Introduction

In response to cellular stress, p53 exerts tumor-suppressive effects, such as apoptosis, cell cycle arrest, and senescence, through the induction of its target genes. Recently, p53 has been shown to control cellular homeostasis by regulating energy metabolism, glycolysis, antioxidant effects, and autophagy. A previous study has reported an association between the p53 expression pattern in immunohistochemical analyses of tumor and the prognosis of renal cell carcinoma (RCC). Although various tumors have p53 mutations, RCC has few p53 mutations. As such, we examined whether a p53 target gene might affect the prognosis of RCC patients. We identified a novel p53 target and examined whether it was useful as a prognostic factor for renal cancer.

Methods

To screen for novel p53 target genes, we conducted a cDNA microarray analysis using mRNAs isolated from HCT116 p53+/+ and HCT116 p53-/- cells that were treated with 2 µg/ml of Adriamycin. To investigate whether mRNA transcription is regulated by p53, we performed a reporter assay and a chromatin immunoprecipitation (ChIP) assay using U373MG cells. To evaluate the effect of the novel p53 target on the biosynthesis of myo-inositol (MI), we performed a MI assay using HEK293T cells that were transfected with a plasmid expressing the novel p53 target, or HCT116 p53+/+ and HCT116 p53-/- cells that were treated with Adriamycin. To examine whether the novel p53 target would be useful as a prognosis factor for renal cancer, we performed a prognostic analysis using the mRNA expression ratio (tumor / normal kidney tissues) of the novel p53 target in RCC.

Results

The results of the cDNA microarray analysis revealed inositol 3-phosphate synthase 1 ( ISYNA1 ) as a novel candidate gene. From the results of the reporter assay and the ChIP assay, we found p53 response elements in the promoter region and the seventh exon. As such, ISYNA1 was identified as a novel p53 target. The results of MI assay showed that ISYNA1 expression increased myo-inositol levels in the cells. In addition, DNA damage significantly increased the intracellular MI content in HCT116 p53+/+ cells, but did not affect the MI content in HCT116 p53-/- cells. The ISYNA1 mRNA expression ratios in renal cancer and renal clear cell carcinoma were significantly correlated with overall survival (p < 0.01).

Conclusions

We identified ISYNA1 as a novel p53 target gene and found that it regulated the intracellular MI content via a p53-ISYNA1 pathway. Our findings show that the ISYNA1 mRNA expression ratio is correlated to overall survival in RCC. Therefore, ISYNA1 might be useful as a novel prognostic factor for renal cancer.

Funding

none