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Within tumors, the cancer stem cell (CSC) population is characterized by enhanced self-renewal, differentiation potential, and migratory capacity. Therefore, CSCs are thought to be responsible for tumor growth, therapy resistance, and metastasis initiation. Novel findings indicating that CSCs undergoing phenotypical and functional adaptations during tumorigenesis, upon microenvironmental changes and under therapeutic pressure. Despite radiotherapy (RT) has the potential to eliminate all malignant cells with clonogenic potential cellular plasticity and intratumor heterogeneity reducing local tumor control rates and patient survival. Within the present study, we investigated cellular plasticity and epigenetic modulation in head and neck squamous cell carcinoma (HNSCC) upon conventional irradiation (IR) with 220 kV photons and 150 MeV protons.

We used 3D colony-formation assay to determine radiosensitivity, γH2AX assay to evaluate DNA damage repair capacity and tumor sphere formation to investigate the self-renewal capacity of several HNSCC cell lines. IR-induced lineage switch from non-CSCs into aldehyde dehydrogenase (ALDH) positive CSCs was demonstrated using color-coded HNSCC cells. These radiobiological and stem cell read-outs were correlated with epigenetic remodeling and metabolic adaptations upon IR. Therefore, we used western blot analysis to illustrate differential histone methylation pattern, histone demethylase activity was determined using EpiQuik/Epigenase assay, and EPIC array to analyze global DNA methylation. Mass spectrometry was applied to analyze tricarboxylic cycle (TCA) metabolites important as co-factors for the regulation of enzymes regulating chromatin modifications. Using a chemical library, we screen > 150 epigenetic targeting compounds for their radiosensitizing, DNA damaging and CSC-targeting potential. The clinical potential of identified and validated chromatin modulators was investigated via gene expression analysis from tumor tissues of patients with locally advanced HNSCC treated with primary and postoperative radiochemotherapy (RCTx) and retrospectively correlated to loco-regional tumor control (LRC), overall survival and distant metastases.

Our data demonstrated that IR is leading to an acquisition of stem-like features in HNSCC cell lines. We identified the histone H3K27 demethylase JMJD3/KDM6B and UTX/KDM6A as key regulator of IR-induced cellular plasticity and demonstrated radiosensitizing and CSC eliminating potential for the corresponding chemical inhibitor GSK-J1. Moreover, both genes are differential expressed in HNSCC patients and have the potential to discriminate a poor prognosis subgroup after RCTx.

Epigenetic biomarkers may have prognostic and therapeutic potential for patients with HNSCC treated with RT. Further functional studies are necessary to characterize the molecular mechanisms for a future clinical translation.