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5-fluorouracil and other fluoropyrimidines in colorectal cancer:

Past, present and future

Sona Vodenkova, Tomas Buchler, Klara Cervena, Veronika

Veskrnova, Pavel Vodicka, Veronika Vymetalkova

PII: S0163-7258(19)30199-8
DOI: https://doi.org/10.1016/j.pharmthera.2019.107447
Reference: JPT 107447

To appear in: Pharmacology and Therapeutics

Please cite this article as: S. Vodenkova, T. Buchler, K. Cervena, et al., 5-fluorouracil and
other fluoropyrimidines in colorectal cancer: Past, present and future, Pharmacology and
Therapeutics(2019), https://doi.org/10.1016/j.pharmthera.2019.107447

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that apply to the journal pertain.

© 2019 Published by Elsevier.

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P&T #23629

5-fluorouracil and other fluoropyrimidines in colorectal cancer: past, present and future

Sona Vodenkova1,2,3, Tomas Buchler4, Klara Cervena1,2, Veronika Veskrnova4, Pavel Vodicka1,2,5,

Veronika Vymetalkova1,2,5*

1
Department of Molecular Biology of Cancer, Institute of Experimental Medicine of the Czech Academy of

Sciences, Videnska 1083, 142 00 Prague, Czech Republic

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2
Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University, Albertov 4, 128 00

Prague, Czech Republic

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3
Department of Medical Genetics, Third Faculty of Medicine, Charles University, Ruska 2411/87, 100 00 Prague,
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Czech Republic

4
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Department of Oncology, First Faculty of Medicine, Charles University and Thomayer Hospital, Videnska 800,

140 59 Prague, Czech Republic

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5
Biomedical Centre, Faculty of Medicine in Pilsen, Charles University, Alej Svobody 76, 323 00 Pilsen, Czech

Republic
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Corresponding author:

*Veronika Vymetalkova
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Department of Molecular Biology of Cancer

Institute of Experimental Medicine of the Czech Academy of Sciences

Videnska 1083, 142 00 Prague, Czech Republic

Tel. +420 241 062 699

Fax. +420 241 062 782

E-mail veronika.vymetalkova@iem.cas.cz

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Abstract

5-Fluorouracil (5-FU) is an essential component of systemic chemotherapy for colorectal cancer (CRC)

in the palliative and adjuvant settings. Over the past four decades, several modulation strategies

including the implementation of 5-FU-based combination regimens and 5-FU pro-drugs have been

developed and tested to increase the anti-tumor activity of 5-FU and to overcome the clinical

resistance.

Despite the encouraging progress in CRC therapy to date, the patients' response rates to therapy

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continue to remain low and the patients' benefit from 5-FU-based therapy is frequently compromised

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by the development of chemoresistance. Inter-individual differences in the treatment response in CRC

patients may originate in the unique genetic and epigenetic make-up of each individual.
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The critical element in the current trend of personalized medicine is the proper comprehension of
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causes and mechanisms contributing to the low or lack of sensitivity of tumor tissue to 5-FU-based
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therapy. The identification and validation of predictive biomarkers for existing 5-FU-based and new

targeted therapies for CRC treatment will likely improve patients' outcomes in the future.
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Herein we present a comprehensive review summarizing options of CRC treatment and the
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mechanisms of 5-FU action at the molecular level, including both anabolic and catabolic ways. The
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main part of this review comprises the currently known molecular mechanisms underlying the

chemoresistance in CRC patients. We also focus on various 5-FU pro-drugs developed to increase the

amount of circulating 5-FU and to limit toxicity. Finally, we propose future directions of personalized

CRC therapy according to the latest published evidence.

Keywords: colorectal cancer, 5-fluorouracil, 5-fluorouracil pro-drugs, oral fluoropyrimidines, drug

resistance, chemoresistance.

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Abbreviations:

5-FU, 5-fluorouracil; 5’-DFUR, 5’-deoxy-5-fluorouridine; 53BP1, P53 binding protein 1; ABC, ATP-binding cassette;

ADAM9, disintegrin and metalloprotease domain-containing protein 9; Bcl, B-cell lymphoma; BER, base excision

repair; CD44v6, CD44 isoform containing variant exon v6; CDHP, 5-chloro-2,4-dihydroxypyridine; CH2THF (also

5,10-CH2THF), 5,10-methylenetetrahydrofolate; CRC, colorectal cancer; CSC, cancer stem cell; CYP2A6,

cytochrome P450 2A6; DIF, DPD inhibitory fluoropyrimidines; dNTP, deoxynucleotide

triphosphate/deoxynucleotide; DPD, dihydropyrimidine dehydrogenase; dTMP, 2’-deoxythymidine-5’-

monophosphate/deoxythymidinemonophosphate; dTTP, 2'-deoxythymidine-5'-triphosphate/deoxythymidine

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triphosphate; dUMP, 2'-deoxyuridine-5'-monophosphate/deoxyuridinemonophosphate; dUTP, 2´-deoxyuridine-

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5´-triphosphate/deoxyuridine triphosphate; dUTPase, deoxyuridine triphosphatase; EGFR, epidermal growth

factor receptor; FBAL, α-fluoro-β-alanine; FdUDP, fluorodeoxyuridine diphosphate; FdUMP, 5-fluoro-2’-

-p
deoxyuridine-5’-monophosphate/fluorodeoxyuridine monophosphate; FdUR, fluorodeoxyuridine; FdUTP, 5-
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fluoro-2'-deoxyuridine-5'-triphosphate; FPs, fluoropyrimidines; FTD, trifluridine; FUDP, fluorouridine

diphosphate; FUMP, 5-fluorouridine monophosphate; FUPA, α-fluoro-β-ureido-propionic acid; FUTP, 5-

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fluorouridine-5’-triphosphate; GSTs, glutathione S-transferases; lncRNAs, long non-coding RNAs; LV, leucovorin;

mCRC, metastatic colorectal cancer; miRNAs (also miRs), microRNAs; MMR, mismatch repair; MRPs, multidrug
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resistance-associated proteins; MSI, microsatellite instable/instability; MSI-H, MSI-high/MMR-deficiency; MSS,

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microsatellite‐stable; MTHFR, methylene tetrahydrofolate reductase; ncRNAs, non-coding RNAs; NER, nucleotide

excision repair; NF-κB, nuclear factor κB; OPRT, orotate phosphoribosyl transferase; OXO, potassium oxonate;
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RNR, ribonucleotide reductase; S-1 (also TS-1), Tegafur + 5-chloro-2,4-dihydroxypyridine (CDHP) and potassium

oxonate (OXO); SLC, solute carrier; SNPs, single nucleotide polymorphisms; TAS-102, Trifluridin-Tipiracil; TK,

thymidine kinase; TP, thymidine phosphorylase; TPI, tipiracil hydrochloride; TS, thymidylate synthase; TYMS,

thymidylate synthetase gene; UMPS, uridine monophosphate synthase; UP, uridine phosphorylase; UTP, uridine-

5'-triphosphate.

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Table of Contents

1. Introduction ................................................................................................................................. 5
1.1. History ..................................................................................................................................... 5
1.2. 5-FU and colorectal cancer ...................................................................................................... 5
1.3. Colorectal cancer treatment ................................................................................................... 6
2. Mechanism of 5-FU action .......................................................................................................... 8
2.1. Anabolic way of 5-FU transformation ..................................................................................... 9
2.2. Catabolic way of 5-FU transformation .................................................................................. 13
3. 5-FU pro-drugs (oral FPs)........................................................................................................... 14
3.1. 5’-deoxy-5-fluorouridine (5’-DFUR) ....................................................................................... 17

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3.2. Capecitabine .......................................................................................................................... 17

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3.3. Tegafur .................................................................................................................................. 18
3.5. S-1 .......................................................................................................................................... 19
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3.6. Emitefur ................................................................................................................................. 20
3.7. Trifluridin-Tipiracil (TAS-102) ................................................................................................ 20
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4. Resistance to 5-FU therapy ....................................................................................................... 21
4.1. Thymidylate synthase (TS)..................................................................................................... 23
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4.2. Methylene tetrahydrofolate reductase (MTHFR) ................................................................. 27

4.3. Dihydropyridine dehydrogenase (DPD) ................................................................................. 28
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4.4. Thymidine phosphorylase (TP) .............................................................................................. 29

4.5. Orotate phosphoribosyl transferase (OPRT) ......................................................................... 31
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4.6. Thymidine kinase (TK) ........................................................................................................... 31

4.7. ATP-binding cassette (ABC) and solute carrier (SLC) transporters ........................................ 32
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4.8. Genes involved in metabolic activation of 5-FU pro-drugs ................................................... 33

4.9. DNA repair ............................................................................................................................. 34
4.10. Non-coding RNAs (ncRNAs) ............................................................................................... 37
4.11. Other relevant genes ......................................................................................................... 40
4.12. Autophagy ......................................................................................................................... 41
5. Future perspectives ................................................................................................................... 42
6. Conclusions ................................................................................................................................ 45
Conflict of Interest Statement ............................................................................................................... 45
Acknowledgements ............................................................................................................................... 46
References ............................................................................................................................................. 46

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1. Introduction

1.1. History

5-Fluorouracil (5-FU) represents an anti-metabolite with substitution of fluorine for hydrogen at the C-

5 position of uracil. The thymine-uracil/5-FU exchange caused by thymine replacement in DNA

consequently leads to the formation of adenine-uracil/5-FU base pairs (Vertessy & Toth, 2009). 5-FU

was one of the first chemotherapeutic drugs reported to have anticancer activity. 5-FU was first

synthesized by Heidelberger et al. (Heidelberger, et al., 1957) 60 years ago in an attempt to utilize the

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increased uptake of uracil by tumors. Already in 1954 Rutman et al. (Rutman, Cantarow, & Paschkis,

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1954) showed that uracil was incorporated into rat hepatomas more rapidly than into non-malignant
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tissues. The reason was that uracil derivative would produce a differential cytotoxicity, because uracil

is preferentially taken up by tumors, although it does not have more specificity for the enzymes from
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tumor tissues. 5-FU was chosen not only because F and H are very similar in molecular weight but also
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because of the exceptional stability of the carbon-F bond. These considerations led Heidelberger et al.

(Ishiba, et al., 2018) to predict that 5-FU would become incorporated into RNA and inhibit DNA
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biosynthesis in vivo as well. In addition, in 1956, Handschumacher et al. (Handschumacher & Welch,
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1956) reported the tumor-inhibitory activity of 6-azauracil. 6-azauracil is not incorporated into RNA

(Cihak, Skoda, & Sorm, 1963; de Kloet, 1968), even though it is converted into structural subunits of
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RNA, such as 6-azauridine and 6-aza uridylic acid. However, 6-azauridine was withdrawn from clinical

use due to the occurrence of serious adverse effects, such as embryotoxicity (Dostal & Jelinek, 1979),

and arterial and venous thromboembolic episodes (Gitel, Grieco, Wessler, & Snyderman, 1979).

1.2. 5-FU and colorectal cancer

Colorectal cancer (CRC) is one of the principal causes of morbidity and mortality with approximately

1.8 million new cases worldwide in 2018. CRC is the second most common cause of cancer-related

mortality, responsible for estimated 880,000 deaths in 2018 (Bray, et al., 2018). Surgery remains the

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most important treatment modality for CRC patients and is the only curative option for patients with

localized and locoregional CRC, as well as for those with resectable distant metastases.

5-FU is a chemotherapy drug commonly used to treat different malignant tumors, including breast,

pancreatic, skin, stomach, esophageal, and head and neck cancers. In CRC, intravenous and oral 5-FU

or other fluoropyrimidines (FPs) have become the mainstay of systemic treatment since the 1990s.

Owing to its unpredictable gastrointestinal absorption and marked variation in pharmacokinetics, use

of oral 5‐FU alone was abandoned early. Since that time, research has focused on the biomodulation

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of 5‐FU to improve its therapeutic effectiveness and cytotoxicity (Chionh, Lau, Yeung, Price, & Tebbutt,

2017). Leucovorin (LV, Folinic Acid), a chemoprotectant inhibiting side effects caused by 5-FU, is used

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for the potentiation of 5-FU effect. In combination with 5-FU, LV has been shown to improve patients'
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survival and the tumor response rate and represents a standard part of all 5-FU-based regimens
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(Thirion, et al., 2004).
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1.3. Colorectal cancer treatment

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Adjuvant (post-operative) chemotherapy is recommended after curative tumor resection for all fit

patients with stage III colon cancer, as well as for patients having stage II colon cancer with high-risk
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features (T4 tumor, tumor perforation, fewer than 12 removed lymph nodes (Stintzing, 2014)). All
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clinical and/or histologic stage II and III rectal cancers should be considered for adjuvant chemotherapy

(Ahn, et al., 2017). Additionally, in rectal cancer, radiotherapy with concomitant FPs is routinely used

as neoadjuvant (prior to surgical resection) treatment.

