AZD6738 promotes the tumor suppressive effects of trifluridine in colorectal cancer cells

Harata,Shinnosuke; Suzuki,Takuya; Takahashi,Hiroki; Hirokawa,Takahisa; Kato,Akira; Watanabe,Kaori; Yanagita,Takeshi; Ushigome,Hajime; Shiga,Kazuyoshi; Ogawa,Ryo; Mitsui,Akira; Kimura,Masahiro; Matsuo,Yoichi; Takiguchi,Shuji

doi:10.3892/or.2023.8489

AZD6738 promotes the tumor suppressive effects of trifluridine in colorectal cancer cells

Authors:
- Shinnosuke Harata
- Takuya Suzuki
- Hiroki Takahashi
- Takahisa Hirokawa
- Akira Kato
- Kaori Watanabe
- Takeshi Yanagita
- Hajime Ushigome
- Kazuyoshi Shiga
- Ryo Ogawa
- Akira Mitsui
- Masahiro Kimura
- Yoichi Matsuo
- Shuji Takiguchi
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Affiliations: Department of Gastroenterological Surgery, Nagoya City University Graduate School of Medical Sciences, Nagoya, Aichi 467‑8601, Japan, Department of Gastroenterological Surgery, Kariya Toyota General Hospital, Kariya, Aichi 448‑8505, Japan
Published online on: February 1, 2023 https://doi.org/10.3892/or.2023.8489
Article Number: 52
Copyright: © Harata et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Ataxia telangiectasia and Rad3‑related (ATR) is a kinase that repairs DNA damage. Although inhibitors that selectively target ATR have been developed, their effectiveness in colorectal cancer has not been widely reported. The present study hypothesized that anticancer agents that effectively act in the S phase before the G₂/M checkpoint may be ideal agents for concomitant use with ATR inhibitors, which act at the G₂/M checkpoint. Therefore, the present study examined the combined effects of AZD6738, an ATR inhibitor, and trifluridine (FTD), which acts in the S phase and has a high DNA uptake rate. In vitro cell viability assays, flow cytometry and western blotting were performed to evaluate cell viability, and changes in cell cycle localization and protein expression. The results revealed that in colorectal cancer cells, the combination of AZD6738 and FTD inhibited cell viability, cell cycle arrest at the G₂/M checkpoint and Chk1 phosphorylation, and increased apoptotic protein expression levels more than that when treated with FTD alone. HT29, a BRAF‑mutant cell line known to be resistant to anticancer drugs, was used to induce tumors in vivo. Since FTD does not have sufficient efficacy when administered orally, it was mixed with tipiracil to prevent degradation; this mixture is known as TAS‑102. TAS‑102 alone exerted minimal tumor suppressive effects; however, when used in combination with AZD6738, tumor suppression was observed, suggesting that AZD6738 may increase the effectiveness of a weakly effective drug. Although ATR inhibitors are effective against p53 mutants, the present study demonstrated that these inhibitors were also effective against the p53 wild‑type HCT116 colorectal cancer cell line. In conclusion, combination therapy with AZD6738 and FTD enhanced the inhibition of tumor proliferation in vitro and in vivo. In the future, we aim to investigate the potentiating effect of AZD6738 on 5‑fluouracil‑resistant cell lines that are difficult to treat.

