AEP-cleaved DDX3X induces alternative RNA splicing events to mediate cancer cell adaptation in harsh microenvironments
Wenrui Zhang, … , Jiayi Chen, Yingying Lin
Wenrui Zhang, … , Jiayi Chen, Yingying Lin
Published November 21, 2023
Citation Information: J Clin Invest. 2024;134(3):e173299. https://doi.org/10.1172/JCI173299.
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Research Article Oncology

AEP-cleaved DDX3X induces alternative RNA splicing events to mediate cancer cell adaptation in harsh microenvironments

Abstract

Oxygen and nutrient deprivation are common features of solid tumors. Although abnormal alternative splicing (AS) has been found to be an important driving force in tumor pathogenesis and progression, the regulatory mechanisms of AS that underly the adaptation of cancer cells to harsh microenvironments remain unclear. Here, we found that hypoxia- and nutrient deprivation–induced asparagine endopeptidase (AEP) specifically cleaved DDX3X in a HIF1A-dependent manner. This cleavage yields truncated carboxyl-terminal DDX3X (tDDX3X-C), which translocates and aggregates in the nucleus. Unlike intact DDX3X, nuclear tDDX3X-C complexes with an array of splicing factors and induces AS events of many pre-mRNAs; for example, enhanced exon skipping (ES) in exon 2 of the classic tumor suppressor PRDM2 leads to a frameshift mutation of PRDM2. Intriguingly, the isoform ARRB1-Δexon 13 binds to glycolytic enzymes and regulates glycolysis. By utilizing in vitro assays, glioblastoma organoids, and animal models, we revealed that AEP/tDDX3X-C promoted tumor malignancy via these isoforms. More importantly, high AEP/tDDX3X-C/ARRB1-Δexon 13 in cancerous tissues was tightly associated with poor patient prognosis. Overall, our discovery of the effect of AEP-cleaved DDX3X switching on alternative RNA splicing events identifies a mechanism in which cancer cells adapt to oxygen and nutrient shortages and provides potential diagnostic and/or therapeutic targets.

Authors

Wenrui Zhang, Lu Cao, Jian Yang, Shuai Zhang, Jianyi Zhao, Zhonggang Shi, Keman Liao, Haiwei Wang, Binghong Chen, Zhongrun Qian, Haoping Xu, Linshi Wu, Hua Liu, Hongxiang Wang, Chunhui Ma, Yongming Qiu, Jianwei Ge, Jiayi Chen, Yingying Lin

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Figure 3

tDDX3X-C aggregates in the nucleus due to the absence of the N-terminal NES, and nuclear DDX3X is positively associated with AEP in HGG.

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tDDX3X-C aggregates in the nucleus due to the absence of the N-terminal ...
(A) 2-dimensional structure diagram of DDX3X. (B and C) HEK293T cells were transfected with mCherry-DDX3X, mCherry-DDX3X mutant NES, mCherry-DDX3X ΔNES, mCherry-tDDX3X-C, and mCherry-NES-tDDX3X-C for 24 hours, fixed, counterstained with DAPI, and finally visualized by confocal microscopy. Representative images are shown. Scale bar: 10 μm. (D) HEK293T cells were transfected with mCherry-DDX3X and mCherry-NES-tDDX3X-C for 24 hours and then treated with LMB, an inhibitor of protein nuclear transport. The follow-up procedure was the same as before. Representative images are shown. Scale bar: 10 μm. (E) Schematic diagram of DDX3X and its series of mutants; the corresponding location shift is shown. (F) HEK293T cells transfected with mCherry-tDDX3X-C mutant NLS1 and 2. The follow-up procedure was the same as before. Representative images are shown. Scale bar: 10 μm. (G) HeLa cells transfected with mCherry-DDX3X-GFP were cotransfected with or without AEP for 24 hours, counterstained with DAPI and visualized by confocal microscopy. Representative images are shown. Scale bar: 10 μm. All images were processed by Leica Imaging System software. (H) Representative images of IHC staining of AEP and DDX3X in glioma tissue microarrays containing different WHO grades. Scale bars: 200 μm for overview; 50 μm for enlargement. (I) Scatter plots of the nuclear DDX3X-positive ratio in HGG and LGG. Quantitative analysis (H-index) of IHC results was performed by Aipathwell (Servicebio, Wuhan). (J) Correlation analysis of AEP expression with the nuclear DDX3X-positive ratio in 2 glioma tissue microarray. Data were plotted as the mean ± SEM. Statistical analysis was performed using an unpaired t test (I) and Spearman correlation test (J). ***P < 0.001. Data shown are representative of 3 independent experiments.