Fitter Mitochondria Are Associated With Radioresistance in Human Head and Neck SQD9 Cancer Cells.

Debora Grasso,Davide Brusa,Pierre Sonveaux,Caroline Bouzin,Philippe Lambin,Marike W Van Gisbergen,Hyllana C D Medeiros,Vincent Grégoire,Vanesa Bol,Marie Dutreix,Hubert Smeets,Alphons P M Stassen,Pierre Danhier,Luca X Zampieri,Ludwig J Dubois

doi:10.3389/fphar.2020.00263

Abstract

The clinical management of head and neck squamous cell carcinoma (HNSCC) commonly involves chemoradiotherapy, but recurrences often occur that are associated with radioresistance. Using human SQD9 laryngeal squamous cell carcinoma cancer cells as a model, we aimed to identify metabolic changes associated with acquired radioresistance. In a top-down approach, matched radiosensitive and radioresistant SQD9 cells were generated and metabolically compared, focusing on glycolysis, oxidative phosphorylation (OXPHOS) and ROS production. The cell cycle, clonogenicity, tumor growth in mice and DNA damage-repair were assessed. Mitochondrial DNA (mtDNA) was sequenced. In a bottom-up approach, matched glycolytic and oxidative SQD9 cells were generated using FACS-sorting, and tested for their radiosensitivity/radioresistance. We found that acquired radioresistance is associated with a shift from a glycolytic to a more oxidative metabolism in SQD9 cells. The opposite was also true, as the most oxidative fraction isolated from SQD9 wild-type cells was also more radioresistant than the most glycolytic fraction. However, neither reduced hexokinase expression nor OXPHOS were directly responsible for the radioresistant phenotype. Radiosensitive and radioresistant cells had similar proliferation rates and were equally efficient for ATP production. They were equally sensitive to redox stress and had similar DNA damage repair, but radioresistant cells had an increased number of mitochondria and a higher mtDNA content. Thus, an oxidative switch is associated with but is not responsible for acquired radioresistance in human SQD9 cells. In radioresistant cells, more abundant and fitter mitochondria could help to preserve mitochondrial functions upon irradiation.

Highlights

Head and neck squamous cell carcinoma (HNSCC) is the 6th most frequent and the 8th deadliest cancer type worldwide, accounting for ∼5% of all malignancies (Ferlay et al, 2015)
We focused on mitochondria that control apoptosis and ATP and reactive oxygen species (ROS) production, and that contain their own DNA that could be a target of radiotherapy
Cell identity was confirmed using short tandem repeat (STR) DNA profiling at the end of treatment. Among these 4 cell lines, only SQD9-wt cells generated radioresistant cells, as validated in vitro with clonogenic assays that revealed that SQD9-res had a higher surviving fraction than SQD9-wt cells after irradiation with increasing doses of γ-rays under normoxia (Figure 1A)

Summary

Introduction

Head and neck squamous cell carcinoma (HNSCC) is the 6th most frequent and the 8th deadliest cancer type worldwide, accounting for ∼5% of all malignancies (Ferlay et al, 2015). When reacting with ROS, in particular with the hydroxyl radical, a DNA radical is formed and water is produced. This reaction is reversible and damage can be promptly repaired unless it is stabilized, typically through a reaction with O2 that forms DNA peroxides (Jordan and Sonveaux, 2012). The oxygen enhancement effect links radiotherapy efficacy to cancer metabolism, as oxidative cancer cells in tumors in vivo and in closed systems in vitro promote hypoxia, radioresistance, whereas glycolytic cancer cells spare oxygen that can be used to stabilize DNA damage (Danhier et al, 2013)

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