Increased biosynthesis of nucleotides (NTPs) and deoxynucleotides (dNTPs) is a hallmark of cancer, driven by the need to support DNA replication and repair, transcription, ribosome biogenesis and post-translational protein glycosylation. Nucleotide biosynthesis encompasses a complex network of enzymes and transporters that can be divided into de novo synthesis pathways and salvage reactions from nucleosides and nucleobases. Wild type p53 plays an important role in nucleotide metabolism regulation inhibiting pentose phosphate pathway and purine de novo synthesis. However, when activated upon DNA damage, p53 can activate dNTP synthesis through RRM2B to favor DNA repair. Moreover, gain of function activities of mutant p53 contribute to nucleotide metabolism reprogramming to support proliferation and invasion. This study aims to define a p53 regulation over some essential elements of nucleoside salvage, such as nucleoside transporters (NTs), which are also responsible for the internalization of nucleoside analog drugs. Our work focuses on Concentrative Nucleoside Transporter 1 (hCNT1, encoded by SLC28A1), a unidirectional pyrimidine transporter whose expression has been reported to be decreased in a wide variety of tumors.

NTs expression was determined after wtp53 restitution by RT-PCR in different models. Possible binding sites for p53 in SLC28A1 promoter were found by bioinformatics approaches. Promoter-luciferase reporter assays and ChIP-RT-PCR were performed to validate them. Induction of hCNT1 expression has been studied in p53 WT and KO HCT-116 cells. In order to induce DNA damage or an imbalance on nucleotide pools, cells were subjected to different treatments, including Etoposide or Brequinar. The role of mutant p53 has been also studied.

Our data indicates that wtp53 overexpression specifically induces hCNT1 expression in a non-tumoral background. Putative binding sites for wtp53 in hCNT1 promoter have been identified and validated. Blockage of pyrimidine de novo synthesis with Brequinar or induction of DNA damage with Etoposide lead to an upregulation of hCNT1 expression largely dependent on p53 activation, suggesting that p53 could play a role in connecting de novo and salvage pathways to maintain nucleotide pools. Interestingly, this regulation induced by wtp53 is not replicated by restoring different mutant p53 expression in a p53 KO model, which could contribute to hCNT1 expression reduction observed in tumors and the consequent decrease in some nucleoside analogs incorporation.

wtp53 regulates nucleoside salvage pathway transporter hCNT1 expression. Besides, in cell contexts where wtp53 is induced by DNA damage or imbalance of nucleotide pools transcription of hCNT1 is potentiated. However, mtp53 fails to induce hCNT1 expression.