Dihydropyrimidinase protects from DNA replication stress caused by cytotoxic metabolites Jihane Basbous, Antoine Aze, Laurent Chaloin, Rana Lebdy, Dana Hodroj, Cyril Ribeyre, Marion Larroque, Caitlin Shepard, Baek Kim, Alain Pruvost, et al. To cite this version: Jihane Basbous, Antoine Aze, Laurent Chaloin, Rana Lebdy, Dana Hodroj, et al.. Dihydropyrimidi- nase protects from DNA replication stress caused by cytotoxic metabolites. Nucleic Acids Research, Oxford University Press, 2020, 48 (4), pp.1886-1904. 10.1093/nar/gkz1162. hal-02556387 HAL Id: hal-02556387 https://hal.archives-ouvertes.fr/hal-02556387 Submitted on 6 Jul 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. 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Distributed under a Creative Commons Attribution - NonCommercial| 4.0 International License 1886–1904 Nucleic Acids Research, 2020, Vol. 48, No. 4 Published online 19 December 2019 doi: 10.1093/nar/gkz1162 Dihydropyrimidinase protects from DNA replication stress caused by cytotoxic metabolites Jihane Basbous1,*, Antoine Aze1, Laurent Chaloin2, Rana Lebdy1, Dana Hodroj1,3, Cyril Ribeyre1, Marion Larroque1,4, Caitlin Shepard5, Baek Kim5, Alain Pruvost6, Jer´ omeˆ Moreaux 1, Domenico Maiorano1, Marcel Mechali1 and Angelos Constantinou 1,* 1Institute of Human Genetics (IGH), CNRS, Universite´ de Montpellier, 34396 Montpellier Cedex 5, France, 2Institut de Recherche en Infectiologie de Montpellier, CNRS, Universite´ de Montpellier, 34293 Montpellier Cedex 5, France, Downloaded from https://academic.oup.com/nar/article-abstract/48/4/1886/5680705 by guest on 06 July 2020 3Cancer Research Center of Toulouse (CRCT), 31037 Toulouse Cedex 1, France, 4Institut du Cancer de Montpellier (ICM),34298 Montpellier Cedex 5, France, 5School of Medicine, Emory University, Atlanta, GA 30322, USA and 6Service de Pharmacologie et Immunoanalyse (SPI), Plateforme SMArt-MS, CEA, INRA, Universite´ Paris-Saclay, 91191 Gif-sur-Yvette Cedex, France Received April 17, 2019; Revised November 27, 2019; Editorial Decision November 28, 2019; Accepted November 29, 2019 ABSTRACT tion (2), accumulating evidence suggests that carcinogene- sis is associated with metabolic rewiring of the pyrimidine Imbalance in the level of the pyrimidine degradation catabolic pathway (3–6). Whether and how rewiring of the products dihydrouracil and dihydrothymine is asso- pyrimidine catabolic pathway supports tumor progression, ciated with cellular transformation and cancer pro- however, remains poorly defined. gression. Dihydropyrimidines are degraded by dihy- In humans, the pyrimidines uracil and thymine are de- dropyrimidinase (DHP), a zinc metalloenzyme that graded in three enzymatic steps (Figure 1A). First, dihy- is upregulated in solid tumors but not in the cor- dropyrimidine dehydrogenase (DPD) reduces the pyrimi- responding normal tissues. How dihydropyrimidine dine ring of uracil and thymine with hydrogen and yields metabolites affect cellular phenotypes remains elu- 5, 6-dihydrouracil and 5, 6-dihydrothymine (dihydropyrim- sive. Here we show that the accumulation of di- idines). Second, the saturated rings between position 3 and ␤ hydropyrimidines induces the formation of DNA– 4 are opened by dihydropyrimidinase (DHP). Third, - ureidopropionase (BUP-1) degrades the ␤-ureidopropionic protein crosslinks (DPCs) and causes DNA replica- acid and ␤-ureidoisobutyric acid products formed by DHP tion and transcriptional stress. We used Xenopus into ␤-alanine and ␤-aminoisobutyric acid. DPD activity egg extracts to recapitulate DNA replication in vitro. has been detected in all tissues examined, but the activity of We found that dihydropyrimidines interfere directly DHP and BUP-1 is essentially restricted to the liver and the with the replication of both plasmid and chromo- kidney (7). In cancer cells, however, the level of pyrimidine somal DNA. Furthermore, we show that the plant degradation activities is considerably altered. Increased ex- flavonoid dihydromyricetin inhibits human DHP ac- pression of DPD has been observed in human hepatocel- tivity. Cellular exposure to dihydromyricetin trig- lular carcinoma (8). Human skin cutaneous melanomas gered DPCs-dependent DNA replication stress in progressing toward metastatic tumors accumulate muta- cancer cells. This study defines dihydropyrimidines tions in DPD and