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• W2103012318 abstract "The receptor tyrosine kinase ErbB2 (HER-2/neu) is overexpressed in up to 30% of breast cancers and is associated with poor prognosis and an increased likelihood of metastasis especially in node-positive tumors. In this proteomic study, to identify the proteins that are associated with the aggressive phenotype of HER-2/neu-positive breast cancer, tumor cells from both HER-2/neu-positive and -negative tumors were procured by laser capture microdissection. Differentially expressed proteins in the two subsets of tumors were identified by two-dimensional electrophoresis and MALDI-TOF/TOF MS/MS. We found differential expression of several key cell cycle modulators, which were linked with increased proliferation of the HER-2/neu-overexpressing cells. Nine proteins involved in glycolysis (triose-phosphate isomerase (TPI), phosphoglycerate kinase 1 (PGK1), and enolase 1 (ENO1)), lipid synthesis (fatty acid synthase (FASN)), stress-mediated chaperonage (heat shock protein 27 (Hsp27)), and antioxidant and detoxification pathways (haptoglobin, aldo-keto reductase (AKR), glyoxalase I (GLO), and prolyl-4-hydrolase β-isoform (P4HB)) were found to be up-regulated in HER-2/neu-positive breast tumors. HER-2/neu-dependent differential expression of PGK1, FASN, Hsp27, and GLO was further validated in four breast cancer cell lines and 12 breast tumors by immunoblotting and confirmed by partially switching off the HER-2/neu signaling in the high HER-2/neu-expressing SKBr3 cell line with Herceptin treatment. Statistical correlations of these protein expressions with HER-2/neu status were further verified by immunohistochemistry on a tissue microarray comprising 97 breast tumors. Our findings suggest that HER-2/neu signaling may result, directly or indirectly, in enhanced activation of various metabolic, stress-responsive, antioxidative, and detoxification processes within the breast tumor microenvironment. We hypothesize that these identified changes in the cellular proteome are likely to drive cell proliferation and tissue invasion and that the key cell cycle modulators involved, when uncovered by future research, would serve as naturally useful targets for the development of therapeutic strategies to negate the metastatic potential of HER-2/neu-positive breast tumors. The receptor tyrosine kinase ErbB2 (HER-2/neu) is overexpressed in up to 30% of breast cancers and is associated with poor prognosis and an increased likelihood of metastasis especially in node-positive tumors. In this proteomic study, to identify the proteins that are associated with the aggressive phenotype of HER-2/neu-positive breast cancer, tumor cells from both HER-2/neu-positive and -negative tumors were procured by laser capture microdissection. Differentially expressed proteins in the two subsets of tumors were identified by two-dimensional electrophoresis and MALDI-TOF/TOF MS/MS. We found differential expression of several key cell cycle modulators, which were linked with increased proliferation of the HER-2/neu-overexpressing cells. Nine proteins involved in glycolysis (triose-phosphate isomerase (TPI), phosphoglycerate kinase 1 (PGK1), and enolase 1 (ENO1)), lipid synthesis (fatty acid synthase (FASN)), stress-mediated chaperonage (heat shock protein 27 (Hsp27)), and antioxidant and detoxification pathways (haptoglobin, aldo-keto reductase (AKR), glyoxalase I (GLO), and prolyl-4-hydrolase β-isoform (P4HB)) were found to be up-regulated in HER-2/neu-positive breast tumors. HER-2/neu-dependent differential expression of PGK1, FASN, Hsp27, and GLO was further validated in four breast cancer cell lines and 12 breast tumors by immunoblotting and confirmed by partially switching off the HER-2/neu signaling in the high HER-2/neu-expressing SKBr3 cell line with Herceptin treatment. Statistical correlations of these protein expressions with HER-2/neu status were further verified by immunohistochemistry on a tissue microarray comprising 97 breast tumors. Our findings suggest that HER-2/neu signaling may result, directly or indirectly, in enhanced activation of various metabolic, stress-responsive, antioxidative, and detoxification processes within the breast tumor microenvironment. We hypothesize that these identified changes in the cellular proteome