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. 2017 Dec;9(12):1646-1659.
doi: 10.15252/emmm.201707767.

Akt inhibition improves long-term tumour control following radiotherapy by altering the microenvironment

Emma J Searle  1   2   3 , Brian A Telfer  1 , Debayan Mukherjee  2   3 , Duncan M Forster  4 , Barry R Davies  5 , Kaye J Williams  1   6   7 , Ian J Stratford  1 , Tim M Illidge  8   3
Affiliations

Akt inhibition improves long-term tumour control following radiotherapy by altering the microenvironment

Emma J Searle et al. EMBO Mol Med. 2017 Dec.

Abstract

Radiotherapy is an important anti-cancer treatment, but tumour recurrence remains a significant clinical problem. In an effort to improve outcomes further, targeted anti-cancer drugs are being tested in combination with radiotherapy. Here, we have studied the effects of Akt inhibition with AZD5363. AZD5363 administered as an adjuvant after radiotherapy to FaDu and PE/CA PJ34 tumours leads to long-term tumour control, which appears to be secondary to effects on the irradiated tumour microenvironment. AZD5363 reduces the downstream effectors VEGF and HIF-1α, but has no effect on tumour vascularity or oxygenation, or on tumour control, when administered prior to radiotherapy. In contrast, AZD5363 given after radiotherapy is associated with marked reductions in tumour vascular density, a decrease in the influx of CD11b+ myeloid cells and a failure of tumour regrowth. In addition, AZD5363 is shown to inhibit the proportion of proliferating tumour vascular endothelial cells in vivo, which may contribute to improved tumour control with adjuvant treatment. These new insights provide promise to improve outcomes with the addition of AZD5363 as an adjuvant therapy following radiotherapy.

Keywords: Akt; microenvironment; radiotherapy.

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Figures

Figure 1
Figure 1. Akt inhibition with AZD5363 does not enhance the radiosensitivity of tumour cells in vitro
  1. A, B

    Cells were treated with 1 μM or 10 μM AZD5363 for 2 h before lysis on ice. (A) Western blots were performed with antibodies detecting pAkt, pGSK3β, pS6 and the housekeeping protein vinculin. A blot representative of three independent experiments is shown in the panel (n = 3 experiments). (B) ELISA was used to measure levels of pPRAS40 (n = 3 experiments).

  2. C

    MTT assay of cells treated with 1–10 μM AZD5363 for 96 h (n = 3 experiments).

  3. D, E

    Clonogenic assay of FaDu (D) and PE/CA PJ34 (E) cells treated with 1 μM or 10 μM AZD5363 for 2 h before, and 24 h after a single dose of RT (2, 4 or 6 Gy) (n = 3 experiments).

  4. F

    Surviving fraction of FaDu cells after a single 4 Gy dose of RT combined with varying schedules of 1 μM AZD5363 (n = 3 experiments).

  5. G

    Cells were treated with 1 μM AZD5363 for 2 h before and 24 h after a single 4 Gy dose of RT before lysis on ice. Western blots were performed with antibodies detecting pAkt, pGSK3β and the housekeeping protein GAPDH. A blot representative of three independent experiments is shown in the panel (n = 3 experiments).

Data information: In (B–F) data are presented as mean ± SEM; n/s P > 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Statistical test in (B) and (F) is one‐way ANOVA with Dunnett's post hoc test (adjusted P = <0.0001–0.005 and P = 0.993–0.998, respectively). In (D) and (E) data are fitted to the linear quadratic model. In (C) data are fitted to a dose‐response curve.
Figure 2
Figure 2. Adjuvant AZD5363 following RT is more effective in improving tumour control in the FaDu head and neck cancer model than other treatment schedules
  1. A

    Diagram displaying the differing sequences of AZD5363 used in combination with RT in FaDu tumour‐bearing mice.

  2. B

    Percentage of mice with tumour control on each experimental day. Plots show the combined data of two independent experiments (n = 6‐13 mice/group).

  3. C–F

    Growth of FaDu tumours treated with AZD5363 (50 mg/kg BD) or 6 Gy RT alone (C), or in combination, with the drug given continuously (D), as an adjuvant (E) or as a neo‐adjuvant (F). Plots show the combined data of two independent experiments (n = 6‐13 mice/group).