In metastatic disease, agents targeting angiogenesis such as bevacizumab, ramucirumab, and

aflibercept or epidermal growth factor receptor (EGFR)-directed treatments including panitumumab

and cetuximab are commonly used along with FPs-based chemotherapy. Regorafenib, an

angiogenesis-targeting tyrosine kinase inhibitor, or trifluridin-tipiracil (a fluoropyrimidine) are

modestly active in late-stage metastatic colorectal cancer (mCRC) (Glynne-Jones, et al., 2017; Labianca,

et al., 2013; Van Cutsem, et al., 2016).

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Despite progress in novel cancer therapies, 5-FU still represents one of the most effective and most

commonly used agents in CRC treatment and the main constituent of chemotherapy combination

regimens (D. Sargent, et al., 2009). Most patients will be exposed to multiple FPs-based regimens

administered sequentially.

Although 5-FU ranks among the safest chemotherapy agents, severe side and toxic effects still occur

in a proportion of CRC patients. The clinical manifestation of 5-FU toxicity includes fever, fatigue,

mucositis, stomatitis, nausea, vomiting, and diarrhea (Latchman, Guastella, & Tofthagen, 2014). Other

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common toxic effects comprise leukopenia, neutropenia, thrombocytopenia, anemia, neuropathy,

skin rash, and hand-foot syndrome (L. Zhang, Xing, Meng, Wang, & Zhong, 2018). Neurologic

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abnormalities such as cerebellar ataxia and changes in cognitive function have also been scarcely
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reported, occurring in less than one percent of the patients (Cordier, et al., 2011; Saif, Syrigos, Mehra,
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Mattison, & Diasio, 2007).
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For increased efficacy, FPs are frequently combined with oxaliplatin and/or irinotecan (Akhtar,

Chandel, Sarotra, & Medhi, 2014). Oxaliplatin, a diaminocyclohexane platinum complex, similarly to
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cisplatin and carboplatin, forms DNA adducts. Irinotecan, (sold, among others, under the brand name

Camptosar, CPT-11, a water-soluble, semisynthetic derivative of camptothecin) inhibits the

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topoisomerase I via the active metabolite SN-38 during replication and/or transcription that eventually
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leads to a permanent DSBs, resulting in cell death (Wellstein A., 2018).

Although it has been reported that the patients' survival has improved after the addition of oxaliplatin

or irinotecan to the 5-FU regimen, the toxicity has also increased (Boige, et al., 2010). The efficacy of

5-FU/LV combined with oxaliplatin (FOLFOX) or irinotecan (FOLFIRI) in the first-line treatment of mCRC

is comparable (Tournigand, et al., 2004). The combination therapies FOLFOX and FOLFIRI have become

established as efficacious cytotoxic regimens for the treatment of mCRC, resulting in overall

improvement in survival of approximately 2 years (Gustavsson, et al., 2015).

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2. Mechanism of 5-FU action

5-FU exerts its antitumor effects mainly through the inhibition of thymidylate synthase (TS) leading to

disrupting the intracellular deoxynucleotide pools required for DNA replication. Other possible sites of

action comprise incorporation into RNA (where it may replace more than 50% of uracil (Horowitz &

Chargaff, 1959)) with the subsequent disruption of RNA synthesis following its anabolism and

incorporation into DNA resulting in its fragmentation (Daher, Harris, & Diasio, 1990).

Nevertheless, it has been demonstrated that only up to 3% of the original dose of 5-FU mediates the

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cytotoxic effects on tumor and non-malignant cells through anabolic actions. A vast majority of 5-FU is

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catabolized to inactive metabolites by dihydropyrimidine dehydrogenase (DPD) in the liver, where it is

abundantly expressed (Diasio & Harris, 1989; Saif, Syrigos, & Katirtzoglou, 2009)). Moreover, when 5-
-p
FU is administrated by infusion, nearly 20% of the dose is directly excreted in the urine. The metabolism
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of 5-FU and its main pro-drugs are summarized in detail in Figure 1.
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Figure 1. 5-FU and its pro-drugs, their metabolism and consequences of their action in cancer cell.

The figure summarizes the metabolism of 5-FU and its main pro-drugs Capecitabine, Tegafur and 5′-deoxy-5-
fluorouridine. On the left-hand side are depicted the anabolic pathways of their transformation into individual

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metabolites in the cancer cell. On the right-hand side, the catabolic pathway in the liver is represented. The figure
is supplemented by the consequences caused by 5-FU active metabolites in the cancer cell.
Abbreviations: BUP-1, β-ureidopropionase; CDA, cytidine deaminase; CES, carboxylesterase; CH2THF, 5,10-
methylenetetrahydrofolate; CYP2A6, cytochrome P450 2A6; DHP, dihydropyrimidinase; DPD, dihydropyrimidine
dehydrogenase; dTMP, deoxythymidinemonophosphate; dUMP, deoxyuridinemonophosphate; FdUMP,
fluorodeoxyuridine monophosphate; MTHFR, methylene tetrahydrofolate reductase; OPRT, orotate
phosphoribosyl transferase; PRPP, phosphoribosyl pyrophosphate; RNR, ribonucleotide reductase; TK, thymidine
kinase; TP, thymidine phosphorylase; TS, thymidylate synthase; UDPK, uridine diphosphate kinase; UK, uridine
kinase; UMPK, uridine monophosphate kinase; UP, uridine phosphorylase.

2.1. Anabolic way of 5-FU transformation

After intravenous administration, 5-FU enters cells promptly by the same transport mechanism as

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uracil (Wohlhueter, McIvor, & Plagemann, 1980). Among all nucleic acid bases, uracil and its 5-FU

derivatives are most easily incorporated into tumor cells due to its structure (Tanaka, Fukuse, Wada,

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& Fukushima, 2000). However, it was also proved that 5-FU could be passively transported through
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paracellular and transcellular route in cancer cell monolayers (Imoto, 2009; Yuasa, Matsuhisa, &
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Watanabe, 1996) or 5-FU rapidly crosses the blood-brain barrier by passive diffusion (Kerr, Zimm,

Collins, O'Neill, & Poplack, 1984). 5-FU itself evinces no cytotoxic effects, the anti-tumor effects are
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produced after its interaction with phosphorylated sugars via enzymatically catalyzed reactions and its
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conversion into active metabolites, such as 5-fluorouridine-5’-triphosphate (FUTP), 5-fluoro-2'-

deoxyuridine-5'-triphosphate (FdUTP) and 5-fluoro-2’-deoxyuridine-5’-monophosphate (FdUMP)

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(Figure 1).
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After entering the cancer cell, 5-FU can be converted into 5-fluorouridine monophosphate (FUMP)

either via a direct mechanism involving orotate phosphoribosyl transferase (OPRT; gene product of

uridine monophosphate synthase (UMPS)) in the presence of phosphoribosyl pyrophosphate (PRPP)

or indirectly by the actions of uridine phosphorylase (UP) and uridine kinase (UK). FUMP is further

phosphorylated to fluorouridine diphosphate (FUDP), which is then either phosphorylated to the

active metabolite FUTP or converted to fluorodeoxyuridine diphosphate (FdUDP) by ribonucleotide

reductase (RNR) (Longley, Harkin, & Johnston, 2003). FdUMP arises from 5-FU due to its indirect

conversion by thymidine phosphorylase (TP) and thymidine kinase (TK). FdUMP is also produced from

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the reduction of FUDP by RNR. In some types of cells, this type of FdUMP formation predominates

compared to its production by TP/TK (Thorn, et al., 2011). Active metabolites FUTP/FdUTP produced

during subsequent processes are incorporated into RNA/DNA instead of uridine-5'-triphosphate/2'-

deoxythymidine-5'-triphosphate (UTP/dTTP, respectively).

FdUMP also inhibits the activity of TS in the ternary complex formed along with 5,10-

methylenetetrahydrofolate (5,10-CH2THF; CH2THF, Figure 2). The ternary complex blocks the access

of 2'-deoxyuridine-5'-monophosphate (dUMP) to the nucleotide-binding site of TS by competition with

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FdUMP and by inhibiting 2’-deoxythymidine-5’-monophosphate (dTMP) synthesis. This ultimately

leads in pool imbalances of deoxynucleotides (dNTPs) (Longley, et al., 2003; Miura, et al., 2010). Since

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TS is a key enzyme in the de novo synthesis of dTMP, it is necessary for DNA replication and repair. If
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DNA synthesis is disrupted via inhibition of TS activity, tumor cell death is induced. Depletion of dTMP
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results in the subsequent depletion of dTTP, which perturbs the levels of the other dNTPs (Jackson,

1984). The pool imbalances of dNTPs disrupt DNA synthesis and repair (Yoshioka, et al., 1987). As a
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result, the inhibition of TS leads to the accumulation of dUMP, which is phosphorylated to 2´-
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deoxyuridine-5´-triphosphate (dUTP) (Mitrovski, Pressacco, Mandelbaum, & Erlichman, 1994). The

increased dUTP/dTTP and 5-FdUTP/dTTP ratios promote uracil misincorporation into DNA due to the
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lack of competition for DNA polymerase between FdUTP and dTTP (Ingraham, Tseng, & Goulian, 1982).
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High levels of uracil in the biosynthetic pool may be bypassed by hydrolyzing dUTP to dUMP and

pyrophosphate by deoxyuridine triphosphatase (dUTPase, also known as DUT). This reaction

additionally supplies TS with its substrate dUMP (Toth, Varga, Kovacs, Malnasi-Csizmadia, & Vertessy,

2007). The physiological function of dUTPase is to reduce dUTP accumulation and prevent

misincorporation of the non-canonical nucleotide into DNA (Hagenkort, et al., 2017). In terms of

treatment, however, this activity could hinder the therapeutic efficacy of 5-FU.

The relatively long retention of FdUTP in DNA might be due to the favorable incorporation conditions,

but also due to inefficient DNA repair. The latter process is catalyzed by uracil-DNA-glycosylases

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(Caradonna & Cheng, 1980; Mauro, De Riel, Tallarida, & Sirover, 1993), specifically by SMUG1 (An,

Robins, Lindahl, & Barnes, 2007), and presumably functions at different rates in the various tumors,

considering the large variation in the incorporation of 5-FU into DNA. It is unlikely that TS inhibition is

related to 5-FU metabolites incorporation into RNA but a prolonged TS inhibition might favor

incorporation of 5-FU derivates into DNA (Noordhuis, et al., 2004).

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Figure 2. Mechanism of thymidylate synthase inhibition by 5-FU metabolites along with its

consequences (adapted from (Longley & Johnston, 2005).

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TS is an enzyme catalyzing the conversion of dUMP to dTMP in the presence of CH2THF which serves as the donor
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of the methyl group. FdUMP, the 5-FU active metabolite, binds to the nucleotide-binding site of TS and forms a
stable ternary complex with TS and CH2THF. This complex blocks the access of dUMP to the nucleotide-binding
site and inhibits dTMP synthesis. This consequently leads in pool imbalances of dNTPs and the accumulation of
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dUMP, which is phosphorylated to dUTP, both of which cause DNA damage. The extent of DNA damage caused
by dUTP is dependent on the levels of the pyrophosphatase dUTPase and UDG. dTMP can be salvaged from
thymidine through the action of TK.
Abbreviations: 5-FU, 5-fluorouracil; CH2THF, 5,10-methylenetetrahydrofolate; dNTPs, deoxynucleotides; dTDP,
deoxythymidine diphosphate; dTMP, deoxythymidine monophosphate; dTTP, deoxythymidine triphosphate;
dUDP, deoxyuridine diphosphate; dUMP, deoxyuridine monophosphate; dUTP, deoxyuridine triphosphate;
FdUMP, fluorodeoxyuridine monophosphate; TDPK, thymidine diphosphate kinase; TK, thymidine kinase; TMPK,
thymidine monophosphate kinase; TS, thymidylate synthase; UDG, uracil-DNA glycosylase; UDPK, uridine
diphosphate kinase; UMPK, uridine monophosphate kinase.

