Genome-wide CRISPR Screening Reveals Pyrimidine Metabolic Reprogramming in 5-FU Chronochemotherapy of Colorectal Cancer

Abstract

ObjectiveDisruption of the circadian rhythm is associated with cancer occurrence, response to chemotherapy, and poor prognosis. Thus, using internal clock-based chronotherapy to optimize the administration time may improve the therapeutic effects of anticancer drugs while reducing the side effects. Chronotherapy with 5-fluorouracil (5-FU) has been observed in colorectal cancer (CRC) for a long time, but its effect is under controversial and the mechanism remains unclear.MethodsGenome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screening and RNA-sequencing were combined to identify the potential genes or pathways involved in 5-FU chronochemotherapy. Genetic deletion or overexpression of pyrimidine metabolic pathway genes were conducted to examine cellular viability with or without 5-FU via flow cytometry. Western blotting, qPCR, chromatin immunoprecipitation, gain-of-function and loss-of-function assays of several CRC cell lines in vitro and in vivo were used to elaborate and validate the mechanism of 5-FU chronotherapeutic effects.ResultsChronochemotherapeutic effects of 5-FU on CRC in vivo were verified. Furthermore, 5-FU chronochemotherapy related genes such as UPP2, UCK2 and UMPS in the pyrimidine metabolic pathway were identified. Disturbance in these genes, especially UMPS, perturbs 5-FU treatment outcomes in CRC cells. Mechanistically, the core circadian gene, brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein-1 (BMAL1), extensively regulate gene expression in pyrimidine metabolic pathway by binding to E-box element in the promoter region of key genes such as UMPS and perturb their enzymatic activities, thereby maintain diurnal efficacy of 5-FU in CRC cells.ConclusionThis study uncovered a new mechanism by which a core circadian gene BMAL1 increases the effectiveness of 5-FU by enhancing the expression and enzymatic activities of key genes in the pyrimidine metabolic pathway in CRC cells. The findings suggest a novel strategy for CRC chemotherapy by targeting chrono-modulated genes of the 5-FU metabolic pathway.

Integrated metabolomics and transcriptomics reveal glyphosate based‐herbicide induced reproductive toxicity through disturbing energy and nucleotide metabolism in mice testes

“…These findings demonstrated that GBH blocked purine and pyrimidine synthesis and further impaired genetic information in testicular cells. In addition, Ak5, Guk1, Nme2, Entpd2, and Upp2 are key genes involved in the nucleotide synthesis and metabolism, which directly or indirectly regulate DNA and RNA synthesis 45–49 . In this study, the expression of Ak5, Guk1, and Nme2 significantly decreased, but Entpd2 and Upp2 increased in purine and pyrimidine metabolism pathways.…”

GPX3-Mediated Oxidative Stress Affects Pyrimidine Metabolism Levels in Stomach Adenocarcinoma via the AMPK/mTOR Pathway

“…It has been shown that when 5-Fu exerts its antitumor mechanism of action, UMPS can convert a part of 5-Fu into components with cytotoxicity, thus inhibiting the activity of the proapoptotic protein, Bok. This allows cancer cells in a nonproliferative state to escape the damaging properties of 5-Fu and develop secondary drug resistance [ 32 – 35 ]. Yu et al also verified the modulation of 5-Fu drug sensitivity by UMPS expression through their findings [ 36 ].…”

Influence of Single-Nucleotide Polymorphisms on Clinical Outcomes of Capecitabine-Based Chemotherapy in Colorectal Cancer Patients: A Systematic Review

“…UMPS is an enzyme that metabolizes 5-FU into other metabolites with cytotoxic activity and is considered to be a major regulator of the cytotoxic effects of 5-FU [ 53 ]. One study included in this review reported an association of the UMPS rs4678145 and rs2279199 SNPs with asthenia and severe nausea/vomiting in CRC patients treated with capecitabine-based regimens [ 25 ].…”