biology Concept Paper 5-Fluorouracil Conversion Pathway Mutations in Gastric Cancer 1, , 2, 2 1 Alessio Biagioni * y , Fabio Staderini y , Sara Peri , Giampaolo Versienti , 1 2 1, 1, Nicola Schiavone , Fabio Cianchi , Laura Papucci y and Lucia Magnelli y 1 Department of Experimental and Clinical Biomedical Sciences “Mario Serio”, University of Florence, Viale G.B. Morgagni 50, 50134 Firenze, Italy; giampaolo.versienti@unifi.it (G.V.); nicola.schiavone@unifi.it (N.S.); laura.papucci@unifi.it (L.P.); lucia.magnelli@unifi.it (L.M.) 2 Department of Experimental and Clinical Medicine, University of Florence, Largo Brambilla 3, 50134 Firenze, Italy; fabio.staderini@unifi.it (F.S.); sara.peri@unifi.it (S.P.); fabio.cianchi@unifi.it (F.C.) * Correspondence: alessio.biagioni@unifi.it These authors contributed equally to this paper. y Received: 23 July 2020; Accepted: 1 September 2020; Published: 2 September 2020 Abstract: To date, 5-Fluorouracil (5FU) is a major component of several chemotherapy regimens, thus its study is of fundamental importance to better understand all the causes that may lead to chemoresistance and treatment failure. Given the evident differences between prognosis in Asian and Caucasian populations, triggered by clear genetic discordances and given the extreme genetic heterogeneity of gastric cancer (GC), the evaluation of the most frequent mutations in every single member of the 5FU conversion and activation pathway might reveal several important results. Here, we exploited the cBioPortal analysis software to query a large databank of clinical and wide-genome studies to evaluate the components of the three major 5FU transformation pathways. We demonstrated that mutations in such ways were associated with a poor prognosis and reduced overall survival, often caused by a deletion in the TYMP gene and amplification in TYMS. The use of prodrugs and dihydropyrimidine dehydrogenase (DPD) inhibitors, which normally catabolizes 5FU into inactive metabolites, improved such chemotherapies, but several steps forward still need to be taken to select better therapies to target the chemoresistant pools of cells with high anaplastic features and genomic instability. Keywords: 5 fluorouracil; chemoresistance; gastric cancer; mutations 1. Introduction The drug 5-fluorouracil (5FU) is one of the most commonly used drugs to treat several kinds of tumors, including breast cancer, head and neck cancers, gastric cancer, colon cancer, and melanoma [1]. Discovered for the first time in 1957 by Charles Heidelberger and colleagues at the University of Wisconsin [2], it has become a major therapeutic option when specific targets are not available and when surgery is not practicable. Indeed, 5FU is actively absorbed by highly proliferating tissues, especially tumor ones, that need uracil for nucleic acid biosynthesis, and thus acts by inhibiting tumor cell division exploiting two different ways: it may inhibit the thymidylate synthase (TYMS), forming a covalent ternary complex with 5,10-methylenetetrahydrofolate, suppressing in that way the DNA synthesis, or it might be incorporated into RNA molecules blocking gene expression [3]. Once internalized into tumor cells, 5FU is converted to fluorouridine monophosphate (FUMP) by the orotate phosphoribosyltransferase (OPRT) and then converted to fluorodeoxyuridine monophosphate (FdUMP) by the ribosyl reductase complexes (RRM1/2), a biochemical process better known as the “OPRT–RR pathway” [4]. While FdUMP inhibits DNA synthesis through its inhibitory action on Biology 2020, 9, 265; doi:10.3390/biology9090265 www.mdpi.com/journal/biology Biology 2020, 9, 265 2 of 16 Biology 2020, 9, x FOR PEER REVIEW 2 of 16 TYMS,action FUMP on TYMS, is largely FUMP responsible is largely forresponsible the inhibition for the of inhibition gene expression of gene expression through its through incorporation its into mRNAs.incorporation Another into mRNAs. conversion Another way conversion is mediated way by is themediated action by of the the action thymidine of the phosphorylasethymidine (TYMP),phosphorylase which is able (TYMP), to convert which 5FU is able into to fluorodeoxyuridine convert 5FU into fluorodeoxyuridine (FdU) and then transformed (FdU) and tothen FdUMP due totransformed the action to of FdUMP the thymidine due to the action kinase of 1 the (TK1), thym inidine a process kinase 1 called(TK1), in “TP–TK a process pathway”. called “TP–TK FdUMP mightpathway”. also be converted FdUMP might back also to FdU be converted by the nucleotidase back to FdU NT5E by the [5 ].nucleotidase Moreover, NT5E uridine [5]. monophosphate Moreover, synthaseuridine (UMPS) monophosphate and phosphoribosyl synthase (UMPS) pyrophosphate and phosphoribosyl amidotransferase pyrophosph (PPAT)ate amidotransferase are also responsible (PPAT) are also responsible for the conversion of 5FU to FUMP, which is in turn processed to for the conversion of 5FU to FUMP, which is in turn processed to fluorouridine diphosphate (FUDP) fluorouridine diphosphate (FUDP) and then to FdUMP by RRM1 and 2 [6,7]. FUMP reservoir can be and thenfueled to also FdUMP by the by transformation RRM1 and 2 of [6 fluorouridin,7]. FUMPe reservoir (FUR) triggered can be by fueled uridine also kinase by the (UK), transformation generated of fluorouridinein turn by (FUR)the conversion triggered of by5FU uridine by the uridine kinase phosphorylase (UK), generated (UP) in. All turn these by steps the conversion are summarized of 5FU by the uridinein the schematic phosphorylase view in (UP).Figure All 1. these steps are summarized in the schematic view in Figure1. FigureFigure 1. A 1. simplified A simplified diagram diagram of of the the 5-fluorouracil 5-fluorouracil (5FU) (5FU) conversion conversion system. system. Uridine Uridine phosphorylase phosphorylase (UP),(UP), uridine uridine kinase kinase (UK), fluorouridine(UK), fluorouridine (FUR), orotate(FUR), phosphoribosyltransferaseorotate phosphoribosyltransferase (OPRT), fluorouridine(OPRT), fluorouridine monophosphate (FUMP), ribosyl reductase complexes (RRM1/2), fluorodeoxyuridine monophosphate (FUMP), ribosyl reductase complexes (RRM1/2), fluorodeoxyuridine monophosphate monophosphate (FdUMP), thymidylate synthase (TYMS), thymidine phosphorylase (TYMP), (FdUMP), thymidylate synthase (TYMS), thymidine phosphorylase (TYMP), fluorodeoxyuridine fluorodeoxyuridine (FdU), thymidine kinase 1 (TK1), nucleotidase (NT5E), uridine monophosphate (FdU), thymidine kinase 1 (TK1), nucleotidase (NT5E), uridine monophosphate synthase (UMPS), synthase (UMPS), phosphoribosyl pyrophosphate amidotransferase (PPAT), and fluorouridine phosphoribosyldiphosphate pyrophosphate(FUDP). amidotransferase (PPAT), and fluorouridine diphosphate (FUDP). In orderIn order to to study study chemoresistance chemoresistance and and its itsside-effects, side-effects, it is important it is important to remark to the remark action of the the action of thedihydropyrimidine dihydropyrimidine dehydrogenase dehydrogenase (DPD), (DPD), which catabolizes which catabolizes 5FU into 5FUinactive into metabolites inactive metabolitesby the by theliver liver [8], producing [8], producing fluoroacetate fluoroacetate and fluorohydrox and fluorohydroxypropionicypropionic acid, which have acid, been which reported have to been reportedinduce to cardiotoxicity induce cardiotoxicity and neurotoxicity and neurotoxicity [9,10]. While 5FU [9, 10is normally]. While administered 5FU is normally intravenously administered to intravenouslyavoid the DPD to avoiddigestion the by DPDthe gut digestion wall, the use by of the oral gut 5FU wall, prodrugs the (capecitabine, use of oral tegafur, 5FU prodrugs and (capecitabine,doxifluridine) tegafur, [11–13] and doxifluridine)and DPD inhibitors[11–13] and(gimeracil, DPD inhibitors uracil, (gimeracil,eniluracil, uracil,5-chloro-2,4- eniluracil, dihydroxypyridine) [14–16] partially resolves the above-described side-effects. Every single mutation 5-chloro-2,4-dihydroxypyridine) [14–16] partially resolves the above-described side-effects. Every in one of the above-described enzymes might affect irreversibly the sensitivity to 5FU leading to the single mutation in one of the above-described enzymes might affect irreversibly the sensitivity to 5FU generation of subpopulations of chemoresistant cancer cells, which cause therapy failure. Indeed, leadinggastric to the cancer generation (GC), together of subpopulations with bladder, of lung chemoresistant and melanoma, cancer is one cells, tumor which histotype cause therapy with the failure. Indeed,highest gastric mutation cancer frequency (GC), together [17]. GC with is bladder,to date th lunge fifth and most melanoma, frequently is diagnosed one tumor cancer histotype and withthe the highestthird mutation leading frequencycause of cancer [17]. GCdeath is toaccording date the to fifth GLOBOCAN most frequently 2018 [18], diagnosed and due cancer to the and lack the of third leadingmacroscopical cause of cancer manifestations death according in the early to GLOBOCAN phases of the 2018 disease, [18], andit is due often to diagnosed the lack of only macroscopical after manifestationsmetastasis in theadvanced early phasesstages, ofwhen the surgery disease, is it no is oftent practicable diagnosed and the only only after therapeutic metastasis option in advanced is stages,chemotherapy. when surgery According is not practicable to the international and the onlyguidelines, therapeutic the first-line option approach is chemotherapy. for therapy According would to the internationalinvolve treating guidelines, HER2 negative