For a long time, limited data were available for the efficacy of 5-FU incorporation into RNA and DNA in

human tumor tissues. Noordhuis et al. (Noordhuis, et al., 2004) therefore performed a measurement

of 5-FU incorporation in human tumor biopsy specimens after administration of 5-FU. The authors

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observed maximal incorporation of 5-FU active metabolites at 24 hours after 5-FU administration

compared to 2 and 48 hours. Incorporation into RNA but not DNA significantly correlated with intra-

tumor 5-FU metabolite levels and incorporation into RNA significantly surpassed incorporation into

DNA. However, incorporation into DNA significantly correlated with incorporation into RNA. In contrast

to TS inhibition, neither the incorporation into RNA nor DNA significantly correlated with response to

5-FU therapy. There is increasing evidence that 5-FU cytotoxicity may be mainly due to the impairment

of RNA processing pathways, specifically due to its incorporation into RNA (reviewed in (Bagrij,

Kralovanszky, Gyergyay, Kiss, & Peters, 1993; Klubes & Leyland-Jones, 1989; Parker & Cheng, 1990)).

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In vitro studies have shown that 5-FU incorporation into RNA but not DNA was associated with cell

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death (Geoffroy, Allegra, Sinha, & Grem, 1994; Pritchard, Watson, Potten, Jackman, & Hickman, 1997).
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It has been shown that uridine, but not thymidine, relieved the cytotoxic and apoptotic effects of 5-FU

(Hoskins & Scott Butler, 2007; Longley, et al., 2003). RNA containing 5-FU has been shown to interfere
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with standard processing and maturation of rRNA, tRNA, and mRNA precursors (Mojardin, Botet,
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Quintales, Moreno, & Salas, 2013). Nevertheless, the significance of these observed effects and their

contribution to the cytotoxicity of 5FU remains unclear.

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Similarly, it was not clear whether the dysfunction of RNA by FUTP, inhibition of DNA synthesis by
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FdUMP or rather the interplay of both above are the main contributors to cytotoxic effects of clinically
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administrated 5-FU. However, it is currently well known that 5-FU cytotoxic activity is highly dependent

on the administration schedule: TS inhibition by FdUMP is prevailing when 5-FU is given as a continuous

infusion. In contrast, FUTP incorporation into RNA is considered as being the main mechanism of action

when 5-FU is administered as a bolus (Aschele, et al., 2002; Humeniuk, et al., 2009; Ibrahim, et al.,

2012; Katsumata, et al., 2003). What are the underlying reasons? Only high 5-FU concentration can

lead to RNA dysfunction (10-100mM, that is 1.3-13mg/ml in vitro) (Spiegelman, et al., 1980), while

DNA synthesis can be inhibited also at the lower 5-FU concentration (0.5-1.0mM) (Parker & Cheng,

1990). Therefore, when 5-FU is administered intravenously by single bolus injection, 5-FU

concentration in blood is temporarily enhanced at a higher level and cytotoxic effects of 5-FU are

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produced mainly by dysfunction of RNA. On the other hand, when 5-FU is administered by intravenous

continuous infusion, relatively low 5-FU concentration is maintained in blood for a long time and anti-

tumor effects of 5-FU are produced mainly by inhibition of DNA synthesis (Aschele, Sobrero, Faderan,

& Bertino, 1992; Parker & Cheng, 1990). Cytotoxic effects of 5-FU are poor even at high concentrations

when it reaches tumor cells for a short time, while enhanced anti-tumor effects can be obtained even

at low concentrations when it contacts tumor cells for a long time. So, it is obvious that 5-FU does not

evince dose-dependent but rather time-dependent chemotherapeutic effects.

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2.2. Catabolic way of 5-FU transformation

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5-FU shows poor bioavailability due to its rapid catabolic degradation to 5,6-dihydro-5-fluorouracil

(DHFU) by DPD, ultimately leading to the formation of α-fluoro-β-ureido-propionic acid (FUPA) and
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then α-fluoro-β-alanine (FBAL), subsequently excreted via the kidneys (Figure 1). The expression of
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DPD, a primary and rate-limiting enzyme involved in 5-FU metabolism, occurs in the liver, gut and
lP

various other tissues (Milano & McLeod, 2000; Miura, et al., 2010). In 1987, Heggie et al. (Heggie,

Sommadossi, Cross, Huster, & Diasio, 1987) investigated the kinetics of 5-FU and its metabolites in
na

cancer patients following intravenous bolus administration of radio-labeled 5-FU. The authors revealed
ur

that approximately 60-90% of 5-FU was excreted in urine as FBAL or as carbon dioxide by exhaled air

within 24 hours (Takiuchi & Ajani, 1998). When 5-FU is administered intravenously by single bolus
Jo

injection at dose range of 600-900 mg/m2, all the administrated 5-FU is eliminated from blood and

bone marrow within 2 hours after the administration (Fraile, Baker, Buroker, Horwitz, & Vaitkevicius,

1980). Half-life of the administered 5-FU in vivo has been reported to be only 10-20 minutes (Tanaka,

et al., 2000).

Oral administration of 5-FU in the form of 5-FU pro-drugs (oral FPs) has been hampered by incomplete

and erratic bioavailability due to substantial variability in the activity of DPD. Therefore, it is associated

with unpredictable levels of 5-FU in the plasma due to pronounced intra- and inter-patient variability

in its adsorption/elimination (Fraile, et al., 1980). In general, the former variability may be partially

13
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explained by the detected circadian variations of DPD activity in humans (Daher, Harris, Willard, &

Diasio, 1991). The inter-patient variability in 5-FU elimination may be associated with the differences

in DPD enzyme activity, which may be genetically determined. Two to four percent of the population

is estimated to be deficient in this enzyme (Lamont & Schilsky, 1999), thus providing a potential

pharmacogenetic basis for 5-FU toxicity (Mattison, Soong, & Diasio, 2002). Cancer patients with DPD

deficiency were shown to be at increased risk of severe toxicities, including diarrhea, mucositis, and

neurotoxicity, as well as death, after administration of standard doses of 5-FU (Saif, Syrigos, et al.,

2009). This phenomenon may be overcome by designing novel 5-FU pro-drugs that avoid its DPD-

of
mediated degradation in the liver.

3. 5-FU pro-drugs (oral FPs)

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-p
5-FU pro-drugs differ from 5-FU in a variety of chemical alterations. Their administration is more
re
convenient by reducing the time spent in an infusion suite or on an infusion pump (Hammond, Swaika,
lP

& Mody, 2016). They are designed to be well absorbed intact through the gastrointestinal mucosa and

subsequently enzymatically converted into 5-FU in the liver or within the tumor itself, in order to
na

expose the tumor to 5-FU for a longer time but at lower concentrations than those observed after an
ur

intravenous bolus, therefore minimizing toxic adverse effects. New oral FPs thus provide protracted 5-

FU delivery, which offers advantages that include schedule flexibility and reductions in professional
Jo

health care resource requirements, administration costs, and toxicity-related hospitalization. These

advantages may reduce the overall cost of treatment (Brito, et al., 1999).

Each agent has been developed according to a specification with a well-defined mechanism for

liberation of the active principle. Some are designed to function alone, and others require co-

administration of a modulator. The aim is to mimic the pharmacokinetics of 5-FU administered by

continuous intravenous infusion, not only by virtue of their chemical structure, but also by careful

choice of dosage (Malet-Martino & Martino, 2002).

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Several modulation strategies have been developed to increase the anti-tumor activity of 5-FU and to

overcome the clinical resistance, primarily with the use of oral FPs that should be at least as active as

a continuous infusion of 5-FU. These strategies include decreasing 5-FU degradation, increasing 5-FU

activation, and enhancing the TS binding activity of FdUMP (Longley, et al., 2003). In practice, this is

achieved by the following approaches: 1. the use of 5-FU analogs/pro-drugs, 2. the use of 5-FU and an

enzyme inhibitor (i.e. DPD inhibitor), and 3. the use of 5-FU pro-drugs in combination with enzyme

inhibitors (reviewed in (Schoffski, 2004)). Table 1 summarizes these three groups of oral FPs which are

in details described in the following subchapters.

of
Table 1. A brief overview of the treatment strategy by oral fluoropyrimidines (updated according to

(Schoffski, 2004)).

ro
-p
Group of Drug Pro-drug Enzyme inhibitor
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fluoropyrimidines
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1. 5-FU analogs/Pro-

drugs only
na

5’-DFUR Yes No
ur

5-FU pro-drug
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Capecitabine Yes No

5′-DFUR pro-drug

Tegafur Yes No

5-FU pro-drug

2. 5-FU and enzyme

inhibitors only

5-FU + Eniluracil No Yes

DIF

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3. 5-FU pro-drugs and

enzyme inhibitors

S-1 Yes Yes

Tegafur/5-FU pro-drug DIF: CDHP and OXO

FT: CDHP: OXO 1:0.4:1

Tegafur-uracil Yes Yes

Tegafur/5-FU pro-drug DIF: Uracil

of
Tegafur-uracil/LV Yes Yes

FT: Uracil 1:4 DIF: uracil

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-p FT/5-FU pro-drug TS inhibitor: LV

Emitefur Yes Yes

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Emitefur/5-FU pro-drug DIF: CNDP 1:1

Trifluridin-tipiracil No Yes
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(TAS-102) TP inhibitor: Tipiracil

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Abbreviations: 5′-DFUR, 5′-deoxy-5-fluorouridine; CDHP, 5-chloro-2, 4-dihydroxypyridine; CNDP, 3-cyano-2,6-

dihydroxypyridine; DIF, DPD inhibitory fluoropyrimidines; FT, Tegafur; LV, Leucovorin; OXO, potassium oxonate;
TP, thymidine phosphorylase; TS, thymidylate synthase.
ur
Jo

Orally administered FPs act either through the catabolic pathway of 5-FU, i.e. DPD inhibitory

fluoropyrimidines (DIF) such as Eniluracil + 5-FU or Tegafur-uracil, or through the anabolic pathway,

i.e. non-DIF/pro-drugs such as 5’-deoxy-5-fluorouridine (5’-DFUR), Capecitabine or Tegafur. They can

also function through both the catabolic and anabolic pathways, i.e. DIF, other enzyme inhibitors, and

non-DIF/pro-drugs such as S-1, Tegafur-uracil/LV, and Emitefur. These pro-drugs are by a sequence of

enzymatic reactions converted to 5-FU in the tumor microenvironment. Several other oral FPs have

been designed and currently are undergoing clinical trials or are used routinely in the clinic.

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3.1. 5’-deoxy-5-fluorouridine (5’-DFUR)

In 1979 and 1980, two research groups of Cook et al. at Hoffmann-La Roche (Switzerland) (Cook,

Holman, Kramer, & Trown, 1979) and Ishitsuka et al. at the Nippon Roche Research Center (Japan)

(Ishitsuka, et al., 1980), respectively, reported the development of 5’-DFUR (5’-deoxy-5-fluorouridine,

5’-fluoro-5’-deoxyuridine, Doxifluridine, Ro 21-9738, Furtulon, etc.). The compound 5’-DFUR is

parenterally and orally effective, and its activity was even better than those other FPs available for CRC

treatment at that period. On the other hand, L1210 leukemia cells were resistant to 5’-DFUR probably

of
due to the lack of the UP (Ishitsuka, et al., 1980). This is because 5’-DFUR is considered to be a depot

form of 5-FU, which can be promptly activated by UP (Ishitsuka, et al., 1980). 5’-DFUR main limitation

ro
lays in its gastrointestinal toxicity which is suggested to be due to the 5-FU release in the small intestine
-p
through the action of TP. Therefore, Capecitabine was subsequently developed as a 5’-DFUR pro-drug,
re
to avoid the metabolic transformation of 5’-DFUR by TP in the intestine (Malet-Martino & Martino,
lP

2002).

3.2. Capecitabine
na

Capecitabine (marketed as Xeloda®) is developed to mimic the continuous infusion of 5-FU while
ur

avoiding complications and inconvenience of intravenous administration (Chintala, Jun 30, 2011).
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Capecitabine is absorbed through the gastrointestinal wall in intact form and, subsequently, it is

converted to 5′-DFUR by carboxylesterase (CES) and cytidine deaminase (CDA) in the liver (Johnston &

Kaye, 2001) (Figure 1). 5′-DFUR is then transformed to 5-FU by TP and/or UP (Cao & Rustum, 2000; P.

