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Identification of novel tumor-associated cell surface sialoglycoproteins in human glioblastoma tumors using quantitative proteomics.

Autelitano F, Loyaux D, Roudières S, Déon C, Guette F, Fabre P, Ping Q, Wang S, Auvergne R, Badarinarayana V, Smith M, Guillemot JC, Goldman SA, Natesan S, Ferrara P, August P - PLoS ONE (2014)

Bottom Line: Glioblastoma multiform (GBM) remains clinical indication with significant "unmet medical need".Among the 843 proteins, 606 (72%) are known cell surface or secreted glycoproteins, including 156 CD-antigens, all major classes of cell surface receptor proteins, transporters, and adhesion proteins.This report presents the comprehensive identification of new biomarkers and therapeutic targets for the treatment of malignant gliomas using quantitative sialoglycoproteomics with clinically relevant, patient derived primary glioma cells.

View Article: PubMed Central - PubMed

Affiliation: Sanofi-Aventis Recherche & Développement, Centre de Toulouse, Toulouse, France.

ABSTRACT
Glioblastoma multiform (GBM) remains clinical indication with significant "unmet medical need". Innovative new therapy to eliminate residual tumor cells and prevent tumor recurrences is critically needed for this deadly disease. A major challenge of GBM research has been the identification of novel molecular therapeutic targets and accurate diagnostic/prognostic biomarkers. Many of the current clinical therapeutic targets of immunotoxins and ligand-directed toxins for high-grade glioma (HGG) cells are surface sialylated glycoproteins. Therefore, methods that systematically and quantitatively analyze cell surface sialoglycoproteins in human clinical tumor samples would be useful for the identification of potential diagnostic markers and therapeutic targets for malignant gliomas. In this study, we used the bioorthogonal chemical reporter strategy (BOCR) in combination with label-free quantitative mass spectrometry (LFQ-MS) to characterize and accurately quantify the individual cell surface sialoproteome in human GBM tissues, in fetal, adult human astrocytes, and in human neural progenitor cells (NPCs). We identified and quantified a total of 843 proteins, including 801 glycoproteins. Among the 843 proteins, 606 (72%) are known cell surface or secreted glycoproteins, including 156 CD-antigens, all major classes of cell surface receptor proteins, transporters, and adhesion proteins. Our findings identified several known as well as new cell surface antigens whose expression is predominantly restricted to human GBM tumors as confirmed by microarray transcription profiling, quantitative RT-PCR and immunohistochemical staining. This report presents the comprehensive identification of new biomarkers and therapeutic targets for the treatment of malignant gliomas using quantitative sialoglycoproteomics with clinically relevant, patient derived primary glioma cells.

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Top 65 cell surface sialoglycoproteins overexpressed in GBM patient tumors (SIDs) compared to human astrocytes.Biostatistics analysis of average differences in sialoglycoprotein mean intensities (“Effect sizes”), between multiple replicate samples of tumor tissues (“tumor experimental group”) and fetal and adult astrocytes (“astrocyte reference group”) was performed using a one-way analysis of variance (ANOVA). Effect sizes (Log2 scale) of proteins which are up-regulated (Effect size >3 and p-value <0,05) in the tumor experimental group versus the astrocyte reference group are plotted against protein median intensities (linear scale) in the tumor experimental group. The x-axis shows the median intensity values on a logarithmic scale. Among the 65 proteins up-regulated in tumors, 52 were detected in all four tumor samples (blue diamonds), while 8 and 5, were found in at least three (red squares) or two (green triangles), among the four tumor samples, respectively.
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pone-0110316-g006: Top 65 cell surface sialoglycoproteins overexpressed in GBM patient tumors (SIDs) compared to human astrocytes.Biostatistics analysis of average differences in sialoglycoprotein mean intensities (“Effect sizes”), between multiple replicate samples of tumor tissues (“tumor experimental group”) and fetal and adult astrocytes (“astrocyte reference group”) was performed using a one-way analysis of variance (ANOVA). Effect sizes (Log2 scale) of proteins which are up-regulated (Effect size >3 and p-value <0,05) in the tumor experimental group versus the astrocyte reference group are plotted against protein median intensities (linear scale) in the tumor experimental group. The x-axis shows the median intensity values on a logarithmic scale. Among the 65 proteins up-regulated in tumors, 52 were detected in all four tumor samples (blue diamonds), while 8 and 5, were found in at least three (red squares) or two (green triangles), among the four tumor samples, respectively.

