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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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Immunohistochemstry and immunocytochemstry and Western blot analysis of cathepsin K in GBM cells and tissues.ICC staining of CatK in U87-MG (A) and U373 (B, D) GBM cells. At high magnification (1000×), a granular pattern of the staining was observed in U373 cells (D), which corresponds to perinuclear endo-lysosomal-like localization of CatK. Strong IHC staining of CatK in GBM tissue (C), and weak staining in control brain tissue (non-malignant brain) (E, F). Mouse jaw with bone tissue (B) with osteoclasts at bone edges (O) was used as positive control for CatK staining (G). Magnifications: A, B, G - 400×; D - 1000×; C, E, F - 200×. (H) Western blot of GBM cells (lanes 7–10) and their conditioned media (CM; lanes 3–6), GBM tissue (lanes 17–25) and non-malignant brain tissue (lanes 11–16) showing positivity for pro-CatK (39 kDa). The active form of the enzyme (27 kDa) was detected only in a small amount in one passage of U87-MG cells (lane 8). At 30 kDa, an intermediate form of CatK was observed (lane 21). Recombinant proform (lane 1) and active CatK (lane 2) were used as positive control. As loading control β-actin was used. Legend: 1 – recombinant pro-CatK, 2 – recombinant active CatK, 3 – U87p38 CM, 4 – U87p39 CM, 5 – U373p41 CM, 6 – U373p42 CM, 7 – U87p45, 8 – U87p48, 9 – U373p45, 10 – U373p48, 11–16 – different non-malignant brain samples, 17–25 – different GBM tissue samples. Please note that Western blotting image does not allow for direct quantitative comparison of CatK expression in control and tumor samples due to variable protein amounts loaded. (I and J) Additional western blot experiments using cell lines U87-MG (I) and U373 (J) and different protease inhibitors. No active form of cathepsin K was observed in any of the conditions tested but in all cases pro-cathepsin K was present. Legend: 1 – without any inhibitor, 2 – 5 µM E-64, 3 – 5 µM CA074, 4 – 5 µM CLIK148, 5 – 5 µM pepstatin A, 6 – 1 mM PMSF, 7 – 20 mM EDTA, 8 – combination of all inhibitors, 9 – control: recombinant pro-CatK, 10 – control: recombinant active CatK.
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pone-0111819-g006: Immunohistochemstry and immunocytochemstry and Western blot analysis of cathepsin K in GBM cells and tissues.ICC staining of CatK in U87-MG (A) and U373 (B, D) GBM cells. At high magnification (1000×), a granular pattern of the staining was observed in U373 cells (D), which corresponds to perinuclear endo-lysosomal-like localization of CatK. Strong IHC staining of CatK in GBM tissue (C), and weak staining in control brain tissue (non-malignant brain) (E, F). Mouse jaw with bone tissue (B) with osteoclasts at bone edges (O) was used as positive control for CatK staining (G). Magnifications: A, B, G - 400×; D - 1000×; C, E, F - 200×. (H) Western blot of GBM cells (lanes 7–10) and their conditioned media (CM; lanes 3–6), GBM tissue (lanes 17–25) and non-malignant brain tissue (lanes 11–16) showing positivity for pro-CatK (39 kDa). The active form of the enzyme (27 kDa) was detected only in a small amount in one passage of U87-MG cells (lane 8). At 30 kDa, an intermediate form of CatK was observed (lane 21). Recombinant proform (lane 1) and active CatK (lane 2) were used as positive control. As loading control β-actin was used. Legend: 1 – recombinant pro-CatK, 2 – recombinant active CatK, 3 – U87p38 CM, 4 – U87p39 CM, 5 – U373p41 CM, 6 – U373p42 CM, 7 – U87p45, 8 – U87p48, 9 – U373p45, 10 – U373p48, 11–16 – different non-malignant brain samples, 17–25 – different GBM tissue samples. Please note that Western blotting image does not allow for direct quantitative comparison of CatK expression in control and tumor samples due to variable protein amounts loaded. (I and J) Additional western blot experiments using cell lines U87-MG (I) and U373 (J) and different protease inhibitors. No active form of cathepsin K was observed in any of the conditions tested but in all cases pro-cathepsin K was present. Legend: 1 – without any inhibitor, 2 – 5 µM E-64, 3 – 5 µM CA074, 4 – 5 µM CLIK148, 5 – 5 µM pepstatin A, 6 – 1 mM PMSF, 7 – 20 mM EDTA, 8 – combination of all inhibitors, 9 – control: recombinant pro-CatK, 10 – control: recombinant active CatK.

Mentions: IHC and ICC were used to determine (sub-)cellular and tissue distribution patterns of CatK protein (Fig. 6). In the cultured GBM cells, staining of the CatK protein was observed in perinuclear area of cells (Fig. 6D). Cryostat sections of 8 GBM samples all stained strongly for CatK, displaying a pattern of high intensity staining around blood vessels. Staining of osteoclasts was taken as positive control (Fig. 6G). Non-malignant brain tissue either did not stain at all for Cat K protein (Fig. 6E) or stained only weakly for CatK (Fig. 6F). In particular, 2 out of 4 hippocampus samples and 2 out of 3 temporal cortex samples showed positive staining (that was considerably weaker than in GBM samples). Staining of Cat K protein suggested that CatK in GBM is not localized at invasive tumor edges.