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1	Yoshino T, Oki E, Nozawa H, Eguchi-Nakajima T, Taniguchi H, Morita S, Takenaka N, Ozawa D and Shirao K: Rationale and design of the TRUSTY study: A randomised, multicentre, open-label phase II/III study of trifluridine/tipiracil plus bevacizumab versus irinotecan, fluoropyrimidine plus bevacizumab as second-line treatment in patients with metastatic colorectal cancer progressive during or following first-line oxaliplatin-based chemotherapy. ESMO Open. 3:e0004112018. View Article : Google Scholar : PubMed/NCBI
2	Hirokawa T, Shiotani B, Shimada M, Murata K, Johmura Y, Haruta M, Tahara H, Takeyama H and Nakanishi M: CBP-93872 inhibits NBS1-mediated ATR activation, abrogating maintenance of the DNA double-strand break-specific G2 checkpoint. Cancer Res. 74:3880–3889. 2014. View Article : Google Scholar : PubMed/NCBI
3	Harper JW and Elledge SJ: The DNA damage response: Ten years after. Mol Cell. 28:739–745. 2007. View Article : Google Scholar : PubMed/NCBI
4	Foote KM, Nissink JWM, McGuire T, Turner P, Guichard S, Yates JWT, Lau A, Blades K, Heathcote D, Odedra R, et al: Discovery and characterization of AZD6738, a potent inhibitor of ataxia telangiectasia mutated and Rad3 related (ATR) kinase with application as an anticancer agent. J Med Chem. 61:9889–9907. 2018. View Article : Google Scholar : PubMed/NCBI
5	Bradbury A, Hall S, Curtin N and Drew Y: Targeting ATR as cancer therapy: A new era for synthetic lethality and synergistic combinations? Pharmacol Ther. 207:1074502020. View Article : Google Scholar : PubMed/NCBI
6	Yap TA, Tan DSP, Terbuch A, Caldwell R, Guo C, Goh BC, Heong V, Haris NRM, Bashir S, Drew Y, et al: First-in-human trial of the oral ataxia telangiectasia and RAD3-related (ATR) inhibitor BAY 1895344 in patients with advanced solid tumors. Cancer Discov. 11:80–91. 2021. View Article : Google Scholar : PubMed/NCBI
7	Yap TA, O'Carrigan B, Penney MS, Lim JS, Brown JS, de Miguel Luken MJ, Tunariu N, Perez-Lopez R, Rodrigues DN, Riisnaes R, et al: Phase I trial of first-in-class ATR inhibitor M6620 (VX-970) as monotherapy or in combination with carboplatin in patients with advanced solid tumors. J Clin Oncol. 38:3195–3204. 2020. View Article : Google Scholar : PubMed/NCBI
8	Kwok M, Davies N, Agathanggelou A, Smith E, Petermann E, Yates E, Brown J, Lau A and Stankovic T: Synthetic lethality in chronic lymphocytic leukaemia with DNA damage response defects by targeting the ATR pathway. Lancet. 385 (Suppl 1):S582015. View Article : Google Scholar : PubMed/NCBI
9	Yap TA, Krebs MG, Postel-Vinay S, El-Khouiery A, Soria JC, Lopez J, Berges A, Cheung SYA, Irurzun-Arana I, Goldwin A, et al: Ceralasertib (AZD6738), an oral ATR kinase inhibitor, in combination with carboplatin in patients with advanced solid tumors: A phase I study. Clin Cancer Res. 27:5213–5224. 2021. View Article : Google Scholar : PubMed/NCBI
10	Wallez Y, Dunlop CR, Johnson TI, Koh SB, Fornari C, Yates JWT, Bernaldo de Quirós Fernández S, Lau A, Richards FM and Jodrell DI: The ATR inhibitor AZD6738 synergizes with gemcitabine in vitro and in vivo to induce pancreatic ductal adenocarcinoma regression. Mol Cancer Ther. 17:1670–1682. 2018. View Article : Google Scholar : PubMed/NCBI
11	Sheng H, Huang Y, Xiao Y, Zhu Z, Shen M, Zhou P, Guo Z, Wang J, Wang H, Dai W, et al: ATR inhibitor AZD6738 enhances the antitumor activity of radiotherapy and immune checkpoint inhibitors by potentiating the tumor immune microenvironment in hepatocellular carcinoma. J Immunother Cancer. 8:e0003402020. View Article : Google Scholar : PubMed/NCBI
