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Tissue transglutaminase mediates the pro-malignant effects of oncostatin M receptor over-expression in cervical squamous cell carcinoma.

Caffarel MM, Chattopadhyay A, Araujo AM, Bauer J, Scarpini CG, Coleman N - J. Pathol. (2013)

Bottom Line: Cervical SCC cells that over-express OSMR show enhanced responsiveness to the major ligand OSM, which induces multiple pro-malignant effects, including increased cell migration and invasiveness.Here, we show that tissue transglutaminase (TGM2) is an important mediator of the ligand-dependent phenotypic effects of OSMR over-expression in SCC cells.We conclude that an OSMR/TGM2/integrin-α5β1/fibronectin pathway is of biological significance in cervical SCC and a candidate for therapeutic targeting.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Cambridge, UK.

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Effect of TGM2 depletion and over-expression on the migration and invasiveness of OSMR over-expressing cervical SCC cells. (A) Western blot showing levels of TGM2 protein in SW756 and CaSki at 24–72 h in control cells treated with vehicle only (C) or in OSM-treated cells (OSM). β-Actin (lower row) was used as the loading control. The graphs below each blot represent the densitometric analysis of protein levels. Results are expressed as optical density (arbitrary units), with control cells set as 1. (B) Levels of TGM2 enzymatic activity in SW756 and CaSki cells after 48 h in the absence (−OSM) or presence (+OSM) of OSM. (C, D) Quantification of cell migration velocity (C) and invasiveness through basement membrane extract (D). Each panel shows data for SW756 (left column) and CaSki (right column) following TGM2 depletion (top row) or TGM2 over-expression (bottom row). In the depletion experiments, cells were assayed after 48 h in the presence (+OSM) or absence (−OSM) of OSM. Cells with TGM2 knock-down (KD) were compared with control cells treated with non-targeting (NT) siRNA. As background levels of TGM2 were barely detectable in CaSki (see A), TGM2 depletion was only performed in OSM-treated CaSki cells. In the over-expression experiments, cells expressing TGM2 and GFP (GFP + TGM2) were compared with control cells expressing GFP alone: ns, non-significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
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fig02: Effect of TGM2 depletion and over-expression on the migration and invasiveness of OSMR over-expressing cervical SCC cells. (A) Western blot showing levels of TGM2 protein in SW756 and CaSki at 24–72 h in control cells treated with vehicle only (C) or in OSM-treated cells (OSM). β-Actin (lower row) was used as the loading control. The graphs below each blot represent the densitometric analysis of protein levels. Results are expressed as optical density (arbitrary units), with control cells set as 1. (B) Levels of TGM2 enzymatic activity in SW756 and CaSki cells after 48 h in the absence (−OSM) or presence (+OSM) of OSM. (C, D) Quantification of cell migration velocity (C) and invasiveness through basement membrane extract (D). Each panel shows data for SW756 (left column) and CaSki (right column) following TGM2 depletion (top row) or TGM2 over-expression (bottom row). In the depletion experiments, cells were assayed after 48 h in the presence (+OSM) or absence (−OSM) of OSM. Cells with TGM2 knock-down (KD) were compared with control cells treated with non-targeting (NT) siRNA. As background levels of TGM2 were barely detectable in CaSki (see A), TGM2 depletion was only performed in OSM-treated CaSki cells. In the over-expression experiments, cells expressing TGM2 and GFP (GFP + TGM2) were compared with control cells expressing GFP alone: ns, non-significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Mentions: As the evidence from human tissue samples suggested an important role for TGM2 in SCC progression, we examined the functional significance of TGM2 in cervical SCC cells in vitro. We used multiple complementary experimental approaches, based on gene depletion and over-expression, to minimize the possibility of non-specific observations. We focused on two representative OSMR-over-expressing cervical SCC cell lines, SW756 and CaSki 4,6. In both, treatment with OSM up-regulated TGM2 at the mRNA (see Supplementary material, Figure S2A) and protein levels (Figure 2A). After 48 h of OSM treatment, levels of TGM2 protein were induced by 360% in CaSki and by 140% in SW756 (in which basal levels of TGM2 were high). In addition, OSM significantly induced TGM2 enzymatic activity in both cell lines, as measured by a colorimetric assay (Figure 2B). This indicated that the OSM-induced TGM2 was functional and catalytically active. We observed mixed effects of exogenous OSM in the cervical SCC cell lines that did not over-express OSMR, MS751 and ME180 6. In MS751, TGM2 was induced after 24 h of OSM treatment, concomitant with up-regulation of OSMR (see Supplementary material, Figure S3). In contrast, in ME180 cells, TGM2 basal levels were undetectable and there was no induction by OSM (see Supplementary material, Figure S3).


