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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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RT-qPCR analysis of expression of selected proteases and protease inhibitors in U87-MG and U373 GBM cells, NHA cells and GBM tissues and non-malignant brain (in vivo).(A) Upregulated expression of seven genes (TFRC, CTSK, GFPT2, ERAP2, GPR56, CD74, PI3) as determined by microarray data was validated in GBM cells in comparison to NHA cells by RT-qPCR, using GAPDH as reference gene. (B) Additional RT-qPCR analysis of expression of the CTSK gene using GBM tissues and cell lines with reference genes TBP and HPRT1 in comparison to NHA cells (NAtotRNA) and non-malignant brain (HBrefRNA). The experiments were performed in triplicate (except for 8 repetitions of GBM tissue and commercial RNA from NHA and normal brain, used in experiment B). Error bars represent standard deviation; * p-value<0.05, ** p-value<0.01, *** p-value<0.001.
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pone-0111819-g003: RT-qPCR analysis of expression of selected proteases and protease inhibitors in U87-MG and U373 GBM cells, NHA cells and GBM tissues and non-malignant brain (in vivo).(A) Upregulated expression of seven genes (TFRC, CTSK, GFPT2, ERAP2, GPR56, CD74, PI3) as determined by microarray data was validated in GBM cells in comparison to NHA cells by RT-qPCR, using GAPDH as reference gene. (B) Additional RT-qPCR analysis of expression of the CTSK gene using GBM tissues and cell lines with reference genes TBP and HPRT1 in comparison to NHA cells (NAtotRNA) and non-malignant brain (HBrefRNA). The experiments were performed in triplicate (except for 8 repetitions of GBM tissue and commercial RNA from NHA and normal brain, used in experiment B). Error bars represent standard deviation; * p-value<0.05, ** p-value<0.01, *** p-value<0.001.

Mentions: RT-qPCR analyses of the GBM cell lines U87-MG, U373 and control NHA cell line confirmed overexpression of all seven candidate genes (TFRC, GFPT2, ERAP2, GPR56, CTSK, CD74 and PI3) in GBM cell lines (Fig. 3A). Among these, the CTSK gene was found to be more than 1000-fold overexpressed in U87-MG cells and more than 10 times overexpressed in U373 cells, when compared to NHA cells. Both protease inhibitor genes (CD74 and PI3) were overexpressed only in U87-MG cells. CD74 and PI3 genes were found to be 4×104 times and 2×105 times overexpressed in U87-MG cells in comparison to NHA cells.


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)

RT-qPCR analysis of expression of selected proteases and protease inhibitors in U87-MG and U373 GBM cells, NHA cells and GBM tissues and non-malignant brain (in vivo).(A) Upregulated expression of seven genes (TFRC, CTSK, GFPT2, ERAP2, GPR56, CD74, PI3) as determined by microarray data was validated in GBM cells in comparison to NHA cells by RT-qPCR, using GAPDH as reference gene. (B) Additional RT-qPCR analysis of expression of the CTSK gene using GBM tissues and cell lines with reference genes TBP and HPRT1 in comparison to NHA cells (NAtotRNA) and non-malignant brain (HBrefRNA). The experiments were performed in triplicate (except for 8 repetitions of GBM tissue and commercial RNA from NHA and normal brain, used in experiment B). Error bars represent standard deviation; * p-value<0.05, ** p-value<0.01, *** p-value<0.001.
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pone-0111819-g003: RT-qPCR analysis of expression of selected proteases and protease inhibitors in U87-MG and U373 GBM cells, NHA cells and GBM tissues and non-malignant brain (in vivo).(A) Upregulated expression of seven genes (TFRC, CTSK, GFPT2, ERAP2, GPR56, CD74, PI3) as determined by microarray data was validated in GBM cells in comparison to NHA cells by RT-qPCR, using GAPDH as reference gene. (B) Additional RT-qPCR analysis of expression of the CTSK gene using GBM tissues and cell lines with reference genes TBP and HPRT1 in comparison to NHA cells (NAtotRNA) and non-malignant brain (HBrefRNA). The experiments were performed in triplicate (except for 8 repetitions of GBM tissue and commercial RNA from NHA and normal brain, used in experiment B). Error bars represent standard deviation; * p-value<0.05, ** p-value<0.01, *** p-value<0.001.
Mentions: RT-qPCR analyses of the GBM cell lines U87-MG, U373 and control NHA cell line confirmed overexpression of all seven candidate genes (TFRC, GFPT2, ERAP2, GPR56, CTSK, CD74 and PI3) in GBM cell lines (Fig. 3A). Among these, the CTSK gene was found to be more than 1000-fold overexpressed in U87-MG cells and more than 10 times overexpressed in U373 cells, when compared to NHA cells. Both protease inhibitor genes (CD74 and PI3) were overexpressed only in U87-MG cells. CD74 and PI3 genes were found to be 4×104 times and 2×105 times overexpressed in U87-MG cells in comparison to NHA cells.

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