M. Wilson, Danenberg, Johnston, Lenz, & Ladner, 2014). The TP enzyme has higher concentrations in

many tumor types compared to adjacent non-malignant tissues (Schuller, et al., 2000). Chintala et al.

(Chintala, Jun 30, 2011) therefore postulated that the higher tumor concentrations of 5-FU might be

expected, thus providing a favorable target-to-non-target ratio for toxicity. Indeed, particularly in

primary CRC tumors, Capecitabine was preferentially activated in the tumor with the average

concentration of 5-FU being 3.2 times higher than in adjacent non-malignant tissue (Schuller, et al.,

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2000). Repeated administration of Capecitabine did not cause significant plasma accumulation of 5-FU

precursors 5’-DFUR (Reigner, Blesch, & Weidekamm, 2001). In phase III clinical trials, mCRC patients

treated with Capecitabine as monotherapy evinced a significantly lower incidence of toxic side effects

in comparison with patients treated with 5-FU/LV (Lamberti, Sauerbruch, & Glasmacher, 2005).

Contrarily, several other studies demonstrated Capecitabine toxicity similar to that of 5-FU. In

combination with irinotecan, its safety profile can be even worse (Aguado, Garcia-Paredes, Sotelo,

Sastre, & Diaz-Rubio, 2014; Garcia-Alfonso, et al., 2009; Garcia-Alfonso, et al., 2010; Kohne, et al., 2008;

Souglakos, et al., 2012). Capecitabine is currently the only one pro-drug used in clinical practice which

of
was approved for the clinical use on humans by the Food and Drug Administration (FDA) in the United

ro
States in 1998 ("U.S. FOOD & DRUG Administration. Xeloda FULL PRESCRIBING INFORMATION.," 2015).
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3.3. Tegafur
re
Tegafur (1-(2-Tetrahydrofuryl)-5-fluorouracil, ftorafur, FT, FT-207, etc.) was developed as a 5-FU pro-
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drug in the Soviet Union during the Cold War (Giller, Zhuk, & Lidak, 1967) and has been clinically used

for over 30 years as an anticancer drug (Blokhina, Vozny, & Garin, 1972). In 1970, the drug was
na

introduced to Taiho Pharmaceuticals (Japan) and started to be produced over the world. The main
ur

utilities of Tegafur include its excellent absorbability from the gastrointestinal tract and its moderate

conversion to 5-FU there in the gastrointestinal tract (Toide, Akiyoshi, Minato, Okuda, & Fujii, 1977).
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Tegafur is activated through two separate metabolic pathways: (i) via hepatic microsomes by

cytochrome P-450 isoenzyme CYP2A6 (Figure 1) and (ii) by systemic soluble enzymes. The principal

adverse effects are gastrointestinal (i.e. nausea, vomiting, diarrhea, and mucositis) and neurological

toxicities (i.e. change in mental status, cerebellar ataxia, and coma) (Lamont & Schilsky, 1999).

Therefore, Tegafur is not well tolerated by patients (Hammond, et al., 2016) and has limited application

of being used alone. Tegafur-uracil, which comprises Tegafur and uracil in molar proportions of 1:4, is

a drug designed to improve the therapeutic index of Tegafur by blocking the DPD-mediated

degradation of fluorouracil´s pyrimidine base. Uracil also competes with 5-FU for DPD activity after

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incorporation into RNA (Diasio, 1998; Rustum, 1997). Both mechanisms result in a prolonged half-life

of 5-FU. It is approved for clinical use on humans in 50 countries worldwide. Wang et al. (T. Wang, et

al., 2011) observed that CYP2A6*4 and novel CYP2A6*1B gene variants play a significant role in the

individual variability towards 5-FU formation activity from Tegafur in the Chinese population. Patients

with the same dosage of Tegafur and a CYP2A6*4 allele did not attain the desired therapeutic effect,

whereas patients with the novel CYP2A6*1B variant suffered from 5-FU-induced toxicity.

3.4. Eniluracil

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Eniluracil (ethynyluracil) represents a uracil analog with an ethynyl substituent at the C-5 position

ro
which irreversibly inhibits DPD. Eniluracil increases the oral bioavailability of 5-FU to 100%, facilitating

uniform absorption and predictable toxicity (Schilsky & Kindler, 2000). The half-life of Eniluracil in
-p
plasma is 4 hours (Baker, et al., 2000). Although Eniluracil is not cytotoxic by itself, it enhances the
re
cytotoxicity of 5-FU when administered to cell lines expressing high levels of DPD. Fischel et al. (Fischel,
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et al., 1995) treated a panel of human tumor cell lines with Eniluracil and 5-FU. The combination of

both drugs produced a one to a five-fold enhancement of cytotoxicity compared to 5-FU alone; the
na

degree of enhancement correlated with pretreatment DPD activity (Fischel, et al., 1995). In humans,

the main side-effects caused by Eniluracil comprise neutropenia, with the 5-day schedule, and
ur

diarrhea, with the 28-day schedule. Based on the negative results from clinical trials on CRC patients,
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the further development of Eniluracil was discontinued in 2000 (Lamont & Schilsky, 1999). However,

Eniluracil has received Orphan Drug status from the FDA for the treatment of hepatocellular cancer in

combination with FPs ("National Center for Biotechnology Information. PubChem Database. Eniluracil,

CID=43157," 2005).

3.5. S-1

S-1 (TS-1) consists of Tegafur (5-FU pro-drug of S-1) and two enzyme inhibitors 5-chloro-2,4-

dihydroxypyridine (CDHP) and potassium oxonate (OXO). The molar ratio of the three constituents of

this combination is 1 (Tegafur): 0.4 (CDHP): 1 (OXO). CDHP is a DPD inhibitor and maintains the

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plasma/serum concentration of 5-FU. CDHP is about 180 times more effective than uracil in inhibiting

of DPD, thus this combination enables higher 5-FU concentrations for the anabolic pathway in cancer

cell (Maehara, 2003; Takechi, Fujioka, Matsushima, & Fukushima, 2002). OXO inhibits the

phosphorylation of 5-FU in the gastrointestinal tract to decrease potential serious gastrointestinal

toxicities (Miyamoto, Sakamoto, Yoshida, & Baba, 2014; Saif, Tytler, Lansigan, Brown, & Husband,

2009). S-1 is now widely used as the adjuvant and palliative chemotherapy for gastric cancer,

pancreatic cancer and CRC in Japan. Furthermore, S-1 is approved for use in advanced gastric cancer

in China, Korea, Taiwan, Singapore, and Thailand. In 2011, the Committee for Medicinal Products for

of
Human Use, a division of the European Medicines Agency issued an opinion recommending the

ro
approval of S-1 in Europe (Miyamoto, et al., 2014).
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3.6. Emitefur
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Emitefur (BOF-A2, 3-[[3-(Ethoxymethyl)-5-fluoro-3 ， 6-dihydro-2 ， 6-dioxo-1(2H)-
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pyrimidinyl]carbonyl]benzoic acid 6-(benzoyloxy)-3-cyano-2-pyridinyl ester) is composed of the 1-

ethoxymethyl derivative of 5-FU and the DPD inhibitor 3-cyano-2,6-dihydroxypyridine (CNDP) in 1:1
na

molar ratio. CNDP prevents the degradation of 5-FU by inhibiting DPD and thereby prolonging the half-
ur

life of 5-FU. In 2000, it was preregistered for CRC treatment in Japan, and in 2001, a phase I of clinical

study has been added to the Cancer adverse events and pharmacokinetics sections ("AdisInsight.
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Emitefur.," 2006). Preclinical investigations of Emitefur confirm antitumor activity in several animal

models and sustained 5-FU levels (Diasio, 1998; Shirasaka, et al., 1990). Most clinical studies to date

have been performed in Japan; the U.S. trials have demonstrated typical 5-FU toxicities, such as

diarrhea; stomatitis; anemia; leukopenia and thrombocytopenia (T. Sasaki, 1997).

3.7. Trifluridin-Tipiracil (TAS-102)

TAS-102, an oral anti-metabolite, consists of two components: trifluridine (FTD) and tipiracil

hydrochloride (TPI) at a molecular ratio of 1:0.5 (Peeters, Cervantes, Moreno Vera, & Taieb, 2018; van

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der Velden, Opdam, & Opdam, 2017). FTD is a thymidine analog that plays a fundamental role in

inducing cytotoxicity through inhibiting TS and incorporation into DNA, resulting in inhibition of cell

proliferation and tumor growth and DNA damage (Peeters, et al., 2018; van der Velden, Opdam, &

Voest, 2016). FTD has been shown to be active against 5-FU sensitive as well as 5-FU resistant CRC cell

lines (Matsuoka, Nakagawa, Kobunai, & Takechi, 2018). TPI inhibits the rapid catabolism of FTD by

inhibiting TP at the first pass by the liver and the intestinal tract, thus increasing systemic exposure to

FTD when TPI is given together with FTD (Suenaga, et al., 2017). Since April 2016, TAS-102 is approved

in Europe for treatment of mCRC progressing on other standard chemotherapies (including 5-FU,

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oxaliplatin, irinotecan, and available targeted therapies). In Japan, it was approved for unresectable

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advanced or recurrent mCRC in March 2014 (Mayer, et al., 2015; Xu, et al., 2018). TAS-102 has
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relatively moderate toxicity profile. Several groups described that development of drug toxicity could

be associated with patients' prolonged survival and particularly the neutropenia was linked with better
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outcome following TAS-102 (Burki, 2018; Cremolini, et al., 2018; Hamauchi, et al., 2017; D. Chen, et al.,
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2018; Kasi, et al., 2016).

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4. Resistance to 5-FU therapy

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Chemoresistance to anti-cancer drugs poses a major barrier in the achievement of the effective cancer

treatment with adequate patients' outcomes. Innate chemoresistance occurs as a consequence of

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genetic changes that are already present in tumor somatic cells at the start of systemic therapy.

Malignant tumors are inherently genetically unstable and often develop acquired chemoresistance

during the course of treatment. Both innate and acquired chemoresistance are important in

determining initial and subsequent lines of treatment (Hammond, et al., 2016).

Chemoresistance may be manifested against a single agent, such as 5-FU, or a class of agents with the

same/similar antineoplastic mechanisms of action (Chang, 2011; T. R. Wilson, Longley, & Johnston,

2006). Multiple mechanisms of acquired resistance have been described for any given cytotoxic

therapy and targeted pathway. However, acquired resistance to one drug often confers resistance to

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other drugs, even if agents have different mechanisms of action, a concept referred to as multidrug

resistance (Gottesman, Fojo, & Bates, 2002). Thus, in the process of acquiring drug resistance, the

tumor may become cross-resistant to a range of chemotherapies, which is believed to be the primary

reason for treatment failure observed in over 90% of patients with metastatic disease (Longley &

Johnston, 2005; T. R. Wilson, et al., 2006).

Chemoresistance is a complex process, resulting from several intrinsic and extrinsic factors that

change, increase, or diminish gene products. Awareness of inter-tumor heterogeneity has existed for

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a long time; however, the extent of intra-tumor heterogeneity has only been recognized in the past

decade (Gerlinger, et al., 2012). Intra-tumor heterogeneity, belonging to intrinsic factors responsible

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for chemoresistance, is caused by various genetic, epigenetic, transcriptomic, and proteomic
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properties of tumor cells (Mansoori, Mohammadi, Davudian, Shirjang, & Baradaran, 2017). Epigenetic
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factors including microRNA, transcriptomic and proteomic heterogeneity may arise due to primary

genotypic variations, but can also reflect cell cycle stage, stochastic variations between cells, or
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hierarchical organization of cells according to the cancer stem cell (CSC) theory (Gupta, et al., 2011;
na

Kreso, et al., 2013).

Cancer cells may also become quiescent or dormant. It is firmly conceived that there must be some
ur

cancer cells to maintain and survive in the patients recovering seemingly or metastasize to distant
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organs and cause the cancer recurrence. These cells are thought to be inherently less sensitive to

conventional systemic treatments (Mansoori, et al., 2017). The quiescence or dormancy of cancer cells

may be therefore critical to evolving malignancies, with implications for understanding cancer

initiation, progression, and treatment resistance (reviewed in (Gao, Zhang, Tang, & Liang, 2017; W.

Chen, Dong, Haiech, Kilhoffer, & Zeniou, 2016; Yeh & Ramaswamy, 2015)).

