www.impactjournals.com/oncotarget/ Oncotarget, 2017, Vol. 8, (No. 32), pp: 53017-53027 Research Paper MicroRNA profiles involved in trifluridine resistance Kenta Tsunekuni1,2,3,*, Masamitsu Konno1,4,*, Ayumu Asai1,2,4, Jun Koseki2, Takashi Kobunai3, Teiji Takechi3, Yuichiro Doki1, Masaki Mori1 and Hideshi Ishii1,2 1Department of Gastroenterological Surgery, Osaka University Graduate School of Medicine, Osaka, 565-0871, Japan 2Department of Medical Data Science, Osaka University Graduate School of Medicine, Osaka, 565-0871, Japan 3Translational Research Laboratory, Taiho Pharmaceutical Co, Ltd, Tokushima City, Tokushima, 771-0194, Japan 4Department of Frontier Science for Cancer and Chemotherapy, Osaka University Graduate School of Medicine, Osaka, 565-0871, Japan *These authors contributed equally to this work Correspondence to: Masaki Mori, email: [email protected] Hideshi Ishii, email: [email protected] Keywords: colorectal cancer, trifluridine, 5-fluorouracil, let-7d-5p, drug resistance Received: March 28, 2017 Accepted: May 09, 2017 Published: May 23, 2017 Copyright: Tsunekuni et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT Trifluridine (FTD) is a key component of the novel oral antitumor drug trifluridine/tipiracil, which is approved for the treatment of patients with metastatic colorectal cancer refractory to standard chemotherapies. A microRNA analysis of three colorectal cell lines was conducted to investigate causes of FTD resistance. Drug resistant sublines of DLD-1, HCT-116, and RKO cells were developed by continuous administration of increasing doses of FTD for 5 months. The let-7d-5p gene, which maps to chromosome 9q22.32, was downregulated in the FTD-resistant DLD-1 sublines. DLD-1 cells became more resistant to FTD when let-7d-5p was knocked down and more sensitive when let-7d-5p was overexpressed. The FTD-resistant sublines were not cross-resistant to 5-fluorouracil (5-FU); 5-FU sensitivity was affected only slightly when let-7d-5p was overexpressed or knocked down. These data indicate that let-7d-5p increases sensitivity of FTD but not 5-FU and that let-7d-5p is a potential clinical marker of treatment sensitivity. INTRODUCTION drugs to infusion regimens has resulted in a median OS of up to 21 months [4], with it being 29 months in patients Colorectal cancer (CRC) was the second leading with additional lines of therapy [5]. Despite these recent cause of cancer death in both men and women in the 35 advances, additional effective options would be especially Organization for Economic Co-operation and Development beneficial for prolonging survival of patients who are countries in North and South America, Europe, and Asia- refractory to current therapies. Pacific in 2015 [1]. The prognosis of metastatic (m)CRC Trifluridine/tipiracil is an oral combination therapy is poor. Between 2004 and 2010 in the USA, the 5-year comprising trifluridine (FTD), which is a thymidine survival of patients with mCRC at diagnosis was only analog, and tipiracil, which is a thymidine phosphorylase 13% [2]. Novel drugs and combination regimens for inhibitor that improves the bioavailability of FTD [6]. management of mCRC have increased median overall FTD is incorporated directly into DNA, thereby interfering survival (OS) from 6 to over 30 months in the past with its function [7]. The 5-FU metabolite fluorodeoxy- two decades. 5-FU-based regimens have become the uridine (FdUrd) is incorporated into DNA and has an preferred mCRC treatment. Continuous infusion of 5-FU, antiproliferative effect; however, the incorporation of FTD leucovorin, and either irinotecan (FOLFIRI) or oxaliplatin into DNA is reported be to significantly higher than that (FOLFOX) have increased median OS to 20 months or of 5-FU [8, 9]. An international, double-blind, placebo- more [3]. The addition of biologically targeted antibody controlled Phase III study confirmed the efficacy of www.impactjournals.com/oncotarget 53017 Oncotarget trifluridine/tipiracil in patients with metastatic colorectal of mRNA expressed by a gene located on chromosome cancer (mCRC) who had previously been treated with 9 (Figure 2B). We therefore speculated that let-7d-5p standard chemotherapy [10]. FTD resistance has been was downregulated by a genome alteration that occurred reported in vitro in colorectal cell lines [11], and the during the acquisition of FTD resistance. involvement of micro (mi)RNAs in chemoresistance has been reported in several cancers [12, 13]. miRNAs are a let-7d expression in FTD-resistant and parental conserved class of noncoding small RNAs that regulate cell lines the expression of genes involved in self renewal, survival, and tumor progression [14]. Several miRNAs that are qPCR was used to compare let-7d-5p expression in aberrantly expressed in CRC have been associated with FTD-resistant and parental sublines. We found that let-7d- clinical outcomes [15]. 