Table_3_Activation of Glucocorticoid Receptor Inhibits the Stem-Like Properties of Bladder Cancer via Inactivating the β-Catenin Pathway.XLSX

Background: Glucocorticoid receptor (GR) signaling pathway has been shown to involve epithelial -to- mesenchymal transition which was implicated in the regulation of bladder cancer stem cells (CSCs) in our previous study. Herein, we aim to figure out how GR affects the stem-like properties of bladder cancer cells.
Methods: We used dexamethasone (DEX) treatment or gene-knockdown/-knockout techniques to activate or silence the GR pathway, respectively. Then we applied immunohistochemical staining and flow cytometry to assess the associations between the expression levels of GR and a stem cell surface marker CD44. Stem-like properties were assessed by reactive oxygen species (ROS), sphere-formation and side population assays. The expression levels of cancer stem cell-associated molecules were assessed by quantitative PCR and Western blotting. Tumor growth was compared using mouse xenograft models.
Results: In GR-positive bladder cancer cells, DEX significantly reduced the expression of CD44 as well as pluripotency transcription factors including β-catenin and its downstream target (C-MYC, Snail, and OCT-4), the rate of sphere formation, and the proportion of side populations, and induced the intracellular levels of ROS. By contrast, GR silencing in bladder cancer cells showed the opposite effects. In xenograft-bearing mice, GR silencing resulted in the enhancement of tumor growth.
Conclusions: These data suggested that GR activity was inversely associated with the stem-like properties of bladder cancer cells, potentially via inactivating the β-catenin pathway.

研究背景：糖皮质激素受体（Glucocorticoid receptor, GR）信号通路已被证实参与上皮间质转化过程，该过程在我们的前期研究中被证实参与膀胱肿瘤干细胞（cancer stem cells, CSCs）的调控。本研究旨在明确GR如何影响膀胱癌细胞的干细胞样特性。
研究方法：本研究分别采用地塞米松（dexamethasone, DEX）处理法与基因敲低/敲除技术，以激活或沉默GR信号通路。随后通过免疫组化染色与流式细胞术，检测GR表达水平与干细胞表面标志物CD44的表达相关性。采用活性氧（reactive oxygen species, ROS）检测、成球实验及侧群细胞分析，评估细胞的干细胞样特性。通过定量聚合酶链反应（quantitative PCR）与蛋白质印迹（Western blotting）检测肿瘤干细胞相关分子的表达水平。利用小鼠异种移植模型比较肿瘤生长情况。
研究结果：在GR阳性膀胱癌细胞中，DEX处理可显著降低CD44及多能性转录因子（包括β-连环蛋白（β-catenin）及其下游靶标C-MYC、Snail与OCT-4）的表达水平，同时抑制细胞成球率与侧群细胞比例，并升高细胞内活性氧水平。与之相反，沉默膀胱癌细胞中的GR则可产生相反的效应。在携带异种移植瘤的小鼠中，GR沉默可促进肿瘤生长。
研究结论：本研究数据表明，GR活性与膀胱癌细胞的干细胞样特性呈负相关，其潜在机制可能为通过灭活β-连环蛋白信号通路。