Identification of specific nuclear structural protein alterations in human breast cancer (original) (raw)
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Electrophoresis, 1998
High-resolution two-dimensional gel electrophoresis (2-DE) and database analysis was used to establish protein expression patterns for cultured normal human mammary epithelial cells and thirteen breast cancer cell lines. The Human Breast Epithelial Cell database contains the 2-DE protein patterns, including relative protein abundances, for each cell line, plus a composite pattern that contains all the common and specifically expressed proteins from all the cell lines. Significant differences in protein expression, both qualitative and quantitative, were observed not only between normal cells and tumor cells, but also among the tumor cell lines. Eight percent (56/727) of the consistently detected proteins were found in significantly (P < 0.001) variable levels among the cell lines. Eight proteins present in normal cultured breast epithelial cells were not detected in any of the tumor cell lines. We identified a subset of the differentially expressed proteins using a combination of immunostaining, protein sequencing, comigration, and subcellular fractionation. These identified proteins include the intermediate filament components vimentin and cytokeratins. The cell lines can be classified into four distinct groups based on their intermediate filament protein profile. We also identified heat shock proteins; hsp27 and hsp60 varied in abundance and in some cases in the relative phosphorylation levels among the cell lines. Many of the differentially expressed proteins we identified have roles in cellular proliferation and differentiation, including annexin V, elongation initiation factor SA, Rho GDP dissociation inhibitor, and prohibitin. We identified inosine-5-monophosphate dehydrogenase in each of the cell lines, and found the levels of this enzyme in the tumor cell lines elevated 2-to 20-fold relative to the levels in normal cells. These results expand the human breast epithelial cell protein database (http:/I www.anl.gov/CMB/PMG) which is being built to assist researchers with the identification of abnormal patterns of expression and pathways associated with malignancy.
PROTEOMICS, 2006
Gene expression analysis has become a promising tool in predicting the clinical course of malignant disease and the response to antineoplastic therapy. Surprisingly, only little is known about the protein expression pattern of human tumors. Recent advances in proteomic analysis allow proteins of interest to be identified by their expression and/or modification pattern in 2-DE rather than using the traditional approach of translating gene expression data. To identify a proteomic pattern that is characteristic for malignant breast epithelium, we performed differential 2-DE analysis in sets of microdissected malignant breast epithelia and corresponding adjacent normal breast epithelia from five patients with invasive breast carcinoma. Thirty-two protein spots were found to be selectively regulated in malignant epithelium, and were subjected to MALDI-TOF and/or immunoblotting for protein identification. Thirteen of the identified proteins had previously not been associated with breast cancer. The validity of these findings was confirmed by literature review and immunohistochemistry for identified proteins in an independent cohort of 50 breast cancer specimens. We here describe, for the first time, a proteomic analysis of matched normal and malignant epithelia from invasive breast carcinomas. This strategy leads to a better understanding of oncogenesis at an operational level and helps to characterize the malignant phenotype of individual tumors, and thereby to identify novel targets for antineoplastic therapy.
Comparative Proteome Analysis of Breast Cancer and Adjacent Normal Breast Tissues in Human
Genomics, Proteomics & Bioinformatics, 2006
Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrixassisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS), incorporated with online database searching, were performed to investigate differential proteins of breast cancer and adjacent normal breast tissues. Considering that serum albumin is abundantly presented in normal control samples, 15 differential spots detected in 11 out of 12 (91.7%) breast cancer samples were identified by online SIENA-2DPAGE database searching and MALDI-TOF/TOF-MS analysis. The results indicate that pathological changes of breast cancer are concerned with augmentation of substance metabolism, promotion of proteolytic activity, decline of activity of some inhibitors of enzymes, and so on. Some important proteins involved in the pathological process of breast cancer with changed expression may be useful biomarkers, such as alpha-l-antitrypsin, EFl-beta, cathepsin D, TCTP, SMTSA, RPS12, and PSMA1, among which SMT3A, RPS12, and PSMAl were first reported for breast cancer in this study. words: biomarker, breast cancer, 2D-PAGE, mass spectrometry, molecular mechanism ' Consequently, 15 differential spots were detected in 11 out of 12 (91.7%) breast cancer samples. These spots were then identified by searching in the online SIENA-2DPAGE database and MALDI-TOF/TOF-MS analysis. Among the 15 spots, 6 were upregulated, 5 were down-regulated, and 4 (<4 folds) were possible to be upregulated in the infiltrating ductal carcinoma samples (Table 1).