are likely to drive cell proliferation and tissue invasion and that the key cell cycle modulators involved, when uncovered by future research, would serve as naturally useful targets for the development of therapeutic strategies to negate the metastatic potential of HER-2/neu-positive breast tumors. Traditional cancer chemotherapy agents designed to block cell division are toxic to healthy cells as well as to cancer cells. Targeting specific metabolic pathways to stop cancer growth is potentially less toxic to normal cells and can improve tolerability considerably. Thus, anticancer drug discovery has shifted from the traditional empiric random screening approach to a more rational and mechanistic, target-based approach whereby specific abnormalities in cell functioning are modulated in a classical drug (ligand)-receptor fashion. The HER/ErbB family of transmembrane receptors is one of the most exciting targets currently under evaluation. This ErbB family of receptor tyrosine kinases includes four closely related members: HER-1/ErbB1 (also known as the epidermal growth factor receptor), HER-2/ErbB2 (also known as HER-2/neu), 1The abbreviations used are: HER-2/neu, human tyrosine kinase-type cell surface receptor, type 2; 2-D, two-dimensional; 2-DE, 2-dimensional electrophoresis; LCM, laser capture microdissection; TMA, tissue microarray; FASN, fatty acid synthase; PGK1, phosphoglycerate kinase 1; GLO, glyoxalase I; Hsp27, heat shock protein 27; AKR, aldo-keto reductase; P4HB, prolyl-4-hydrolase β-subunit; ENO1, enolase 1; TPI, triose-phosphate isomerase; PI3K, phosphatidylinositol 3-kinase; MAPK, mitogen-activated protein kinase; MDR, multidrug resistance; PTEN, phosphatase and tensin homologue deleted on chromosome 10; DAB+, 3,3′-diaminobenzidine; PEA3, polyomavirus enhancer activaton 3. 1The abbreviations used are: HER-2/neu, human tyrosine kinase-type cell surface receptor, type 2; 2-D, two-dimensional; 2-DE, 2-dimensional electrophoresis; LCM, laser capture microdissection; TMA, tissue microarray; FASN, fatty acid synthase; PGK1, phosphoglycerate kinase 1; GLO, glyoxalase I; Hsp27, heat shock protein 27; AKR, aldo-keto reductase; P4HB, prolyl-4-hydrolase β-subunit; ENO1, enolase 1; TPI, triose-phosphate isomerase; PI3K, phosphatidylinositol 3-kinase; MAPK, mitogen-activated protein kinase; MDR, multidrug resistance; PTEN, phosphatase and tensin homologue deleted on chromosome 10; DAB+, 3,3′-diaminobenzidine; PEA3, polyomavirus enhancer activaton 3. HER-3/ErbB3, and HER-4/ErbB4. These receptors initiate signals by forming ligand-induced combinations of homo- and heterodimers (1Yarden Y. Sliwkowski M.X. Untangling the ErbB signalling network.Nat. Rev. Mol. Cell. Biol. 2001; 2: 127-137Google Scholar) and play a critical role in the pathogenesis of breast cancer. HER-2/neu, one of the most well characterized breast cancer oncogenes, is amplified in about 20–30% of all human breast cancers (2Isola J. Chu L. DeVries S. Matsumura K. Chew K. Ljung B.M. Waldman F.M. Genetic alterations in ERBB2-amplified breast carcinomas.Clin. Cancer Res. 1999; 5: 4140-4145Google Scholar, 3Zhang D.-H. Salto-Tellez M. Do E. Putti T.C. Koay E.S.C. Evaluation of HER-2/neu oncogene status in breast tumors on tissue microarrays.Hum. Pathol. 2003; 34: 362-368Google Scholar) and is also overexpressed in a variety of other human tumors, including ovarian, lung, gastric, and oral cancers. It appears not to bind to any known ligand but is the preferred heterodimerization partner for the other family members (4Citri A. Skaria K.B. Yarden Y. The deaf and the dumb: the biology of ErbB-2 and ErbB-3.Exp. Cell Res. 2003; 284: 54-65Google Scholar). The ErbB2/3 heterodimer efficiently activates the phosphatidylinositol 3-kinase (PI3K)/AKT/PTEN pathway and the Ras/Raf/mitogen-activated protein kinase (MAPK) pathway, which are essential in cellular survival, by phosphorylating and inactivating growth-inhibitory and proapoptotic proteins. Aberrant expression of ErbB receptors triggers the activation of multiple downstream signal transduction pathways and plays a key role in inducing increased cell proliferation and differentiation, decreasing apoptosis, and enhancing tumor cell motility and angiogenesis. Clinically accumulated evidence has shown that HER-2/neu overexpression in tumors is associated with a poor prognosis and a more aggressive phenotype. In particular, tumors with HER-2/neu overexpression are known to be refractory to various types of chemo- and endocrine therapy and to be associated with shortened overall survival (5Pietras R.J. Arboleda J. Reese D.M. Wongvipat N. Pegram M.D. Ramos L. Gorman C.M. Parker M.G. Sliwkowski M.X. Slmon D.J. HER-2 tyrosine kinase pathway targets estrogen receptor and promotes hormone-independent growth in human breast cancer cells.Oncogene. 