P53 represses pyrimidine catabolic gene dihydropyrimidine dehydrogenase (DPYD) expression in response to thymidylate synthase (TS) targeting

Prashanth Gokare,Jenny Dai,Phillip H Abbosh,Wafik S El-Deiry,Maureen E Murphy,Niklas K Finnberg

doi:10.1038/s41598-017-09859-x

Abstract

Nucleotide metabolism in cancer cells can influence malignant behavior and intrinsic resistance to therapy. Here we describe p53-dependent control of the rate-limiting enzyme in the pyrimidine catabolic pathway, dihydropyrimidine dehydrogenase (DPYD) and its effect on pharmacokinetics of and response to 5-fluorouracil (5-FU). Using in silico/chromatin-immunoprecipitation (ChIP) analysis we identify a conserved p53 DNA-binding site (p53BS) downstream of the DPYD gene with increased p53 occupancy following 5-FU treatment of cells. Consequently, decrease in Histone H3K9AC and increase in H3K27me3 marks at the DPYD promoter are observed concomitantly with reduced expression of DPYD mRNA and protein in a p53-dependent manner. Mechanistic studies reveal inhibition of DPYD expression by p53 is augmented following thymidylate synthase (TS) inhibition and DPYD repression by p53 is dependent on DNA-dependent protein kinase (DNA-PK) and Ataxia telangiectasia mutated (ATM) signaling. In-vivo, liver specific Tp53 loss increases the conversion of 5-FU to 5-FUH2 in plasma and elicits a diminished 5-FU therapeutic response in a syngeneic colorectal tumor model consistent with increased DPYD-activity. Our data suggest that p53 plays an important role in controlling pyrimidine catabolism through repression of DPYD expression, following metabolic stress imposed by nucleotide imbalance. These findings have implications for the toxicity and efficacy of the cancer therapeutic 5-FU.

Highlights

Dihydropyrimidine dehydrogenase (DPYD) is the initial rate-limiting enzyme of the pyrimidine catabolic pathway that catalyzes the reduction of the nucleotide bases uracil and thymine[1]
We show for the first time that the p53 tumor suppressor protein controls 5-FU catabolism by repressing the expression of the key rate limiting enzyme in pyrimidine degradation dihydropyrimidine dehydrogenase (DPYD). p53 has a well-documented function in the cell death response following 5-FU treatment in pre-clinical experiments in vitro and in vivo[19, 43]
A critical determinant of the efficacy of 5-FU treatment directly relates to the expression of key metabolic genes that are responsible for the biotransformation of the drug

Summary

Introduction

Dihydropyrimidine dehydrogenase (DPYD) is the initial rate-limiting enzyme of the pyrimidine catabolic pathway that catalyzes the reduction of the nucleotide bases uracil and thymine[1]. Canonical p53 signaling involves induced transcription of genes involved in cell cycle arrest, DNA damage repair and programmed forms of cell death. In order to investigate how p53 selectively modulates the cellular response to 5-FU we performed an in-silico screen for p53 DNA-binding sites (p53BS) in the proximity of or within genes involved in nucleotide metabolism. By combining this analysis with chromatin immunoprecipitation (ChIP), expression analysis in vitro and in vivo we show that the expression of DPYD is negatively regulated by p53 in the context of inhibition of thymidylate synthase (TS). Our results have implications for the toxicity of 5-FU as well as its efficacy in the treatment of cancer

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Results

Conclusion