Abstract PR03: P53 inhibits the expression of the pyrimidine catabolic gene Dihydropyrimidine dehydrogenase (DPYD)
Authors: Gokare Prashanth Ravishankar , Finnberg Niklas , Dai Jenny , El-Deiry Wafik 
Published: May 2016

Abstract
Fluorouracil (5-FU) a widely used chemotherapeutic drug whose unpredictable pharmacokinetics is controlled by the pyrimidine catabolic gene dihydropyrimidine dehydrogenase (DPYD), that has recently also been shown to be a gatekeeper of the epithelial-to-mesenchymal transition (EMT) in breast cancer. Relatively little is known about the transcriptional control of DPYD and here we show for the first time an interaction between p53 and DPYD (involved in catabolism of pyrimidines as well as 5-FU) where p53 represses both the base-line expression of DPYD and that following 5-FU administration in vitro and in vivo. This mechanism affects the catabolic conversion of 5-FU to 5-FUH2 in mice in vivo. Using an in-silico approach we also identified several putative p53 binding sites (P53DBS) in and around ~20Kb upstream and downstream of the mouse DPYD gene. In-vivo ChIP from mice livers identified a key p53DBS binding site downstream (chr3: 119451237-11941257) of the gene to which p53 binds to at about 1.8 ± 0.05 fold over untreated control following a single IV bolus of 5-FU (150 mg/kg bw). Interestingly DPYD mRNA and protein levels were decreased by 1.8 and 1.5 fold respectively (P&lt; 0.0005) in a p53-dependent manner in the liver. Similarly DPYD-repression was documented at both the mRNA and protein levels in the wild-type (WT) p53-expressing cancer cell lines H460, HCT-116, and A569. To further characterize the functional effect of DPYD repression in vivo, we utilized mice with a liver specific deletion of p53 (Albcre p53Δ/Δ) which showed ~2-fold (p≤0.03 wilcoxon rank-sum test) decrease plasma ratio of 5-FUH2/5-FU to mice with an intact allele of p53 in their livers (Albcre p53Δ/+). Increased plasma concentrations of 5-FU caused a significant tumor growth delay (TGD) in mouse colon cancer cells implanted in syngeneic Albcre p53Δ/+ versus Albcre p53Δ/Δ mice (TGD 11 vs 8.93 days). Our data also suggest a possible feedback mechanism of DPYD repression by p53 due to thymidylate synthase (TS) inhibition, a key mediator for supplying thymidine for DNA replication and itself a target for regulation by 5-FU. The addition of exogenous thymidine abrogated the p53-dependent repression of DPYD expression following siRNA-mediated targeting of TS. Further cross-validation experiments using the specific TS inhibitors raltitrexed and methotrexate showed similar effects. Furthermore treatment with the ATM (KU-55933) or DNA-PK (NU7026) inhibitors reversed the p53-dependent repression of DPYD-expression indicating a specific DNA damage feedback mechanism following TS inhibition. Taken together our data suggest that p53 plays an important role in controlling pyrimidine catabolism, particularly following metabolic stress imposed by nucleotide imbalances. In light of more recent discoveries of the role of DPYD in malignant progression, it is plausible that the relationship between p53 and DPYD can have implications beyond influencing the pharmacokinetics of 5-FU and may extend to control of EMT and metastasis. Thus our current efforts are focused on exploring short- and long-term consequences of p53-dependent DPYD-repression as part of p53⊠s tumor suppressor function as well as understanding cellular context for the observed link in vitro and in vivo.
Citation Format: Prashanth Ravishankar Gokare, Niklas Finnberg, Jenny Dai, Wafik El-Deiry. P53 inhibits the expression of the pyrimidine catabolic gene Dihydropyrimidine dehydrogenase (DPYD). [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr PR03.