Mentions: Of the 606 cell surface proteins identified and quantified with high confidence (p-value <0.05), 65 were significantly up-regulated (Effect size >3) in the tumor group compared to the astrocyte group (Table S3), of which 6 (ABCB6, AKR1A1, AKT2, CRYAB, SLC1A3 and UCHL1) were not annotated as N-glycoproteins in the UniProtKB Knowledgebase. Among the 65 proteins up-regulated in tumor cells, 51 (78%) have been described as integral or peripheral plasma membrane proteins and 14 (22%) as extracellular space proteins. Amongst suitable antigen characteristics that are critical for the identification of new glioma-specific surface antigens as putative targets for targeted toxins, abundant and homogeneous antigen expression on the external surface of all tumor cells in the majority of patients, and limited or preferably no antigen expression in vital normal tissue, are essential prerequisites. Fig. 6 shows a scatter plot of the median protein intensity in the tumor group versus the Effect size for the 65 cell surface proteins up-regulated in the tumor group. Levels of the 65 detected proteins spanned approximately five orders of magnitude. Of these, 52 (80%) were detected in all four tumor samples indicative for common expression of these proteins among GBM tumors, while 8 (12%) and 5 (8%), were found in at least three or two, among the four tumor samples, respectively. The 52 tumor-associated proteins extend over almost the entire distribution of the proteome expression. Clusterin (CLU) was the protein with the highest expression value, while protocadherin 18 (PCDH18) was the least abundant protein. Several functional protein classes were found in the PANTHER database for the 52 proteins up-regulated in the four tumor samples (Tables 3 and S3). A total of 72 molecular function hits were allotted to these proteins (Fig. S1).


Identification of novel tumor-associated cell surface sialoglycoproteins in human glioblastoma tumors using quantitative proteomics.

Autelitano F, Loyaux D, Roudières S, Déon C, Guette F, Fabre P, Ping Q, Wang S, Auvergne R, Badarinarayana V, Smith M, Guillemot JC, Goldman SA, Natesan S, Ferrara P, August P - PLoS ONE (2014)

Top 65 cell surface sialoglycoproteins overexpressed in GBM patient tumors (SIDs) compared to human astrocytes.Biostatistics analysis of average differences in sialoglycoprotein mean intensities (“Effect sizes”), between multiple replicate samples of tumor tissues (“tumor experimental group”) and fetal and adult astrocytes (“astrocyte reference group”) was performed using a one-way analysis of variance (ANOVA). Effect sizes (Log2 scale) of proteins which are up-regulated (Effect size >3 and p-value <0,05) in the tumor experimental group versus the astrocyte reference group are plotted against protein median intensities (linear scale) in the tumor experimental group. The x-axis shows the median intensity values on a logarithmic scale. Among the 65 proteins up-regulated in tumors, 52 were detected in all four tumor samples (blue diamonds), while 8 and 5, were found in at least three (red squares) or two (green triangles), among the four tumor samples, respectively.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4216004&req=5