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)

Immunohistochemstry and immunocytochemstry and Western blot analysis of cathepsin K in GBM cells and tissues.ICC staining of CatK in U87-MG (A) and U373 (B, D) GBM cells. At high magnification (1000×), a granular pattern of the staining was observed in U373 cells (D), which corresponds to perinuclear endo-lysosomal-like localization of CatK. Strong IHC staining of CatK in GBM tissue (C), and weak staining in control brain tissue (non-malignant brain) (E, F). Mouse jaw with bone tissue (B) with osteoclasts at bone edges (O) was used as positive control for CatK staining (G). Magnifications: A, B, G - 400×; D - 1000×; C, E, F - 200×. (H) Western blot of GBM cells (lanes 7–10) and their conditioned media (CM; lanes 3–6), GBM tissue (lanes 17–25) and non-malignant brain tissue (lanes 11–16) showing positivity for pro-CatK (39 kDa). The active form of the enzyme (27 kDa) was detected only in a small amount in one passage of U87-MG cells (lane 8). At 30 kDa, an intermediate form of CatK was observed (lane 21). Recombinant proform (lane 1) and active CatK (lane 2) were used as positive control. As loading control β-actin was used. Legend: 1 – recombinant pro-CatK, 2 – recombinant active CatK, 3 – U87p38 CM, 4 – U87p39 CM, 5 – U373p41 CM, 6 – U373p42 CM, 7 – U87p45, 8 – U87p48, 9 – U373p45, 10 – U373p48, 11–16 – different non-malignant brain samples, 17–25 – different GBM tissue samples. Please note that Western blotting image does not allow for direct quantitative comparison of CatK expression in control and tumor samples due to variable protein amounts loaded. (I and J) Additional western blot experiments using cell lines U87-MG (I) and U373 (J) and different protease inhibitors. No active form of cathepsin K was observed in any of the conditions tested but in all cases pro-cathepsin K was present. Legend: 1 – without any inhibitor, 2 – 5 µM E-64, 3 – 5 µM CA074, 4 – 5 µM CLIK148, 5 – 5 µM pepstatin A, 6 – 1 mM PMSF, 7 – 20 mM EDTA, 8 – combination of all inhibitors, 9 – control: recombinant pro-CatK, 10 – control: recombinant active CatK.
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pone-0111819-g006: Immunohistochemstry and immunocytochemstry and Western blot analysis of cathepsin K in GBM cells and tissues.ICC staining of CatK in U87-MG (A) and U373 (B, D) GBM cells. At high magnification (1000×), a granular pattern of the staining was observed in U373 cells (D), which corresponds to perinuclear endo-lysosomal-like localization of CatK. Strong IHC staining of CatK in GBM tissue (C), and weak staining in control brain tissue (non-malignant brain) (E, F). Mouse jaw with bone tissue (B) with osteoclasts at bone edges (O) was used as positive control for CatK staining (G). Magnifications: A, B, G - 400×; D - 1000×; C, E, F - 200×. (H) Western blot of GBM cells (lanes 7–10) and their conditioned media (CM; lanes 3–6), GBM tissue (lanes 17–25) and non-malignant brain tissue (lanes 11–16) showing positivity for pro-CatK (39 kDa). The active form of the enzyme (27 kDa) was detected only in a small amount in one passage of U87-MG cells (lane 8). At 30 kDa, an intermediate form of CatK was observed (lane 21). Recombinant proform (lane 1) and active CatK (lane 2) were used as positive control. As loading control β-actin was used. Legend: 1 – recombinant pro-CatK, 2 – recombinant active CatK, 3 – U87p38 CM, 4 – U87p39 CM, 5 – U373p41 CM, 6 – U373p42 CM, 7 – U87p45, 8 – U87p48, 9 – U373p45, 10 – U373p48, 11–16 – different non-malignant brain samples, 17–25 – different GBM tissue samples. Please note that Western blotting image does not allow for direct quantitative comparison of CatK expression in control and tumor samples due to variable protein amounts loaded. (I and J) Additional western blot experiments using cell lines U87-MG (I) and U373 (J) and different protease inhibitors. No active form of cathepsin K was observed in any of the conditions tested but in all cases pro-cathepsin K was present. Legend: 1 – without any inhibitor, 2 – 5 µM E-64, 3 – 5 µM CA074, 4 – 5 µM CLIK148, 5 – 5 µM pepstatin A, 6 – 1 mM PMSF, 7 – 20 mM EDTA, 8 – combination of all inhibitors, 9 – control: recombinant pro-CatK, 10 – control: recombinant active CatK.
Mentions: IHC and ICC were used to determine (sub-)cellular and tissue distribution patterns of CatK protein (Fig. 6). In the cultured GBM cells, staining of the CatK protein was observed in perinuclear area of cells (Fig. 6D). Cryostat sections of 8 GBM samples all stained strongly for CatK, displaying a pattern of high intensity staining around blood vessels. Staining of osteoclasts was taken as positive control (Fig. 6G). Non-malignant brain tissue either did not stain at all for Cat K protein (Fig. 6E) or stained only weakly for CatK (Fig. 6F). In particular, 2 out of 4 hippocampus samples and 2 out of 3 temporal cortex samples showed positive staining (that was considerably weaker than in GBM samples). Staining of Cat K protein suggested that CatK in GBM is not localized at invasive tumor edges.

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