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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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Related in: MedlinePlus

p63 directly transactivates the caspase-1 promoter. (a) Schematic map of the human caspase-1 promoter region with the p53-like RE. The insert shows the sequence of the p53-RE, located between −94 and −117 bp upstream of the transcription-start site. (b) Both TAp63α and ΔNp63α isoforms transactivate the caspase-1 promoter at 48 h. Caspase-1 promoter activity was evaluated after co-transfection with pcDNA vector, TAp63α ΔNp63α plasmids. The luciferase assay was performed after 48 and 72 h of co-transfection in 293T cells and was normalized by co-transfection of Renilla vector. The graphs show a mean±S.D. of three different experiments. **P<0.001; *P<0.05; ^P=0.1. (c) Western blot analysis from the same lysates as the luciferase assay performed as control to show the p63 protein expression. (d) SaOs-2 cells were induced with 4μg/ml Doxy for 24 h. The sonicated chromatin was bound to TAp63α-HA and amplified by PCR with caspase-1 primer that recognizes the p53-response element (from −94 to −117 bp). ChIP on MDM2 promoter was performed as a positive control. Mouse IgG antibody was used as a negative control of the ChIP procedure
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fig2: p63 directly transactivates the caspase-1 promoter. (a) Schematic map of the human caspase-1 promoter region with the p53-like RE. The insert shows the sequence of the p53-RE, located between −94 and −117 bp upstream of the transcription-start site. (b) Both TAp63α and ΔNp63α isoforms transactivate the caspase-1 promoter at 48 h. Caspase-1 promoter activity was evaluated after co-transfection with pcDNA vector, TAp63α ΔNp63α plasmids. The luciferase assay was performed after 48 and 72 h of co-transfection in 293T cells and was normalized by co-transfection of Renilla vector. The graphs show a mean±S.D. of three different experiments. **P<0.001; *P<0.05; ^P=0.1. (c) Western blot analysis from the same lysates as the luciferase assay performed as control to show the p63 protein expression. (d) SaOs-2 cells were induced with 4μg/ml Doxy for 24 h. The sonicated chromatin was bound to TAp63α-HA and amplified by PCR with caspase-1 primer that recognizes the p53-response element (from −94 to −117 bp). ChIP on MDM2 promoter was performed as a positive control. Mouse IgG antibody was used as a negative control of the ChIP procedure

Mentions: As the above results indicated a transcriptional effect of TAp63α and ΔNp63α on the caspase-1 promoter, we next explored whether p63 isoforms were able to directly bind a responsive element (RE) in the promoter region. As a first approach, we performed a bioinformatics analysis to identify putative consensus p53 REs in the promoter sequence of human caspase-1. We used MatInspector Professional software, which allows identification of p53-like REs. Within the first 1600 bp, we identified one putative binding site, which was located between −94 bp and −117 bp from the transcriptional start of the caspase-1 gene (Figure 2a). To experimentally validate our hypothesis, we utilized a reporter gene vector, which contained this promoter region upstream of the luciferase reporter gene. 293T cells were then co-transfected with reporter plasmid and TAp63α or ΔNp63α isoform expressing plasmids. To verify transfection efficiency, western blotting for TAp63α and ΔNp63α was performed on the same protein extracts as used in the luciferase assays (Figure 2c). Consistent with our hypothesis, after 48 and 72 h of transfection the luciferase assays showed significant increase of luciferase activity in presence of one of the two p63 isoforms (Figure 2b), suggesting a direct ability of p63 to affect transcriptional regulation of caspase-1. These data strongly suggest that caspase-1 might represent a transcriptional target of p63.


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 directly transactivates the caspase-1 promoter. (a) Schematic map of the human caspase-1 promoter region with the p53-like RE. The insert shows the sequence of the p53-RE, located between −94 and −117 bp upstream of the transcription-start site. (b) Both TAp63α and ΔNp63α isoforms transactivate the caspase-1 promoter at 48 h. Caspase-1 promoter activity was evaluated after co-transfection with pcDNA vector, TAp63α ΔNp63α plasmids. The luciferase assay was performed after 48 and 72 h of co-transfection in 293T cells and was normalized by co-transfection of Renilla vector. The graphs show a mean±S.D. of three different experiments. **P<0.001; *P<0.05; ^P=0.1. (c) Western blot analysis from the same lysates as the luciferase assay performed as control to show the p63 protein expression. (d) SaOs-2 cells were induced with 4μg/ml Doxy for 24 h. The sonicated chromatin was bound to TAp63α-HA and amplified by PCR with caspase-1 primer that recognizes the p53-response element (from −94 to −117 bp). ChIP on MDM2 promoter was performed as a positive control. Mouse IgG antibody was used as a negative control of the ChIP procedure
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: p63 directly transactivates the caspase-1 promoter. (a) Schematic map of the human caspase-1 promoter region with the p53-like RE. The insert shows the sequence of the p53-RE, located between −94 and −117 bp upstream of the transcription-start site. (b) Both TAp63α and ΔNp63α isoforms transactivate the caspase-1 promoter at 48 h. Caspase-1 promoter activity was evaluated after co-transfection with pcDNA vector, TAp63α ΔNp63α plasmids. The luciferase assay was performed after 48 and 72 h of co-transfection in 293T cells and was normalized by co-transfection of Renilla vector. The graphs show a mean±S.D. of three different experiments. **P<0.001; *P<0.05; ^P=0.1. (c) Western blot analysis from the same lysates as the luciferase assay performed as control to show the p63 protein expression. (d) SaOs-2 cells were induced with 4μg/ml Doxy for 24 h. The sonicated chromatin was bound to TAp63α-HA and amplified by PCR with caspase-1 primer that recognizes the p53-response element (from −94 to −117 bp). ChIP on MDM2 promoter was performed as a positive control. Mouse IgG antibody was used as a negative control of the ChIP procedure
Mentions: As the above results indicated a transcriptional effect of TAp63α and ΔNp63α on the caspase-1 promoter, we next explored whether p63 isoforms were able to directly bind a responsive element (RE) in the promoter region. As a first approach, we performed a bioinformatics analysis to identify putative consensus p53 REs in the promoter sequence of human caspase-1. We used MatInspector Professional software, which allows identification of p53-like REs. Within the first 1600 bp, we identified one putative binding site, which was located between −94 bp and −117 bp from the transcriptional start of the caspase-1 gene (Figure 2a). To experimentally validate our hypothesis, we utilized a reporter gene vector, which contained this promoter region upstream of the luciferase reporter gene. 293T cells were then co-transfected with reporter plasmid and TAp63α or ΔNp63α isoform expressing plasmids. To verify transfection efficiency, western blotting for TAp63α and ΔNp63α was performed on the same protein extracts as used in the luciferase assays (Figure 2c). Consistent with our hypothesis, after 48 and 72 h of transfection the luciferase assays showed significant increase of luciferase activity in presence of one of the two p63 isoforms (Figure 2b), suggesting a direct ability of p63 to affect transcriptional regulation of caspase-1. These data strongly suggest that caspase-1 might represent a transcriptional target of p63.

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