up-regulate the expression of the genes as potentially cytotoxic metabolites that may offer an encoding DPD and DHP (6). The accumulation of 5, 6- dihydrouracil is a distinct metabolic feature of early lung opportunity for therapeutic-targeting of DHP activity adenocarcinoma (5). Intriguingly, an increase in the con- in solid tumors. centration of dihydropyrimidines in epithelial breast can- cer cells supports the acquisition of aggressive mesenchymal INTRODUCTION characteristics (4). How dihydropyrimidines affect cellular Adjustments in metabolic activities are required to satisfy phenotypes, however, remains elusive. Pioneering studies high metabolic demands associated with cancer cell pro- have identified DHP activity as a good marker of tumori- liferation (1). Alterations in nucleotide metabolisms are genicity and a target for cancer therapy (3). Whereas hardly emerging as distinctive features of cancers cells. While nu- detectable in normal extrahepatic and kidney tissues (van cleotide bases may be limiting for cancer cells prolifera- Kuilenburg et al., (7)), the activity of DHP is strikingly high *To whom correspondence should be addressed. Tel: +33 4 34 35 98 14; Fax: +33 4 34 35 99 01; Email: [email protected] Correspondence may also be addressed to Jihane Basbous. Email: [email protected] C The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] Nucleic Acids Research, 2020, Vol. 48, No. 4 1887 Downloaded from https://academic.oup.com/nar/article-abstract/48/4/1886/5680705 by guest on 06 July 2020 Figure 1. Depletion of DHP impairs the proliferation of epithelial cancer cells. (A) Schematic representation of the pyrimidine degradation pathway. (B) DHP was probed by western blotting in the indicated transformed cells. When indicated, DHP was knocked down using anti-DHP siRNA or shRNA molecules with distinct target sequences. Ponceau staining was used as loading control. * non-specific signal. One representative experimentwn issho from three to six biological replicates. (C) U-2 OS cells were transfected with control or anti-DHP siRNA (siDHP-sp) and their viability was assessed during 4 days using the MTT cell growth assay. Mean viability is representative of three independent biological replicates. Error bars represent ±S.D. (D) Colony-forming assay of U-2 OS cells after transfection with control or anti-DHP siRNA (siDHP-1). A representative image is shown. An histogram represents the quantification of colony formation. Data shown are averages over three independent biological replicates with two technicaltes replica for each. Error bars represent ±S.D. P-values were calculated using a regression model with Poisson distribution: ***P < 0.0001. Bottom panel: the efficiency of DHP knockdown was assessed by western blotting. (E) Histogram representing the percentage of U-2 OS cells, 72 h after transfection with control or anti-DHP siRNA (siDHP-1) in G0/G1, S and G2/M phases. Data shown are averages over three independent biological replicates. Error bars represent ±S.D. P-values were calculated using a regression model with Poisson distribution: ***P < 0.0001. 1888 Nucleic Acids Research, 2020, Vol. 48, No. 4 in human carcinomas of the lung, colon, pancreas, salivary HMCLs were routinely maintained in RPMI 1640 and gland and stomach (3). 10% fetal calf serum (FCS; Biowittaker, Walkersville, Another hallmark of cancer is DNA replication stress MD), supplemented with 3 ng/ml IL-6 (Peprotech, Rocky causing genomic instability (1,9). Stress in the context of Hill, NJ, USA) for IL6 dependent cell lines. HMCLs DNA replication is defined as the slowing or stalling of were authenticated according to their short tandem re- DNA chain elongation (10). Known sources of DNA repli- peat profiling and their gene expression profiling using cation stress include nucleotides insufficiency (11), mis- Affymetrix U133 plus 2.0 microarrays deposited in the incorporated nucleotides (12,13), replication/transcription ArrayExpress public database under accession numbers conflicts (14–16) and DNA lesions caused by reactive E-TABM-937 and E-TABM-1088. Dihydrouracil, Uracil, metabolic products such as reactive oxygen species and Dihydromyricetin, Aphidicolin and Roscovitine were pur- aldehydes (17–19). Furthermore, cellular metabolites can chased from Sigma-Aldrich. Formaldehyde was purchased yield structurally