The influence of PI3K inhibition on the radiotherapy response of head and neck cancer cells

Mary Glorieux, Rüveyda Dok, Sandra Nuyts, Mary Glorieux, Rüveyda Dok, Sandra Nuyts

Abstract

Radiotherapy has a central role in the treatment of head and neck squamous cell carcinoma (HNSCC). Activation of the PI3K/AKT/mTOR pathway can decrease the efficiency of radiotherapy via the promotion of cell survival and DNA repair. Here, the influence of PI3K pathway inhibition on radiotherapy response was investigated. Two PI3K inhibitors were investigated and both BKM120 and GDC0980 effectively inhibited cellular and clonogenic growth in 6 HNSCC cells, both HPV-positive as well as HPV-negative. Despite targeted inhibition of the pathway and slight increase in DNA damage, PI3K inhibition did not show significant radiosensitization. Currently only one clinical trial is assessing the effectiveness of combining BKM120 with RT in HNSCC (NCT02113878) of which the results are eagerly awaited.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
(A) Dose response curves of BKM120 (left) and GDC0980 (right). Cells were treated for 72 h with the indicated concentrations of BKM120 (left) and for 24 h with GDC0980 (right). Data is presented as the mean ± SEM of three HPV-positive cell lines (SCC154, SCC104 and SCC47) and three HPV-negative cell lines (SQD9, SCC61, CAL27) HNSCC cell lines of which 3 independent experiments were performed per cell line. One-way Anova analysis plus posthoc t-testing with Bonferroni correction showed the statistical significant differences. (B) The effect of PI3K inhibition monotherapy on plating efficiency of HNSCC cell lines. Colony assay of HNSCC cells treated with BKM120 for 72 h (left) or with GC0980 for 24 h (right). Colony formation was tested in three HPV-positive cell lines (SCC154, SCC104 and SCC47) and three HPV-negative cell lines (SQD9, SCC61, CAL27). Data are represented as the mean plating efficiency ± SEM for three independent performed experiments. One-way Anova analysis plus posthoc t-testing with Bonferroni correction showed the statistical significant differences.
Figure 2
Figure 2
The effect of PI3K inhibition on the clonogenic survival of HNSCC cell lines. (A) Colony assay of HNSCC cells treated with BKM120. Cells were irradiated 2 h after drug exposure and the drug was removed 70 h after radiotherapy. (B) Colony assay of HNSCC cells treated with GDC0980. Cells were irradiated 2 h after drug exposure and the drug was removed 22 h after radiotherapy. Data are represented as the mean ± SEM for three independent performed experiments. The fitted curves were compared with Anova analysis to show the statistical significant differences.
Figure 3
Figure 3
The effect of BKM120 and GDC0980 on the PI3K pathway. (A) Cells were treated with BKM120 (1 µM) 2 h before radiotherapy (6 Gy). Control condition cells are treated with equal concentrations of DMSO. After 24 h, total cell lysates were prepared to perform western blotting. (B) Cells were treated with GDC0980: 2.5 µM for HPV-positive cell lines (SCC154 and SCC104) and 1 µM for HPV-negative cells (SQD9 and SCC61). Control condition cells are treated with equal concentrations of DMSO. GDC0980 was administered 2 h before radiotherapy (6 Gy). After 24 h of drug treatment, total cell lysates were prepared to perform western blotting. Densitometry quantification was done and relative expression levels were corrected to the loading control and to the non-phosphorylated protein. Uncropped images are provided in the supplementary information.
Figure 4
Figure 4
(A) The effect of BKM120 on the cell cycle. The percentage (%) of SCC104 HPV-positive (left) and SQD9 HPV-negative (right) HNSCC cells distributed in cell cycle phases after treatment for 24 h with 1 µM BKM120 and/or 6 Gy. Data are represented as the mean ± SEM for three independent performed experiments. One-way Anova plus posthoc t-testing with Bonferroni correction showed the statistical differences. (B) The effect of PI3K inhibition on the DNA damage response. (A) HPV-positive (SCC104) and HPV-negative (SQD9) cells were exposed to 1 µM of BKM120 and/or 6 Gy of radiotherapy to assess gH2AX foci. Fixation was done after 2 or 24 h to represent kinetics of DNA repair. Data are represented as the mean ± SEM for three independent performed experiments. T-testing showed the significant difference (p = 0.0454).

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