Data information: In (B) data is presented as percentage mice with tumour control (tumour volume less than 700 mm3); *, significance compared to control mice; +, significance compared to RT monotherapy; #, significance compared to adjuvant group: +/# P < 0.05, **/++ P < 0.01, ***P < 0.001. In (C‐F) data are presented as mean tumour volumes ± SEM; ++ P < 0.01. Statistical test in (B) is Gehan‐Breslow‐Wilcoxon test (P = 0.002), and in (E) is one‐way ANOVA with Dunnett's post hoc test (P = 0.0001 and P = 0.038).Source data are available online for this figure.
Figure 3
Figure 3. Optimal tumour control is achieved in the PE/CA PJ34 model with adjuvant AZD5363 following RT
PE/CA PJ34 tumour‐bearing mice were treated with 6 Gy RT either alone or in combination with AZD5363 (n = 6–13 mice/group).
  1. A

    Percentage of mice with tumour control on each experimental day.

  2. B–D

    Tumour volumes for single‐agent AZD5363 and RT (B), neo‐adjuvant (C) or adjuvant (D) AZD5363 (50 mg/kg BD).

Data information: In (A) data are presented as percentage mice with tumour control (tumour volume less than 700 mm3); *, significance when compared to control mice; +, significance when compared to RT monotherapy: + P < 0.05, ***P < 0.001. In (C, D) data are presented as mean tumour volumes ± SEM; *P < 0.05. Statistical test in (A) is Gehan‐Breslow‐Wilcoxon test (+ P = 0.044 and ***P = 0.009), and in (D) is one‐way ANOVA with Dunnett's post hoc test (adjusted *P = 0.043). Source data are available online for this figure.
Figure EV1
Figure EV1. A reduction in total vessel length occurs 7 days post‐RT in mice receiving adjuvant AZD5363
Two mice bearing FaDu tumours grown within dorsal windows were treated with 6 Gy RT and adjuvant AZD5363 (50 mg/kg BD), commencing on the first‐day substantial tumour vasculature was visualised post‐tumour inoculation (termed day 0).

  1. Mice were imaged using bright‐field microscopy on days 2, 4 and 7 following RT.

  2. Total vessel length estimated with the assistance of computer software.

Figure 4
Figure 4. When given after RT, AZD5363 causes a reduction in tumour vessel density, which is associated with an increase in tumour hypoxia
Histological analysis of FaDu tumours 7 days after treatment with 6 Gy RT alone, or in combination with neo‐adjuvant or adjuvant AZD5363 (50 mg/kg BD). Tumour sections were stained with antibodies to detect CD31 or pimonidazole. Three sections were stained with each antibody, per tumour (n = 4–5 mice/group).

  1. Sections demonstrating CD31 staining to allow visualisation of tumour vessels; 10× magnification.

  2. Vessel density as measured in tumour sections.

  3. Sections demonstrating pimonidazole staining to allow assessment of hypoxia; 10× magnification.

  4. Percentage of pimonidazole‐positive viable tumour cells.

Data information: (B, D) show mean levels ± SEM; *P < 0.05, ***P < 0.001. Statistical test in (B) and (D) is Kruskal‐Wallis with Dunn's post hoc test (adjusted *P = 0.047, ***P = 0.0004).
Figure 5
Figure 5. AZD5363 does not reduce vascularity or increase hypoxia when given as a single agent
FaDu tumour‐bearing mice were treated with AZD5363 (50 mg/kg BD) or vehicle. On day 6 of treatment (after 11 doses of drug/vehicle), mice underwent tumour imaging with FAZA PET/CT. After 7 days of treatment (14 doses), tumours were assessed histologically for HIF‐1α, CD31 and pimonidazole, and by ELISA for VEGF. Three sections were stained with each antibody per tumour (n = 4–5/group).
  1. A

    Representative tumour sections stained with anti‐HIF‐1α antibody (10× magnification).

  2. B

    HIF‐1α levels relative to vehicle control mice.

