Abstract 2886: ChIP-seq analysis to explore DNA replication profile in trifluridine-treated human colorectal cancer cells in vitro
Authors: Kobunai Takashi , Matsuoka Kazuaki , Takechi Teiji 
Published: August 2018

Background: Trifluridine (FTD) is a key component of the novel oral antitumor drug trifluridine/tipiracil, which was approved for the treatment of patients with metastatic colorectal cancer refractory to standard chemotherapies. FTD, an antineoplastic thymidine analogue, is efficiently incorporated into the genomic DNA of tumor cells. However, the molecular mechanisms underlying its cytotoxic effects remain unclear. Therefore, the DNA replication profile in FTD-treated cells was comprehensively analyzed to explore the mode of FTD incorporation into the genomic DNA. Method: We generated the DNA replication profile in HCT-116 human colorectal cancer cells as follows: asynchronous cultures of HCT-116 were exposed to 5 μM 5-bromo-2′-deoxyuridine (BrdU), a non-cytotoxic control drug or FTD, for 4 h, harvested, and subjected to DNA immunoprecipitation using an anti-BrdU antibody. An input and immunoprecipitated DNA from each experiment were sequenced, and the sequence reads generated using Illumina sequencing were aligned uniquely to the human genome (hg19), allowing up to two mismatches using the BWA algorithm with default settings. The density of aligned reads along the genome was calculated and stored in a bigWig file for visualization in genome browsers. Peak finding and gene ontology analysis were performed using MACS and Genomic Regions Enrichment of Annotations Tool (GREAT). The 60-bp sequences surrounding the summits of the top 1000 peaks were analyzed for conserved motifs using the online program MEME. Results: In an antibody validation step, we observed significant FTD peaks over input DNA. We identified 6043 and 5080 high confident FTD and BrdU peaks in HCT-116 cells, respectively. Interestingly, 2911 of 6043 FTD peaks were uncommon to BrdU peaks, while 3232 peaks were common in FTD and BrdU. The FTD and BrdU peaks were not randomly distributed throughout the genome. Most FTD and BrdU peaks were ±5 kb or further away from transcription start sites. We identified the genes associated with distant peaks of FTD using GREAT. FTD peaks occurred near genes involved in extracellular matrix structural composition such as ACAN, BGN, CHAD, and COL12A1, while BrdU peaks were near genes involved in cysteine-type endopeptidase inhibitor activity such as ARRB1, BIRC7, LCN1, and CST1. Dinucleotide repeats such as TGTGTG were observed as conserved motifs in FTD-peak sequences. Conclusion: Global FTD incorporation patterns can delineate active replication loci, namely FTD preferentially incorporating loci, in human cancer cells. The DNA replication profile in FTD-treated cells differed from that in BrdU-treated cells. Thus, differences in the incorporation patterns of FTD and BrdU into the genomic DNA revealed the mechanisms of antitumor activities of FTD.

Citation Format: Takashi Kobunai, Kazuaki Matsuoka, Teiji Takechi. ChIP-seq analysis to explore DNA replication profile in trifluridine-treated human colorectal cancer cells in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2886.