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.
Highlights
The incidence of head and neck squamous cell carcinoma (HNSCC) has been reported to be~800,000 cases per year, and linked with this is the increased rise in oropharyngeal tumours associated with human papillomavirus type 16 (HPV) infection [1,2,3]
There are some discrepancies in relation to the specific double-strand break (DSB) repair defect, as the reduced expression of proteins involved in both non-homologous end joining (NHEJ; 53BP1 and DNA-Pkcs) and homologous recombination (HR; BRCA2 and RAD51) have been observed
In combination with photon irradiation, we demonstrate that there was a significant impact in reducing cell survival of HPV-negative HNSCC cells in the presence of either ATM inhibitor (ATMi), ATR inhibitor (ATRi) or DNA-Pkcsi (1 h pre-incubation, followed by a further treatment for 24 h post-irradiation) versus the DMSO control (Figure 2A–D; see Figure S6A–D for linear scale graphs and data fitting), with dose enhancement ratios (DER) of 1.91–2.39 (Table 1)
Summary
The incidence of head and neck squamous cell carcinoma (HNSCC) has been reported to be~800,000 cases per year, and linked with this is the increased rise in oropharyngeal tumours associated with human papillomavirus type 16 (HPV) infection [1,2,3]. It has been clearly demonstrated that patients with HPV-positive squamous cell carcinoma of the oropharynx display improved outcomes and survival rates in comparison to patients with HPV-negative disease [4,5,6,7], which is largely due to the increased responsiveness of HPV-positive tumours to radiotherapy and chemotherapy. This difference in radiotherapy response between HPV-positive and HPV-negative HNSCC has been observed in cultured cells derived from patients [8,9,10]. Studies conducted at the genomic level have identified significant genome instability in HPV-positive HNSCC cells and tissues, including alterations in DNA repair genes [13,14,15]
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