1995; 10: 2435-2446Google Scholar, 6Alaoui-Jamali M.A. Paterson J. Al Moustafa A.-E. Yen L. The role of ErbB2 tyrosine kinase receptor in cellular intrinsic chemoresistance: mechanisms and implications.Biochem. Cell Biol. 1997; 75: 315-325Google Scholar). The genes/proteins regulated by HER-2/neu-mediated signal transduction pathways have been investigated recently (7Wilson K.S. Roberts H. Leek R. Harris A.L. Geradts J. Differential gene expression patterns in HER2/neu-positive and -negative breast cancer cell lines and tissues.Am. J. Pathol. 2002; 161: 1171-1185Google Scholar, 8Mackay A. Jones C. Dexter T. Silva R.L. Bulmer K. Jones A. Simpson P. Harris R.A. Jat P.S. Neville A.M. Reis L.F. Lakhani S. O’Hare M.J. cDNA microarray analysis of genes associated with ErbB2 (HER2/neu) overexpression in human mammary luminal epithelial cells.Oncogene. 2003; 22: 2680-2688Google Scholar, 9White S.L. Gharbi S. Bertani M.F. Chan H.-L. Waterfield M.D. Timms J.F. Cellular responses to ErbB-2 overexpression in human mammary luminal epithelial cells: comparison of mRNA and protein expression.Br. J. Cancer. 2004; 90: 173-181Google Scholar, 10Kumar-Sinha C. Ignatoski K.W. Lippman M.E. Ethier S.P. Chinnaiyan A.M. Transcriptome analysis of HER2 reveals a molecular connection to fatty acid synthesis.Cancer Res. 2003; 63: 132-139Google Scholar). Transcriptome analysis revealed that a large number of these differentially regulated genes were involved in cell-matrix interactions, cell proliferation, and transformation (8Mackay A. Jones C. Dexter T. Silva R.L. Bulmer K. Jones A. Simpson P. Harris R.A. Jat P.S. Neville A.M. Reis L.F. Lakhani S. O’Hare M.J. cDNA microarray analysis of genes associated with ErbB2 (HER2/neu) overexpression in human mammary luminal epithelial cells.Oncogene. 2003; 22: 2680-2688Google Scholar). Many interferon-inducible genes were found to be down-regulated, consistent with increased proliferation of the ErbB2-overexpressing cells, whereas the key cell cycle modulators, including cyclin D2, were up-regulated in the breast cell lines exposed to heregulin β1, an ErbB-specific growth factor (9White S.L. Gharbi S. Bertani M.F. Chan H.-L. Waterfield M.D. Timms J.F. Cellular responses to ErbB-2 overexpression in human mammary luminal epithelial cells: comparison of mRNA and protein expression.Br. J. Cancer. 2004; 90: 173-181Google Scholar). Kumar-Sinha et al. (10Kumar-Sinha C. Ignatoski K.W. Lippman M.E. Ethier S.P. Chinnaiyan A.M. Transcriptome analysis of HER2 reveals a molecular connection to fatty acid synthesis.Cancer Res. 2003; 63: 132-139Google Scholar) reported that HER-2/neu regulated fatty acid synthase (FASN) expression through the PI3K pathway, whereas Menendez et al. (11Menendez J.A. Vellon L. Mehmi I. Oza B.P. Ropero S. Colomer R. Lupu R. Inhibition of fatty acid synthase (FAS) suppresses HER2/neu (erbB-2) oncogene overexpression in cancer cells.Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 10715-10720Google Scholar) showed that FASN modulated HER-2/neu transcription and suggested HER-2/neu could function as a sensor of energy imbalance in the FASN-overexpressing tumor cells. Several other targets of the ErbB2/PI3K signal transduction cascade have been implicated in tumor angiogenesis and metastasis as well (12Laughner E. Taghavi P. Chiles K. Mahon P.C. Semenza G.L. HER2 (neu) signaling increases the rate of hypoxia-inducible factor-1α (HIF-1 α) synthesis: novel mechanism for HIF-1-mediated vascular endothelial growth factor expression.Mol. Cell. Biol. 2001; 21: 3995-4004Google Scholar, 13Hernan R. Fasheh R. Calabrese C. Frank A.J. Maclean K.H. Allard D. Barraclough R. Gilbertson R.J. ErbB2 up-regulates S100A4 and several other prometastatic genes in medulloblastoma.Cancer Res. 2003; 63: 140-148Google Scholar), leading to stimulated expression of vascular endothelial growth factor (12Laughner E. Taghavi P. Chiles K. Mahon P.C. Semenza G.L. HER2 (neu) signaling increases the rate of hypoxia-inducible factor-1α (HIF-1 α) synthesis: novel mechanism for HIF-1-mediated vascular endothelial growth factor expression.Mol. Cell. Biol. 2001; 21: 3995-4004Google Scholar) and enhanced cytoskeletal reorganization and tumor cell motility (14Adam L. Vadlamudi R. Kondapaka S.B. Chernoff J. Mendelsohn J. Kumar R. Heregulin regulates cytoskeletal reorganization and cell migration through the p21-activated kinase-1 via phosphatidylinositol 3-kinase.J. Biol. Chem. 1998; 273: 28238-28246Google Scholar). The prometastatic gene S100A4 is another gene shown to be up-regulated by the HER-2/neu signaling network that includes the PI3K, AKT1, and extracellular signal-regulated kinase 1/2 pathways (13Hernan R. Fasheh R. Calabrese C. Frank A.J. Maclean K.H. Allard D. Barraclough R. Gilbertson R.J. ErbB2 up-regulates S100A4 and several other prometastatic genes in medulloblastoma.Cancer Res. 2003; 63: 140-148Google Scholar). HER-2/neu itself is strongly inhibited by a tumor suppressor protein, ARF, an alternative reading frame protein, which inhibits HER-2/neu-mediated oncogenic growth by negating its effect in blocking cancer cells from undergoing programmed cell death or apoptosis (15Zhang Y. Yang H.-Y. Zhang X.-C. Yang H. Tsai M. Lee M.-H. Tumor suppressor ARF inhibits HER-2/neu-mediated oncogenic growth.Oncogene. 2004; 23: 7132-7143Google Scholar). Recent advances in proteomic technology have provided powerful analytical tools to identify the differentially expressed and/or post-translationally modified proteins as potential biomarkers in tumors (16Chen J. He Q.-Y. Yuen A. P.-W. Chiu J.-F. Proteomics of buccal squamous cell carcinoma: the involvement of multiple pathways in tumorigenesis.Proteomics. 2004; 4: 2465-2475Google Scholar, 17Tomonaga T. Matsushita K. Yamaguchi S. Oh-Ishi M. Kodera Y. Maeda T. Shimada H. Ochiai T. Nomura F. Identification of altered protein expression and post-translational modification in primary colorectal cancer by using agarose two-dimensional gel electrophoresis.Clin. Cancer Res. 2004; 10: 2007-2014Google Scholar) and blood (18Ahmed N. Barker G. Oliva K.T. Hoffmann P. Riley C. Reeve S. Smith A.I. Kemp B.E. Quinn M.A. Rice G.E. Proteomic-based identification of haptoglobin-1 precursor as a novel circulating biomarker of ovarian cancer.Br. J. Cancer. 2004; 91: 129-140Google Scholar, 19Juan H.-F. Chen J.-H. Hsu W.-T. Huang S.-C. Chen S.-T. Lin J. Y.-C. Chang Y.-W. Chiang C.-Y. Wen L.-L. Chan D.-C. Liu Y.-C. Chen Y.-J. Identification of tumor-associated plasma biomarkers using proteomic techniques: from mouse to human.Proteomics. 2004; 4: 2766-2775Google Scholar). Two-dimensional electrophoresis (2-DE), as the principal tool in proteomics, is able to resolve thousands of proteins in one experiment and provide the highest resolution in protein separation. The aggregate protein information attainable from such proteomic analyses would provide a vast resource for a better understanding of the translated protein milieu and dynamic protein-protein interactions that occur within the tumor microenvironment during oncogenesis and pathogenesis. Deciphering the changes in the proteome in the tumor cells compared with the normal untransformed counterparts also provides a basis for the identification of potential targets for early disease detection and for the rational designs of diagnostic and therapeutic methods. A few studies on HER-2/neu-induced changes in protein expression have been reported using breast cancer cell lines (20Gharbi S. Gaffney P. Yang A. Zvelebil M.J. Cramer R. Waterfield M.D. Timms J.F. Evaluation of two-dimensional differential gel electrophoresis for proteomic expression analysis of a model breast cancer cell system.Mol. Cell. Proteomics. 2002; 1: 91-98Google Scholar), but none, to our knowledge, have been performed on HER-2/neu-positive and -negative clinical breast tumor tissues to discover the deregulated proteins and/or phosphoproteins involved in the HER-2/neu signal transduction pathways in the tumor microenvironment. The development of tumor-specific biomarkers and discovery of novel target-based drugs have been challenged by the heterogeneous populations of cells present in human breast tumor tissues. To overcome this problem, we used laser capture microdissection (LCM) to procure relatively homogeneous cell populations from both the HER-2/neu-positive and -negative breast tumors to generate well defined differential protein profiles and to identify the deregulated proteins associated with the HER-2/neu oncogene. In a previous study (21Zhang D.