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

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.

Figure 1

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

Figure 2

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

Figure 3

p63 and caspase-1 expression directly correlate in human prostate and bladder cancers. (a and b) Expression data from the Oncomine website (http://www.oncomine.org). We used the following filters: gene ‘TP63' Analysis Type: ‘Coexpression analysis' Cancer Type: Prostate Cancer (a) or Bladder Cancer (b). The color changes according to a weaker (blue) or higher (red) expression, passing by white, with fluctuating color intensity. The reporters indicate probes used for the analysis. (a) Considering the maximum correlation 1, caspase-1 has a correlation of 0.446, in Arredouani and 0.859, in Varambally prostate cancers. The Arredouani data set was performed on mRNA samples of 8 normal prostates and 13 prostate cancers. In the Varambally data set, mRNA of six hormone-refractory metastatic prostate carcinoma samples, seven primary prostate carcinoma samples, and six normal prostate gland samples were analyzed. Both data sets were performed with Human Genome U133 Plus 2.0 Array and 19 574 genes were measured. (b) In the Stransky bladder data set, caspase-1 shows a correlation with TP63 of 0.368; in this data set, mRNA from 8603 genes on 25 superficial bladder cancer and 32 infiltrating bladder urothelial carcinomas were analyzed using Affymetrix U95A microarrays. (c and d) The graphs are obtained from the expression values of TP63 and caspase-1 on the prostate (c) and bladder (d) data sets; the straight lines indicate that the correlation between caspase-1 and TP63 genes is direct. All three data sets are selected using as threshold a P-value of 0.01

Figure 4

Positive p63/caspase-1 correlation represents a prognostic factor for good patient survival. (a–c) Three different human breast cancer data sets have been analyzed: GSE3494 (a), GSE11121 (b), GSE2034 (c). Left and central panels represent p63 and caspase-1 expression profiles in the cohorts 1 (positive correlation) and cohorts 2 (negative or absent correlation). Right panels represent patient survival estimation of p63/caspase-1-positive correlation groups compared with negative or absent correlation groups