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Transl Oncol. 2018 Jun;11(3):755-763. doi: 10.1016/j.tranon.2018.04.001. Epub 2018 Apr 24.

Global Effects of DDX3 Inhibition on Cell Cycle Regulation Identified by a Combined Phosphoproteomics and Single Cell Tracking Approach.

Author information

1
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.
2
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.
3
Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands.
4
McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University, School of Medicine, Baltimore, MD, USA.
5
Mass Spectrometry and Proteomics Core, Johns Hopkins University, School of Medicine, Baltimore, MD, USA.
6
Department of Molecular Biology and Genetics, Johns Hopkins University, School of Medicine, Baltimore, MD, USA.
7
Department of Radiology and Radiological Sciences, Johns Hopkins University, School of Medicine, Baltimore, MD, USA; Department of Oncology, Johns Hopkins University, School of Medicine, Baltimore, MD, USA.
8
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; Department of Oncology, Johns Hopkins University, School of Medicine, Baltimore, MD, USA. Electronic address: vraman2@jhmi.edu.

Abstract

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.

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