-H. Tai L.K. Wong L.L. Sethi S.K. Koay E.S.C. Proteomics of breast cancer: enhanced expression of cytokeratin19 in HER-2/neu-positive breast tumors.Proteomics. 2005; 5: 1797-1805Google Scholar), we identified seven of the 21 spots with at least 5-fold changes in spot volume in HER-2/neu-positive tumors using the Ettan MALDI-TOF mass spectrometer. In this study, the remaining spots were analyzed with the MALDI-TOF/TOF MS/MS tandem mass spectrometer, and nine more proteins with high Mascot scores were identified. Four differentially expressed proteins were further validated using a combination of immunoblotting, monoclonal antibody inhibition of HER-2/neu signaling, and immunohistochemistry, the latter performed on 97 breast tumors compiled into a breast tumor tissue microarray (TMA). We present data to suggest that these proteins may represent some of the key enzymes or non-enzyme mediators in the signal transduction cascade that are involved in the underlying mechanisms that collectively confer the poor prognosis associated with HER-2/neu overexpression in breast tumors. Four commercially available breast carcinoma cell lines, MDA-MB-231, MCF-10A, BT474, and SKBr3 (kindly given by Dr. Q. C. Lau, Oncology Research Institute, National University of Singapore), were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, and 100 mg/ml streptomycin. Two of these cell lines, BT474 and SKBr3, were known to be high expressors of HER-2/neu oncoprotein, whereas the MDA-MB-231 and MCF-10A cell systems had not demonstrated any HER-2/neu expression. The adherent cells in culture were harvested by trypsinization before reaching confluence; the cells were treated at 37 °C for 5–10 min with trypsin-EDTA solution (Sigma) and washed with PBS buffer. The proteins were extracted using the Mammalian Protein Extraction Reagent (M-PER, Pierce). To determine the influence or effect of HER-2/neu signaling on the protein expression of various differentially expressed proteins, the HER-2/neu-overexpressing SKBr3 cell line was treated with Herceptin at a final concentration of 200 μg/ml for 48 h at 37 °C (10Kumar-Sinha C. Ignatoski K.W. Lippman M.E. Ethier S.P. Chinnaiyan A.M. Transcriptome analysis of HER2 reveals a molecular connection to fatty acid synthesis.Cancer Res. 2003; 63: 132-139Google Scholar) to inhibit HER-2/neu expression. These analyses were done in triplicates. Total proteins were extracted and used for Western blotting. Stringent selection of the tumor tissue samples and the procurement of homogeneous populations of tumor cells using the PixCell II laser capture system for proteomic analyses were as described previously (21Zhang D.-H. Tai L.K. Wong L.L. Sethi S.K. Koay E.S.C. Proteomics of breast cancer: enhanced expression of cytokeratin19 in HER-2/neu-positive breast tumors.Proteomics. 2005; 5: 1797-1805Google Scholar). Briefly 10 HER-2/neu-positive and 15 HER-2/neu-negative frozen tumor tissues and their matched normal tissues were obtained from the Tissue Repository of the Singapore National University Hospital. The tumors were obtained following regulations set by our Institutional Review Board for tissue usage, including informed patient consent and anonymization prior to release for research use. For the proteomic studies, which were carried out in triplicates to ensure reproducibility of results, we carefully selected six tumors, controlling for histological type or grade (all of Grade 3), nodal status (all node-positive), and estrogen receptor content (all estrogen receptor-negative), thus ensuring that HER-2/neu status remains the major discerning variable. Three of the selected tumors were HER-2/neu-positive, and the remaining three were HER-2/neu-negative. The decision to work with proteins pooled from a small number of well matched tumors was made to minimize the misinterpretation of protein profiles arising from random differences in gene expression of different tumors and to obtain sufficient materials for downstream analyses. Multiple 5-μm-thick sections were cut from the frozen tissues fixed in 70% ethanol and embedded in paraffin, further stained in hematoxylin in the presence of protease inhibitor mixture tablets (Complete Mini, Roche Applied Science), and dehydrated in xylene. Cells were microdissected using the PixCell II LCM system (Arcturus Engineering Inc., Mountain View, CA) with 10,000 shots, corresponding to 50,000–70,000 cells, within the set maximum time period of 30 min per slide. The cells were immediately lysed in the appropriate protein extraction buffer. Protein sample preparation and separation by 2-DE were performed as stated in our previous work (21Zhang D.