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Expression analysis of all protease genes reveals cathepsin K to be overexpressed in glioblastoma.

Verbovšek U, Motaln H, Rotter A, Atai NA, Gruden K, Van Noorden CJ, Lah TT - PLoS ONE (2014)

Bottom Line: We performed transcriptome analysis of all known genes of peptidases also called proteases and their endogenous inhibitors in glioblastoma multiforme (GBM), which is one of the most aggressive and deadly types of brain cancers, where unbalanced proteolysis is associated with tumor progression.Cathepsin K immunohistochemical staining and western blotting showed that only proteolytically inactive proforms of cathepsin K were overexpressed in GBM tissues and cells.The presence of high levels of inactive proforms of cathepsin K in GBM tissues and cells indicate that in GBM the proteolytic/collagenolytic role is not its primary function but it plays rather a different yet unknown role.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Ljubljana, Slovenia.

ABSTRACT

Background: Cancer genome and transcriptome analyses advanced our understanding of cancer biology. We performed transcriptome analysis of all known genes of peptidases also called proteases and their endogenous inhibitors in glioblastoma multiforme (GBM), which is one of the most aggressive and deadly types of brain cancers, where unbalanced proteolysis is associated with tumor progression.

Methods: Comparisons were performed between the transcriptomics of primary GBM tumors and unmatched non-malignant brain tissue, and between GBM cell lines (U87-MG and U373) and a control human astrocyte cell line (NHA). Publicly-available data sets and our own datasets were integrated and normalized using bioinformatics tools to reveal protease and protease inhibitor genes with deregulated expression in both malignant versus non-malignant tissues and cells.

Results: Of the 311 protease genes identified to be differentially expressed in both GBM tissues and cells, 5 genes were highly overexpressed, 2 genes coding for non-peptidase homologues transferrin receptor (TFRC) and G protein-coupled receptor 56 (GPR56), as well as 3 genes coding for the proteases endoplasmic reticulum aminopeptidase 2 (ERAP2), glutamine-fructose-6-phosphate transaminase 2 (GFPT2) and cathepsin K (CTSK), whereas one gene, that of the serine protease carboxypeptidase E (CPE) was strongly reduced in expression. Seventy five protease inhibitor genes were differentially expressed, of which 3 genes were highly overexpressed, the genes coding for stefin B (CSTB), peptidase inhibitor 3 (PI3 also named elafin) and CD74. Seven out of 8 genes (except CSTB) were validated using RT-qPCR in GBM cell lines. CTSK overexpression was validated using RT-qPCR in GBM tissues as well. Cathepsin K immunohistochemical staining and western blotting showed that only proteolytically inactive proforms of cathepsin K were overexpressed in GBM tissues and cells.

Conclusions: The presence of high levels of inactive proforms of cathepsin K in GBM tissues and cells indicate that in GBM the proteolytic/collagenolytic role is not its primary function but it plays rather a different yet unknown role.

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Venn diagrams of protease and protease inhibitor genes upregulated in both GBM tissue and GBM cells.In total, 669 protease and 242 protease inhibitor genes were checked for deregulation in GBM tissues and cells in comparison to non-malignant brain tissue and NHA cells. Venn diagrams include upregulated genes in GBM tissues and cells with PFP >0.05. Candidate genes appear in the intersections of Venn diagrams. Comparisons of protease genes upregulated in GBM tissue and in U87-MG cells (A) and upregulated in GBM tissue and in U373 cells (B) across Illumina and Affymetrx platforms and within the Affymetrix platform only are shown. The other 2 Venn diagrams show comparisons of protease inhibitor genes upregulated in GBM and in U87-MG cells (C) and upregulated in GBM and in U373 cells (D) across Illumina and Affymetrx platforms and within the Affymetrix platform only.
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pone-0111819-g002: Venn diagrams of protease and protease inhibitor genes upregulated in both GBM tissue and GBM cells.In total, 669 protease and 242 protease inhibitor genes were checked for deregulation in GBM tissues and cells in comparison to non-malignant brain tissue and NHA cells. Venn diagrams include upregulated genes in GBM tissues and cells with PFP >0.05. Candidate genes appear in the intersections of Venn diagrams. Comparisons of protease genes upregulated in GBM tissue and in U87-MG cells (A) and upregulated in GBM tissue and in U373 cells (B) across Illumina and Affymetrx platforms and within the Affymetrix platform only are shown. The other 2 Venn diagrams show comparisons of protease inhibitor genes upregulated in GBM and in U87-MG cells (C) and upregulated in GBM and in U373 cells (D) across Illumina and Affymetrx platforms and within the Affymetrix platform only.