12	Nam AR, Jin MH, Park JE, Bang JH, Oh DY and Bang YJ: Therapeutic targeting of the DNA damage response using an ATR inhibitor in biliary tract cancer. Cancer Res Treat. 51:1167–1179. 2019. View Article : Google Scholar : PubMed/NCBI
13	Min A, Im SA, Jang H, Kim S, Lee M, Kim DK, Yang Y, Kim HJ, Lee KH, Kim JW, et al: AZD6738, a novel oral inhibitor of ATR, induces synthetic lethality with ATM deficiency in gastric cancer cells. Mol Cancer Ther. 16:566–577. 2017. View Article : Google Scholar : PubMed/NCBI
14	Smith J, Tho LM, Xu N and Gillespie DA: The ATM-Chk2 and ATR-Chk1 pathways in DNA damage signaling and cancer. Adv Cancer Res. 108:73–112. 2010. View Article : Google Scholar : PubMed/NCBI
15	Toczyski DP, Galgoczy DJ and Hartwell LH: CDC5 and CKII control adaptation to the yeast DNA damage checkpoint. Cell. 90:1097–1106. 1997. View Article : Google Scholar : PubMed/NCBI
16	Fan S, Smith ML, Rivet DJ II, Duba D, Zhan Q, Kohn KW, Fornace AJ Jr and O'Connor PM: Disruption of p53 function sensitizes breast cancer MCF-7 cells to cisplatin and pentoxifylline. Cancer Res. 55:1649–1654. 1995.PubMed/NCBI
17	Reaper PM, Griffiths MR, Long JM, Charrier JD, Maccormick S, Charlton PA, Golec JM and Pollard JR: Selective killing of ATM-or p53-deficient cancer cells through inhibition of ATR. Nat Chem Biol. 7:428–430. 2011. View Article : Google Scholar : PubMed/NCBI
18	Pabla N, Huang S, Mi QS, Daniel R and Dong Z: ATR-Chk2 signaling in p53 activation and DNA damage response during cisplatin-induced apoptosis. J Biol Chem. 283:6572–6583. 2008. View Article : Google Scholar : PubMed/NCBI
19	Iacopetta B: TP53 mutation in colorectal cancer. Hum Mutat. 21:271–276. 2003. View Article : Google Scholar : PubMed/NCBI
20	Suzuki T, Hirokawa T, Maeda A, Harata S, Watanabe K, Yanagita T, Ushigome H, Nakai N, Maeda Y, Shiga K, et al: ATR inhibitor AZD6738 increases the sensitivity of colorectal cancer cells to 5-fluorouracil by inhibiting repair of DNA damage. Oncol Rep. 47:782022. View Article : Google Scholar : PubMed/NCBI
21	Matsuoka K, Iimori M, Niimi S, Tsukihara H, Watanabe S, Kiyonari S, Kiniwa M, Ando K, Tokunaga E, Saeki H, et al: Trifluridine induces p53-dependent sustained G2 phase arrest with its massive misincorporation into DNA and Few DNA strand breaks. Mol Cancer Ther. 14:1004–1013. 2015. View Article : Google Scholar : PubMed/NCBI
22	Tanaka N, Sakamoto K, Okabe H, Fujioka A, Yamamura K, Nakagawa F, Nagase H, Yokogawa T, Oguchi K, Ishida K, et al: Repeated oral dosing of TAS-102 confers high trifluridine incorporation into DNA and sustained antitumor activity in mouse models. Oncol Rep. 32:2319–2326. 2014. View Article : Google Scholar : PubMed/NCBI
23	Ohashi S, Kikuchi O, Nakai Y, Ida T, Saito T, Kondo Y, Yamamoto Y, Mitani Y, Nguyen Vu TH, Fukuyama K, et al: Synthetic lethality with trifluridine/tipiracil and checkpoint kinase 1 inhibitor for esophageal squamous cell carcinoma. Mol Cancer Ther. 19:1363–1372. 2020. View Article : Google Scholar : PubMed/NCBI
24	Mayer RJ, Van Cutsem E, Falcone A, Yoshino T, Garcia-Carbonero R, Mizunuma N, Yamazaki K, Shimada Y, Tabernero J, Komatsu Y, et al: Randomized trial of TAS-102 for refractory metastatic colorectal cancer. N Engl J Med. 372:1909–1919. 2015. View Article : Google Scholar : PubMed/NCBI
