Laboratory of Chemical Biology and Genomics, Korea Research Institute of Bioscience and Biotechnology, 125 Gwahakro, Daejeon 34141, Republic of Korea.
Laboratory of Chemical Biology and Genomics, Korea Research Institute of Bioscience and Biotechnology, 125 Gwahakro, Daejeon 34141, Republic of Korea; University of Science and Technology in Korea, 217 Gajeongro, Daejeon 34113, Republic of Korea.
Biochem Pharmacol. 2020 May;175:113920. doi: 10.1016/j.bcp.2020.113920. Epub 2020 Mar 19.
To identify signal transducer and activator of transcription factor 3 (STAT3) inhibitors, we generated STAT3-dependent gene expression signature by analyzing gene expression profiles of DU145 cancer cells treated with STAT3 inhibitor, piperlongumine and 2-hydroxycinnamaldehyde. Then we explored gene expression signature-based strategies using a connectivity map database and identified several STAT3 inhibitors, including ethacrynic acid (EA). EA is currently used as a diuretic drug. EA inhibited STAT3 activation in DU145 prostate cancer cells and consequently decreased the levels of STAT3 target genes such as cyclin A and MCL-1. Furthermore, EA treatment inhibited tumor growth in mice xenografted with DU145 cells and decreased p-STAT3 expression in tumor tissues. Knockdown of Src homology region 2 domain-containing phosphatase-2 (SHP2) or Protein tyrosine phosphatase 1B (PTP1B) gene expression by siRNA suppressed the ability of EA to inhibit STAT3 activation. When EA was combined with an activator of SHP2 or PTP1B, p-STAT3 expression was synergistically decreased; when EA was combined with an inhibitor of SHP2 or PTP1B, p-STAT3 expression was rescued. By using an affinity pulldown assay with biotinyl-EA, EA was shown to associate with SHP2 and PTP1B in vitro. Additionally, the drug affinity responsive target stability (DARTS) assay confirmed the direct binding of EA to SHP2 and PTP1B. SHP2 is activated by EA through active phosphorylation at Y580 and direct binding to SHP2. Collectively, our results suggest that EA inhibits STAT3 activity through the modulation of phosphatases such as SHP2 and PTP1B and may be a potential anticancer drug to target STAT3 in cancer progression.
为了鉴定信号转导和转录激活因子 3（STAT3）抑制剂，我们通过分析用 STAT3 抑制剂、胡椒碱和 2-羟基肉桂醛处理的 DU145 癌细胞的基因表达谱，生成了 STAT3 依赖性基因表达特征。然后，我们使用连接图谱数据库探索了基于基因表达特征的策略，并鉴定了几种 STAT3 抑制剂，包括依他尼酸（EA）。EA 目前用作利尿剂药物。EA 抑制 DU145 前列腺癌细胞中的 STAT3 激活，从而降低 STAT3 靶基因如细胞周期蛋白 A 和 MCL-1 的水平。此外，EA 处理抑制了 DU145 细胞异种移植小鼠的肿瘤生长，并降低了肿瘤组织中 p-STAT3 的表达。siRNA 下调Src 同源结构域 2 区磷酸酶-2（SHP2）或蛋白酪氨酸磷酸酶 1B（PTP1B）基因表达，抑制了 EA 抑制 STAT3 激活的能力。当 EA 与 SHP2 或 PTP1B 的激活剂联合使用时，p-STAT3 的表达协同降低；当 EA 与 SHP2 或 PTP1B 的抑制剂联合使用时，p-STAT3 的表达得到恢复。通过使用生物素化-EA 的亲和拉下测定，EA 被证明在体外与 SHP2 和 PTP1B 结合。此外，药物亲和反应靶标稳定性（DARTS）测定法证实了 EA 与 SHP2 和 PTP1B 的直接结合。EA 通过 Y580 的活性磷酸化和与 SHP2 的直接结合来激活 SHP2。总之，我们的结果表明，EA 通过调节 SHP2 和 PTP1B 等磷酸酶来抑制 STAT3 活性，并且可能是针对癌症进展中 STAT3 的潜在抗癌药物。