The extrinsic factors comprise pH, hypoxia, and paracrine signaling interactions with stromal and other

tumor cells. Moreover, growing evidence supports the key role of tumor microenvironment in drug

resistance (Gatenby, Gillies, & Brown, 2010; Junttila & de Sauvage, 2013).

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Clearly, the understanding of molecular features of chemoresistance to develop modified and/or new

and effective strategies are therefore urgently needed. For instance, it has been documented that up

to 40% of patients receiving 5-FU-based adjuvant chemotherapy following the resection of stage II and

III colon cancer experience recurrence or die within 8 years of follow-up (Pereira, et al., 2016; D.

Sargent, et al., 2009). Furthermore, almost half of the patients diagnosed with mCRC are resistant to

5-FU-based chemotherapy and their five-year survival rate is only slightly over 12% (Douillard, et al.,

2000; Giacchetti, et al., 2000).

of
In the following sections, we emphasized the most explored enzymes involved in the chemoresistance

to 5-FU and other FPs (summarized in Table 2) as well as other intensively studied possible molecular

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mechanisms underlying poor treatment response of CRC patients.
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4.1. Thymidylate synthase (TS)
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TS, the primary target of the metabolite FdUMP (Figure 2), represents an enzyme encoded by TYMS
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gene. Both the level of TYMS and TS expression represent a prognostic marker of the treatment
na

efficacy of several types of cancer. Increased expression of TS is one of the most well-established

mechanisms of resistance towards 5-FU and other antifolates. Thus, the sensitivity profile of cells to 5-
ur

FU may be affected by genetic variants of the TYMS gene, expression levels of TYMS gene / TS protein,
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and intracellular concentrations of dNTPs and CH2THF (Zhou, et al., 2012). Several studies indicated

that low TS expression in CRC patients with advanced tumors was associated with increased individual

sensitivity to 5-FU-based therapy (Iyevleva, et al., 2007; Lenz, et al., 1998; Qiu, et al., 2008; Salonga, et

al., 2000). In contrast, studies using CRC cell lines and tumor tissues demonstrated that 5-FU therapy

contributes to the induction of TS expression (Chu, Koeller, Johnston, Zinn, & Allegra, 1993; Longley,

et al., 2002). This seems to be a consequence of a negative feedback loop in which ligand-free TS binds

to its own mRNA and inhibits its own translation (Chu, et al., 1994). When stably bound by FdUMP, TS

can no longer bind its own mRNA and suppress translation, resulting in increased protein expression.

This constitutes a potentially important resistance mechanism, as acute increases in TS would facilitate

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the recovery of enzyme activity (Longley, et al., 2002). Low TS expression may lead to serious DNA

damage. Since cancer cells are characterized by a higher degree of proliferation compared to non-

malignant cells, low TS expression in tumor tissue may lead to a decrease in the dTMP substrate

necessary for DNA synthesis, which would inhibit its replication and proliferation. Therefore, the low

level of TS expression in tumor cells may be associated with a less aggressive course of the disease and

a more favorable prognosis of patients. According to the studies of Aprile et al. and Allegra et al., a low

level of TS expression may be prognostic rather than a predictor of FPs effectiveness (Allegra, et al.,

2003; Aprile, Mazzer, Moroso, & Puglisi, 2009).

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Although the reason for the ontogenetic variations in TS expression is still not clear, one of the

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possibilities lies in the link between TYMS gene polymorphisms and TS expression. As it is now, some
-p
of the described polymorphisms affect inter-individual differences in patient sensitivity to 5-FU
re
treatment (Gosens, et al., 2008; Gusella, et al., 2009; Paez, et al., 2010). Polymorphism of the variable

number of tandem repeats located in the TYMS gene sequence is one of the intensively studied genetic
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variants with clinical relevance as a predictive marker for the effectiveness of 5-FU treatment (Gusella,
na

et al., 2009; Lecomte, et al., 2004; Marcuello, et al., 2004). Patients bearing double repeats in the TYMS

promoter region and a 6- bp variation in the 3’UTR were significantly associated with a high risk of
ur

severe side effects related to 5-FU administration (Lecomte, et al., 2004). More details about all
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common polymorphisms of the TYMS gene and other genes, such as MTHFR, DPYD, and UMPS, which

may potentially impact the function of proteins associated with the pharmacology of 5-FU are

reviewed by Panczyk et al. (Panczyk, 2014). A meta-analysis of 13 clinical trials comprising 887 patients

with advanced CRC carried out by Popat et al. (Popat, Matakidou, & Houlston, 2004) demonstrated

that patients with low TS expression in tumor tissue evinced longer overall survival than those with

higher expression. In another meta-analysis including 24 clinical trials with more than 1100 CRC

patients, the pooled relative risk of overall response rate indicated that the group with lower TS

expression had higher sensitivity to FP-based chemotherapy than patients with high TS expression level

(Qiu, et al., 2008).

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Table 2. The key enzymes involved in chemoresistance in colorectal cancer reported for 5-FU and other

FPs (updated according to (Hammond, et al., 2016)).

Chemotherapy Enzyme Mechanism of resistance Reference

drug involved

5-FU Thymidylate Genetic amplification of TYMS (Z. Sun, et al., 2015;

synthase (TS) leads to increased expression of Wang, et al., 2004)

TS

of
Down-regulation of miRNA leads (Z. Sun, et al., 2015)

to increased expression of TYMS

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Increased TYMS expression in (Libra, et al., 2004)
-p
liver metastasis leads to 5-FU
re
resistance

Changes in the status of p53 alter (Etienne, et al., 2002;

lP

sensitivity to TS inhibitors Giovannetti, et al.,

na

2007)

Insertion in TYMS gene (TS 3’UTR (Naghibalhossaini, et

ur

6-bp) leads to increased al., 2017)

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expression of TS

Low expression of TS correlates (Soong, et al., 2008)

with increased response to 5-FU

therapy

Methylene Polymorphisms (C677T, A1298C) (Cohen, et al., 2003;

tetrahydrofola lead to increased expression of TS Kristensen, Pedersen,

te reductase Melsen, Ellehauge, &

(MTHFR) Mejer, 2010;

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Naghibalhossaini, et

al., 2017; Sohn,

Croxford, Yates,

Lucock, & Kim, 2004)

Thymidine Low TP expression correlates with (Lindskog, Derwinger,

phosphorylase increased response to 5-FU Gustavsson, Falk, &

(TP) therapy Wettergren, 2014;

Soong, et al., 2008)

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High TP expression correlates (Lindskog, et al., 2014)

ro
with increased response to 5-FU

therapy
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Orotate High OPRT activity is associated (Muhale, Wetmore,
re

phosphoribosy with greater survival Thomas, & McLeod,

lP

l transferase 2011)

(OPRT)
na

Dihydropyridin Low DPD expression correlates (Kristensen, et al.,

ur

e with increased response to 5-FU 2010; Kunicka, et al.,

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dehydrogenas therapy 2016; L. H. Li, et al.,

e (DPD) 2013; Salonga, et al.,

2000; Soong, et al.,

2008)

Thymidine Phosphorylation of FdUR by TK (Grem & Fischer,

kinase (TK) permit repletion of dTMP and 1989)

dTTP pools, thereby bypassing the

TS blockade

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Ribonucleotide Low RNR expression correlates (Fukushima, Fujioka,

reductase with resistance to 5-FU therapy Uchida, Nakagawa, &

(RNR) Takechi, 2001)

Deoxyuridine Low dUTPase protein expression is (Ladner, et al., 2000)

triphosphatase associated with response to 5-FU-

(dUTPase) based chemotherapy

Capecitabine Thymidine Higher TP expression is associated (Meropol, et al., 2006;

phosphorylase with resistance to capecitabine Petrioli, et al., 2010)

of
(TP)

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Dihydropyridin Higher DPD expression is (Koopman, et al.,

e
-p
associated with resistance to 2007; Vallbohmer,

dehydrogenas capecitabine Yang, et al., 2007)

re

e (DPD)
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Abbreviations: 5-FU, 5-fluorouracil; dTMP, 2'-deoxythymidine-5'-monophosphate; dTTP, 2'-deoxythymidine-5'-

triphosphate; FdUR, fluorodeoxyuridine; miRNA, micro RNA; TYMS, thymidylate synthetase gene.
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4.2. Methylene tetrahydrofolate reductase (MTHFR)

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The use of LV in combination with 5-FU has become a standard in the treatment of advanced CRC for
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more than 30 years. The intracellular metabolic balance of LV is regulated by methylene

tetrahydrofolate reductase (MTHFR), catalyzing the irreversible conversion of CH2THF to 5-

methyltetrahydrofolate (CH3THF). Several single nucleotide polymorphisms (SNPs) have been shown

to affect the activity of MTHFR, from which especially SNPs 677C>T and 1298A>C are of the

importance. A variant 677TT genotype is responsible for a 30% reduction in enzymatic activity

compared to a wild-type 677CC genotype associated with reduced thermolability observed in vitro

(Frosst, et al., 1995). An important consequence of the MTHFR 677T variant allele presence is the

tendency to accumulate CH2THF in the cells, which may have a significant effect on the

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pharmacological efficacy of 5-FU. Sohn et al. (Sohn, et al., 2004) in both in vitro and in vivo studies

observed that the presence of the variant 677T allele is responsible for pronounced 5-FU

chemosensitivity in colon cancer cells, suggesting its utility as a pharmacogenetic factor to assess the

effectiveness of 5-FU-based chemotherapy.

However, clinical studies published in recent years have led to contradictory and inconsistent

conclusions. In patients with advanced CRC undergoing 5-FU-based therapy, the presence of the 677T

variant allele was associated with a higher percentage of positive responses (Cohen, et al., 2003;

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Etienne, et al., 2004; Jakobsen, Nielsen, Gyldenkerne, & Lindeberg, 2005), while the results of another

study did not confirm such a relationship (Marcuello, Altes, Menoyo, Rio, & Baiget, 2006). Although it

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seems intuitive that increased activity of this enzyme should lead to chemoresistance, the correlation
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of up-regulation or amplification of the MTHFR gene or protein product with chemoresistance was not
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declared by the studies. Another frequent SNP of the MTHFR gene, 1298A>C, results in the substitution

of glutamine amino acid by alanine. Similarly, to the SNP 677C>T, 1298A>C polymorphism contributes
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to the reduction in enzymatic activity of MTHFR but it shows no association with the thermolabile
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proteins. Some of the published studies on the SNP 1298A>C suggested that the presence of the 1298C

variant allele has no impact on the percentage of positive responses to 5-FU-based treatment
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(Jakobsen, et al., 2005; Marcuello, et al., 2006; R. Sharma, et al., 2008), while two studies proposed its
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association with significantly decreased patients' survival (Etienne, et al., 2004; W. Zhang, et al., 2007).

4.3. Dihydropyridine dehydrogenase (DPD)

Partial or total loss of DPD activity may be associated with the presence of genetic determinants of the

DPYD gene encoding DPD, such as SNPs, deletion mutations and methylation. Several researchers have

investigated the impact of genetic variations in the DPYD gene on DPD expression levels in tumor cells

as predictive markers for both the effectiveness of 5-FU treatment and its toxicity, reviewed in

(Panczyk, 2014). DPYD*2A polymorphism was found in 50% of patients with the 4th stage of

neutropenia as a result of 5-FU treatment (van Kuilenburg, et al., 2000). Moreover, promoter

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hypermethylation of the DPYD gene is considered as a possible mechanism of variable DPD activity

(Amstutz, Farese, Aebi, & Largiader, 2008; J. Yu, McLeod, Ezzeldin, & Diasio, 2006). Low DPD expression

level should lead to reduced 5-FU catabolism, and thus to the more effective accumulation of the drug

inside cells (Kunicka, et al., 2016). On the other hand, high DPD activity in tumor tissue might be

responsible for the development of drug resistance by reducing the cytotoxic effects of 5-FU (Panczyk,

2014). Intrinsic over-expression of DPD by malignant cells has been shown to enhance the resistance

to 5-FU in vitro (Longley & Johnston, 2005; Takebe, et al., 2001). High levels of DPD mRNA expression

in CRC cells have also been associated with 5-FU resistance (Salonga, et al., 2000). This has been

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demonstrated as an intrinsic mechanism of resistance but the available data about DPD as a cause of

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acquired resistance are not yet conclusive (Hammond, et al., 2016). In addition, DPYD gene expression
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has been investigated as a biomarker of treatment resistance but the results of several studies on

mCRC patients treated with FPs suggested this correlation as weak or concluded that there is no
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evidence of an association between the expression of DPYD and effectiveness of chemotherapy
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(Meropol, et al., 2006; Vallbohmer, et al., 2006; Yanagisawa, et al., 2007). A correlation between low

expression of the DPYD gene and better response to 5-FU-based therapy was found in patients with
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advanced CRC treated with first-line Capecitabine (Vallbohmer, et al., 2006). Shorter progression-free
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survival and a lower response rate in patients treated with Capecitabine were observed in patients

with higher DPD mRNA in tumor tissue (Vallbohmer, Marjoram, et al., 2007). The clinical data have
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confirmed the predictive value of DPYD expression levels in order to predict the efficacy of 5-FU-based

therapy in CRC patients (Scartozzi, et al., 2011).