5p was downregulated in DLD-1/FTD compared with its In this study, we analyzed miRNA expression in parental cell line (Figure 3C), but that let-7d-5p expression CRC parental cell lines and FTD-resistant sublines to was not significantly different in either HCT-116/FTD or investigate the relationship of miRNAs and FTD treatment RKO/FTD cells compared with the respective parental effectiveness. The miRNA let-7d-5p was associated with cells (Figure 3A, 3B). FTD resistance; both miRNA and mRNA expression differed in FTD-resistant and parental cells. miRNA and let-7d-5p controls sensitivity to FTD mRNA clustered in a locus on chromosome 9 (miRNA at 9p22.32) were downregulated in FTD-resistant cells, one We assessed the effects of let-7d-5p expression of which was the miRNA let-7d-5p, a member of the let-7 on FTD sensitivity by treating DLD-1 cells with anti- family that is known to target oncogenes and several genes let-7d-5p oligo RNA. qPCR assay found that let-7d-5p regulating the cell cycle and cell proliferation [16]. expression was lower in cells treated with anti-let-7d-5p than those treated with an oligo RNA-negative control RESULTS (Figure 4A). Anti-let-7d-5p-treated DLD-1 cells were less sensitive to FTD than DLD-1 negative control cells and had IC values of 16.8 μM and 7.6 μM, respectively (IC FTD-resistant colorectal cancer cell lines 50 50 fold change = 2.2, Figure 4B and Supplementary Table 2). We established FTD-resistant sub lines of DLD-1 5-FU sensitivity was slightly changed in anti-let-7d-5p- and HCT-116, and RKO CRC cells by exposure to step- treated cells (Figure 4B, and Supplementary Table 2). wise increasing concentrations of FTD over 3–5 days We assessed the effects of let-7d-5p overexpression starting at 1 µM and ending at 400 µM. FTD resistance on FTD sensitivity by treating DLD-1 with a let-7d-5p mimic. The expression of let-7d-5p was assayed by qPCR, was estimated as the ratio of the IC50 of each resistant line to that of the respective parent cell line after exposure to which showed that let-7d-5p expression was higher in various concentrations of FTD for 3 days. Each of the cell cells treated with the let-7d-5p mimic than in those treated with an oligo RNA-negative control (DLD-1 in Figure 5A; lines had become highly resistant to FTD, with IC50 ratios DLD-1/FTD in Figure 5D). Contrary to the results of that were 22.5- to > 40-fold higher than the IC50 of the parent cell lines (Figure 1A–1C, Supplementary Table 1). treatment with the let-7d-5p inhibitor, DLD-1 cells treated FTD-resistant sublines were not cross-resistant to 5-FU with the let-7d-5p mimic which induced overexpression (Figure 1D–1F, Supplementary Table 1). These results of let-7d-5p, were more sensitive to FTD than negative suggest that different mechanisms of action were involved controls were. The IC50 values were 13.9 μM and 3.7 μM, in development of FTD and 5-FU resistance. respectively (IC50 fold change of 0.27), (Figure 5B and Supplementary Table 2). 5-FU sensitivity was slightly miRNA and mRNA expression in FTD-resistant changed in cells treated with the let-7d-5p mimic compared cell lines with negative controls (Figure 5C and Supplementary Table 2). Both parental DLD-1 and FTD-resistant DLD- To identify the miRNA associated with FTD 1/FTD cells were sensitized to FTD (Figure 5E and resistance, we compared miRNA expression in resistant Supplementary Table 2) and slightly sensitized to 5-FU and sensitive cell lines using 3D-Gene human miRNA and (Figure 5F and Supplementary Table 2) by the let-7d-5p mRNA oligo chips (Toray). Probe sets that downregulated mimic. These data suggest that let-7d-5p was more closely with a fold-change greater than two were counted, and associated with FTD than with 5-FU sensitivity. Fisher’s exact probability test results with p-values of 2 × 10−3, were considered as chromosomal regions with Aurora B, a let-7d target, controls FTD a significant expression change. Of the three cell lines, sensitivity DLD-1/FTD was the most resistant to FTD, and had acquired a significant downregulation of two miRNAs TargetScan target prediction software indicated that encoded by a gene located on 9q22.32 including let-7d-5p Aurora B was a possible let-7d-5p target (Supplementary (Figure 2A). We also found significant downregulation Table 4). We found that let-7d-5p was downregulated www.impactjournals.com/oncotarget 53018 Oncotarget and Aurora B upregulated, in DLD-1/FTD compared treated with FTD or 5-FU, and cell viability was assayed.