Mentions: Differentially-expressed protease and protease inhibitor genes (Files S6 and S7) differed both in their number and identity when comparing GBM tissue and cell lines, as shown in the heatmap (Fig. 1B), implying discrepancies between gene expression in tissues and cell lines and between different cell lines. Cross-platform comparison (Illumina versus Affymetrix) gave different results in comparison with same-platform comparison (Affymetrix versus Affymetrix). In case of protease inhibitors U87-MG and U373 cell lines clustered together independently from microarray technology. Five protease genes (Fig. 2A,B), and 3 protease inhibitor genes (Fig. 2C,D) appeared in the intersection of the Venn diagrams of overexpressed genes (drawn on the basis of all lists of upregulated protease and protease inhibitor genes) and matched the criteria for candidate gene selection as is described in the Data analysis section. These criteria were valid for 2 metalloprotease homologous genes: transferrin receptor (TFRC) and endoplasmic reticulum aminopeptidase 2 (ERAP2), a serine protease homologous G-protein-coupled receptor 56 (GPR56), and two genes with homology to cysteine proteases: the glutamine-fructose-6-phosphate transaminase 2 (GFPT2) and cathepsin K (CTSK). The 3 inhibitor genes identified were stefin B (CSTB), peptidase inhibitor 3 (PI3; also called elafin) and CD74 (CD74; MHC class II chaperone). The TFRC gene was the only gene found to be overexpressed in GBM tissue and in both selected GBM cell lines, across both microarray platforms.


Expression analysis of all protease genes reveals cathepsin K to be overexpressed in glioblastoma.

Verbovšek U, Motaln H, Rotter A, Atai NA, Gruden K, Van Noorden CJ, Lah TT - PLoS ONE (2014)

Venn diagrams of protease and protease inhibitor genes upregulated in both GBM tissue and GBM cells.In total, 669 protease and 242 protease inhibitor genes were checked for deregulation in GBM tissues and cells in comparison to non-malignant brain tissue and NHA cells. Venn diagrams include upregulated genes in GBM tissues and cells with PFP >0.05. Candidate genes appear in the intersections of Venn diagrams. Comparisons of protease genes upregulated in GBM tissue and in U87-MG cells (A) and upregulated in GBM tissue and in U373 cells (B) across Illumina and Affymetrx platforms and within the Affymetrix platform only are shown. The other 2 Venn diagrams show comparisons of protease inhibitor genes upregulated in GBM and in U87-MG cells (C) and upregulated in GBM and in U373 cells (D) across Illumina and Affymetrx platforms and within the Affymetrix platform only.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4214761&req=5

