DNA methylation analysis of radioresistant and ALDH+ Head and Neck Squamous Cell Carcinoma cells and their parental counterparts. DNA methylation analysis of radioresistant and ALDH+ Head and Neck Squamous Cell Carcinoma cells and their parental counterparts

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.

研究背景：头颈部鳞状细胞癌（head and neck squamous cell carcinoma, HNSCC）对电离辐射等治疗手段的敏感性，主要由具有高克隆形成潜能及干细胞样特征的肿瘤细胞数量决定。上述细胞特征会随治疗进程发生动态调控，可能富集出具有癌干细胞（cancer stem cell, CSC）表型的放射抵抗细胞。表观遗传调控机制——尤其是DNA与组蛋白甲基化——是基因特异性转录及细胞可塑性的关键调控因子。据此我们提出假说：靶向特定表观遗传位点可阻断辐射诱导的细胞可塑性，进而使HNSCC细胞对放射治疗增敏。

研究方法：我们对比了放射抵抗型FaDu、Cal33细胞系与其亲本对照的DNA甲基化组及三羧酸循环代谢物胞内浓度，同时也分析了醛脱氢酶（aldehyde dehydrogenase, ALDH）阳性癌干细胞与阴性对照的相关指标。此外，我们开展了联合放射治疗的表观遗传调控酶化学文库筛选，通过克隆形成存活实验、球体形成实验及DNA修复能力检测，筛选同时具备放射增敏与癌干细胞靶向潜力的化合物。

研究结果：我们筛选得到靶向UTX（KDM6A）与JMJD3（KDM6B）的组蛋白去甲基化酶抑制剂GSK-J1，该抑制剂可提升H3K27三甲基化水平、促进异染色质形成并介导基因沉默。通过siRNA介导的双基因敲低实验，可使Cal33与SAS细胞系获得放射增敏效果。此外，对137例初始放化疗及187例术后放化疗的HNSCC患者组织切片分析显示，高表达KDM6A的患者局部区域控制率更佳；反之，高表达KDM6B的患者总生存期更短，提示其为不良预后因子。

研究结论：本研究针对放射抵抗的关键诱因——辐射诱导的细胞可塑性——开展细胞与分子机制探究，重点聚焦表观遗传改变。我们证实UTX（KDM6A）可作为接受放射治疗的HNSCC患者潜在的预后与治疗靶点。

实验整体设计：本研究使用人源头颈部鳞状细胞癌（HNSCC）细胞系FaDu(DD)（RRID:CVCL_VP44, ATCC）与Cal33（RRID:CVCL_1108, 德国微生物与细胞培养保藏中心DSMZ GmbH）。放射抵抗（radioresistant, RR）亚细胞系通过至少15次4Gy剂量的辐射筛选获得，与同龄对照细胞共同开展后续实验。此外，我们按照Stem Cell Technologies公司的Aldefluor试剂盒操作说明，从FaDu与Cal33细胞中分离得到癌干细胞（CSC）。本研究共收集23份细胞群体样本，使用Qiamp DNA Mini Kit（Qiagen, 德国希尔德）按照厂商说明书提取基因组DNA。对48份亚硫酸氢盐转化后的DNA样本，采用Illumina Infinium人类甲基化EPIC V1芯片进行杂交检测。