DNA Replication Stress Induced by Trifluridine Determines Tumor Cell Fate According to p53 Status.

DNA replication stress (DRS) is a predominant cause of genome instability, a driver of tumorigenesis and malignant progression. Nucleoside analogue-type chemotherapeutic drugs introduce DNA damage and exacerbate DRS in tumor cells. However, the mechanisms underlying the antitumor effect of these drugs are not fully understood. Here, we show that the fluorinated thymidine analogue trifluridine (FTD), an active component of the chemotherapeutic drug trifluridine/tipiracil, delayed DNA synthesis by human replicative DNA polymerases by acting both as an inefficient deoxyribonucleotide triphosphate source (FTD triphosphate) and as an obstacle base (trifluorothymine) in the template DNA strand, which caused DRS. In cells, FTD decreased the thymidine triphosphate level in the dNTP pool and increased the FTD triphosphate level, resulting in the activation of DRS-induced cellular responses during S-phase. In addition, replication protein A-coated single-stranded DNA associated with FancD2 and accumulated after tumor cells completed S-phase. Finally, FTD activated the p53-p21 pathway and suppressed tumor cell growth by inducing cellular senescence via mitosis skipping. In contrast, tumor cells that lost wild-type p53 underwent apoptotic cell death via aberrant late mitosis with severely impaired separation of sister chromatids. These results demonstrate that DRS induced by a nucleoside analogue-type chemotherapeutic drug suppresses tumor growth irrespective of p53 status by directing tumor cell fate toward cellular senescence or apoptotic cell death according to p53 status. IMPLICATIONS: Chemotherapeutic drugs that increase DRS during S-phase but allow tumor cells to complete S-phase may have significant antitumor activity even when functional p53 is lost.

Cytotoxicity of trifluridine correlates with the thymidine kinase 1 expression level.

Trifluridine (FTD), a tri-fluorinated thymidine analogue, is a key component of the oral antitumor drug FTD/TPI (also known as TAS-102), which is used to treat refractory metastatic colorectal cancer. Thymidine kinase 1 (TK1) is thought to be important for the incorporation of FTD into DNA, resulting in DNA dysfunction and cytotoxicity. However, it remains unknown whether TK1 is essential for FTD incorporation into DNA and whether this event is affected by the expression level of TK1 because TK1-specific-deficient human cancer cell lines have not been established. Here, we generated TK1-knock-out human colorectal cancer cells using the CRISPR/Cas9 genome editing system and validated the specificity of TK1 knock-out by measuring expression of AFMID, which is encoded on the same locus as TK1. Using TK1-knock-out cells, we confirmed that TK1 is essential for cellular sensitivity to FTD. Furthermore, we demonstrated a correlation between the TK1 expression level and cytotoxicity of FTD using cells with inducible TK1 expression, which were generated from TK1-knock-out cells. Based on our finding that the TK1 expression level correlates with sensitivity to FTD, we suggest that FTD/TPI might efficiently treat cancers with high TK1 expression.

Thymidine Kinase 1 Loss Confers Trifluridine Resistance without Affecting 5-Fluorouracil Metabolism and Cytotoxicity.

Acquired resistance to therapeutic drugs is a serious problem for patients with cancer receiving systemic treatment. Experimentally, drug resistance is established in cell lines in vitro by repeated, continuous exposure to escalating concentrations of the drug; however, the precise mechanism underlying the acquired resistance is not always known. Here, it is demonstrated that the human colorectal cancer cell line DLD1 with acquired resistance to trifluridine (FTD), a key component of the novel, orally administered nucleoside analogue-type chemotherapeutic drug trifluridine/tipiracil, lacks functional thymidine kinase 1 (TK1) expression because of one nonsense mutation in the coding exon. Targeted disruption of the TK1 gene also conferred severe FTD resistance, indicating that the loss of TK1 protein expression is the primary cause of FTD resistance. Both FTD-resistant DLD1 cells and DLD1-TK1 -/- cells exhibited similar 5-fluorouracil (5-FU) sensitivity to that of the parental DLD1 line. The quantity of cellular pyrimidine nucleotides in these cells and the kinetics of thymidylate synthase ternary complex formation in 5-FU-treated cells is similar to DLD1 cells, indicating that 5-FU metabolism and cytotoxicity were unaffected. The current data provide molecular-based evidence that acquired resistance to FTD does not confer 5-FU resistance, implying that 5-FU-based chemotherapy would be effective even in tumors that become refractory to FTD during trifluridine/tipiracil treatment. Mol Cancer Res; 16(10); 1483-90. ©2018 AACR.

