A Lack of Effectiveness in the ATM-Orchestrated DNA Damage Response Contributes to the DNA Repair Defect of HPV-Positive Head and Neck Cancer Cells

Abstract

Patients with human papillomavirus-positive squamous cell carcinoma of the head and neck (HPV+ HNSCC) have a favorable prognosis compared to those with HPV-negative (HPV-) ones. We have shown previously that HPV+ HNSCC cell lines are characterized by enhanced radiation sensitivity and impaired DNA double-strand break (DSB) repair. Since then, various publications have suggested a defect in homologous recombination (HR) and dysregulated expression of DSB repair proteins as underlying mechanisms, but conclusions were often based on very few cell lines. When comparing the expression levels of suggested proteins and other key repair factors in 6 HPV+ vs. 5 HPV- HNSCC strains, we could not confirm most of the published differences. Furthermore, HPV+ HNSCC strains did not demonstrate enhanced sensitivity towards PARP inhibition, questioning a general HR defect. Interestingly, our expression screen revealed minimal levels of the central DNA damage response kinase ATM in the two most radiosensitive HPV+ strains. We therefore tested whether insufficient ATM activity may contribute to the enhanced cellular radiosensitivity. Irrespective of their ATM expression level, radiosensitive HPV+ HNSCC cells displayed DSB repair kinetics similar to ATM-deficient cells. Upon ATM inhibition, HPV+ cell lines showed only a marginal increase in residual radiation-induced γH2AX foci and induction of G2 cell cycle arrest as compared to HPV- ones. In line with these observations, ATM inhibition sensitized HPV+ HNSCC strains less towards radiation than HPV- strains, resulting in similar levels of sensitivity. Unexpectedly, assessment of the phosphorylation kinetics of the ATM targets KAP-1 and Chk2 as well as ATM autophosphorylation after radiation did not indicate directly compromised ATM activity in HPV-positive cells. Furthermore, ATM inhibition delayed radiation induced DNA end resection in both HPV+ and HPV- cells to a similar extent, further suggesting comparable functionality. In conclusion, DNA repair kinetics and a reduced effectiveness of ATM inhibition clearly point to an impaired ATM-orchestrated DNA damage response in HPV+ HNSCC cells, but since ATM itself is apparently functional, the molecular mechanisms need to be further explored.

Keywords: DNA damage response (DDR); DNA double-strand break repair; ataxia telangiectasia mutated (ATM); head and neck cancer; human papillomavirus (HPV); radiation sensitivity.

Figure 1

Sensitivity towards PARP inhibition and expression of DNA repair factors. (A) Exponentially growing cells were seeded at low, defined numbers and, on the next day, treated with olaparib as indicated. After one week, the medium was exchanged and the cells were incubated without the inhibitor until the formation of colonies. Colored curves represent group means. (B) Expression of DNA repair factors in HPV+ and HPV− HNSCC cell lines as determined by Western blot analysis from exponentially growing cells. Note that TRIP12 expression was also tested but no interpretable results were obtained. (C) Quantification and comparison of DNA repair factors by HPV status. Individual expression is depicted as normalized to the average expression of the whole cohort (dotted line). An asterisk indicates significance (p <0.05, two-tailed Student’s t-test). (D) Association of ATM expression and cell survival in 5 HPV+ and 5 HPV− HNSCC cell lines at 6 Gy as reported previously by us (6).

Figure 2

Radiation-induced G2 arrest. Cells were treated for 30 min with the ATM inhibitor before irradiation, as indicated. 24 h after irradiation the cells were fixed and the cell cycle distribution assessed by DAPI staining and flow cytometry. Statistical evaluation was performed for changes in the fraction of G2 phase cells upon ATM inhibition. Asterisks depict significant differences with *, **, and *** indicating p < 0.05, p < 0.01, and p < 0.001 (two-tailed Student’s t-test).

Figure 3

Residual double-strand breaks and radiation sensitivity. (A) DSB repair kinetics of HPV+ and HPV− HNSCC cell lines. For each cell line, the values for nuclear foci were normalized to the first time point (30 min) after irradiation with 2 Gy. (B) Quantification of nuclear γH2AX foci at 24 h after 2 Gy irradiation with and without ATM inhibition. Counts of the respective non-irradiated controls were subtracted. (C) Fraction of cells with three or less nuclear γH2AX foci at 24 h after 0 or 2 Gy. (D) Cell survival as determined by colony formation assays with and without ATM inhibitor treatment. In (B–D) cells were generally treated with ATM inhibitor or solvent 30 min prior to and for 24 h after irradiation. In (B, C), S phase cells (EdU+, Geminin+) were excluded from quantification. In the case of UM-SCC-47, 53BP1 foci were assessed instead of γH2AX. Statistical evaluation was performed for changes upon ATM inhibition in (B, C), asterisks depict significant differences with *, ** and *** indicating p < 0.05, p < 0.01 and p < 0.001, respectively (two-tailed Student’s t-test).

Figure 4

Effect of ATM inhibition on DSB repair pathways. Reporter strains with stable integration of the respective repair constructs were transfected with an I-SceI expression vector. At 6 h and again at 24 h after transfection, the medium was exchanged and the inhibitor added. Forty-eight hours after transfection, the fraction of GFP-positive cells was assessed by flow cytometry. (A, C) Schemes of the NHEJ reporter construct pEJ and the HR reporter construct pGC. (B) Effect of ATM inhibition on NHEJ efficacy and (D) on HR efficacy. Results were normalized to the respective solvent controls of the individual experiments. Statistical evaluation was performed for changes upon ATM inhibition. Asterisks depict significant differences, with ** indicating p < 0.01 (two-tailed Student’s t-test).

Figure 5

Phosphorylation kinetics of ATM target proteins. Exponentially growing cells were irradiated with 6 Gy and harvested in sample buffer at the indicated time points. (A) Western blot signals. (B) Quantification. Data points represent the mean of two independent experiments. Left: whole protein level. Right: phosphorylation. Dotted lines indicate the normalized signal intensity of the non-irradiated samples.

Figure 6

Impact of ATM on radiation-induced DNA end resection. Cells were treated with or without an ATM inhibitor for 30 min before irradiation with 6 Gy. At the indicated time points, cells were pre-extracted before fixation and flow cytometric assessment of chromatin-bound RPA. (A) Gating strategy for G2-phase cells with normal (low) amounts of chromatin-bound RPA. (B) Quantification of the fraction of G2 phase cells with normal levels of chromatin-bound RPA at the indicated time points after radiation. To reduce variation through differences in the starting fractions of G2-phase cells, values in all individual experiments were normalized to the respective untreated controls harvested at the time of DMSO/ATM inhibitor treatment, which is represented by the dotted line (Y = 1). Asterisks depict significant differences, with *, **, and *** indicating p < 0.05, p < 0.01, and p < 0.001, respectively (two-tailed Student’s t-test).