Chip-Seq Analysis to Explore DNA Replication Profile in Trifluridine
Total Page：16
File Type：pdf, Size：1020Kb
ANTICANCER RESEARCH 39 : 3565-3570 (2019) doi:10.21873/anticanres.13502 ChIP-seq Analysis to Explore DNA Replication Profile in Trifluridine-treated Human Colorectal Cancer Cells In Vitro TAKASHI KOBUNAI 1* , KAZUAKI MATSUOKA 2* and TEIJI TAKECHI 1 1Taiho Pharmaceutical Co., Ltd., Translational Research Laboratory, Tokyo, Japan; 2Taiho Pharmaceutical Co., Ltd., Translational Research Laboratory (Tokushima office), Tokushima, Japan Abstract. Background/Aim: Trifluridine (FTD) is a key efficacy and a tolerable safety profile for previously treated component of the novel oral antitumor drug trifluridine/ metastatic colorectal cancer (2). tipiracil that has been approved for the treatment of metastatic In basic research, FTD, the active antitumor component of colorectal cancer. In this study, a comprehensive analysis of FTD/TPI, is an antineoplastic thymidine analog (3), efficiently DNA replication profile in FTD-treated colon cancer cells was incorporated into genomic DNA in tumor cells (4-7). FTD performed. Materials and Methods: HCT-116 cells were transported into the cytoplasm of tumor cells by equilibrative exposed to BrdU or FTD and subjected to DNA immuno- nucleoside transporter 1 and 2 is phosphorylated to mono- precipitation. Immunoprecipitated DNA was sequenced; the phosphate, diphosphate, and triphosphate (FTD-TP) forms by density of aligned reads along the genome was calculated. Peak thymidine kinase 1 and nucleoside diphosphate kinase, finding, gene ontology, and motif analysis were performed using respectively. DNA polymerase α incorporates FTD-TP into MACS, GREAT, and MEME, respectively. Results: We identified DNA at sites aligned with adenine on the opposite strand (8). 6,043 and 5,080 high-confidence FTD and BrdU peaks in HCT- FTD/TPI exerts its antitumor activity in vivo predominantly 116 cells, respectively. Of 6,043 FTD peaks, 2,911 peaks were due to its DNA incorporation (7), rather than as a result of uncommon to BrdU. We observed that FTD was preferentially thymidilate synthase inhibition (6). However, the detailed incorporated into genomic regions containing simple repeats, molecular mechanisms underlying the cytotoxic effects after CpG islands, and gene bodies. Conserved motifs in FTD peaks FTD incorporation into DNA remain uncertain. ’ contained dinucleotide repeats such as (GT)n. Conclusion: An approach that combines 5-bromo-2 -deoxyuridine Global FTD incorporation patterns delineated FTD, (BrdU)-labeled DNA immunoprecipitation with DNA preferentially incorporating loci in cancer cells. microarray or next generation sequencing (ChIP-seq) to enable the genome-wide identification of BrdU-labeled Trifluridine/tipiracil (FTD/TPI), also known as TAS-102, is chromosomal DNA has been used to study DNA replication, approved for the treatment of patients with metastatic DNA repair, and DNA metabolism (9, 10). Recently, Kitao colorectal cancer and advanced gastric cancer. A phase III et al. reported that several antibodies against BrdU clinical study in patients with metastatic colorectal cancer specifically cross-react with FTD incorporated into DNA (CRC) refractory to standard chemotherapies showed that (11). Therefore, we used ChIP-seq analysis to assess DNA FTD/TPI significantly improved overall and progression-free replication profiles in FTD treated cells in order to explore survival compared to those of the placebo group (1). the mode of FTD incorporation into genomic DNA in Moreover, this combination of drugs showed an encouraging comparison with BrdU, which is a non-cytotoxic compound. Materials and Methods This article is freely accessible online. Chemicals and antibody. FTD, BrdU, and Anti-BrdU antibody 3D4 *These Authors contributed equally to this study. (Cat#555627) were purchased from Yuki Gosei Kogyo (Tokyo, Japan), Tokyo Chemical Industry (Tokyo, Japan), and BD Correspondence to: Takashi Kobunai, Ph.D., Taiho Pharmaceutical Biosciences (San Jose, CA, USA), respectively. Co., Ltd., Translational Research Laboratory, 1-2-4 Uchikanda, Chiyoda-ku, Tokyo 101-0047, Japan. Tel.: +81 332935394, Fax: Cell lines. The human colorectal cancer cell line HCT-116 was +81 332934798, e-mail: [email protected] obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured at 37˚C in a humidified Key Words: Trifluridine, trifluridine/tipiracil (TAS-102), ChIP-seq, atmosphere with 5% CO 2 in Dulbecco’s modified Eagle medium colorectal cancer. (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal 3565 ANTICANCER RESEARCH 39 : 3565-3570 (2019) bovine serum (Sigma-Aldrich), 100 U/ml penicillin (Nacalai Tesque, confidence FTD and BrdU active regions, respectively, in Kyoto, Japan), and 100 mg/ml streptomycin (Nakcalai Tesque). These HCT-116 cells (Table I). Interestingly, 2,911 out of 6,043 cells were authenticated in 2014 by analyzing short tandem repeats. FTD active regions were uncommon to those of BrdU, while ChIP-sequencing and data analyses. We obtained the DNA the remaining 3,132 