[Evolving 5-Fluorouracil Therapy to Achieve Enhanced Efficacy-Past and Current Efforts of Researchers].

5-fluorouracil(5-FU)therapy has advanced greatly over the past 50 years, achieving enhanced therapeutic effects and reduced adverse effects. By taking advantage of the metabolism of 5-FU, researchers have made efforts to develop prodrugs, combination drug products, and combination therapy regimens via biochemical modulation(BCM)with alteration of the drug metabolism. Examples include the advent of the prodrug tegafur(FT), followed by tegafur-uracil(UFT)and tegafurgimeracil- potassium oxonate(S-1)as combined products based on BCM. In the current standard treatment for gastrointestinal cancers, anticancer 5-FU derivatives serve as a platform for combination regimens with other cytotoxic agents or molecular- targeted drugs. To provide further improvements in anticancer therapy outcomes, novel molecular-targeted agents, immune checkpoint inhibitors, and other drugs are being developed, but 5-FU remains an attractive target that shows further potential for increased efficacy. In the future, the evolution of anticancer therapy with 5-FU derivatives is expected to continue via a variety of approaches.

[The Molecular Aspect of the Antitumor Effect of Oxaliplatin in Combination with 5-FU].

Platinum-based chemotherapeutic drugs as a component of combination chemotherapy are widely used in the treatment of cancer. In particular, oxaliplatin(L-OHP), one such platinum-based chemotherapeutic drug, has a synergistic effect in combination with 5-FU and Leucovorin for the treatment of advanced colorectal cancer. However, the underlying molecular mechanism of this synergistic effect has not been fully clarified yet. In this review, we summarize several updates about the in vitro action of oxaliplatin in human tumor cells and discuss the underlying mechanism of its synergistic effect with 5-FU.

The antibodies against 5-bromo-2'-deoxyuridine specifically recognize trifluridine incorporated into DNA.

Trifluridine (FTD) is a key component of the novel oral antitumor drug TAS-102 (also named TFTD), which consists of FTD and a thymidine phosphorylase inhibitor. FTD is supposed to exert its cytotoxicity via massive misincorporation into DNA, but the underlying mechanism of FTD incorporation into DNA and its correlation with cytotoxicity are not fully understood. The present study shows that several antibodies against 5-bromo-2'-deoxyuridine (BrdU) specifically cross-react with FTD, either anchored to bovine serum albumin or incorporated into DNA. These antibodies are useful for several biological applications, such as fluorescence-activated cell sorting, fluorescent immunostaining and immunogold detection for electron microscopy. These techniques confirmed that FTD is mainly incorporated in the nucleus during S phase in a concentration-dependent manner. In addition, FTD was also detected by immunohistochemical staining in paraffin-embedded HCT-116 xenograft tumors after intraperitoneal administration of FTD. Intriguingly, FTD was hardly detected in surrounding matrices, which consisted of fibroblasts with marginal expression of the nucleoside transporter genes SLC29A1 and SLC29A2. Thus, applications using anti-BrdU antibodies will provide powerful tools to unveil the underlying mechanism of FTD action and to predict or evaluate the efficacy and adverse effects of TAS-102 clinically.

The 1,2-Diaminocyclohexane Carrier Ligand in Oxaliplatin Induces p53-Dependent Transcriptional Repression of Factors Involved in Thymidylate Biosynthesis.