-H. Tai L.K. Wong L.L. Sethi S.K. Koay E.S.C. Proteomics of breast cancer: enhanced expression of cytokeratin19 in HER-2/neu-positive breast tumors.Proteomics. 2005; 5: 1797-1805Google Scholar). Generally the microdissected cells were lysed in a 40 mm Tris-HCl buffer containing 7 m urea, 2 m thiourea, 4% CHAPS, 1% Mega-10, 0.5% Triton X-100, 50 mm dithiothreitol, 1% IPG buffer (pH 3–10 nonlinear), and 2 mm tributylphosphine. The protein concentrations were determined using the PlusOne 2-D Quantitation kit (Amersham Biosciences) after cleaning up using the PlusOne 2-D Clean-up kit (Amersham Biosciences). The pooled proteins from the three HER-2/neu-positive tumors and pooled proteins from the three HER-2/neu-negative tumors were separately mixed with 350 μl of rehydration solution, applied for IEF using the Immobiline IPG DryStrips (180 mm, pH 3–10 nonlinear) (Amersham Biosciences), and separated by SDS-PAGE (9%) using the PROTEAN® II xi Cell system (Bio-Rad). Each pooled sample was run in triplicates. The pooled samples from HER-2/neu-negative tumors were used to prepare the triplicate reference gels and to create a reference matched set. In the matched set, only spots that were present in all three reference gels were included. The same was done to obtain the HER-2/neu-positive matched set. The two matched sets were compared using the ImageMaster 2-D Elite software (Amersham Biosciences) for image acquisition and analysis. Spot quantities were normalized to remove nonexpression-related variations in spot intensity, and the results were evaluated as spot optical density. The criterion of a differential expression of any particular protein between the two subsets of tumors was set as at least a 5-fold change in spot volume between the two matched sets in triplicates. Most of the protein spots that were included fulfilled the above criterion, but there were some that showed more than 5-fold changes in volume in only two of the three replicates. All these spots were selected for analysis by MALDI-TOF/TOF MS/MS. The protein spots were excised from the gel and digested with trypsin. Briefly silver-stained protein spots were destained with 30 mm potassium ferricyanide and 100 mm sodium thiosulfate (1:1) and washed with Millipore-Q water. The gel pieces were dehydrated with ACN, dried in a SpeedVac centrifuge, and reswelled with 20–40 μl of digestion solution containing 20 mm ammonium bicarbonate and 20 ng/μl sequencing grade trypsin (Promega, Madison, WI). After the tryptic digestion at 37 °C for at least 2 h, the resultant peptides were extracted, desalted with ZipTip C18 columns (Millipore Corp., Bedford, MA), and eluted with 2.5 μl of 50% ACN containing 0.5% TFA and 3 mg/ml α-cyano-4-hydroxycinnamic acid. Samples were spotted onto stainless steel MALDI sample target plates, and peptide mass spectra were obtained by the Applied Biosystems 4700 Proteomics Analyzer MALDI-TOF/TOF mass spectrometer (Applied Biosystems, Framingham, MA) in the positive ion reflector mode. The subsequent MS/MS analysis was performed in a data-dependent manner, and the 10 most abundant ions fulfilling certain preset criteria were subjected to high energy CID analysis. The collision energy was set to 1 keV, and nitrogen was used as the collision gas. For the database search, known contamination peaks such as keratin and autoproteolysis peaks were removed prior to database search. Spectra were processed and analyzed using the Mascot software (Matrix Science, London, UK) to search for the peptide mass fingerprints and MS/MS data in the National Center for Biotechnology Information nonredundant (NCBInr) database. The mass accuracy was considered to be within 50 ppm for the