FTD active regions were common to replication profile of HCT-116 cells as follows: asynchronous BrdU. The ChIP fragment densities along the genome are cultures of HCT-116 were exposed to 5 μM BrdU, a non-cytotoxic shown in Figure 1. The examples of FTD-specific ChIP control drug, or FTD for 4 h. The cells (4 ×10 7 cells per sample) peaks and common ChIP peaks between FTD and BrdU are were then harvested, and subjected to DNA immunoprecipitation shown in Figure 2. The number of FTD ChIP peaks were using an anti-BrdU antibody as described previously (12). Briefly, significantly correlated to the number of CpG islands cell lysates were sonicated and the DNA was sheared to an average (Spearman’s Rho r=0.77, p< 0.001), but not to chromosomal length of 300-500 bp. Genomic DNA (Input) was prepared by p= treating aliquots of chromatin with RNase, and proteinase K, size (r=0.28, 0.19) and the number of simple repeats followed by ethanol precipitation. An aliquot of chromatin (25 μg) (r=0.33, p= 0.12). Besides, the number of BrdU ChIP peaks was pre-cleared with protein A agarose beads (Invitrogen, Carlsbad, was significantly correlated to the number of CpG islands CA, USA). Genomic DNA regions of interest were isolated using 3 (r=0.67, p= 0.0004), the number of simple repeats (r=0.50, μl of anti-BrdU antibody. Complexes were washed, eluted from the p= 0.013), and chromosomal size (r=0.46, p= 0.025). These beads with SDS buffer, and subjected to RNase and proteinase K results suggested that the FTD peaks were not randomly treatment. ChIP DNA was purified by phenol-chloroform extraction distributed throughout the genome and some sequence and ethanol precipitation. Illumina sequencing libraries were prepared from the ChIP and Input DNAs by the standard preferences exist during FTD incorporation into DNA strand. consecutive enzymatic steps of end-polishing, dA-addition, and adaptor ligation. After a final PCR amplification step, the resulting ChIP peak distributions. The median length of FTD or BrdU DNA libraries were quantified and sequenced on Illumina’s NextSeq active regions was 500 bp or 501 bp, respectively. Therefore, 500 (Illumina, San Diego, CA, USA). Sequence analysis was we generated two sets of 50,000 random control regions of performed as follows: The 75-nt length sequence reads were 500-bp length over the entire hg19 genome as a control to mapped to the human genome (hg19) using the BWA algorithm compare with those of FTD or BrdU peaks. We analyzed FTD (13). Only reads that passed Illumina’s purity filter, aligned with no more than two mismatches, and mapped uniquely to the genome or BrdU active regions across each target region. Overlapping were used in the subsequent analysis. Also, duplicate reads were percentages of the active regions with genomic annotations are removed from the analysis. Determination of Fragment Density was presented in Table II. Overlapping between FTD active performed as follows: To identify the density of fragments, the regions and genomic regions such as simple repeat ( p< 0.05), g’enome was divided into 32-nt bins and the number of tags and, gene and gene prediction (including coding exon and intron) 5 -ends of the aligned reads in each bin was determined. Peak (p< 0.05) were significantly higher than those of BrdU active finding was performed using the MACS algorithm, which identifies regions. When compared to the random control, the significant enrichments in the ChIP-seq data file when compared to the Input data file (~random background). To compare peak metrics overlapping percentage of FTD active regions was 11 times between two samples, overlapping intervals are grouped into “active higher for simple repeat ( p< 0.0001), 3.6 times higher for the regions,” which are defined by the start coordinate of the most replication origin ( p< 0.0001), and 2.5 times higher for the upstream interval and the end coordinate of the most downstream CpG islands ( p< 0.0001), respectively. Simple repeats in hg19 interval (=union of overlapping intervals; “merged peaks”). In genome consist of 6.6% of mono-nucleotide repeat, 47% of locations where only one sample has an interval, this interval di-nucleotide repeats, 10.5% of tri-nucleotide repeats, 20.6% defines the active region. A comparison of active regions along of tetra-nucleotide repeats, 9.5% of penta-nucleotide repeats, genomic coordinates was performed using Strand NGS software ver.3.2 (Strand Life Science. Bangalore, India). The 60-bp and 5.8% of hexa-nucleotide repeats. FTD active regions sequences surrounding the summits of the top 1000 peaks were overlapped mono-nucleotide repeats by 0.02%, di-nucleotide analyzed for conserved motifs using the online program MEME repeats by 45.2%, tri-nucleotide repeats by 9.1%, and tetra- Suite ver. 5.0.4 (14). Gene ontology analysis was performed using nucleotide repeats by 22.3%, respectively (Table III). These Genomic Regions Enrichment of Annotations Tool (GREAT) (15). findings also supported that the distribution of FTD peaks along the genome showed specific sequence preferences.