Platinum-based chemotherapeutic drugs are widely used as components of combination chemotherapy in the treatment of cancer. One such drug, oxaliplatin, exerts a synergistic effect against advanced colorectal cancer in combination with 5-fluorouracil (5-FU) and leucovorin. In the p53-proficient colorectal cancer cell line HCT116, oxaliplatin represses the expression of deoxyuridine triphosphatase (dUTPase), a ubiquitous pyrophosphatase that catalyzes the hydrolysis of dUTP to dUMP and inhibits dUTP-mediated cytotoxicity. However, the underlying mechanism of this activity has not been completely elucidated, and it remains unclear whether factors other than downregulation of dUTPase contribute to the synergistic effect of 5-FU and oxaliplatin. In this study, we found that oxaliplatin and dachplatin, platinum-based drugs containing the 1,2-diaminocyclohexane (DACH) carrier ligand, repressed the expression of nuclear isoform of dUTPase (DUT-N), whereas cisplatin and carboplatin did not. Oxaliplatin induced early p53 accumulation, upregulation of primary miR-34a transcript expression, and subsequent downregulation of E2F3 and E2F1. Nutlin-3a, which activates p53 nongenotoxically, had similar effects. Introduction of miR-34a mimic also repressed E2F1 and DUT-N expression, indicating that this miRNA plays a causative role. In addition to DUT-N, oxaliplatin repressed, in a p53-dependent manner, the expression of genes encoding enzymes involved in thymidylate biosynthesis. Consequently, oxaliplatin significantly decreased the level of dTTP in the dNTP pool in a p53-dependent manner. These data indicate that the DACH carrier ligand in oxaliplatin triggers signaling via the p53-miR-34a-E2F axis, leading to transcriptional regulation that ultimately results in accumulation of dUTP and reduced dTTP biosynthesis, potentially enhancing 5-FU cytotoxicity.

Trifluridine Induces p53-Dependent Sustained G2 Phase Arrest with Its Massive Misincorporation into DNA and Few DNA Strand Breaks.

Trifluridine (FTD) is a key component of the novel oral antitumor drug TAS-102, which consists of FTD and a thymidine phosphorylase inhibitor. Like 5-fluoro-2'-deoxyuridine (FdUrd), a deoxynucleoside form of 5-fluorouracil metabolite, FTD is sequentially phosphorylated and not only inhibits thymidylate synthase activity, but is also incorporated into DNA. Although TAS-102 was effective for the treatment of refractory metastatic colorectal cancer in clinical trials, the mechanism of FTD-induced cytotoxicity is not completely understood. Here, we show that FTD as well as FdUrd induce transient phosphorylation of Chk1 at Ser345, and that this is followed by accumulation of p53 and p21 proteins in p53-proficient human cancer cell lines. In particular, FTD induced p53-dependent sustained arrest at G2 phase, which was associated with a proteasome-dependent decrease in the Cyclin B1 protein level and the suppression of CCNB1 and CDK1 gene expression. In addition, a p53-dependent increase in p21 protein was associated with an FTD-induced decrease in Cyclin B1 protein. Although numerous ssDNA and dsDNA breaks were induced by FdUrd, few DNA strand breaks were detected in FTD-treated HCT-116 cells despite massive FTD misincorporation into genomic DNA, suggesting that the antiproliferative effect of FTD is not due to the induction of DNA strand breaks. These distinctive effects of FTD provide insights into the cellular mechanism underlying its antitumor effect and may explain the clinical efficacy of TAS-102.

Cdc6 protein obstructs apoptosome assembly and consequent cell death by forming stable complexes with activated Apaf-1 molecules.

Cdc6 is the bifunctional AAA+ ATPase that assembles prereplicative complexes on origins of replication and activates p21(CIP1)- or p27(KIP1)-bound Cdk2. During the G(1)-S transition, the Cdc6 gene essential for chromosomal replication is activated by the E2F transcriptional factor. Paradoxically, Apaf-1 encoding the central component of the apoptosome is also activated at the same time and by E2F. Consequently, genes for antipodal life and death are regulated in the same manner by the same transcriptional factor. Here we report a striking solution to this paradox. Besides performing prereplicative complex assembly and Cdk2 activation, Cdc6 obstructed apoptosome assembly by forming stable complexes very likely with a monomer of cytochrome c-activated Apaf-1 molecules. This function depended on its own ATPase domain but not on the cyclin-binding motif. In proliferating rodent fibroblasts, Cdc6 continued to block apoptosome assembly induced by a non-cytochrome c or some other mechanism, suppressing seemingly unintended apoptosis when promoting cell proliferation. Thus, Cdc6 is an AAA+ ATPase with three functions, all working for life.