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Caspase-1 is a novel target of p63 in tumor suppression.

Celardo I, Grespi F, Antonov A, Bernassola F, Garabadgiu AV, Melino G, Amelio I - Cell Death Dis (2013)

Bottom Line: We show that both p63 isoforms promote caspase-1 expression by physical binding to its promoter.Consistent with our in vitro findings, we also identified a direct correlation between p63 and caspase-1 expression in human cancer data sets.In addition, survival estimation analysis demonstrated that functional interaction between p63 and caspase-1 represents a predictor of positive survival outcome in human cancers.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council, Toxicology Unit, Leicester University, Leicester, UK.

ABSTRACT
p63 is a p53 family transcription factor, which besides unique roles in epithelial development, shares tumor suppressive activity with its homolog p53. The p63 gene has different transcriptional start sites, which generate two N-terminal isoforms (transactivation domain (TA)p63 and amino terminal truncated protein(ΔN)p63); in addition alternative splicing at the 5'-end give rise to at least five C-terminal isoforms. This complexity of gene structure has probably fostered the debate and controversy on p63 function in cancer, with TP63-harboring two distinctive promoters, codifying for the TAp63 and ΔNp63 isoforms, and having discrete functions. However, ΔNp63 also drives expression of target genes that have a relevant role in cancer and metastasis. In this study, we identified a novel p63 transcriptional target, caspase-1. Caspase-1 is proinflammatory caspase, which functions in tumor suppression. We show that both p63 isoforms promote caspase-1 expression by physical binding to its promoter. Consistent with our in vitro findings, we also identified a direct correlation between p63 and caspase-1 expression in human cancer data sets. In addition, survival estimation analysis demonstrated that functional interaction between p63 and caspase-1 represents a predictor of positive survival outcome in human cancers. Overall, our data report a novel p63 target gene involved in tumor suppression, and the clinical analysis underlines the biological relevance of this finding and suggests a further clinically predictive biomarker.

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p63 upregulates protein and mRNA levels of caspase-1. (a and b) Caspase-1 protein is induced by both p63 isoforms in a time-dependent manner. TAp63α (a) and ΔNp63α (b) SaOs-2 inducibile cells were treated with 4 μg/ml Doxy to induce p63 expression. After 3, 6, 12, 24 (h) of p63 induction, a western blot analysis was performed using the indicated antibodies. GAPDH levels were evaluated as a control. The experiment shown is representative of three independent experiments. (c and d) mRNA expression of caspase-1 was induced by p63. Levels of caspase-1 were evaluated by real-time qPCR following TAp63α (c) and ΔNp63α (d) induction at the same time indicated for western blot analysis. p21 was used as positive control. Relative expression of caspase-1 and p21 were normalized against TBP and calculated as fold induction. Data represent mean±S.D. of three different experiments analyzed in triplicate. *P<0.05
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fig1: p63 upregulates protein and mRNA levels of caspase-1. (a and b) Caspase-1 protein is induced by both p63 isoforms in a time-dependent manner. TAp63α (a) and ΔNp63α (b) SaOs-2 inducibile cells were treated with 4 μg/ml Doxy to induce p63 expression. After 3, 6, 12, 24 (h) of p63 induction, a western blot analysis was performed using the indicated antibodies. GAPDH levels were evaluated as a control. The experiment shown is representative of three independent experiments. (c and d) mRNA expression of caspase-1 was induced by p63. Levels of caspase-1 were evaluated by real-time qPCR following TAp63α (c) and ΔNp63α (d) induction at the same time indicated for western blot analysis. p21 was used as positive control. Relative expression of caspase-1 and p21 were normalized against TBP and calculated as fold induction. Data represent mean±S.D. of three different experiments analyzed in triplicate. *P<0.05

Mentions: p63 is a transcriptional factor involved in cancer and metastasis. In many cancer types, the loss of p63 expression is associated with increased tumorigenesis48 and metastasis.49 Caspase-1 knockout mice show enhanced tumor formation associated with increased cell proliferation and reduced apoptosis.44 Therefore, we decided to evaluate a possible association between p63 function and caspase-1 expression. To this end, we used SaOs-2 Tet-On cell lines, which carry inducible expression systems for TAp63α or ΔNp63α. As shown in Figures 1a and b, 4 μg/ml doxycycline (Doxy) treatment strongly induced expression of both p63 isoforms. Western blot analysis showed that in both TAp63- and ΔNp63-expressing cell lines caspase-1 protein was induced in a time-dependent fashion, in parallel with the expression of the transcriptional factor (Figures 1a and b). To evaluate whether caspase-1 upregulation was associated with induced transcription of the casp-1 gene, we performed additional analysis by real-time quantitative polymerase chain reaction (qPCR) in SaOs-2 Tet-On cell lines. qPCR confirmed upregulation of caspase-1 mRNA with a kinetics consistent with the well-known p63 transcriptional target, p21 (Figures 1c and d).22 Collectively, this data demonstrate that both p63 isoforms can regulate caspase-1 expression and also suggest that p63 might directly act on the caspase-1 promoter regulating its expression at the transcriptional level.