4.4. Thymidine phosphorylase (TP)

TYMP is the gene encoding TP, a cytosolic enzyme that catalyzes the conversion of thymidine or

deoxyuridine to thymine or uracil, respectively, via the addition of phospho group to the deoxyribose.

TP is thus essential for the nucleotide salvage pathway that recovers pyrimidine nucleosides formed

during DNA or RNA degradation (Bronckaers, Gago, Balzarini, & Liekens, 2009). Several studies

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suggested TP as an initiator of tumor growth and metastasis by inhibiting apoptosis and induction of

angiogenesis in cancer cells (Pang & Poon, 2006). On the other hand, TP expression is significantly

higher in tumor cells (approximately four times than in adjacent non-malignant tissue) allowing

targeted activation of 5-FU to minimize the toxicity caused by such therapy. Moreover, the enzymatic

activity of TP is essential to obtain an adequate concentration of an active form of Capecitabine. The

measurement of its expression may thus represent a useful marker for predicting the effectiveness of

5-FU-based chemotherapy (Meropol, et al., 2006). There is accumulating evidence suggesting that TP

is key factor in determining response to chemotherapy. Soong et al. (Soong, et al., 2008) observed on

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945 CRC patients treated with 5-FU that the low level of TP expression may be associated with the

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improved treatment outcomes and represent a predictive marker for good response to 5-FU
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chemotherapy. Similarly, Metzger et al. (Metzger, et al., 1998) and Gustavsson et al. (Gustavsson, et

al., 2009) noticed that low TP expression was independently associated with the prediction of better
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response to 5-FU based chemotherapy and with improved overall survival. However, these results
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differ from those obtained by Meropol et al. (Meropol, et al., 2006). Patients with higher TP expression

were associated with a significantly longer time to progression in comparison with patients with a low
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level of TP expression. Most likely, the cells with higher TP expression may exhibit increased sensitivity
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to 5-FU due to the enhancement of FdUMP concentration as a consequence of increased 5-FU

activation (Panczyk, 2014). Also, patients with higher TP mRNA levels evinced better responses to
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Capecitabine, while loss-of-function conferred the resistance (Stark, Bram, Akerman, Mandel-

Gutfreund, & Assaraf, 2011). In terms of patients' prognosis, high TP expression is suggested to be

associated with the aggressiveness of cancer cells and poor prognosis, despite there are conflicting

reports documenting this statement (Bronckaers, et al., 2009; Elamin, Rafee, Osman, KJ, & Gately,

2016).

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4.5. Orotate phosphoribosyl transferase (OPRT)

The product of UMPS gene is the enzyme OPRT which converts 5-FU into FUMP, a common substrate

for the production of FUTP and FdUTP (the latter needs to be additionally converted by RNR), two

cytotoxic metabolites that target RNA and DNA, respectively. Muhale et al. (Muhale, et al., 2011)

observed that UMPS was the only gene in the 5-FU anabolic pathway that produced a cellular

phenotype of 5-FU chemoresistance. It suggests that the misincorporation of 5-FU metabolites into

RNA and DNA are important steps in the initiation of 5-FU cytotoxicity. In addition, high OPRT activity

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or increased UMPS mRNA expression were associated with better survival (Ishikawa, Miyauchi, &

Kashiwagi, 2008; Koizumi, et al., 2010), suggesting that UMPS/ORTP may have a clinical relevance

regarding 5-FU-based chemotherapy regimens.

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-p
4.6. Thymidine kinase (TK)
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The other 5-FU activation pathway involves TP catalyzing the conversion of 5-FU to fluorodeoxyuridine
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(FdUR). FdUR is further converted by phosphorylation by TK to FdUMP which either serves as a

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substrate for FdUTP synthesis or forms the ternary complex along with TS and CH2THF and thus inhibits

the synthesis of dTMP. TK should optimize FdUMP formation within tumor cells and is also involved in
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the pyrimidine salvage pathway of cancer cells treated with anti-TS drugs, aiming at replenishing
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thymidine pools and alleviating the effects of TS deficiency (Broet, et al., 2001; Longley & Johnston,

2005). This salvage pathway represents a potential mechanism of resistance to 5-FU (Grem & Fischer,

1989) (Figure 2). Increased expression of TK was observed in 5-FU-resistant human gastric carcinoma

cells SNU638 (Chung, et al., 2000). The authors suggested that blocking of 5-FU metabolites

incorporation into nucleic acids and TK over-expression may play a major role in 5-FU resistance.

Fanciullino et al. (Fanciullino, et al., 2006) studied HT29 CRC cell lines over-expressing TK and their

sensitivity to 5-FU. The authors observed that sensitivity to 5-FU was markedly decreased, thus

suggesting that high TK levels are associated with drug resistance. They proposed that the

enhancement of 5-FU activation towards the cytotoxic FdUMP metabolite and, subsequently, TS

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inhibition with markedly increased sensitivity was only achieved through modulation of TP activity. The

authors concluded that TP, and not TK, is the critical, limiting step in the optimization of 5-FU activation

and its efficacy as an anti-cancer agent.

4.7. ATP-binding cassette (ABC) and solute carrier (SLC) transporters

The transport-based cellular mechanisms of chemoresistance mainly refer to the drugs which are

effluxed out of cancer cells through a variety of membrane transporter proteins, thereby leading to

decreased intracellular availability of anticancer drugs and chemotherapy failure (Hu, Li, Gao, & Cho,

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2016). Membrane transporters are responsible for the control of the transport of their substrates (e.g.

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ions, amino acids, peptides, lipids, sugars, and xenobiotic) into and out of the cells (International

Transporter, et al., 2010). To date, more than 400 membrane transporters have been annotated in the
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human genome, and they are divided into two major superfamilies: ATP-binding cassette (ABC) and
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solute carrier (SLC) transporters. Representatives of ABC transporters include P-glycoprotein (P-gp,
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ABCB1 or MDR1), breast cancer resistance protein (BCRP/ABCG2, also named as ABCP or MXR1), and

multidrug resistance-associated proteins (MRPs). Transporters such as the organic anion transporters,
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organic cation transporters, and organic anions belong to the SLC superfamily (Duan, et al., 2015;
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International Transporter, et al., 2010). One of the most commonly observed mechanism underlying

drug resistance in cancer cells is the over-expression of ABC transporters on the plasma membrane
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(Szakacs, Paterson, Ludwig, Booth-Genthe, & Gottesman, 2006), thus the inhibition of these

transporters is suggested to be an effective approach to sensitize drug-resistant cancer cells to

chemotherapeutic agents (Szakacs, et al., 2006).

P-gp over-expression has been observed in different types of hematologic and solid cancers, such as

leukemia, neuroblastomas, ovarian and breast cancers (Deeley & Cole, 2006). Only a few studies

documenting ABC transporters in link with CRC chemoresistance were published up to date. Up-

regulation of MRP1 was found during the development of drug resistance in the HT29 colon cancer cell

lines (Klappe, Hinrichs, Kroesen, Sietsma, & Kok, 2004). MRP2 was reported to be important for the

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oxaliplatin resistance in CRC (Hinoshita, et al., 2000) and its level was increased in SW620 and LoVo

cells resistant to oxaliplatin as well (Liu, et al., 2010). ABCB5 expression was substantially enhanced in

CRC patients after 5-FU-based chemotherapy, thus contributing to the development of 5-FU resistance

(B. J. Wilson, et al., 2011). Oguri et al. (Oguri, et al., 2007) discovered that expression of the ABCC11 is

induced by 5-FU and that ABCC11 is directly involved in 5-FU resistance by the efflux transport of the

active metabolite FdUMP in human small-cell lung cancer cell lines. Hlavata et al. (Hlavata, et al., 2012)

observed that the high ABCC11 expression in CRC tumors before chemotherapy may limit further

ABCC11 induction by 5-FU treatment. In the study of Guo et al. (Guo, Tang, Wang, & Feng, 2003),

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ABCC11 was found to be up-regulated in the chemotherapy-naive tumors from patients who

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subsequently achieved a good response to the chemotherapy in both palliative and adjuvant settings.
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Deregulation of ABCC11 was shown to be a resistance factor for FPs. In addition to ABC transporters,

HES1, a downstream transcriptional factor of Notch signaling pathway, promotes CRC cell
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chemoresistance to 5-FU by prompting epithelial-mesenchymal transition and inducing transcription
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of several ABC transporter genes (L. Sun, et al., 2017).

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4.8. Genes involved in metabolic activation of 5-FU pro-drugs

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CYP2A6 gene, P-450 cytochrome isoenzyme, is involved in the metabolic activation of Tegafur. Wang

et al. (H. Wang, et al., 2011) showed that the presence of the variant CYP2A6*4 affects the decrease
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in CYP2A6 gene expression and, therefore, it is the main determinant contributing to the reduction of

5-FU formed from Tegafur. The authors concluded that variants CYP2A6*4 in addition with CYP2A6*1B

are major genetic factors responsible for the inter-individual variation of Tegafur activation to 5-FU.

Glutathione S-transferases (GSTs) belong to the phase II metabolic enzymes involved in drug

metabolism and protect cellular macromolecules from damage by catalyzing the conjugation of toxic

and carcinogenic electrophilic molecules with glutathione. The resulting complex is less toxic and more

readily excreted into bile or urine. The over-expression of GSTs can, therefore, facilitate the anticancer

drug detoxification in cancer cells and decrease in their therapeutic effects. In contrast to the other

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members of the GSTs family, such as GSTT1, GSTM1, and GSTA1, the variant GSTP1 is predominantly

expressed in the colon (Schwarzenbach, 2010). The impact of GSTP1 on 5-FU metabolism is still

controversial. Although Zhan and Liu (Zhan, 1999) showed a decrease of GSTP1 levels after 5-FU

treatment, Nishiyama et al. (Nishiyama, et al., 1999) noticed an increase in GSTP1 expression.

Currently, it is unknown if GSTP1 participates in the detoxification of 5-FU. According to Stoehlmacher

et al. (Stoehlmacher, et al., 2002), the GSTP1 Ile105Val polymorphism was associated with increased

survival of patients with advanced CRC receiving 5-FU/oxaliplatin chemotherapy.

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Apart to the CRC, Sharma et al. (A. Sharma, Rajappa, Satyam, & Sharma, 2010) have evaluated

differences in the circulating prooxidants in advanced cervical cancer patients before and after 5-FU

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based-neoadjuvant chemoradiation and observed a mild increase in glutathione (GSH) and antioxidant
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enzyme (SOD, CAT, GSHPx) activities after chemotherapy in the complete responders in comparison
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with partial responders and non-responders.
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4.9. DNA repair

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DNA repair machinery identifies and corrects the damage in the DNA molecules, either induced by

endogenous (reactive oxygen species) or exogenous (ultraviolet radiation, x- and gamma rays, plant
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toxins, mutagenic chemicals, and chemotherapeutic agents) substances. DNA repair mechanisms, such
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as base excision repair (BER) (Lord & Ashworth, 2012) and mismatch repair (MMR) (Iwaizumi, Tseng-

Rogenski, & Carethers, 2011) may not only influence tumor characteristics (cancer phenotype) and

patients' prognosis but may also be involved in response to chemotherapy (Slyskova, et al., 2015).

While BER recognizes and removes uracil and 5-FU from DNA, MMR drives 5-FU-induced cytotoxicity.

Several authors observed an association between inappropriate BER and increased tumor

aggressiveness and poor outcomes in CRC patients (summarized in (Leguisamo, et al., 2017; Wallace,

2014)). However, the mechanism of how BER affects tumor properties still remains to be defined as

conflicting results are often reported on the expression of a limited number of BER genes (Leguisamo,

et al., 2017; Slyskova, et al., 2012; Vodenkova, et al., 2018; Vodicka, 2019).

