Background: The sensitivity of head and neck squamous cell carcinoma (HNSCC) to ionizing radiation, among others, is determined by the number of cells with high clonogenic potential and stem-like features.
More...Background: The sensitivity of head and neck squamous cell carcinoma (HNSCC) to ionizing radiation, among others, is determined by the number of cells with high clonogenic potential and stem-like features. These cellular characteristics are dynamically regulated in response to treat-ment and may lead to an enrichment of radioresistant cells with a cancer stem cell (CSC) pheno-type. Epigenetic mechanisms, particularly DNA and histone methylation, are key regulators of gene-specific transcription and cellular plasticity. Therefore, we hypothesized that specific epi-genetic targeting may prevent irradiation-induced plasticity and may sensitize HNSCC cells to radiotherapy. Methods: We compared the DNA methylome and intracellular concentrations of tricarboxylic acid cycle metabolites in radioresistant FaDu and Cal33 cell lines with their parental controls, as well as aldehyde dehydrogenase (ALDH)-positive CSCs with negative controls. Moreover, we conducted a screen of a chemical library targeting enzymes in-volved in epigenetic regulation in combination with irradiation and analyzed the clonogenic potential, sphere formation, and DNA repair capacity to identify compounds with both radiosensi-tizing and CSC-targeting potential. Results: We identified the histone demethylase inhibitor GSK-J1, which targets UTX (KDM6A) and JMJD3 (KDM6B), leading to increased H3K27 tri-methylation, heterochromatin formation, and gene silencing. The clonogenic survival assay after siRNA-mediated knock-down of both genes radiosensitized Cal33 and SAS cell lines. Moreover, high KDM6A expression in tissue sections of patients with HNSCC was associated with improved locoregional control after primary (n = 137) and post-operative (n = 187) radio/chemotherapy. Conversely, high KDM6B expression was a prognostic factor for reduced overall survival. Conclusions: Within this study, we investigated cellular and molecular mechanisms underlying irradiation-induced cellular plasticity, a key inducer of radioresistance, with a focus on epigenetic alterations. We identified UTX (KDM6A) as a putative prognostic and therapeutic target for HNSCC patients treated with radiotherapy.
Overall design: Within this study, the human head and neck squamous cell carcinoma (HNSCC) cell lines FaDu(DD) (RRID:CVCL_VP44, ATCC) and Cal33 (RRID:CVCL_1108, Deutsche Sammlung von Mikroorganismen und Zellkulturen DSMZ GmbH) were used. Radioresistant (RR) sublines were generated from indicated parental cell lines via selection with at least 15 fractions of 4 Gy and analyzed together with age-matched controls. Moreover, cancer stem cells (CSC) were isolated from FaDu and Cal33 cells by Aldefluor assay (Stem cell technologies) according to the manufacture’s recommendation. Genomic DNA was isolated from each cell population (23 samples in total) with the Qiamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s recommendations. Bisulphite converted DNA from the 48 samples were hybridised to the Illumina Infinium Human MethylationEPIC V1 Beadchip.
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