Background: Trifluridine (FTD) is a key component of the antitumor drug combination trifluridine/tipiracil (FTD/TPI, also known as TAS-102), which is approved for the treatment of patients with metastatic colorectal cancer refractory to standard chemotherapies. Recently, a global phase III Trial (TAGS trial) revealed that FTD/TPI also prolongs overall survival in patients with metastatic gastric cancer. In this study, we explored the efficacy of FTD/TPI in 5-fluorouracil (5-FU)-resistant gastric cancer cell lines in vitro and in vivo, revealing their unique genomic signatures by a multi-omics profiling approach.

Method: 5-FU-resistant cell lines were established by continuously exposing the parent cell lines (MKN45, MKN74, and KATOIII) to escalating concentrations of 5-FU over a 1-year period. The sensitivities of the cell lines to FTD were evaluated by a cell growth inhibitory assay using crystal violet staining. Total RNA was purified using a silica membrane spin column-based centrifugation procedure. The extracted RNA was used to establish a cDNA library for transcriptome sequencing using a HiSeq 2500 platform. The resulting fastq files were used for mapping, gene mutation, and expression analysis. We especially focused on “pyrimidine metabolism” in Kyoto Encyclopedia of Genes and Genomes analysis.

Results: The KATOIII/5FU, MKN74/5FU, and MKN45/5FU were 2.4-fold, 4.7-fold, and 14.3-fold more resistant to 5-FU than their parent cell lines, respectively. MKN74/5FU and KATOIII/5FU did not show any cross-resistance to FTD, while TS-overexpressing MKN45/5FU showed partial cross-resistance to FTD in vitro. FTD/TPI exhibited significant antitumor activity in both MKN45/5FU and MKN74/5FU cells in vivo. As compared with corresponding parental cell lines, the transcriptome analysis revealed that ENTPD3 gene expression level changed by more than 2-fold in only MKN45/5FU, while ENTPD8 and ENPP3 gene expression changed by less than 0.5-fold in both MKN74/5FU and KATOIII/5FU. In addition, there are genes (UMPS, POLR2B, POLR2D, POLR2H, POLR2L, POLR2J2, POLR3G, PRIM1, POLE, and UPP1) that had non-synonymous mutation within their coding sequences. Interestingly, mutation status of POLR2L and PRIM1 in MKN45/5FU, of UMPS, POLR2D, and POLR2H in MKN74/5FU, of POLR2B, POLR2J2, and POLR3G in KATOIII/5FU differed from those of parental cell lines.

Conclusions: FTD/TPI was effective against 5-FU-resistant gastric cancer cells harboring various genetic alterations in vitro and in vivo. This might be a reason why FTD/TPI is expected to show significant benefits against heterogeneous metastatic gastric cancer in a clinical setting.

Citation Format: Kazuaki Matsuoka, Takashi Kobunai, Teiji Takechi. Trifluridine/tipiracil can overcome the resistance of gastric 5-fluorouracil-refractory cancer cells with various cancer driver-genes alterations in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2067.