  3. C, D

    ELISA measurement of human and mouse VEGF, respectively.

  4. E

    Example FAZA PET/CT of a FaDu tumour‐bearing mouse.

  5. F

    18F‐FAZA accumulation in tumours as measured by standardised uptake values (SUV) mean.

  6. G

    Tumour vessel density (CD31 staining).

  7. H

    Tumour hypoxia levels (pimonidazole staining).

Data information: Column charts give mean values ± SEM; n/s P > 0.05, *P < 0.05, **P < 0.01. In (B‐H) statistical test is Mann–Whitney (*P = 0.01, **P = 0.004).
Figure EV2
Figure EV2. AZD5363 does not reduce vascularity, increase hypoxia or alter tumour cell proliferation when given as a single agent
FaDu tumour‐bearing mice were treated with AZD5363 (50 mg/kg) for 7 days (14 doses) after which mice were culled and the tumours excised and prepared for histological analysis. Tumour sections were stained with antibodies to detect CD31, pimonidazole and Ki‐67. Three sections were stained with each antibody, per tumour; n = 4–5/group.

  1. CD31 staining to allow visualisation of tumour vessels, 10× magnification.

  2. Pimonidazole staining to allow estimation of tumour hypoxia, 10× magnification.

  3. Ki‐67 staining to assess tumour cell proliferation, 10× magnification.

Figure 6
Figure 6. Treatment with AZD5363 reduces the proportion of proliferating vascular endothelial cells and following RT, the influx of CD11b+ bone marrow‐derived cells
  1. A–E

    Tumours from FaDu xenograft‐bearing mice treated with AZD5363 (50 mg/kg BD) or vehicle for 7 days were harvested for histological analysis and stained for co‐expression of CD31 and mouse Ki‐67 (A–D), or human Ki‐67 (E); 3 consecutive sections stained and analysed; n = 4‐5/group. (A) Example of a whole (left) and magnified (right) section stained for CD31, mouse Ki‐67 and DAPI (20× magnification). (B) Percentage of CD31+ cells co‐expressing mouse Ki‐67. (C) Number of CD31+ cells. (D) Number of mouse Ki‐67+ cells. (E) Percentage of human Ki‐67+ viable tumour cells.

  2. F

    Typical section of an RT control tumour stained with anti‐CD11b antibody; 100× magnification.

  3. G

    Number of CD11b+ bone marrow‐derived cells, per unit area of viable tumour; 3 consecutive sections stained and analysed; n = 4–5/group.

Data information: In (B–E) and (G) data are presented as mean levels ± SEM; n/s P > 0.05, *P < 0.05, ***P < 0.001, ****P < 0.0001. Statistical test in (B‐E) is Mann–Whitney test (P = < 0.0001) and in (G) is Kruskal‐Wallis with Dunn's post hoc test (adjusted *P = 0.025, ***P < 0.0004).
Figure EV3
Figure EV3. AZD5363 reduces human vascular endothelial cell proliferation but does not cause a greater than additive effect on proliferation after RT

  1. HUVEC cells (1,000 cells/well) were seeded in a gelatin‐coated 96‐well plate and treated with doses of AZD5363 ranging from 0.1 to 10 μM for either 48 or 96 h. BrdU was added overnight, and an ELISA then performed to detect BrdU incorporation. A greater than 50% reduction in proliferation is produced with AZD5363 at a dose of between 2 and 3 μM (n = 3 experiments).

  2. A BrdU assay was performed as in (A) but with the addition of 1 μM AZD5363 for 2 h before, 2 h before and 96 h after, or for 96 h after, a single 6‐Gy dose of RT. None of the treatment schedules produced a greater than additive effect on the proliferation of vascular endothelial cells 96 h after a single 6‐Gy dose of RT.

Data information: In (A) results are shown as the mean ± SEM, normalised to DMSO‐treated control data and fitted to a dose‐response curve. In (B) data is shown as the mean ± SEM, normalised to the relevant AZD5363 alone treated control; n/s = P > 0.05. Statistical test is Kruskal‐Wallis with Dunn's post hoc test (adjusted P = 0.523 to >0.9999).
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