Chen Syue-Ting, Chang Kang-Shuo, Lin Wei-Yin, Hsu Shu-Yuan, Sung Hsin-Ching, Lin Yu-Hsiang, Feng Tsui-Hsia, Hou Chen-Pang, Juang Horng-Heng
Department of Anatomy, College of Medicine, Chang Gung University, Taoyuan, Taiwan; Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan; Department of Urology, Chang Gung Memorial Hospital at Linkou, Taoyuan, Taiwan.
Department of Anatomy, College of Medicine, Chang Gung University, Taoyuan, Taiwan.
Biomed J. 2024 Jun 27;48(2):100756. doi: 10.1016/j.bj.2024.100756.
The functions of activating transcription factor 3 (ATF3) within the human bladder remain unexplored. This study delves into the expressions, functions, and regulatory mechanisms of ATF3 in human bladder cancer.
Gene expressions were determined by immunoblot, RT-qPCR, and reporter assays. Assays of Ki67, colony formation, Matrigel invasion, and the xenograft animal study were used to assess the cell proliferation, invasion, and tumorigenesis in vitro and in vivo. Silico analysis from TCGA database examined the correlations between GDF15 and ATF3 expressions, clinicopathologic features, and progression-free survival rates.
Silico analysis confirmed that ATF3 is an antitumor gene, and the expression positively correlates with GDF15 in bladder cancer tissues. Multivariate analysis revealed that low ATF3/GDF15 but not a single low expression of ATF3 is an independent prognostic factor for progression-free survival of bladder cancer patients. Ectopic overexpression of ATF3 downregulated cell proliferation and invasion in bladder cancer cells in vitro, while ATF3-knockdown reversed these results. Knockdown of ATF3 upregulated EMT markers to enhance cell invasion in vitro and downregulated GDF15, NDRG1, and KAI-1 to elevate tumor growth in vivo. The activation of metformin on ATF3 and GDF15 in bladder cancer cells was blocked by SB431542, a TGFβ receptor inhibitor. ATF3 positively regulated GDF15 expression in bladder cancer cells through a feedback loop.
Our results identify that ATF3 is a metformin-upregulated antitumor gene. Results of Silico analysis align with cell-based studies suggesting that low ATF3/GDF15 could be a negative prognostic marker for bladder cancer.
激活转录因子3（ATF3）在人膀胱中的功能尚未得到探索。本研究深入探讨了ATF3在人膀胱癌中的表达、功能及调控机制。
通过免疫印迹、逆转录定量聚合酶链反应（RT-qPCR）和报告基因检测来确定基因表达。使用Ki67检测、集落形成检测、基质胶侵袭检测及异种移植动物研究来评估体外和体内的细胞增殖、侵袭及肿瘤发生情况。来自癌症基因组图谱（TCGA）数据库的计算机分析检查了生长分化因子15（GDF15）与ATF3表达、临床病理特征及无进展生存率之间的相关性。
计算机分析证实ATF3是一种抗肿瘤基因，且其表达与膀胱癌组织中的GDF15呈正相关。多变量分析显示，低ATF3/GDF15而非单一的低ATF3表达是膀胱癌患者无进展生存的独立预后因素。ATF3的异位过表达在体外下调了膀胱癌细胞的增殖和侵袭，而ATF3基因敲低则逆转了这些结果。敲低ATF3上调上皮-间质转化（EMT）标志物以增强体外细胞侵袭，并下调GDF15、N-myc下游调节基因1（NDRG1）和钾离子通道四聚体化结构域包含蛋白1（KAI-1）以提高体内肿瘤生长。转化生长因子β（TGFβ）受体抑制剂SB431542阻断了二甲双胍对膀胱癌细胞中ATF3和GDF15的激活作用。ATF3通过反馈环正向调节膀胱癌细胞中GDF15的表达。
我们的结果表明ATF3是二甲双胍上调的抗肿瘤基因。计算机分析结果与基于细胞的研究结果一致，提示低ATF3/GDF15可能是膀胱癌的不良预后标志物。