Cancer research, 2001
Application of SEREX (serological analysis of recombinant tumor cDNA expression libraries) to different tumor types has led to the identification of several categories of human tumor antigens. In this study, the analysis of a breast cancer library with autologous patient serum led to the isolation of seven genes, designated NY-BR-1 through NY-BR-7. NY-BR-1, representing 6 of 14 clones isolated, showed tissue-restricted mRNA expression in breast and testis but not in 13 other normal tissues tested. Among tumor specimens, NY-BR-1 mRNA expression was found in 21 of 25 breast cancers but in only 2 of 82 nonmammary tumors. Structural analysis of NY-BR-1 cDNA and the corresponding genomic sequences in the recently released working draft of human genome indicated that NY-BR-1 is composed of 37 exons and has an open reading frame of 4.0-4.2 kb, encoding a peptide of Mr 150,000-160,000. A bipartite nuclear localization signal motif indicates a nuclear site for NY-BR-1, and the presence of a ...
Novel Molecular Markers of Malignancy in Histologically Normal and Benign Breast
Pathology Research International, 2011
To detect the molecular changes of malignancy in histologically normal breast (HNB) tissues, we recently developed a novel 117gene-malignancy-signature. Here we report validation of our leading malignancy-risk-genes, topoisomerase-2-alpha (TOP2A), minichromosome-maintenance-protein-2 (MCM2) and "budding-uninhibited-by-benzimidazoles-1-homolog-beta" (BUB1B) at the protein level. Using our 117-gene malignancy-signature, we classified 18 fresh-frozen HNB tissues from 18 adult female breast cancer patients into HNB-tissues with low-grade (HNB-LGMA; N = 9) and high-grade molecular abnormality (HNB-HGMA; N = 9). Archival sections of additional HNB tissues from these patients, and invasive ductal carcinoma (IDC) tissues from six other patients were immunostained for these biomarkers. TOP2A/MCM2 expression was assessed as staining index (%) and BUB1B expression as H-scores (0-300). Increasing TOP2A, MCM2, and BUB1B protein expression from HNB-LGMA to HNB-HGMA tissues to IDCs validated our microarray-based molecular classification of HNB tissues by immunohistochemistry. We also demonstrated an increasing expression of TOP2A protein on an independent test set of HNB/benign/reductionmammoplasties, atypical-ductal-hyperplasia with and without synchronous breast cancer, DCIS and IDC tissues using a custom tissue microarray (TMA). In conclusion, TOP2A, MCM2, and BUB1B proteins are potential molecular biomarkers of malignancy in histologically normal and benign breast tissues. Larger-scale clinical validation studies are needed to further evaluate the clinical utility of these molecular biomarkers. *
Quantitative Proteomic Profiling of Matched Normal and Tumor Breast Tissues
Journal of Proteome Research, 2010
Proteomic analysis of breast cancer tissue has proven difficult due to its inherent histological complexity. This pilot study presents preliminary evidence for the ability to differentiate adenoma and invasive carcinoma by measuring changes in proteomic profile of matched normal and disease tissues. A dual lysis buffer method was used to maximize protein extraction from each biopsy, proteins digested with trypsin, and the resulting peptides iTRAQ labeled. After combining, the peptide mixtures they were separated using preparative IEF followed by RP nanoHPLC. Following MALDI MS/MS and database searching, identified proteins were combined into a nonredundant list of 481 proteins with associated normal/tumor iTRAQ ratios for each patient. Proteins were categorized by location as blood, extracellular, and cellular, and the iTRAQ ratios were normalized to enable comparison between patients. Of those proteins significantly changed (upper or lower quartile) between matched normal and disease tissues, those from two invasive carcinoma patients had >50% in common with each other but <22% in common with an adenoma patient. In invasive carcinoma patients, several cellular and extracellular proteins that were significantly increased (Periostin, Small breast epithelial mucin) or decreased (Kinectin) have previously been associated with breast cancer, thereby supporting this approach for a larger diseasestage characterization effort.