mass measurement and within 0.5 Da for CID experiments. Searches were performed without constraining protein molecular weight (Mr) or pI and species and allowed for carbamidomethylation of cysteine and partial oxidation of methionine residues. Up to one missed tryptic cleavage was considered for all tryptic mass searches. Protein scores greater than 75 were considered as significant (p < 0.05). Further confirmation of protein identifications was obtained by duplicating the protein identification using the MS-Fit software (prospector.ucsf.edu). For 2-D immunodetection, the same protein samples used for the protein profile analysis were tested. The pooled proteins were first separated using IPG DryStrips (7 cm, pH 3–10) and further separated by 10% SDS-PAGE. For Western blotting, two HER-2/neu-overexpressing cell lines, BT474 and SKBr3, and two HER-2/neu-negative cell lines, MDA-MB-231 and MCF10A, as well as 12 clinical specimens, six each from HER-2/neu-positive and HER-2/neu-negative tumor cells procured by LCM, were analyzed. The protein mixture from each case was individually separated by 10% SDS-PAGE and electrotransferred onto PVDF membranes using the SemiDry apparatus (Bio-Rad). The membranes were serially probed with one of the following primary antibodies: anti-FASN (1:500, BD Biosciences), anti-Hsp27 (1:1000, Santa Cruz Biotechnology, Inc.), anti-PGK1 (1:500, Santa Cruz Biotechnology, Inc.), anti-GLO (1:500, kindly given by Dr. K. D. Tew from Institute of Cancer Research, University of London), and anti-β-actin (1:5000, Sigma). Goat anti-rabbit IgG horseradish peroxidase (1:10,000, Zymed Laboratories Inc.) or goat anti-mouse IgG horseradish peroxidase (1:5000, Molecular Probes) in TBS-Tween 20 buffer were used as secondary antibodies. The chemiluminescent signals were detected using SuperSignal® West Pico Chemiluminescent Substrate (Pierce). Signals were captured with the MULTI GENIUS Bio Imaging System (Syngene, Frederick, MD), and the signal intensities were analyzed using GeneTools software (Syngene). Two breast cancer TMAs consisting of 97 tumors and corresponding matched normal tissues, respectively, were constructed as reported previously (22Zhang D.-H. Salto-Tellez M. Putti T.C. Do E. Koay E.S.C. Reliability of tissue microarrays in detecting protein expression and gene amplification in breast cancer.Mod. Pathol. 2003; 16: 79-84Google Scholar). The DAKO Envision system was used for the immunostaining of the TMA sections. Briefly sections were deparaffinized in xylene and rehydrated in graded alcohols. Antigen unmasking was undertaken using DAKO® Target Retrieval Solution in a microwave oven. Endogenous peroxidases were removed using 3% hydrogen peroxide in methanol. Sections were incubated for 1 h with anti-FASN (1:500), anti-Hsp27 (1:500), anti-GLO (1:200), and anti-PGK1 (1:200) at room temperature followed by detection with labeled dextran polymer conjugated with peroxidase and DAB+-substrate chromogen solution. Nuclei were lightly stained with Mayer’s hematoxylin. Each antibody was applied to three sections. The staining level was scored as negative (0), weak (1), moderate (2), and strong (3) by a pathologist and a research scientist independently, according to the staining intensity of the tumor cells. Cases with discrepant scores were rescored by the same or additional scorers to obtain a consensus score, failing which the scores were not included in the data analysis. All the sections were examined without prior knowledge of the clinical information for any of the cases. A Fisher’s exact test was used to compare the expression of FASN, Hsp27, PGK1, and GLO between HER-2/neu-positive and -negative tumors. A p value (two-sided) of 0.05 was considered statistically significant. To identify the proteins whose expressions were strongly associated with HER-2/neu status in breast cancer, we compared the protein profiles between pooled HER-" @default.
• W2103012318 created "2016-06-24" @default.
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• W2103012318 date "2005-11-01" @default.
• W2103012318 modified "2023-10-18" @default.
• W2103012318 title "Proteomic Study Reveals That Proteins Involved in Metabolic and Detoxification Pathways Are Highly Expressed in HER-2/neu-positive Breast Cancer" @default.
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