER- breast cancer tissues might reveal possible of the predictive markers including progesterone re- target(s) and identify putative biomarkers or tumor ceptor (PR), Her-2/neu, and ER for breast cancer. Yet, suppressors for pro- and pre-aggressive breast cancer the anti-estrogen drug tamoxifen is often ineffective stages. Here, we compare glycoproteins in breast even after estrogen receptor positivity [2]. Previous cancer ER+ tissue and those in ER- tissue, the more research has revealed that the tumorigenic epithelial aggressive breast cancer, after sample cells in the breast duct are ER positive (ER+) cells N-deglycosylation to compare glycosylation differ- overlying an intact myoepithelial membrane, but ences with controls and double staining of the some adjacent cells overlying a focally disrupted two-dimensional electrophoresis (2-DE) gels. Glyco- myoepithelial cell layer have lost their response to proteins were identified by reversed-phase anti-estrogen drugs because they no longer express nano-liquid chromatography coupled with a hybrid the ER. Moreover, the loss of the ER of those once linear quadrupole ion trap/ Fourier transform ion tumorigenic cells at the invasion site contributes to cyclotron resonance mass spectrometer their higher potential for aggressiveness and invasion (LC-LTQ/FT-ICR MS) and the glycosylation type for [4-6]. Therefore, it is important to search for new selected breast cancer-related proteins was verified by prognostic and predictive markers for breast cancer Western blotting. progression from ER+ to ER-. Many fundamental cellular processes such as Materials and Methods cellular differentiation, control of cell division, Chemicals and materials cell–cell recognition, adhesion, and malignant trans- Ready immobilized pH gradient (IPG) Strips, pH formation are controlled by oligosaccharides [7-10]. In 4-7, 11 cm long, were purchased from Bio-Rad. Dithi- most cases, oligosaccharides are conjugated with othreitol was purchased from Promega Corporation other biomolecules known as glycol-conjugates. This (Madison, WI). Bromophenol blue, Tris, SDS, Tricine, process is observed within the endoplasmic reticu- Ponseau S solution, bovine serum albumin (BSA), and lum, Golgi apparatus, nucleus, and the cytoplasm of a cholamidopropyl dimethylammonio propanesul- cell. More than half of all proteins are glycosylated, fonate hydrate (CHAPS) were purchased from Sig- either N- and/or O-linked [11]. Within the endo- ma-Aldrich. Glacial acetic acid was purchased from plasmic reticulum and Golgi apparatus, O-linked EMD chemicals Inc. Urea was purchased from Fisher glycans are usually attached to the peptide chain by a Scientific. Primary polyclonal anti-human rabbit an- serine or threonine hydroxyl group. Mostly within the tibodies against ER-α MC-20 (MW 66 kDa), calmodu- endoplasmic reticulum, N-linked glycans, the pre- lin FL-149 (MW 17 kDa), and superoxide dismutase dominant carbohydrate attachment in mammalian FL-222 (MW 25 kDa) were purchased from Santa Cruz cells [12], are linked to the side chain by means of an Biotechnology, Inc. (Santa Cruz, CA). Polyclonal rab- amide bond to asparagine in a 60-65% conserved bit anti-human alpha-1-antitrypsin A0012 (MW ~57 Asn-Xxx-Ser/Thr motif (in which Xxx can be any kDa) was purchased from DakoCytomation, Den- amino acid except proline) [11, 13-14]. N-linked car- mark. Rabbit polyclonal anti-human alpha-1-acid bohydrates are frequently found together with glycoprotein ab440-2 (MW ~50 kDa) was bought from O-linked oligosaccharides in proteins such as fetuin Abcam. Anti-rabbit IgG (whole molecule) peroxidase [15], insulin receptor [16], IgG immunoglobulins [17], antibody A0545 produced in goat and monoclonal and O-linked glycoproteins include collagens [18], mouse anti-β-actin antibody A5316 (MW 42 kDa) mucins [19], and proteoglycans [20]. were purchased from Sigma-Aldrich. Protease and In breast cancer cells, changes in O-linked [21-26] phosphatase inhibitor cocktails were purchased from and N-linked [27-28] oligosaccharide structures are Thermo-Pierce (Rockford, IL). frequently correlated with cancerous transformation. For example, breast carcinomas may display altered Sample preparation expression of secreted and membrane-bound mucins, Human ductal breast carcinoma tissue samples such as MUC1 and MUC2 [29]. When epithelial were obtained from the Cooperative Human Tissue cell-surface mucin MUC1 is abnormally glycosylated, Network-Midwestern Division. Tissues were cut into its expression shows up-regulation in breast cancer 1 mm cubes on ice [33]. Tissues were suspended in [30]. As a result, new epitopes are exposed leading to cold tissue lysis buffer in 10 mM Tris pH 7.5, 130 mM humoral and cell-mediated immune responses [31]. NaCl, 1% octylglucopyranoside (OG), 1% CHAPS, 10 http://www.jcancer.org Journal of Cancer 2012, 3 271 mM sodium phosphate, 10 mM sodium fluoride, and teins, and antibodies were diluted in 1% BSA in TBST. 1x phosphatase and protease inhibitor (EDTA-free). For multiple probing on the same nitrocellulose, the Samples were centrifuged for 15 min at 14,000 rpm at membrane was treated with 30% H2O2 for 15 min at 37 4 ˚C to remove insoluble material. The supernatant ºC while covered, then rinsed with TBST, followed by layer was transferred to a new tube, and the pellet and blocking and normal probing procedure [35]. The the top lipid layer were discarded. Samples were then H2O2 inactivates the