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CRCs can also be classified according to their MMR capacity into MMR‐deficient and MMR‐proficient

tumors, also termed as microsatellite instability-high (MSI-H) or microsatellite‐stable (MSS) cancers.

Microsatellites are repetitive DNA sequences, typically 1–5 bp repeated 15-30 times, and instability in

these regions due to either insertion or deletion of repeated units causes alteration in the DNA

replication process. Deficiency in or the loss of the protein products of the hMSH2, hMLH1 and hMSH6

MMR genes causes diffuse errors in microsatellites, resulting from loss of scanning and recognizing

errors during DNA replication and failure to edit these errors to maintain an intact genetic code (Fink,

Aebi, & Howell, 1998). The MMR deficiency pathway induces hypermutated MSI cancers that differ

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from MSS tumors in their mutation profile and biological behavior. The frequency of mutations in MMR

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deficient cells is increased 100- to 700-fold compared to cells with an efficient MMR system (Wierzbicki
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PM, 2009). Cancers with high mutation burden (generally defined as more than 10 mutations per

megabase) underlies the pathogenesis of 15-20% of all CRC, and, most often, develop in the presence
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of defects in MMR system. MSI is an indirect marker of the replication error phenotype and underlies
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the pathogenesis of approximately 15% of all CRC cases (Valle, et al., 2019). MSI frequency is higher

among stage II CRC (20%) (Cook, et al., 1979; Kawakami, Zaanan, & Sinicrope, 2015) compared to mCRC
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(4%) (Koopman, et al., 2009).

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In general, defective MMR contributes to chemoresistance in CRC (Leguisamo, et al., 2017). The loss
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of detection of mismatched and unpaired bases is thought to be a primary mechanism of inherent

resistance to FPs. 5-FU forms mismatched base pairs that are recognized by MMR and can stimulate

apoptosis (Carethers, et al., 2004). However, the importance of this process is cell line-specific, and the

clinical importance of MMR status for FPs response may be due to alternative processes including

immune response as well (Viale, Trapani, & Curigliano, 2017). Better survival benefit after 5-FU

treatment was observed in MMR-proficient patients, while MMR-deficiency was not associated with

survival benefit after 5-FU treatment (D. J. Sargent, et al., 2010). The cytotoxic action of 5-FU may be

dependent on recognition of the modified DNA by DNA mismatch repair molecules. Since the binding

of MMR proteins is impaired in MSI-H tumors, cell death is not triggered. (Jo & Carethers, 2006).

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However, there are MMR independent cell death mechanisms via which also MMR-deficient CRC cells

undergo apoptosis in response to FPs (L. S. Li, et al., 2009). MSI-H status has been associated with

detrimental survival in stage II patients treated with adjuvant 5-FU regimen in a pivotal study by Ribic

et al. (Ribic, et al., 2003). However, much less is known about the efficacy of 5-FU-based chemotherapy,

especially chemotherapy combinations, in the metastatic setting. On the other hand, a MMR-deficient

status appears to lose its prognostic value in recurrent and metastatic disease and it has been

associated with worse clinical outcomes (Alex, et al., 2017). Interestingly, the metastatic patterns of

MMR-deficient cancers are also considerably different from the remaining spectrum of CRC, with a

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higher frequency of peritoneal dissemination rather than hematogenous spread to liver or lungs

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(Fujiyoshi, et al., 2017). Currently, MSI testing is recommended for all patients with stage II CRC,
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although a recent studies found that the MMR status is neither prognostic nor predictive biomarker

for patients with high-risk stage II colon cancer (Baek, et al., 2019; Kim, 2014). MSI-H status of the
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tumor is associated with good prognosis and lack of benefit from 5-FU-based adjuvant chemotherapy
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(Copija, Waniczek, Witkos, Walkiewicz, & Nowakowska-Zajdel, 2017). MSI may also be associated with

favorable prognosis and a better overall survival in CRC patients with advanced stage of the disease,
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probably due to immune response to plentiful neoantigens generated in the absence of functional
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MMR. The fact that a pronounced lymphocytic infiltration is a hallmark of MMR-deficient CRCs has

prompted scientists to examine the immunogenic character of these tumors with their more favorable
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clinical behavior. Galon et al. (Galon, et al., 2012) developed ImmunoscoRe®, a tool aimed at improving

the prognostic stratification of CRC patients, particularly through the identification of high-risk II

patients that might benefit from adjuvant therapy. These patients also uniquely respond to treatment

with checkpoint inhibitors (Overman, et al., 2017).

So far, only a few studies have examined nucleotide excision repair (NER) in relation to 5-FU therapy

response and patients' survival. Slyskova et al. (Slyskova, et al., 2015) collected samples from the same

CRC patients three times in 6-months intervals, starting at the diagnosis, and compared their NER

determinants with healthy controls. One year after the diagnosis and successful 5-FU-based treatment,

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the NER down-regulation was not detectable anymore, and the patients exhibited similar molecular

pattern of NER to that of healthy controls.

4.10. Non-coding RNAs (ncRNAs)

Several recent studies have shown that microRNAs (miRNAs, miRs), a class of non-coding RNAs

(ncRNAs), may play an important role in the regulation of chemoresistance in CRC by controlling

relevant signaling pathways. These include cell cycle, proliferation, apoptosis, DNA damage repair,

drug metabolism, and transport pathways (Fanale, Castiglia, Bazan, & Russo, 2016; Raza, Zhang, &

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Sahin, 2014; Wu, Sheng, Zhang, Yang, & Wang, 2018). Additionally, there are miRNAs directly linked to

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cancer, referred to as "oncomiRs" and “anti-oncomiRs”. OncomiRs act as oncogenes and inhibit the

expression of target tumor suppressor genes, while anti-oncomiRs have a role as tumor suppressors
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and lead to inhibition of the expression of oncogenes. The inactivation of oncomiRs and activation of
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anti-oncomiRs may represent the important intervention in regulating the expression of specific genes
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able to restore drug sensitivity (Donzelli, et al., 2014). For this reason, miRNA-based research could

contribute to an innovative therapeutic strategy and development of proper anticancer therapy.

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Furthermore, miRNAs could serve as prognostic and predictive biomarkers, adding important
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information to personalized medicine.

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Several studies have demonstrated that miR-192 and miR-215 may directly contribute to the

mechanism of chemoresistance to FPs and antifolates in CRC (Song, et al., 2008; Vychytilova-

Faltejskova, et al., 2017; Xi, Shalgi, Fodstad, Pilpel, & Ju, 2006) as they target both TS and dihydrofolate

reductase (DHFR) mRNA. The reduction of TS and DHFR levels with elevated expression of miR-215

leads to the enhanced G2/M cell cycle checkpoint control and reduced cell proliferation (Song, et al.,

2010). Yu et al. (Y. Yu, Sarkar, & Majumdar, 2013) suggested that down-regulation of miR-21 might

represent an effective therapeutic strategy for chemotherapy-resistant colon cancer by regulating

CSCs differentiation and subsequently rendering them susceptible to therapeutic agents. Borralho et

al. (Borralho, et al., 2009) observed that over-expression of miR-143 in the human colon HCT116 cell

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line significantly decreased cell viability and increased cell death after 5-FU treatment via inhibiting

expression of ERK5, NF-κB and Bcl-2.

Ras proteins are small GTPases responding to upstream signaling, subsequently acting as a trigger for

the Raf/MEK/MAPK kinases cascade that represents a fundamental pathway required for cell growth

and proliferation. Mutations in genes of Ras/MAPK cascade and its activation have been implicated in

CRC development and acquiring of treatment resistance (Zhao, et al., 2017). Let-7 family, a target of

Ras pathway, comprises 9 members so far: let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g, let-7i, and

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miR-98. Various miRNAs from let-7 family were observed to be deregulated in relation to resistance

against different anti-cancer agents in CRC patients; however, the results are not uniform. Weidhaas

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et al. (Weidhaas, et al., 2007) referred to the role of let-7 in improving the radiosensitivity of cancer
-p
cells by targeting Ras protein. Increased let-7g expression was found to be significantly associated with
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the chemoresistance against S-1-based therapy in CRC patients (Nakajima, et al., 2006). Mizuno et al.

(Mizuno, Kawada, & Sakai, 2018) observed that the down-regulation of let-7 family members
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contributes to the chemoresistance against S-1 in CRC patients. In connection with these results, the
na

authors postulated the mechanism of action which assumes that the decreased levels of let-7 family

members induce the expression of ERCC1 protein involved in NER pathway which contributes to the
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resistance against cisplatin or 5-FU (Mizuno, et al., 2018). Recently, Tie et al. (Tie, et al., 2018) observed
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that let-7f-5p expression was elevated in chemotherapy-resistant CRC tissues compared to those

chemotherapy-sensitive. The authors demonstrated that let-7f-5p-promoted chemotherapeutic

resistance repressed the expression of several pro-apoptotic proteins, including tumor-suppressor

protein p53, tumor protein p53-inducible nuclear protein 1, tumor protein p53-inducible nuclear

protein 2 and caspase-3.

For the first time, Fu et al. (Fu, et al., 2017) determined the expression of miR-20b and the levels of a

disintegrin and metalloprotease domain-containing protein 9 (ADAM9) and EGFR in malignant and

adjacent non-malignant tissues from 5-FU-sensitive or -resistant colon cancer patients, supplemented

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by the experiments on 5-FU-sensitive (HCT116) and -resistant (HCT116-R) cells. The results showed

that miR-20b was expressed at lower levels in the 5-FU-resistant tissues and cells than in the 5-FU-

sensitive tissues and cells. The opposite was the case in expression of ADAM9 and EGFR. In addition,

they demonstrated that ADAM9 is a direct target of miR-20b and that miR-20b decreased the 5-FU

resistance of HCT116-R cells. Their findings suggested that miR-20b reduced 5-FU resistance to induce

apoptosis in vitro by suppressing ADAM9/EGFR in colon cancer cells.

Most recently it has been postulated that miRNAs also offer an advantage to overcome the

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chemoresistance of colon CSCs as a therapeutic agent (Fesler, Guo, Liu, Wu, & Ju, 2017). Since the

chemoresistance of colon CSCs involves several different mechanisms, their rapid adaptation via

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miRNAs may be a major factor in tackling this issue as miRNA can target many genes at once. Some
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miRNAs have already shown their potential to regulate colon CSCs. For instance, miR-145 and miR-
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450b target SOX2 and have been found to enhance sensitivity to 5-FU and reduce stem cell-like

characteristics in colon cancer cells (Iliou, et al., 2014; Jin, et al., 2016; Y. Yu, et al., 2015). In addition,
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targeting of miR-21 leads to a reduced number of colon CSCs during 5-FU treatment (Y. Yu, et al., 2013).
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All these miRNAs regulate important stemness-related genes and may be useful in increasing the

chemosensitivity by disrupting their resistance mechanisms.

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Long non-coding RNAs (lncRNAs) represent other class of ncRNA with a length greater than
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200 nucleotides, with limited or no protein-coding capacity. Some lncRNAs have been functionally

characterized in patients’ tissues and serum and act similarly to genes to mediate tumor-suppressing

or oncogenic effects (Di Gesualdo, Capaccioli, & Lulli, 2014). Several studies have shown that lncRNAs

also play an important role in epigenetic, transcriptional, and post-transcriptional gene expression,

thereby mediating 5-FU drug resistance (Deng, Wang, Lei, Lei, & Xiong, 2017; Shi, et al., 2015; Yang, et

al., 2013). Xiong et al. (Xiong, et al., 2015) observed that the expression levels of many lncRNAs are

altered following the 5-FU treatment in colon cancer cells. Majority of aberrantly expressed lncRNAs

were involved in the PI3KAKT and NF-κB signaling pathways. Lee et al. (Lee, et al., 2014) analyzed two

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5-FU-resistant cell lines to determine the expression of lncRNAs. The authors showed that lncRNA

called snaR is down-regulated in 5-FU-resistant cells and that loss of snaR increases cell viability after

5-FU treatment. These results suggest that snaR may be a negative regulator of colon cancer cell

growth in response to 5-FU. Moreover, Xiao et al. (Xiao, Yurievich, & Yosypovych, 2017) identified

lncRNA XIST as considerable diagnostic biomarker due to its up-regulated levels in non-responding

patients. Besides, increased serum XIST level was associated with poor therapy response and lower

survival rate in CRC patients receiving 5FU-based treatment.

