Abstract 2556: Trifluridine induces p53-dependent sustained G2 phase arrest with its massive misincorporation into DNA and few DNA strand breaks
Authors: Matsuoka Kazuaki , Iimori Makoto , Niimi Shinichiro , Tsukihara Hiroshi , Watanabe Sugiko , Kiyonari Shinichi , Kiniwa Mamoru , Ando Koji , Tokunaga Eriko , Saeki Hiroshi , Oki Eiji , Kitao Hiroyuki , Maehara Yoshihiko 
Published: August 2015

Abstract
Background: TAS-102 is a combination of an antineoplastic thymidine-based nucleoside analogue, trifluridine (FTD), and a thymidine phosphorylase inhibitor, tipiracil (TPI), at a molar ratio of 1:0.5. FTD is the active antitumor component of TAS-102, and its triphosphate form is incorporated into the DNA in tumor cells. TPI, a potent inhibitor of the FTD-degrading enzyme thymidine phosphorylase, maintains adequate plasma concentrations of orally administered FTD, thereby potentiating its antitumor activity. In an international multicenter randomized double blind Phase III study (RECOURSE), TAS-102 administration resulted in a significant improvement in overall survival and progression-free survival and a favorable safety profile compared to placebo treatment in patients with metastatic colorectal cancer refractory to standard chemotherapies. In this study, we investigated the mechanism of FTD-induced cytotoxicity in colorectal cancer and other cell lines.
Method: The colorectal cancer cell lines RKO and HCT-116, the subline HCT-116 p53−/−, and the lung adenocarcinoma cell line A549 were used. Cells were treated with FTD or FdUrd using the IC50 values for 12 to 72 h. Molecules involved in DNA damage and the cell cycle were evaluated by western blot and RT-PCR. Cell cycle analysis and DNA strand-breaks were evaluated by flow cytometry and alkaline- and neutral-comet assays.
Results: FTD and FdUrd activated similar DNA damage response pathways via transient phosphorylation of Chk1 at Ser345, followed by accumulation of p53 and p21 in HCT-116 cells. A similar biochemical response was observed using the IC50 concentration of FdUrd or FTD in RKO and A549 cells. FTD repressed G2 to M phase progression and the cells accumulated in G2 phase 72 h after treatment, with a 35% and 29% decrease in the CDK1 and CCNB1 mRNA levels compared to the control. The G2 phase arrest by FTD was commonly induced in each p53 proficient cell lines (HCT-116, RKO, and A549). In contrast, FTD-induced G2 phase arrest was not observed when p53 expression was suppressed. Furthermore, the p53-dependent G2 phase arrest by FTD was associated with the downregulation of Cyclin B1, both through its transcriptional repression and proteasomal degradation. Although FdUrd induced numerous single- and double-strand DNA breaks, FTD did not induce detectable DNA strand breaks. FTD was massively incorporated into DNA (41.4 pmol/μg DNA), whereas less FdUrd was incorporated (0.6 pmol/μg DNA) 72 h after treatment.
Conclusion: Our analyses revealed that FTD induced sustained G2 phase arrest and few DNA strand breaks, in spite of massive incorporation into DNA. These results suggest that the mechanism of FTD action is unique at the cellular level, resulting in the clinical efficacy of TAS-102.
Citation Format: Kazuaki Matsuoka, Makoto Iimori, Shinichiro Niimi, Hiroshi Tsukihara, Sugiko Watanabe, Shinichi Kiyonari, Mamoru Kiniwa, Koji Ando, Eriko Tokunaga, Hiroshi Saeki, Eiji Oki, Hiroyuki Kitao, Yoshihiko Maehara. Trifluridine induces p53-dependent sustained G2 phase arrest with its massive misincorporation into DNA and few DNA strand breaks. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2556. doi:10.1158/1538-7445.AM2015-2556