Franck Brichory

Global Profiling of the Cell Surface Proteome of Cancer Cells Uncovers an Abundance of Proteins with Chaperone Function

There is currently limited data available pertaining to the global characterization of the cell surface proteome. We have implemented a strategy for the comprehensive profiling and identification of surface membrane proteins. This strategy has been applied to cancer cells, including the SH-SY5Y neuroblastoma, the A549 lung adenocarcinoma, the LoVo colon adenocarcinoma, and the Sup-B15 acute lymphoblastic leukemia (B cell) cell lines and ovarian tumor cells. Surface membrane proteins of viable, intact cells were subjected to biotinylation then affinity-captured and purified on monomeric avidin columns. The biotinylated proteins were eluted from the monomeric avidin columns as intact proteins and were subsequently separated by two-dimensional PAGE, transferred to polyvinylidene difluoride membranes, and visualized by hybridization with streptavidin-horseradish peroxidase. Highly reproducible, but distinct, two-dimensional patterns consisting of several hundred biotinylated proteins were obtained for the different cell populations analyzed. Identification of a subset of biotinylated proteins among the different cell populations analyzed using matrix-assisted laser desorption ionization and tandem mass spectrometry uncovered proteins with a restricted expression pattern in some cell line(s), such as CD87 and the activin receptor type IIB. We also identified more widely expressed proteins, such as CD98, and a sushi repeat-containing protein, a member of the selectin family. Remarkably, a set of proteins identified as chaperone proteins were found to be highly abundant on the cell surface, including GRP78, GRP75, HSP70, HSP60, HSP54, HSP27, and protein disulfide isomerase. Comprehensive profiling of the cell surface proteome provides an effective approach for the identification of commonly occurring proteins as well as proteins with restricted expression patterns in this compartment.

An immune response manifested by the common occurrence of annexins I and II autoantibodies and high circulating levels of IL-6 in lung cancer

The identification of circulating tumor antigens or their related autoantibodies provides a means for early cancer diagnosis as well as leads for therapy. The purpose of this study was to identify proteins that commonly induce a humoral response in lung cancer by using a proteomic approach and to investigate biological processes that may be associated with the development of autoantibodies. Aliquots of solubilized proteins from a lung adenocarcinoma cell line (A549) and from lung tumors were subjected to two-dimensional PAGE, followed by Western blot analysis in which individual sera were tested for primary antibodies. Sera from 54 newly diagnosed patients with lung cancer and 60 patients with other cancers and from 61 noncancer controls were analyzed. Sera from 60% of patients with lung adenocarcinoma and 33% of patients with squamous cell lung carcinoma but none of the noncancer controls exhibited IgG-based reactivity against proteins identified as glycosylated annexins I and/or II. Immunohistochemical analysis showed that annexin I was expressed diffusely in neoplastic cells in lung tumor tissues, whereas annexin II was predominant at the cell surface. Interestingly, IL-6 levels were significantly higher in sera of antibody-positive lung cancer patients compared with antibody-negative patients and controls. We conclude that an immune response manifested by annexins I and II autoantibodies occurs commonly in lung cancer and is associated with high circulating levels of an inflammatory cytokine. The proteomic approach we have implemented has utility for the development of serum-based assays for cancer diagnosis as we report in this paper on the discovery of antiannexins I and/or II in sera from patients with lung cancer.

A Distinct Repertoire of Autoantibodies in Hepatocellular Carcinoma Identified by Proteomic Analysis

