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, 107 (2), 291-9

Targeting Radiation-Resistant Hypoxic Tumour Cells Through ATR Inhibition

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Targeting Radiation-Resistant Hypoxic Tumour Cells Through ATR Inhibition

I M Pires et al. Br J Cancer.

Abstract

Background: Most solid tumours contain regions of sub-optimal oxygen concentration (hypoxia). Hypoxic cancer cells are more resistant to radiotherapy and represent the most aggressive fraction of a tumour. It is therefore essential that strategies continue to be developed to target hypoxic cancer cells. Inhibition of the DNA damage response (DDR) might be an effective way of sensitising hypoxic tumour cells to radiotherapy.

Methods: Here, we describe the cellular effects of pharmacological inhibition of the apical DDR kinase ATR (Ataxia Telangiectasia and Rad 3 related) with a highly selective inhibitor, VE-821, in hypoxic conditions and its potential as a radiosensitiser.

Results: VE-821 was shown to inhibit ATR-mediated signalling in response to replication arrest induced by severe hypoxia. In these same conditions, VE-821 induced DNA damage and consequently increased Ataxia Telangiectasia Mutated-mediated phosphorylation of H2AX and KAP1. Consistently, ATR inhibition sensitised tumour cell lines to a range of oxygen tensions. Most importantly, VE-821 increased radiation-induced loss of viability in hypoxic conditions. Using this inhibitor we have also demonstrated for the first time a link between ATR and the key regulator of the hypoxic response, HIF-1. HIF-1 stabilisation and transcriptional activity were both decreased in response to ATR inhibition.

Conclusion: These findings suggest that ATR inhibition represents a novel strategy to target tumour cells in conditions relevant to pathophysiology and enhance the efficacy of radiotherapy.

Figures

Figure 1
Figure 1
VE-821 radiosensitises various cell lines. RKO, HCT116, DLD1, SKmel28, IGR39, RCC4, HeLa, U2OS, SQ20B, MDA-MB-231, MDA-MB-468 and VmCub1 cells were treated with DMSO or 1 μℳ VE-821 for 6 h and then exposed to a range of radiation doses. Colonies were counted after 8–12 days. Cells with reported WT TP53 are indicated with ‡. Significance values: *P<0.05; **P<0.005; ***P<0.0005.
Figure 2
Figure 2
VE-821 inhibits the growth of a 3D tumour model. Spheroids were grown from MG-U87 cells to average size of 400 μm and grown for further 9 days. (A) Spheroids were treated with either DMSO or 1 μℳ VE-821 and were replaced every 2 days for 9 days. The graph represents the average volume of at least 12 spheroids throughout treatment. (B) Before fixing, spheroids were treated with EF5. Spheroids were then fixed, sectioned and stained for EF5, demonstrating the presence of hypoxic regions within the spheroids. EF5 regular stain – ELK5-A8 antibody, EF5-competed stain – ELK5-A8+EF5. Significance values: *P<0.05.
Figure 3
Figure 3
VE-821 inhibits hypoxia-induced ATR-mediated signaling, while inducing DNA damage and affecting replication kinetics. (A) RKO cells were exposed to ⩽0.02% O2 for the times indicated in the presence of DMSO or 1 μℳ VE-821. Western blotting was carried out as indicated. Representative blots of n=3 experiments are shown. The levels of β-actin were determined to ensure equal loading. Quantification is shown in Supplementary Figure S2. (B) RKO cells were treated with DMSO or 1 μℳ VE-821 as shown. Norm-6 h; Hyp-6 h ⩽0.02% O2. The percentage of cells with more than six nuclear 53BP1 foci is shown. (CF) VE-821 decreases replication fork progression and increases origin firing. RKO cells were treated with DMSO or 1 μℳ VE-821 and were treated as indicated, either in normoxia or ⩽0.02% O2 for 6 h and subsequently reoxygenated for 1 h. DNA fibres were then produced and scored. (C, D) The percentage of different replication structures is shown. (E, F) The distribution of the fork rates for the second label (IdU) for the on-going forks (average speeds are indicated). Significance values: *P<0.01, **P<0.001, ***P<0.0001.
Figure 4
Figure 4
VE-821 sensitises cells to hypoxia. Clonogenic assays were carried out using RKO cells in the presence of DMSO or 1 μℳ VE-821. Cells were exposed to ⩽0.02% O2 (A) and 2% O2 (B) for the indicated time points and grown for 10 days. (C) Sensitisation of cells to 1 μℳ VE-821 vs DMSO after 24 h of exposure to various oxygen levels. (D) RKO cells were treated with DMSO or 1 μℳ VE-82 and exposed to 24 h normoxia (Norm), 24 h 1% O2 (Hyp) or 24 h 1% O2 and reoxygenated for 4 or 24 h (Reox 4 h) (Reox 24 h). Graph represents the percentage of cells with more than six 53BP1 foci. Significance values: *P<0.05; **P<0.005; ***P<0.0005.
Figure 5
Figure 5
VE-821 is also a radiosensitiser in hypoxic conditions. RKO cells were treated with DMSO or 1 μℳ VE-821 and irradiated as indicated in the following oxygen tensions, 2% O2 (A), 1% O2 (B) or 0.5% O2 (C). Colony survival assays were carried out. The radiation response of RKO cells in normal O2 conditions (20% O2) is shown in Figure 1. (D) Spheroids were grown from MG-U87 cells as before and treated with combinations of DMSO or 1 μℳ VE-821 and mock per 1 Gy irradiation (RT) at the indicated times for 12 days. Average spheroid size is shown. Significance values: *P<0.05; **P<0.005; ***P<0.0005. (E) Western blots were carried out for HIF-1α, GLUT-1 and β-actin (loading control) on samples generated from spheroids at day 12 (lanes 3–6). Lanes 1–2 correspond to MG-U87 monolayer samples exposed to normoxia (Con) or 2% O2 for 30 min (Hyp).
Figure 6
Figure 6
VE-821 inhibits HIF-1 signalling. (A) RKO cells were exposed to ⩽0.02% O2 for the times indicated in the presence of DMSO or 1 μℳ VE-821. Western blotting was carried out for HIF-1α and β-actin (loading control). Representative blots of n=3 experiments are shown. (B) The levels of GLUT-1 mRNA were determined by qRT–PCR. 18S was used as the control. (C) RKO cells were transiently transfected with a HIF-1 reporter construct and exposed to hypoxia as indicated. The levels of luciferase relative to the renilla control are shown. (D) MDA-MB-231 cells were transfected with either Scr (scramble) or HIF-1 siRNA and scratch wound assays were carried out in the presence of DMSO or 1 μℳ VE-821. Western blot showing efficiency of knockdown is shown in inset. The graph represents the percentage of wound closure after 18 h exposure to hypoxia (⩽0.02% O2). Significance values: *P<0.05, **P<0.01.

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