pone-0110316-g006: Top 65 cell surface sialoglycoproteins overexpressed in GBM patient tumors (SIDs) compared to human astrocytes.Biostatistics analysis of average differences in sialoglycoprotein mean intensities (“Effect sizes”), between multiple replicate samples of tumor tissues (“tumor experimental group”) and fetal and adult astrocytes (“astrocyte reference group”) was performed using a one-way analysis of variance (ANOVA). Effect sizes (Log2 scale) of proteins which are up-regulated (Effect size >3 and p-value <0,05) in the tumor experimental group versus the astrocyte reference group are plotted against protein median intensities (linear scale) in the tumor experimental group. The x-axis shows the median intensity values on a logarithmic scale. Among the 65 proteins up-regulated in tumors, 52 were detected in all four tumor samples (blue diamonds), while 8 and 5, were found in at least three (red squares) or two (green triangles), among the four tumor samples, respectively.
Mentions: Of the 606 cell surface proteins identified and quantified with high confidence (p-value <0.05), 65 were significantly up-regulated (Effect size >3) in the tumor group compared to the astrocyte group (Table S3), of which 6 (ABCB6, AKR1A1, AKT2, CRYAB, SLC1A3 and UCHL1) were not annotated as N-glycoproteins in the UniProtKB Knowledgebase. Among the 65 proteins up-regulated in tumor cells, 51 (78%) have been described as integral or peripheral plasma membrane proteins and 14 (22%) as extracellular space proteins. Amongst suitable antigen characteristics that are critical for the identification of new glioma-specific surface antigens as putative targets for targeted toxins, abundant and homogeneous antigen expression on the external surface of all tumor cells in the majority of patients, and limited or preferably no antigen expression in vital normal tissue, are essential prerequisites. Fig. 6 shows a scatter plot of the median protein intensity in the tumor group versus the Effect size for the 65 cell surface proteins up-regulated in the tumor group. Levels of the 65 detected proteins spanned approximately five orders of magnitude. Of these, 52 (80%) were detected in all four tumor samples indicative for common expression of these proteins among GBM tumors, while 8 (12%) and 5 (8%), were found in at least three or two, among the four tumor samples, respectively. The 52 tumor-associated proteins extend over almost the entire distribution of the proteome expression. Clusterin (CLU) was the protein with the highest expression value, while protocadherin 18 (PCDH18) was the least abundant protein. Several functional protein classes were found in the PANTHER database for the 52 proteins up-regulated in the four tumor samples (Tables 3 and S3). A total of 72 molecular function hits were allotted to these proteins (Fig. S1).

Bottom Line: Glioblastoma multiform (GBM) remains clinical indication with significant "unmet medical need".Among the 843 proteins, 606 (72%) are known cell surface or secreted glycoproteins, including 156 CD-antigens, all major classes of cell surface receptor proteins, transporters, and adhesion proteins.This report presents the comprehensive identification of new biomarkers and therapeutic targets for the treatment of malignant gliomas using quantitative sialoglycoproteomics with clinically relevant, patient derived primary glioma cells.

View Article: PubMed Central - PubMed

Affiliation: Sanofi-Aventis Recherche & Développement, Centre de Toulouse, Toulouse, France.

ABSTRACT
Glioblastoma multiform (GBM) remains clinical indication with significant "unmet medical need". Innovative new therapy to eliminate residual tumor cells and prevent tumor recurrences is critically needed for this deadly disease. A major challenge of GBM research has been the identification of novel molecular therapeutic targets and accurate diagnostic/prognostic biomarkers. Many of the current clinical therapeutic targets of immunotoxins and ligand-directed toxins for high-grade glioma (HGG) cells are surface sialylated glycoproteins. Therefore, methods that systematically and quantitatively analyze cell surface sialoglycoproteins in human clinical tumor samples would be useful for the identification of potential diagnostic markers and therapeutic targets for malignant gliomas. In this study, we used the bioorthogonal chemical reporter strategy (BOCR) in combination with label-free quantitative mass spectrometry (LFQ-MS) to characterize and accurately quantify the individual cell surface sialoproteome in human GBM tissues, in fetal, adult human astrocytes, and in human neural progenitor cells (NPCs). We identified and quantified a total of 843 proteins, including 801 glycoproteins. Among the 843 proteins, 606 (72%) are known cell surface or secreted glycoproteins, including 156 CD-antigens, all major classes of cell surface receptor proteins, transporters, and adhesion proteins. Our findings identified several known as well as new cell surface antigens whose expression is predominantly restricted to human GBM tumors as confirmed by microarray transcription profiling, quantitative RT-PCR and immunohistochemical staining. This report presents the comprehensive identification of new biomarkers and therapeutic targets for the treatment of malignant gliomas using quantitative sialoglycoproteomics with clinically relevant, patient derived primary glioma cells.

Show MeSH
Related in: MedlinePlus