Chronic infections with hepatitis B (HBV) and hepatitis C (HCV) viruses are major risk factors for hepatocellular carcinoma (HCC). We have utilized a proteomic approach to determine whether a distinct repertoire of autoantibodies can be identified in HCC. Sera from 37 patients with HCC and 31 subjects chronically infected with HBV or HCV without HCC were investigated. Sera from 116 patients with other cancers, three patients with systemic lupus erythematosus, and 24 healthy subjects were utilized as controls. We report the identification of eight proteins, for each of which autoantibodies were detected in sera from more than 10% of patients with HCC but not in sera from healthy individuals (p < 0.05). Autoantibodies to four of these proteins were detected at a comparable frequency in sera from patients with chronic hepatitis. The other four proteins, which consisted of calreticulin isoforms, cytokeratin 8, nucleoside diphosphate kinase A, and F1-ATP synthase β-subunit, induced autoantibodies among patients with HCC, independently of their HBV/HCV status. Calreticulin, and a novel truncated form of calreticulin (Crt32) we have identified, most commonly elicited autoantibodies among patients with HCC (27%). We conclude that a distinct repertoire of autoantibodies is associated with HCC that may have utility in early diagnosis of HCC among high risk subjects with chronic hepatitis.

Protein based microarrays: A tool for probing the proteome of cancer cells and tissues

A novel proteomic approach for probing cell and tissue proteome, which combines liquid phase protein separations with microarray technology has been developed. Proteins in cell and tissue lysates or in cellular subfractions are separated using any one of a number of separation modes which may consist of ion exchange liquid chromatography (LC), reverse phase LC, carrier ampholyte based separations, e.g. the use of Rotofor, affinity based separations, or gel based separations. Each first‐dimension fraction obtained using one separation mode can be further resolved using one or more of the other separation modes to yield either purified protein in solution or liquid fractions with substantially reduced protein complexity. The advantage of a liquid based separation system is that proteins in hundreds of individual fractions can be arrayed directly and used as targets for a variety of probes. Constituent proteins in reactive fractions are identified by mass spectrometry and may be further resolved to determine the nature of the reactive protein(s). We present in this report initial data based on microarray analysis of individual Rotofor fractions obtained from lung adenocarcinoma cell line A549 lysates which have been probed with antibodies against specific proteins.

A database of protein expression in lung cancer

We have developed a comprehensive approach to identifying molecular changes in lung cancer that includes both genomic and proteomic analyses. The related effort has produced a large amount of data pertaining to gene expression at the RNA and protein levels. As a result, we have constructed a database that contains protein expression data on lung cancer as well as other relevant data including DNA microarray derived data. A large number of proteins that are expressed in different types of lung cancer have been identified and have been correlated with the expression measures for their corresponding genes at the RNA level. The database is intended to facilitate our effort at developing novel classification schemes for lung cancer and the identification of novel markers for early diagnosis.

A proteomic approach to the identification of lung cancer markers

We have developed a comprehensive approach to the identification of protein markers in lung cancer that includes profiling of tumor tissue and cell lines as well as the analysis of serum for autoantibodies to lung tumor antigens. A large number of proteins that are differentially expressed in the major subtypes of lung cancer have been identified by mass spectrometry. A database of protein expression in lung cancer and other types of cancer has been constructed that integrates two-dimensional gel profiles, mass spectrometry data, quantitative protein data and gene expression data at the RNA level, that serves as a resource for biomarker identification. Analysis of the serological response in lung cancer has led to the identification of novel markers detectable in serum of lung cancer patients at the time of diagnosis. The proteomic approach is likely to yield novel classification schemes and novel markers for early diagnosis of lung cancer.

Profiling of pathway-specific changes in gene expression following growth of human cancer cell lines transplanted into mice