25	Chou TC and Talalay P: Quantitative analysis of dose-effect relationships: The combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul. 22:27–55. 1984. View Article : Google Scholar : PubMed/NCBI
26	Fukushima M, Suzuki N, Emura T, Yano S, Kazuno H, Tada Y, Yamada Y and Asao T: Structure and activity of specific inhibitors of thymidine phosphorylase to potentiate the function of antitumor 2′-deoxyribonucleosides. Biochem Pharmacol. 59:1227–1236. 2000. View Article : Google Scholar : PubMed/NCBI
27	Emura T, Suzuki N, Fujioka A, Ohshimo H and Fukushima M: Potentiation of the antitumor activity of alpha, alpha, alpha-trifluorothymidine by the co-administration of an inhibitor of thymidine phosphorylase at a suitable molar ratio in vivo. Int J Oncol. 27:449–455. 2005.PubMed/NCBI
28	Iwata T, Uchino T, Koyama A, Johmura Y, Koyama K, Saito T, Ishiguro S, Arikawa T, Komatsu S, Miyachi M, et al: The G2 checkpoint inhibitor CBP-93872 increases the sensitivity of colorectal and pancreatic cancer cells to chemotherapy. PLoS One. 12:e01782212017. View Article : Google Scholar : PubMed/NCBI
29	Emura T, Suzuki N, Yamaguchi M, Ohshimo H and Fukushima M: A novel combination antimetabolite, TAS-102, exhibits antitumor activity in FU-resistant human cancer cells through a mechanism involving FTD incorporation in DNA. Int J Oncol. 25:571–578. 2004.PubMed/NCBI
30	Vendetti FP, Lau A, Schamus S, Conrads TP, O'Connor MJ and Bakkenist CJ: The orally active and bioavailable ATR kinase inhibitor AZD6738 potentiates the anti-tumor effects of cisplatin to resolve ATM-deficient non-small cell lung cancer in vivo. Oncotarget. 6:44289–44305. 2015. View Article : Google Scholar : PubMed/NCBI
31	Checkley S, MacCallum L, Yates J, Jasper P, Luo H, Tolsma J and Bendtsen C: Bridging the gap between in vitro and in vivo: Dose and schedule predictions for the ATR inhibitor AZD6738. Sci Rep. 5:135452015. View Article : Google Scholar : PubMed/NCBI
32	Dillon MT, Barker HE, Pedersen M, Hafsi H, Bhide SA, Newbold KL, Nutting CM, McLaughlin M and Harrington KJ: Radiosensitization by the ATR inhibitor AZD6738 through generation of acentric micronuclei. Mol Cancer Ther. 16:25–34. 2017. View Article : Google Scholar : PubMed/NCBI
33	Kataoka Y, Iimori M, Niimi S, Tsukihara H, Wakasa T, Saeki H, Oki E, Maehara Y and Kitao H: Cytotoxicity of trifluridine correlates with the thymidine kinase 1 expression level. Sci Rep. 9:79642019. View Article : Google Scholar : PubMed/NCBI
34	Rothkamm K, Christiansen S, Rieckmann T, Horn M, Frenzel T, Brinker A, Schumacher U, Stein A, Petersen C and Burdak-Rothkamm S: Radiosensitisation and enhanced tumour growth delay of colorectal cancer cells by sustained treatment with trifluridine/tipiracil and X-rays. Cancer Lett. 493:179–188. 2020. View Article : Google Scholar : PubMed/NCBI
35	Wilson Z, Odedra R, Wallez Y, Wijnhoven PWG, Hughes AM, Gerrard J, Jones GN, Bargh-Dawson H, Brown E, Young LA, et al: ATR inhibitor AZD6738 (Ceralasertib) exerts antitumor activity as a monotherapy and in combination with chemotherapy and the PARP inhibitor olaparib. Cancer Res. 82:1140–1152. 2022. View Article : Google Scholar : PubMed/NCBI
36	Goto H, Izawa I, Li P and Inagaki M: Novel regulation of checkpoint kinase 1: Is checkpoint kinase 1 a good candidate for anti-cancer therapy? Cancer Sci. 103:1195–1200. 2012. View Article : Google Scholar : PubMed/NCBI
37	Bernabe-Ramirez C, Patel R, Chahal J and Saif MW: Treatment options in BRAF-mutant metastatic colorectal cancer. Anticancer Drugs. 31:545–557. 2020. View Article : Google Scholar : PubMed/NCBI