Department of Nuclear Medicine, Hunan Cancer Hospital, Changsha, Hunan, 410008, People's Republic of China.
Department of Pediatrics, Xiangya Hospital, Central South University, Changsha, Hunan, 410008, People's Republic of China.
J Exp Clin Cancer Res. 2019 Jul 22;38(1):325. doi: 10.1186/s13046-019-1328-3.
Sodium/iodide symporter (NIS)-mediated iodide uptake plays an important physiological role in regulating thyroid gland function, as well as in diagnosing and treating Graves' disease and thyroid cancer. High-mobility group box 1 (HMGB1), a highly conserved nuclear protein, is a positive regulator of autophagy conferring resistance to chemotherapy, radiotherapy and immunotherapy in cancer cells. Here the authors intended to identify the role of HMGB1 in Hank's balanced salt solution (HBSS)-induced autophagy, explore NIS protein degradation through a autophagy-lysosome pathway in thyroid cancer cells and elucidate the possible molecular mechanisms.
Immunohistochemical staining and reverse transcription-polymerase chain reaction (RT-PCR) were performed for detecting the expression of HMGB1 in different tissues. HMGB1 was knocked down by lentiviral transfection in FTC-133/TPC-1 cells. Autophagic markers LC3-II, p62, Beclin1 and autophagosomal formation were employed for evaluating HMGB1-mediated autophagy in HBSS-treated cells by Western blot, immunofluorescence and electron microscopy. Western blot, quantitative RT-PCR and gamma counter analysis were performed for detecting NIS expression and iodide uptake in HMGB1-knockdown cells after different treatments. The reactive oxygen species (ROS) level, ROS-mediated LC3-II expression and HMGB1 cytosolic translocation were detected by fluorospectrophotometer, flow cytometry, Western blot and immunofluorescence. HMGB1-mediated AMPK, mTOR and p70S6K phosphorylation (p-AMPK, p-mTOR & p-p70S6K) were detected by Western blot. Furthermore, a nude murine model with transplanted tumor was employed for examining the effect of HMGB1-mediated autophagy on imaging and biodistribution of TcO4. NIS, Beclin1, p-AMPK and p-mTOR were detected by immunohistochemical staining and Western blot in transplanted tumor samples.
HMGB1 was a critical regulator of autophagy-mediated NIS degradation in HBSS-treated FTC-133/TPC-1 cells. And HMGB1 up-regulation was rather prevalent in thyroid cancer tissues and closely correlated with worse overall lymph node metastasis and clinical stage. HMGB1-knockdown dramatically suppressed autophagy, NIS degradation and boosted iodide uptake in HBSS-treated cells. Moreover, HBSS enhanced ROS-sustained autophagy and promoted the cytosolic translocation of HMGB1. A knockdown of HMGB1 suppressed LC3-II conversion and NIS degradation via an AMPK/mTOR-dependent signal pathway through a regulation of ROS generation, rather than ATP. Furthermore, these data were further supported by our in vivo experiment of xenografts formed by HMGB1 knockdown cells reverting the uptake of TcO4 as compared with control shRNA-transfected cells in hunger group.
Acting as a critical regulator of autophagy-mediated NIS degradation via ROS/AMPK/mTOR pathway, HMGB1is a potential intervention target of radioiodine therapy in thyroid cancer.
钠/碘同向转运体（NIS）介导的碘摄取在调节甲状腺功能以及诊断和治疗格雷夫斯病和甲状腺癌方面发挥着重要的生理作用。高迁移率族蛋白 B1（HMGB1）是一种高度保守的核蛋白，是自噬的正调节剂，使癌细胞对化疗、放疗和免疫治疗产生耐药性。作者旨在确定 HMGB1 在 Hank 平衡盐溶液（HBSS）诱导的自噬中的作用，探索甲状腺癌细胞中 NIS 蛋白通过自噬溶酶体途径降解，并阐明可能的分子机制。
通过免疫组织化学染色和逆转录-聚合酶链反应（RT-PCR）检测 HMGB1 在不同组织中的表达。通过慢病毒转染在 FTC-133/TPC-1 细胞中敲低 HMGB1。通过 Western blot、免疫荧光和电子显微镜检测 HBSS 处理细胞中 HMGB1 介导的自噬的自噬标记物 LC3-II、p62、Beclin1 和自噬体形成。Western blot、定量 RT-PCR 和伽马计数器分析检测 HMGB1 敲低细胞在不同处理后的 NIS 表达和碘摄取。通过荧光分光光度计、流式细胞术、Western blot 和免疫荧光检测活性氧（ROS）水平、ROS 介导的 LC3-II 表达和 HMGB1 细胞质易位。通过 Western blot 检测 HMGB1 介导的 AMPK、mTOR 和 p70S6K 磷酸化（p-AMPK、p-mTOR 和 p-p70S6K）。此外，在移植肿瘤的裸鼠模型中研究了 HMGB1 介导的自噬对 TcO4 成像和分布的影响。通过免疫组织化学染色和 Western blot 检测移植瘤样本中的 NIS、Beclin1、p-AMPK 和 p-mTOR。
HMGB1 是 HBSS 处理的 FTC-133/TPC-1 细胞中 NIS 降解的自噬的关键调节剂。HMGB1 在甲状腺癌组织中上调更为常见，与总体淋巴结转移和临床分期较差密切相关。HMGB1 敲低显著抑制 HBSS 处理细胞中的自噬、NIS 降解和碘摄取增加。此外，HBSS 增强了持续的 ROS 自噬，并促进了 HMGB1 的细胞质易位。HMGB1 敲低通过调节 ROS 产生而不是通过 ATP 抑制 AMPK/mTOR 依赖性信号通路抑制 LC3-II 转化和 NIS 降解。此外，我们在饥饿组中，通过 HMGB1 敲低细胞恢复 TcO4 的摄取，与对照 shRNA 转染细胞相比，进一步支持了这些数据在异种移植中的体内实验结果。
HMGB1 作为自噬介导的 NIS 降解的关键调节剂，通过 ROS/AMPK/mTOR 途径，是甲状腺癌放射性碘治疗的潜在干预靶点。