pone-0111819-g002: Venn diagrams of protease and protease inhibitor genes upregulated in both GBM tissue and GBM cells.In total, 669 protease and 242 protease inhibitor genes were checked for deregulation in GBM tissues and cells in comparison to non-malignant brain tissue and NHA cells. Venn diagrams include upregulated genes in GBM tissues and cells with PFP >0.05. Candidate genes appear in the intersections of Venn diagrams. Comparisons of protease genes upregulated in GBM tissue and in U87-MG cells (A) and upregulated in GBM tissue and in U373 cells (B) across Illumina and Affymetrx platforms and within the Affymetrix platform only are shown. The other 2 Venn diagrams show comparisons of protease inhibitor genes upregulated in GBM and in U87-MG cells (C) and upregulated in GBM and in U373 cells (D) across Illumina and Affymetrx platforms and within the Affymetrix platform only.
Mentions: Differentially-expressed protease and protease inhibitor genes (Files S6 and S7) differed both in their number and identity when comparing GBM tissue and cell lines, as shown in the heatmap (Fig. 1B), implying discrepancies between gene expression in tissues and cell lines and between different cell lines. Cross-platform comparison (Illumina versus Affymetrix) gave different results in comparison with same-platform comparison (Affymetrix versus Affymetrix). In case of protease inhibitors U87-MG and U373 cell lines clustered together independently from microarray technology. Five protease genes (Fig. 2A,B), and 3 protease inhibitor genes (Fig. 2C,D) appeared in the intersection of the Venn diagrams of overexpressed genes (drawn on the basis of all lists of upregulated protease and protease inhibitor genes) and matched the criteria for candidate gene selection as is described in the Data analysis section. These criteria were valid for 2 metalloprotease homologous genes: transferrin receptor (TFRC) and endoplasmic reticulum aminopeptidase 2 (ERAP2), a serine protease homologous G-protein-coupled receptor 56 (GPR56), and two genes with homology to cysteine proteases: the glutamine-fructose-6-phosphate transaminase 2 (GFPT2) and cathepsin K (CTSK). The 3 inhibitor genes identified were stefin B (CSTB), peptidase inhibitor 3 (PI3; also called elafin) and CD74 (CD74; MHC class II chaperone). The TFRC gene was the only gene found to be overexpressed in GBM tissue and in both selected GBM cell lines, across both microarray platforms.

Bottom Line: We performed transcriptome analysis of all known genes of peptidases also called proteases and their endogenous inhibitors in glioblastoma multiforme (GBM), which is one of the most aggressive and deadly types of brain cancers, where unbalanced proteolysis is associated with tumor progression.Cathepsin K immunohistochemical staining and western blotting showed that only proteolytically inactive proforms of cathepsin K were overexpressed in GBM tissues and cells.The presence of high levels of inactive proforms of cathepsin K in GBM tissues and cells indicate that in GBM the proteolytic/collagenolytic role is not its primary function but it plays rather a different yet unknown role.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Ljubljana, Slovenia.

ABSTRACT

Background: Cancer genome and transcriptome analyses advanced our understanding of cancer biology. We performed transcriptome analysis of all known genes of peptidases also called proteases and their endogenous inhibitors in glioblastoma multiforme (GBM), which is one of the most aggressive and deadly types of brain cancers, where unbalanced proteolysis is associated with tumor progression.

Methods: Comparisons were performed between the transcriptomics of primary GBM tumors and unmatched non-malignant brain tissue, and between GBM cell lines (U87-MG and U373) and a control human astrocyte cell line (NHA). Publicly-available data sets and our own datasets were integrated and normalized using bioinformatics tools to reveal protease and protease inhibitor genes with deregulated expression in both malignant versus non-malignant tissues and cells.

Results: Of the 311 protease genes identified to be differentially expressed in both GBM tissues and cells, 5 genes were highly overexpressed, 2 genes coding for non-peptidase homologues transferrin receptor (TFRC) and G protein-coupled receptor 56 (GPR56), as well as 3 genes coding for the proteases endoplasmic reticulum aminopeptidase 2 (ERAP2), glutamine-fructose-6-phosphate transaminase 2 (GFPT2) and cathepsin K (CTSK), whereas one gene, that of the serine protease carboxypeptidase E (CPE) was strongly reduced in expression. Seventy five protease inhibitor genes were differentially expressed, of which 3 genes were highly overexpressed, the genes coding for stefin B (CSTB), peptidase inhibitor 3 (PI3 also named elafin) and CD74. Seven out of 8 genes (except CSTB) were validated using RT-qPCR in GBM cell lines. CTSK overexpression was validated using RT-qPCR in GBM tissues as well. Cathepsin K immunohistochemical staining and western blotting showed that only proteolytically inactive proforms of cathepsin K were overexpressed in GBM tissues and cells.

Conclusions: The presence of high levels of inactive proforms of cathepsin K in GBM tissues and cells indicate that in GBM the proteolytic/collagenolytic role is not its primary function but it plays rather a different yet unknown role.

Show MeSH
Related in: MedlinePlus