BackgroundTumor cells cultured in vitro are widely used to investigate the molecular biology of cancers and to evaluate responses to drugs and other agents. The full extent to which gene expression in cancer cells is modulated by extrinsic factors and by the microenvironment in which the cancer cells reside remains to be determined. Two cancer cell lines (A549 lung adenocarcinoma and U118 glioblastoma) were transplanted subcutaneously into immunodeficient mice to form tumors. Global gene-expression profiles of the tumors were determined, based on analysis of expression of human genes, and compared with expression profiles of the cell lines grown in culture.ResultsA bioinformatics approach associated genes that showed changes in their expression levels with functional classes as defined by either the GO gene annotations or MeSH terms in the literature. The classes of genes expressed at higher levels in cells grown in vitro indicated increased cell division and metabolism, reflecting the more favorable environment for cell proliferation. In contrast, in vivo tumor growth resulted in upregulation of a significant number of genes involved in the extracellular matrix (ECM), cell adhesion, cytokine and metalloendopeptidase activity, and neovascularization. When placed in comparable tissue environments, the U118 cells and the A549 cells expressed different sets of ECM and cell adhesion-related genes, suggesting different mechanisms of extracellular interaction at work in the different cancers.ConclusionsStudies of this type allow us to examine the specific contribution of cancer cells to gene expression patterns within an in vivo tumor mixed with non-cancerous tissue.

Two messenger RNAs and five isoforms for Po66-CBP, a galectin-8 homolog in a human lung carcinoma cell line

Galectins are animal proteins which specifically bind beta-D-galactoside residues and their specific cellular function is not yet clearly established. However, these proteins seem to play a role in neoplastic transformations. Po66 is a murine monoclonal antibody directed against a protein from human lung carcinoma, Po66 Carbohydrate-Binding-Protein (Po66-CBP), which belongs to the galectin-8 family. Our results show that the Po66-CBP gene generates five transcripts by alternative splicing, which could give rise to five proteins: two proteins belong to the tandemly repeated galectin family and three belong to the single carbohydrate recognition domain galectins. All these proteins are encoded by a unique gene located in 1q42. Experiments carried out by reverse transcriptase-polymerase chain reaction show that the levels of expression of these five galectin-8 isoforms are variable during the culture time in SK-MES-1, a human lung squamous carcinoma cell line. Cancer Genome Anatomy Project database analysis confirms the presence of Po66-CBP in lung cancer and its absence in healthy lung.

Identification of Tumor-Associated Antigens Using Proteomics

In the post-genomic era, the identification of tumor-associated antigens that elicit a humoral response is allowed at the protein level using proteomics. Indeed, the screening of autoantibodies using 2-D Western blot experiments with sera from cancer patients, followed by the subsequent identification of the target protein by mass spectrometry and database search has permitted the exploitation of the B-cell repertoire of patients with cancer. Applied to several types of cancer, a proteomic-based approach has revealed a high frequency of autoantibodies in sera from patients. Several of the antigenic proteins identified may constitute novel cancer markers and may have clinical utility in diagnosis or in establishing prognosis. Furthermore, the approach has allowed to distinguish isoforms that may help to define epitopes. On the other hand, the analysis of the expression levels of some of the antigenic proteins has revealed differential expression in tumors as compared with healthy tissues that might explain antigenicity.

Immunohistochemical Expression of the Intracellular Component of Galectin-8 in Squamous Cell Metaplasiaof the Bronchial Epitheliumin Neoplastic and Benign Processes

Specified galectins are known to play a role in regulating cell proliferation, differentiation, adhesion and migration. Po66, a mouse IgG1 monoclonal antibody produced by immunization against squamous cell cancer, reacts against a carbohydrate-binding protein (Po66-CBP), recently shown to be a member of the galectin family with a strong homology with galectin-8 (PCTA-1), identified as a human tumor-associated antigen. We studied Po66 in squamous metaplasia of the bronchi in order to determine whether it could be specifically involved in neoplastic conditions and if so, if it would be helpful in distinguishing metaplasia at risk of cancer. Twenty-eight formalin-fixed, paraffin-embedded archival tissues of 17 metaplasias with SCC, 3 metaplasia with distant neoplastic disease and 8 metaplasias with an inflammatory process, were immunostained using a streptavidin biotin peroxydase method. The squamous metaplasias were positively stained in non-neoplastic disease as well as in neoplastic processes. Expression was also observed in stromal and normal cells. Po66-CBP was not associated with a pre-neoplastic character. We discussed the expression of this intra-cellular component of galectin-8 according to the functions of galectins in cellular differentiation, host reaction against tumor, and inflammation.