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4.11. Other relevant genes

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Activation of NF-κB, MAP3K8 and AKT is thought to protect cancer cells against therapy-induced

cytotoxicity. Körber et al. (Korber, Staribacher, Ratzenbock, Steger, & Mader, 2016) demonstrated NF-
-p
κB p65 subunit was selectively activated in 5-FU-resistant CRC cell lines. Consequently, several
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inhibitors of NF-κB, MAP3K8, and AKT effectively circumvented this chemoresistance. The authors
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described that as a cellular reaction, NF-κB inhibition may trigger a feedback loop resulting in activation

of extracellular signal-regulated kinase. Their results suggested that chemoresistance to 5-FU in CRC
na

cells was strongly dependent on NF-κB activation. The efficacy of MAP3K8 inhibition in their model
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potentially uncovered a new mechanism to bypass 5-FU resistance.

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Loss of P53 binding protein 1 (53BP1) is considered as a poor prognostic factor in CRC. In the study of

Yao et al. (Yao, et al., 2017), it has been demonstrated that 53BP1 down-regulation resulted in reduced

time to progression after first-line 5-FU chemotherapy in mCRC patients. In addition, 53BP1 silencing

increased proliferation, inhibited apoptosis and induced S phase arrest in HCT116 and HT29 CRC cells

after 5-FU treatment. Moreover, 53BP1 knockdown also reduced the protein levels of ATM-CHK2-P53

apoptotic pathway effectors, caspase 9 and caspase 3, while increasing Bcl-2 expression. In vivo, 53BP1

silencing accelerated tumor proliferation in nude mice and enhanced resistance to 5-FU. These findings

confirmed that 53BP1 loss might be a negative factor for 5-FU treatment efficacy, promoting cell

proliferation and inhibiting apoptosis by suppressing ATM-CHK2-P53 signaling.

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4.12. Autophagy

The modulation of autophagy is becoming increasingly important in setting up effective treatment for

gastrointestinal cancer patients in clinical practice (Tang, Feng, Liang, & Cai, 2016). Autophagy is

activated by cellular stress or increased metabolic demand to degrade damaged organelles or particles

in the cells and recycle amino or fatty acids via autophagosome formation. This occurs after cells

suffered from nutrient starvation, hypoxia and chemotherapy agents (Zheng, 2017). Autophagy

adaptation can promote cell survival and cause tumor growth and therapeutic resistance. Therefore,

of
autophagic inhibition restores chemosensitivity and increases cancer cell death using

hydroxychloroquine or it derives (Ojha, Bhattacharyya, & Singh, 2015).

ro
The link between autophagy in various cancer types and chemoresistance to different chemotherapy
-p
drugs has been observed. Regarding CRC, previous studies have demonstrated that inhibition of
re
autophagy augments anticancer effects of 5-FU (K. Sasaki, et al., 2012; K. Sasaki, et al., 2010) and
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autophagy responds to 5-FU through the regulation of anti-apoptotic proteins Bcl-2 and Bcl-xL (J. Li, et

al., 2009; Sui, et al., 2014). It has been suggested that the induction of autophagic cell death might be
na

better suited to the strategies focusing on the inhibition of Bcl-2 protein family for overcoming 5-FU
ur

resistance (Hersey & Zhang, 2003). More recently, a critical role of the p38MAPK signaling pathway

was found in the cellular response to 5-FU by controlling the balance between apoptosis and
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autophagy. The genotoxic stress induced by 5-FU is mediated by ATM and ATR related proteins and

increases p53 expression in CRC cells, which also promotes the activation of the signaling axis, MAPK

kinase 6/3-p38MAPK-p53 driven apoptosis (de la Cruz-Morcillo, et al., 2012). Another mechanism that

may participate in the 5-FU-induced autophagy response is p53-AMPK-mTOR pathway; p53 positively

regulates autophagy by activation of AMPK and subsequent inhibition of mTOR (Hu, et al., 2015; J. Li,

Hou, Faried, Tsutsumi, & Kuwano, 2010).

The CD44 antigen is a cell-surface glycoprotein involved in cell-cell interactions, cell adhesion and

migration. The CD44 isoform containing variant exon v6 (CD44v6) plays an important role in the

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progression, metastasis, and prognosis of CRC. Recently, it was found that CD44v6 is involved in

acquired drug resistance. Lv et al. (Lv, et al., 2016) studied the molecular mechanism of CD44v6 in the

resistance of CRC cells to 5-FU and oxaliplatin on SW480 CRC cells and human CRC tissues. The authors

revealed that the overexpression of CD44v6 contributes to chemoresistance in cell lines under

cytotoxic stress via the modulation of autophagy, epithelial-mesenchymal transition, activation of the

PI3K-AKT, and MAPK-Ras-Erk pathways. Moreover, the high CD44v6 expression in the primary tumor

was closely associated with an early recurrence in CRC patients who underwent curative surgery and

adjuvant chemotherapy.

of
Pharmacologic interference with these interactions might provide a novel therapeutic strategy

ro
targeting CRC cells with high 5‑FU treatment resistance. In fact, the combination of
-p
oxaliplatin/bevacizumab with hydroxychloroquine is currently being investigated in clinical trials
re
(summarized in (Tang, et al., 2016)).
lP

5. Future perspectives
na

Personalized medicine aims to provide therapies specifically tailored for each patient according to not

only his/her clinical and histopathological status but also genomic/epigenomic/proteomic

ur

characteristics. Unfortunately, our ability to predict the clinical efficacy of anticancer drugs based on
Jo

preclinical research or clinical responses in relation to tumor mutational characteristics is still limited

(Niederst, et al., 2015). Chemoresistance remains the main obstacle to CRC therapy, and delaying or

overcoming it emerges as a major challenge. Although the number of prognostic/predictive molecular

biomarkers for CRC is increasing, only MSI status, RAS-mutation and possibly BRAF-mutation status

currently influence clinical decision-making. Early trials of personalized therapy based on mutation

status of KRAS, BRAF, PI3KCA, and expression of Topo-I, ERCC1, TS, and TP did not show any

improvement in progression-free survival (Cubillo, et al., 2014). However, some authors have proposed

using KRAS, BRAF, PI3KCA, and PTEN mutation status as a signature to guide personalized therapy

(Bardelli & Siena, 2010; Sartore-Bianchi, et al., 2009).

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An ideal biomarker should meet the following criteria: it should be reliable, sensitive, specific, robust,

accurate, reproducible, and ideally non-invasive. With this respect, circulating nucleic-acid based

biomarkers (or so-called “liquid biopsy”) are currently extensively studied in cancer research. Besides

detection of specific DNA mutations for predicting response to anti-EGFR therapies (Strickler, et al.,

2018), measuring the plasma/serum concentrations of cell-free DNA (cfDNA) and/or circulating tumor

DNA (ctDNA) emerged to be an effective indirect predictive biomarker for 5-FU efficacy in CRC patients

(reviewed in (Vymetalkova, Cervena, Bartu, & Vodicka, 2018)).

of
The main limitation of studies dealing with characterization drug tailoring in relation to response lay in

the fact that the large majority of differences in responses to particular drug are not attributed to

ro
individual molecular features and the majority of associations between drug activity and genetic
-p
features are relatively weak (Barretina, et al., 2012; Garnett, et al., 2012). Therefore, international
re
collaborations and innovative study designs are warranted to drive progress in the clinical

development of subgroup-specific treatments (Punt, Koopman, & Vermeulen, 2017).

lP

The challenges associated with intra-tumor heterogeneity are immense and include minimal residual
na

disease and the emergence of therapy resistance. Intra-tumor heterogeneity relates to genetic,

functional, and non-genetic (such as epigenetic) heterogeneity. Heterogeneity also exists between
ur

genetically identical cancer cells. The most-critical distinction is between fully differentiated, non-
Jo

clonogenic cancer cells that have lost the ability to contribute to tumor growth, and CSCs, which are

believed to fuel long-term cancer growth and metastasis (Relton, Torgerson, O'Cathain, & Nicholl,

2010). Important prominent issues that need to be addressed include a fast turnaround time for the

use of new model systems and genomic assays, as well as a better understanding of the influence of

intra-tumor variation and how this relates to the sensitivity of drug testing using these methods (Punt,

et al., 2017).

The QUASAR (QUick And Simple And Reliable) trial was designed to provide large-scale randomized

evidence on the value of adjuvant chemotherapy with 5-FU and LV in both colon and rectal cancer and,

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in particular, in stage II disease (Quasar Collaborative, et al., 2007). That particular adjuvant

chemotherapy produces a small (approximately 3%) survival benefit in stage II colon cancer, which

must be balanced with its toxicity, including toxic deaths (approximately 0.5%). However, post hoc

analysis showed no significant difference in the survival between adjuvant therapy and surgery-alone

groups in patients with stage II CRC. This narrow therapeutic index underlines the importance of

selecting the suitable patients for adjuvant therapy. In the absence of strong evidence, clinical

guidelines by the National Comprehensive Cancer Network ("National Comprehensive Cancer Network

Clinical Practice Guidelines in Oncology (NCCN Guidelines). Colon Cancer.," 2019), the American

of
Society of Clinical Oncology (Benson, et al., 2004), and the European Society for Medical Oncology

ro
(Labianca, et al., 2013) recommend the use of adjuvant therapy in stage II CRC with specific high-risk
-p
features. However, the results of studies on the survival improvement with adjuvant therapy in high-

risk stage II CRC patients are inconclusive (Jalaeikhoo, et al., 2019).

re

According to the Clinical Trials Database that is maintained by the National Library of Medicine (NLM)
lP

at the National Institutes of Health (NIH), there are hundreds of currently recruiting studies using 5-FU
na

and other FPs for CRC (https://clinicaltrials.gov/, 2019). Specifically, there are 141 and 107 of those

studies for 5-FU and Capecitabine, respectively. Sixteen, six, and three studies are ongoing on newer
ur

FPs such as TAS-102, S-1, and Tegafur, respectively. None of currently recruiting studies are for 5’-
Jo

DFUR, Eniluracil, and Emitefur for CRC. From all the currently recruiting studies using 5-FU and other

FPs, we selected those in phase III and are presented in Supplementary Table 1.

In current practice, clinical and pathologic markers (for example intestinal perforation/obstruction,

pathologic stage T4, presence of lymphatic/vascular invasion, high tumor grade, < 12 nodes examined)

can identify a minority of patients with stage II disease with higher recurrence risk, but they do not

adequately assess recurrence risk for individual patients. To address this issue, the use of molecular

markers, such as MSI/MMR, loss of heterozygosity at 18q, and levels of expression of individual genes

or groups of genes has been investigated (O'Connell, et al., 2010). It is generally known that MSI-H may

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identify a small percentage (approximately 15%) of patients with stage II disease who receive little

benefit from FU/LV (Richman, 2015).

6. Conclusions

FPs continue to be the mainstay of systemic therapy for CRC. Recent advances in molecular medicine

have significantly expanded our knowledge of mechanisms underlying resistance to 5-FU and other

FPs in CRC.

Despite the ongoing development of novel antitumor agents and therapeutic principles as we enter

of
the era of personalized cancer medicine, systemic chemotherapy involving infusional 5-FU/LV

ro
continues to be the cornerstone of treatment for CRC patients. The significant progress in the
-p
exploration of causes of drug resistance in the CRC treatment has been achieved; nevertheless, there
re
are still several barriers which prevent the implementation of personalized medicine to clinical

practice. The implementation of molecular technologies, such as microarrays, next generation

lP

sequencing, and proteomics in recent years has led to the accumulation of vast amounts of genomic
na

and proteomic data. Inter-individual differences in the treatment response in CRC patients are caused

by the unique genetic and epigenetic characteristics of each individual. CRC is a heterogeneous disease
ur

and therefore, future studies taking into consideration this heterogeneity, also in interplay with tumor
Jo

microenvironment, are warranted. This approach might contribute to identifications of reliable links

between specific molecular features of the tumor and effectiveness of 5-FU-based chemotherapy.

Conflict of Interest Statement

TB received honoraria from Roche and Servier (unrelated to the present article). Other authors declare

that there are no conflicts of interest.

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Acknowledgements

This project was supported by Grant Agency of the Ministry of Health of the Czech Republic (AZV 17-

30920A and NV19-09-00237), by Charles University Research Centre program UNCE/MED/006

“University Center of Clinical and Experimental Liver Surgery” and National Sustainability Program I

(NPU I) Nr. LO1503 provided by the Ministry of Education Youth and Sports of the Czech Republic, and

by Grant Agency of the Charles University (GAUK 302119).

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