Breast Cancer Research, 2000
The two-dimensional gel electrophoresis represents a unique tool of analysing expression levels of a thousand proteins simultaneously and to compare the protein profiles of a given cell in a worldwide dimension. In addition, the recent programs developed for cross-comparing gel images, even from different laboratories, offer the possibility to evaluate homologies and differences between affine maps. By Melanie-assisted analyses and access to Swiss-2DPAGE database, we have detected the proteomic profile of the 8701-BC cell line, deriving from a primary DIC, which represents the most common type of breast cancer. The cell-line map was compared by gel matching with that of DIC biopsy available on line, and within the large area of overlapping, some of the corresponding spots were identified and quantified. To our knowledge this is the first attempt to correlate the pattern of protein expression of a cell line derived from a primary tumor with the corresponding tissue. The results are of great interest from at least two point of view: firstly, the 8701-BC cell line retains the dominant luminal phenotype of the breast epithelium, which expresses predominantly cytokeratins -8 and -18; secondly the proteomic profile of the cell line appears highly homologous to the "in vivo" counterpart. In particular, a panel of 28 peptides, showing a higher expression in the DIC tissue with respect to the normal counterpart, displays the same, or even more robust signature in the cancer cells. This group of peptides, mainly belonging to stress protein family and to metabolic enzymes, may be considered as tumor markers of peculiar pertinence of neoplastic cells, rather than of stromal components, and representative, if not exclusive, of breast carcinoma.
International Journal of Research in Pharmaceutical Sciences
Incidence of breast cancer in the age group of 30 to 50 is increasing at an alarming rate globally. Moreover, existing treatments are either marginally effective or resistance developed, hence development of safe and potent pharmacological agents is immediately required. However, establishment of a disease progression marker for early detection, development of safe and effective treatment agents requires identification of key proteins that are exclusively expressed in advanced malignant breast tumors. 2-Dimensional gel electrophoresis (2-DGE) is one of the early methods used for the identification of deregulated proteins in cancers. Therefore, the proteins of benign andmalignant tumors as well as breast cancer cell lines MCF-7, SKBR-3 and MDAMB- 468, were subjected to 2-DGE and relative expression calculated. Analysis of the data showed a significant increase in the intensity of 7 protein spots compared to malignant tissues and benign ones. Further, comparison of cell line proteins ...
Identification of Stage-Specific Breast Markers Using Quantitative Proteomics
Journal of Proteome Research, 2013
Matched healthy and diseased tissues from breast cancer patients were analysed by quantitative proteomics. By comparing proteomic profiles of fibroadenoma (benign tumors, 3 patients), DCIS (noninvasive cancer, 3 patients) and invasive ductal carcinoma (4 patients) we identified protein alterations which correlated with breast cancer progression. Three 8-plex iTRAQ experiments generated an average of 826 proteins identifications, of which 402 were common. After excluding those originating from blood, 59 proteins were significantly changed in tumor compared to normal tissues with the majority associated with invasive carcinomas. Bioinformatics analysis identified relationships between proteins in this subset including roles in redox regulation, lipid transport, protein folding and proteasomal degradation, with a substantial number increased in expression due to Myc oncogene activation. Three target proteins, cofilin-1 and p23 (increased in invasive carcinoma) and membrane copper amine oxidase 3 (decreased in invasive carcinoma), were subject to further validation. All three were observed in phenotype-specific breast cancer cell lines, normal (non-transformed) breast cell lines and primary breast epithelial cells by Western blotting, but only cofilin-1 and p23 were detected by multiple reaction monitoring mass spectrometry analysis. All three proteins were detected by both analytical approaches in matched tissue biopsies emulating the response observed with proteomics analysis. Tissue microarray analysis (361 patients) indicated cofilin-1 staining positively correlating with tumor grade and p23 staining with ER positive status, both therefore merit further investigation as potential biomarkers.