Enhancing treatment response to radiation therapy in HPV negative head and neck cancer

Abstract

Head and neck squamous-cell carcinoma (HNSCC) is a disease characterised by its aggressive nature and a high risk of recurrence. Radiotherapy has been widely applied to the primary and adjuvant treatment of HNSCC. However, the efficacy of radiotherapy is often compromised under hypoxic conditions. One relatively unexplored strategy to alleviate tumour hypoxia is to reduce oxygen consumption by targeting the oxidative phosphorylation (OXPHOS) pathway, consequently increasing tumour oxygenation. In this thesis, we evaluated the action of three typical electron transport chain (ETC) complex inhibitors (atovaquone, papaverine and metformin) on energy metabolism and their impact on the radiation response. Several potential mechanisms contributing to ETC inhibitors induced radiosensitisation were also explored. Finally, we investigated the impact of endoplasmic reticulum (ER) stress response caused by OXPHOS inhibition and its role in influencing the radiation response under hypoxic conditions.

Through bioenergetic profiling of HSNCC cells in response to atovaquone, papaverine or metformin, we found that treatment with ETC inhibitors greatly inhibited oxygen consumption rate (OCR) and concomitantly stimulated an increase in extracellular acidification (ECAR). These ETC inhibitors markedly inhibited mitochondrial respiration, generating a significant reduction in basal respiration, maximal respiration, and ATP levels. The clonogenic assay revealed that treatment with atovaquone induced a significant increase in radiosensitivity in FaDu cells under both normoxia and hypoxia. Importantly, treatment with radiation and atovaquone not only significantly increased ROS levels, but also caused G2/M cell cycle arrest under normoxic conditions. 53BP1 DNA damage repair studies demonstrated that the combination of atovaquone and radiation impaired DNA damage repair.

Furthermore, in this thesis we also demonstrated the combination of ETC inhibitors and AuNPs augmented radiation sensitivity, resulting a greater effect than achieved by either AuNPs or ETC inhibitor alone. We found that combination of atovaquone (or papaverine) and AuNPs significantly induced S-phase accumulation under normoxia. Additionally, we also found that combined treatment with atovaquone and AuNPs induced the highest proportion of apoptotic cells than achieved by AuNPs and atovaquone alone. In addition, studies evaluating the effect of atovaquone, AuNPs and combination on radiation induced DNA damage demonstrated a significant increase in the number of residual unresolved DNA damage in the combination group than that achieved by AuNPs alone.

The activation of ER stress was previously linked with a radioresistant phenotype in HNSCC. We revealed that treatment with atovaquone induced the activation of UPR signalling via an increase in eIF2 alpha phosphorylation. Treatment with atovaquone or papaverine greatly suppressed the expression of cyclin D1 and HIF-1 alpha protein expression. More importantly, we also found that treatment with atovaquone promotes the induction of autophagy under hypoxic conditions. Using a genetic knockdown approach, we demonstrated that eIF2 alpha suppression significantly enhanced the radiosensitising potential of atovaquone.
In summary, the finding of this thesis confirmed that atovaquone acts as an ideal radiosensitiser for the treatment of hypoxic tumours. We have also identified an adaptive protective response in response to atovaquone treatment which plays a key role in mediating a pro-survival response under hypoxic conditions.

Thesis is embargoed until 31 July 2025.

Date of Award	Jul 2023
Original language	English
Awarding Institution	Queen's University Belfast
Supervisor	Jonathan Coulter (Supervisor) & Wafa Al-Jamal (Supervisor)