Cancer Res Treat.  2020 Jul;52(3):945-956. 10.4143/crt.2020.080.

Inhibition of ATR Increases the Sensitivity to WEE1 Inhibitor in Biliary Tract Cancer

• Nam AR
• Jin MH
• Bang JH
• Oh KS
• Seo HR
• Oh DY
• Bang YJ

• KMID: 2504473
• DOI: http://doi.org/10.4143/crt.2020.080

Abstract

Purpose
Currently, the DNA damage response (DDR) pathway represents a key target for new cancer drug development. Advanced biliary tract cancer (BTC) has a poor prognosis because of the lack of efficacious treatment options. Although DNA repair pathway alterations have been reported in many patients with BTC, little is known regarding the effects of DDR-targeted agents against BTC.
Materials and Methods
In this study, nine BTC cell lines were exposed to the WEE1 inhibitor (AZD1775). In vitro, MTT assay, colony-forming assay, cell cycle analysis, phospho-histone H3 staining assay, Transwell migration assay, and western blot were performed. Then, to enhance the antitumor effect of AZD1775, the combination treatment of WEE1 inhibitor and ataxia telangiectasia mutated and Rad3 related (ATR) inhibitor (AZD6738) was conducted using MTT assay and comet assay. Finally, HuCCT-1 and SNU2670 xenograft models were established to confirm the anti-tumor effect of AZD1775 alone. Furthermore, the combination treatment was also evaluated in SNU2670 xenograft models.
Results
AZD1775 blocked the phosphorylation of CDC2 and CDC25C in all cell lines, but significantly increased apoptosis and S phase arrest in sensitive cells. However, increased p-ATR and phosphorylated ataxia telangiectasia mutated levels were observed in less sensitive cells. In addition, in vitro and in vivo data illustrated that AZD1775 combined with AZD6738 exerted more potent anti-tumor effects than either drug alone. Although WEE1 inhibition has promising anti-tumor effects in some BTC cells, the addition of ATR inhibitors could enhance its efficacy.
Conclusion
Taken together, this study supports further clinical development of DDR-targeted strategies as monotherapy or combination regimens for BTC.

Keyword

Biliary tract neoplasms; DNA damage response; WEE1; ATR

Figure

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  Fig. 1. Anti-proliferative effect of AZD1775 in biliary tract cancer (BTC) cells. (A) The anti-proliferative effects of AZD1775 in nine BTC cell lines were evaluated using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay after 72 hours. (B) The half-maximal inhibitory concentration (IC50) of AZD1775 in the MTT assay was calculated. (C) Western blot analyses of the effects of AZD1775 on signaling pathways in four BTC cell lines. Cells were treated with increasing concentrations of AZD1775 (0, 0.1, 0.5, and 1 μM) for 24 hours.

• 

  Fig. 2. The effect of AZD1775 alone in biliary tract cancer (BTC) cells. (A) Cell cycle analyses were performed by flow cytometry after treatment with increasing concentrations of AZD1775 (0, 0.1, 0.5, and 1 μM) for 24 hours. The data represent three independent experiments. *p < 0.05. (B) The phospho-histone H3 (p-HH3)–positive population in S phase was analyzed using flow cytometry after treatment with AZD1775 (0 and 1 μM) for 24 hours. The data represent three independent experiments. *p < 0.05. (C) Western blot analyses of the effects of AZD1775 on cell cycle pathways in four BTC cell lines. Cells were treated with increasing concentrations of AZD1775 (0, 0.1, 0.5, and 1 μM) for 24 hours. PARP, poly(ADP-ribose) polymerase. (D, E) Transwell migration assays were conducted with or without AZD1775 treatment (1 μM). The image was captured at 24 hours after treatment. The percentage of migratory cells was analyzed after images were captured. Experiments were repeated three times. *p < 0.05.

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  Fig. 3. Combination effects of WEE1 and ataxia telangiectasia mutated and Rad3 related (ATR) inhibitors in biliary tract cancer. (A) Western blotting was performed after treated with various concentrations of AZD1775 (0, 0.1, 0.5, and 1 μM) for 24 hours. ATM, ataxia telangiectasia mutated. (B) The combination indices (CIs) in the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay after combination treatment with AZD1775 (0, 0.001, 0.01, 0.1, 1, and 10 μM) and AZD0156 (0, 0.001, 0.01, 0.1, 1, and 10 μM)/AZD6738 (0, 0.001, 0.01, 0.1, 1, and 10 μM) for 72 hours. CI value was analyzed using CalcuSyn software. CI > 1, 1, and < 1 indicate antagonistic, additive, and synergistic effects, respectively. (C, D) Comet assays were conducted after treatment with AZD1775 (1 μM), AZD6738 (1 μM), or both for 24 hours. The tail intensity and moment were analyzed using the Comet Assay IV program. Experiments were repeated three times. *p < 0.05, **p < 0.01. (E) Cells were incubated with AZD1775 (1 μM), AZD6738 (1 μM), or both for 24 hours, and then western blotting was performed.

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  Fig. 4. Anti-tumor effects of AZD1775 alone and in combination with AZD6738 in xenograft mouse models. (A) HuCCT-1 and SNU2670 xenograft mice were administrated vehicle or AZD1775 (25 or 90 mg/kg) for 3 weeks (5 days on/2 days off). Each group contained five mice. *p < 0.05. (B) SNU2670 xenograft mice were treated with vehicle, AZD1775 (60 mg/kg), AZD6738 (25 mg/kg), or both AZD1775 and AZD6738 for 3 weeks (5 days on/2 days off). Each group contained five mice. TE, end of treatment. *p < 0.05. (C) H&E staining (×400) and immunohistochemical analysis (×400) of p-ataxia telangiectasia mutated and Rad3 related (ATR), Ki-67, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) expression were performed using isolated HuCCT-1 and SNU2670 tumors. (D) Western blot analysis of various proteins in isolated tumors.

Reference

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