Center of Oral Medicine, Qingdao Municipal Hospital, #5 Donghai Middle Road, Qingdao, 266000, PR China; School of Stomatology, Qingdao University, #19 JiangSu Road, Qingdao, 266000, PR China.
Department of Oral and Maxillo-facial Surgery, Weifang Medical University Affiliated Qingdao Stomatological Hospital, #17 Dexian Road, Qingdao, 266000, PR China.
Mol Cell Probes. 2020 Oct;53:101577. doi: 10.1016/j.mcp.2020.101577. Epub 2020 Apr 22.
Ionizing radiation (IR) confers a survival advantage in tongue squamous cell carcinoma (TSCC), however, IR resistance limits its efficacy. Although Yin Yang 1 (YY1) has been reported to play a role in genotoxic drug resistance by accelerating DNA repair, its role in TSCC radioresistance remains unclear. In this study, we examined YY1 mRNA and protein expression in human tongue cancer samples using qRT-PCR and western blotting, respectively. DNA array data identified YY1 mRNA expression in IR sensitivity or resistance cell lines and tissues. Tongue carcinoma primary cells and CAL27 cells with YY1 stably overexpressed or knocked-down were exposed to IR and evaluated for cell proliferation and apoptosis by CCK8-assay and caspase-3 assay, respectively. We also examined DNA damage- or repair-related indicators, such as YY1, p-H2AX, nuclear PTEN, p-PTEN, and Rad51 through Western blot analysis. Additionally, we explored the mechanism of IR-induced PTEN nuclear translocation by introducing a series of PTEN phosphorylation site mutations and co-IP assay. We observed that YY1 mRNA and protein are highly expressed in TSCC tissues, which was correlated with worse overall survival. Moreover, higher expression of YY1 and Rad51 was observed in radioresistant cells and tissues, overexpression of YY1 led to IR resistance in TSCC cells, whereas YY1 knockdown sensitized TSCC cells to IR. The underlying mechanism showed that the overexpression of YY1 upregulated nuclear PTEN and Rad51 expression, which is essential for DNA repair. IR upregulated YY1, nuclear PTEN, and Rad51; thus, knockdown of YY1 completely blocked IR-induced upregulation of nuclear PTEN/Rad51. IR upregulated PTEN phosphorylation, and mutation of the phosphorylation site of Ser380 nearly completely blocked IR-induced PTEN nuclear translocation. Furthermore, the phosphatase PP2A negatively regulated pS380-PTEN, and knockdown of YY1 completely blocked IR-induced pS380-PTEN through PP2A. In conclusion, knockdown of YY1 enhanced TSCC radiosensitivity through PP2A-mediated dephosphorylation of PTEN Ser380; thus, antagonizing the IR-induced nuclear PTEN/Rad51 axis and targeting YY1 may reverse IR resistance in TSCC.
电离辐射（IR）赋予舌鳞状细胞癌（TSCC）生存优势，但 IR 抗性限制了其疗效。虽然 Yin Yang 1（YY1）已被报道通过加速 DNA 修复在细胞毒性药物耐药性中发挥作用，但它在 TSCC 放射抗性中的作用尚不清楚。在这项研究中，我们使用 qRT-PCR 和 Western blot 分别检测了人舌癌样本中的 YY1 mRNA 和蛋白表达。DNA 阵列数据鉴定了 IR 敏感性或抗性细胞系和组织中的 YY1 mRNA 表达。用 YY1 稳定过表达或敲低的舌癌细胞原代细胞和 CAL27 细胞暴露于 IR 下，并通过 CCK8 测定和 caspase-3 测定分别评估细胞增殖和细胞凋亡。我们还通过 Western blot 分析检测了 YY1、p-H2AX、核 PTEN、p-PTEN 和 Rad51 等与 DNA 损伤或修复相关的指标。此外，我们通过引入一系列 PTEN 磷酸化位点突变和共免疫沉淀（co-IP）实验来探索 IR 诱导的 PTEN 核转位的机制。我们观察到 YY1 mRNA 和蛋白在 TSCC 组织中高表达，与总生存率较差相关。此外，在耐药细胞和组织中观察到更高表达的 YY1 和 Rad51，YY1 的过表达导致 TSCC 细胞对 IR 产生抗性，而 YY1 的敲低则使 TSCC 细胞对 IR 敏感。潜在机制表明，YY1 的过表达上调核 PTEN 和 Rad51 的表达，这对 DNA 修复至关重要。IR 上调 YY1、核 PTEN 和 Rad51；因此，YY1 的敲低完全阻断了 IR 诱导的核 PTEN/Rad51 的上调。IR 上调 PTEN 磷酸化，而 Ser380 磷酸化位点的突变几乎完全阻断了 IR 诱导的 PTEN 核转位。此外，磷酸酶 PP2A 负调控 pS380-PTEN，而 YY1 的敲低通过 PP2A 完全阻断了 IR 诱导的 pS380-PTEN。总之，通过 PP2A 介导的 PTEN Ser380 去磷酸化，敲低 YY1 增强了 TSCC 的放射敏感性；因此，拮抗 IR 诱导的核 PTEN/Rad51 轴并靶向 YY1 可能逆转 TSCC 中的 IR 抗性。
