Targeting DNA Double-Strand Break Repair Enhances Radiosensitivity of HPV-Positive and HPV-Negative Head and Neck Squamous Cell Carcinoma to Photons and Protons

Abstract

The response of head and neck squamous cell carcinoma (HNSCC) to radiotherapy depends on human papillomavirus type 16 (HPV) status, and where improved outcome and survival is observed in HPV-positive disease. However, strategies to further radiosensitise the tumours, particularly relatively radioresistant HPV-negative HNSCC, are actively being sought. The impact of targeting the major protein kinases involved in the signaling of DNA double-strand break (DSB) repair, namely ataxia telangiectasia-mutated (ATM), ataxia telangiectasia and Rad3-related (ATR), and the catalytic subunit of DNA-dependent protein kinase (DNA-Pkcs), on the radiosensitisation of HNSCC cells was examined. The response to both conventional photon radiotherapy, but also proton beam therapy, was analysed by clonogenic assays and 3D spheroid growth. We observed that inhibition of ATM, ATR, and particularly DNA-Pkcs, caused a significant reduction in HNSCC cell survival post-irradiation with both photons and protons, with less of an impact on the most radiosensitive HPV-positive cell line. The inhibition of DNA-Pkcs and, to a lesser extent ATM, in combination with radiation was also more effective at inhibiting the growth of 3D spheroids derived from relatively radioresistant HPV-negative HNSCC. Similar effects of the inhibitors were observed comparing photon and proton irradiation, demonstrating the potential for targeting DSB repair as an effective combination treatment for HNSCC.

Keywords: ATM; ATR; DNA repair; DNA-PKcs; ionising radiation; proton beam therapy.

Figure 1

Comparative radiosensitivity of human papillomavirus type 16 (HPV)-negative and HPV-positive head and neck squamous cell carcinoma (HNSCC) cells in response to photons and protons. (A) Whole cell extracts from HNSCC cells were prepared and analysed by immunoblotting with the indicated antibodies. Clonogenic survival of HNSCC cells following treatment with increasing doses of (B,C) x-rays or (D,E) protons was analysed from three to four biologically independent experiments. (B,D) Shown is the surviving fraction ± S.E. (C,E) Representative images of colonies in non-irradiated and irradiated plates (the latter were seeded with four times and eight times the number of cells, accordingly). Statistical analysis using a one sample t-test of surviving fractions at a 2 Gy dose of x-rays reveals significant differences of p < 0.03 (UMSCC6 vs. UPCI-SCC090), p < 0.005 (UMSCC74A vs. UPCI-SCC090); and at a 4 Gy dose of protons of p < 0.04 (UMSCC6 vs. UPCI-SCC090), p < 0.04 (UMSCC74A vs. UPCI-SCC090). The uncropped blots and molecular weight markers of Figure 1 are shown in Figure S1.

Figure 2

Inhibition of ataxia telangiectasia-mutated (ATM), ataxia telangiectasia and Rad3-related (ATR) and DNA-dependent protein kinase (DNA-Pkcs) can enhance sensitivity of HNSCC cells to photon irradiation. Clonogenic survival of HNSCC cells following treatment with increasing doses of x-rays in the presence of DMSO (Control), ATMi (10 µM), ATRi (1 µM) and DNA-Pkcsi (1 µM) was analysed from three biologically independent experiments. (A,C,E,G) Shown is the surviving fraction ± S.E. (B,D,F,H) representative images of colonies in non-irradiated and irradiated plates (the latter of which were seeded with four times the number of cells).

Figure 3

Inhibition of ATM, ATR and DNA-Pkcs can enhance sensitivity of HNSCC cells to proton irradiation. Clonogenic survival of HNSCC cells following treatment with increasing doses of protons in the presence of DMSO (Control), ATMi (10 µM), ATRi (1 µM) and DNA-Pkcsi (1 µM) was analysed from four biologically independent experiments. (A,C,E,G) Shown is the surviving fraction ± S.E. (B,D,F,H) representative images of colonies in non-irradiated and irradiated plates (the latter of which were seeded with four times the number of cells).

Figure 4

Inhibition of ATM, ATR and DNA-Pkcs in combination with photons can decrease growth of HNSCC 3D spheroids. Spheroids were allowed to develop for 48 h, pretreated with DMSO (Control), ATMi (10 µM), ATRi (1 µM) and DNA-Pkcsi (1 µM), and irradiated with a single dose (1 Gy) of x-rays. Spheroid growth of (A,D,G) UMSCC74A, (B,E,H) UMSCC6 and (C,F,I) UPCI-SCC090 was measured by microscopy and analysed from three biologically independent experiments. Solid blue line is DMSO only, dashed blue lines are DMSO plus 1 Gy x-rays, solid red line is inhibitor only, dashed red lines are inhibitors plus 1 Gy x-rays. Shown is the spheroid volume ± S.E.

Figure 5

Inhibition of ATM, ATR and DNA-Pkcs in combination with protons can decrease growth of HNSCC 3D spheroids. Spheroids were allowed to develop for 48 h, pretreated with DMSO (Control), ATMi (10 µM), ATRi (1 µM) and DNA-Pkcsi (1 µM), and irradiated with a single dose (2 Gy) of protons. Spheroid growth of (A,D,G) UMSCC74A, (B,E,H) UMSCC6 and (C,F,I) UPCI-SCC090 was measured by microscopy and analysed from three biologically independent experiments. Solid blue line is DMSO only, dashed blue lines are DMSO plus 2 Gy protons, solid red line is inhibitor only, dashed red lines are inhibitors plus 2 Gy protons. Shown is the spheroid volume ± S.E.

Figure 6

Inhibition of ATM, ATR and DNA-Pkcs in combination with photons and protons can decrease growth of HPV-negative HNSCC 3D spheroids. Spheroids were allowed to develop for 48 h, pretreated with DMSO (Control), ATMi (10 µM), ATRi (1 µM) and DNA-Pkcsi (1 µM), and irradiated with a single dose of (A–F) x-rays at 1 Gy or (G–L) protons at 2 Gy. Spheroid growth of (A,C,E,G,I,K) hypopharynx (FaDu) and (B, D, F, H, J and L) A253 was measured by microscopy and analysed from three biologically independent experiments. Solid blue line is DMSO only, dashed blue lines (A–F) are DMSO plus 1 Gy x-rays or (G–L) 2 Gy protons, solid red lines are inhibitor only, dashed red lines are inhibitor plus (A–F) 1 Gy x-rays or (G–L) 2 Gy protons. Shown is the spheroid volume ± S.E.