Heerma van Voss Marise R, Kammers Kai, Vesuna Farhad, Brilliant Justin, Bergman Yehudit, Tantravedi Saritha, Wu Xinyan, Cole Robert N, Holland Andrew, van Diest Paul J, Raman Venu
Department of Radiology and Radiological Sciences, Johns Hopkins University, School of Medicine, Baltimore, MD, USA; Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands.
Division of Biostatistics and Bioinformatics, Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, The Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Transl Oncol. 2018 Jun;11(3):755-763. doi: 10.1016/j.tranon.2018.04.001. Epub 2018 Apr 24.
DDX3 is an RNA helicase with oncogenic properties. The small molecule inhibitor RK-33 is designed to fit into the ATP binding cleft of DDX3 and hereby block its activity. RK-33 has shown potent activity in preclinical cancer models. However, the mechanism behind the antineoplastic activity of RK-33 remains largely unknown. In this study we used a dual phosphoproteomic and single cell tracking approach to evaluate the effect of RK-33 on cancer cells. MDA-MB-435 cells were treated for 24 hours with RK-33 or vehicle control. Changes in phosphopeptide abundance were analyzed with quantitative mass spectrometry using isobaric mass tags (Tandem Mass Tags). At the proteome level we mainly observed changes in mitochondrial translation, cell division pathways and proteins related to cell cycle progression. Analysis of the phosphoproteome indicated decreased CDK1 activity after RK-33 treatment. To further evaluate the effect of DDX3 inhibition on cell cycle progression over time, we performed timelapse microscopy of Fluorescent Ubiquitin Cell Cycle Indicators labeled cells after RK-33 or siDDX3 exposure. Single cell tracking indicated that DDX3 inhibition resulted in a global delay in cell cycle progression in interphase and mitosis. In addition, we observed an increase in endoreduplication. Overall, we conclude that DDX3 inhibition affects cells in all phases and causes a global cell cycle progression delay.
DDX3是一种具有致癌特性的RNA解旋酶。小分子抑制剂RK-33被设计用于契合DDX3的ATP结合裂隙，从而阻断其活性。RK-33在临床前癌症模型中已显示出强大的活性。然而，RK-33抗肿瘤活性背后的机制在很大程度上仍不为人知。在本研究中，我们使用了双磷酸蛋白质组学和单细胞追踪方法来评估RK-33对癌细胞的影响。用RK-33或溶剂对照处理MDA-MB-435细胞24小时。使用等压质量标签（串联质量标签）通过定量质谱分析磷酸肽丰度的变化。在蛋白质组水平，我们主要观察到线粒体翻译、细胞分裂途径以及与细胞周期进程相关的蛋白质的变化。磷酸蛋白质组分析表明，RK-33处理后CDK1活性降低。为了进一步评估随着时间推移DDX3抑制对细胞周期进程的影响，我们在RK-33或siDDX3处理后对荧光泛素细胞周期指示剂标记的细胞进行了延时显微镜观察。单细胞追踪表明，DDX3抑制导致间期和有丝分裂期细胞周期进程整体延迟。此外，我们观察到核内复制增加。总体而言，我们得出结论，DDX3抑制会影响所有阶段的细胞，并导致整体细胞周期进程延迟。
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