Caspase-1 is a novel target of p63 in tumor suppression.

Celardo I, Grespi F, Antonov A, Bernassola F, Garabadgiu AV, Melino G, Amelio I - Cell Death Dis (2013)

p63 upregulates protein and mRNA levels of caspase-1. (a and b) Caspase-1 protein is induced by both p63 isoforms in a time-dependent manner. TAp63α (a) and ΔNp63α (b) SaOs-2 inducibile cells were treated with 4 μg/ml Doxy to induce p63 expression. After 3, 6, 12, 24 (h) of p63 induction, a western blot analysis was performed using the indicated antibodies. GAPDH levels were evaluated as a control. The experiment shown is representative of three independent experiments. (c and d) mRNA expression of caspase-1 was induced by p63. Levels of caspase-1 were evaluated by real-time qPCR following TAp63α (c) and ΔNp63α (d) induction at the same time indicated for western blot analysis. p21 was used as positive control. Relative expression of caspase-1 and p21 were normalized against TBP and calculated as fold induction. Data represent mean±S.D. of three different experiments analyzed in triplicate. *P<0.05
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3674380&req=5

fig1: p63 upregulates protein and mRNA levels of caspase-1. (a and b) Caspase-1 protein is induced by both p63 isoforms in a time-dependent manner. TAp63α (a) and ΔNp63α (b) SaOs-2 inducibile cells were treated with 4 μg/ml Doxy to induce p63 expression. After 3, 6, 12, 24 (h) of p63 induction, a western blot analysis was performed using the indicated antibodies. GAPDH levels were evaluated as a control. The experiment shown is representative of three independent experiments. (c and d) mRNA expression of caspase-1 was induced by p63. Levels of caspase-1 were evaluated by real-time qPCR following TAp63α (c) and ΔNp63α (d) induction at the same time indicated for western blot analysis. p21 was used as positive control. Relative expression of caspase-1 and p21 were normalized against TBP and calculated as fold induction. Data represent mean±S.D. of three different experiments analyzed in triplicate. *P<0.05
Mentions: p63 is a transcriptional factor involved in cancer and metastasis. In many cancer types, the loss of p63 expression is associated with increased tumorigenesis48 and metastasis.49 Caspase-1 knockout mice show enhanced tumor formation associated with increased cell proliferation and reduced apoptosis.44 Therefore, we decided to evaluate a possible association between p63 function and caspase-1 expression. To this end, we used SaOs-2 Tet-On cell lines, which carry inducible expression systems for TAp63α or ΔNp63α. As shown in Figures 1a and b, 4 μg/ml doxycycline (Doxy) treatment strongly induced expression of both p63 isoforms. Western blot analysis showed that in both TAp63- and ΔNp63-expressing cell lines caspase-1 protein was induced in a time-dependent fashion, in parallel with the expression of the transcriptional factor (Figures 1a and b). To evaluate whether caspase-1 upregulation was associated with induced transcription of the casp-1 gene, we performed additional analysis by real-time quantitative polymerase chain reaction (qPCR) in SaOs-2 Tet-On cell lines. qPCR confirmed upregulation of caspase-1 mRNA with a kinetics consistent with the well-known p63 transcriptional target, p21 (Figures 1c and d).22 Collectively, this data demonstrate that both p63 isoforms can regulate caspase-1 expression and also suggest that p63 might directly act on the caspase-1 promoter regulating its expression at the transcriptional level.

Bottom Line: We show that both p63 isoforms promote caspase-1 expression by physical binding to its promoter.Consistent with our in vitro findings, we also identified a direct correlation between p63 and caspase-1 expression in human cancer data sets.In addition, survival estimation analysis demonstrated that functional interaction between p63 and caspase-1 represents a predictor of positive survival outcome in human cancers.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council, Toxicology Unit, Leicester University, Leicester, UK.

ABSTRACT
p63 is a p53 family transcription factor, which besides unique roles in epithelial development, shares tumor suppressive activity with its homolog p53. The p63 gene has different transcriptional start sites, which generate two N-terminal isoforms (transactivation domain (TA)p63 and amino terminal truncated protein(ΔN)p63); in addition alternative splicing at the 5'-end give rise to at least five C-terminal isoforms. This complexity of gene structure has probably fostered the debate and controversy on p63 function in cancer, with TP63-harboring two distinctive promoters, codifying for the TAp63 and ΔNp63 isoforms, and having discrete functions. However, ΔNp63 also drives expression of target genes that have a relevant role in cancer and metastasis. In this study, we identified a novel p63 transcriptional target, caspase-1. Caspase-1 is proinflammatory caspase, which functions in tumor suppression. We show that both p63 isoforms promote caspase-1 expression by physical binding to its promoter. Consistent with our in vitro findings, we also identified a direct correlation between p63 and caspase-1 expression in human cancer data sets. In addition, survival estimation analysis demonstrated that functional interaction between p63 and caspase-1 represents a predictor of positive survival outcome in human cancers. Overall, our data report a novel p63 target gene involved in tumor suppression, and the clinical analysis underlines the biological relevance of this finding and suggests a further clinically predictive biomarker.

Show MeSH
Related in: MedlinePlus