Tissue transglutaminase mediates the pro-malignant effects of oncostatin M receptor over-expression in cervical squamous cell carcinoma.

Caffarel MM, Chattopadhyay A, Araujo AM, Bauer J, Scarpini CG, Coleman N - J. Pathol. (2013)

Effect of TGM2 depletion and over-expression on the migration and invasiveness of OSMR over-expressing cervical SCC cells. (A) Western blot showing levels of TGM2 protein in SW756 and CaSki at 24–72 h in control cells treated with vehicle only (C) or in OSM-treated cells (OSM). β-Actin (lower row) was used as the loading control. The graphs below each blot represent the densitometric analysis of protein levels. Results are expressed as optical density (arbitrary units), with control cells set as 1. (B) Levels of TGM2 enzymatic activity in SW756 and CaSki cells after 48 h in the absence (−OSM) or presence (+OSM) of OSM. (C, D) Quantification of cell migration velocity (C) and invasiveness through basement membrane extract (D). Each panel shows data for SW756 (left column) and CaSki (right column) following TGM2 depletion (top row) or TGM2 over-expression (bottom row). In the depletion experiments, cells were assayed after 48 h in the presence (+OSM) or absence (−OSM) of OSM. Cells with TGM2 knock-down (KD) were compared with control cells treated with non-targeting (NT) siRNA. As background levels of TGM2 were barely detectable in CaSki (see A), TGM2 depletion was only performed in OSM-treated CaSki cells. In the over-expression experiments, cells expressing TGM2 and GFP (GFP + TGM2) were compared with control cells expressing GFP alone: ns, non-significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
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fig02: Effect of TGM2 depletion and over-expression on the migration and invasiveness of OSMR over-expressing cervical SCC cells. (A) Western blot showing levels of TGM2 protein in SW756 and CaSki at 24–72 h in control cells treated with vehicle only (C) or in OSM-treated cells (OSM). β-Actin (lower row) was used as the loading control. The graphs below each blot represent the densitometric analysis of protein levels. Results are expressed as optical density (arbitrary units), with control cells set as 1. (B) Levels of TGM2 enzymatic activity in SW756 and CaSki cells after 48 h in the absence (−OSM) or presence (+OSM) of OSM. (C, D) Quantification of cell migration velocity (C) and invasiveness through basement membrane extract (D). Each panel shows data for SW756 (left column) and CaSki (right column) following TGM2 depletion (top row) or TGM2 over-expression (bottom row). In the depletion experiments, cells were assayed after 48 h in the presence (+OSM) or absence (−OSM) of OSM. Cells with TGM2 knock-down (KD) were compared with control cells treated with non-targeting (NT) siRNA. As background levels of TGM2 were barely detectable in CaSki (see A), TGM2 depletion was only performed in OSM-treated CaSki cells. In the over-expression experiments, cells expressing TGM2 and GFP (GFP + TGM2) were compared with control cells expressing GFP alone: ns, non-significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001
Mentions: As the evidence from human tissue samples suggested an important role for TGM2 in SCC progression, we examined the functional significance of TGM2 in cervical SCC cells in vitro. We used multiple complementary experimental approaches, based on gene depletion and over-expression, to minimize the possibility of non-specific observations. We focused on two representative OSMR-over-expressing cervical SCC cell lines, SW756 and CaSki 4,6. In both, treatment with OSM up-regulated TGM2 at the mRNA (see Supplementary material, Figure S2A) and protein levels (Figure 2A). After 48 h of OSM treatment, levels of TGM2 protein were induced by 360% in CaSki and by 140% in SW756 (in which basal levels of TGM2 were high). In addition, OSM significantly induced TGM2 enzymatic activity in both cell lines, as measured by a colorimetric assay (Figure 2B). This indicated that the OSM-induced TGM2 was functional and catalytically active. We observed mixed effects of exogenous OSM in the cervical SCC cell lines that did not over-express OSMR, MS751 and ME180 6. In MS751, TGM2 was induced after 24 h of OSM treatment, concomitant with up-regulation of OSMR (see Supplementary material, Figure S3). In contrast, in ME180 cells, TGM2 basal levels were undetectable and there was no induction by OSM (see Supplementary material, Figure S3).

Bottom Line: Cervical SCC cells that over-express OSMR show enhanced responsiveness to the major ligand OSM, which induces multiple pro-malignant effects, including increased cell migration and invasiveness.Here, we show that tissue transglutaminase (TGM2) is an important mediator of the ligand-dependent phenotypic effects of OSMR over-expression in SCC cells.We conclude that an OSMR/TGM2/integrin-α5β1/fibronectin pathway is of biological significance in cervical SCC and a candidate for therapeutic targeting.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Cambridge, UK.

Show MeSH
Related in: MedlinePlus