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 5

AEP promotes oncogenic splicing of widespread pre-mRNAs via tDDX3X-C.

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AEP promotes oncogenic splicing of widespread pre-mRNAs via tDDX3X-C. (A...
(A and B) GSEA of the expression profile of the NOD-like signaling pathway and Toll-like receptor signaling pathway in U87-MG cells (AEP KD or AEP KD/tDDX3X-C res). (C) Heatmap for gene sets of the Toll-like signaling pathway and the NOD-like signaling pathway regulated by AEP/tDDX3X-C. (D) Summary of differential splicing analysis performed using U87-MG cells in the following groups: NC, AEP KD, and AEP KD/tDDX3X-C res. The numbers of identified AS events in each category upon AEP/tDDX3X-C regulation are indicated. (E) Pie charts showing the percentages of changed AS events identified in U87-MG cells (left); pie charts representing the distribution of AEP/tDDX3X-C-regulated splicing events among different splicing profiles (right). (F) Representative heatmap for changed AS events from skipped exons (SEs) positively regulated by AEP/tDDX3X. (G) RNA-Seq results of alternative sites in the indicated genes using IGV software analysis. (H) PCR and AGE analyses for PRDM2 exon 2 and ARRB1 exon 13 regulated by AEP/tDDX3X-C in U87-MG and MDA-MB-231 cells. The middle panels show schematic diagrams of the indicated AS exons. Right panels show the quantification of percent spliced in (PSI). (I) PCR and AGE analyses for PRDM2 exon 2 and ARRB1 exon 13 in U87-MG and MDA-MB-231 cells with or without hypoxia and nutrient deprivation stimulus for 4 hours. (J) RNA-IP results of U87-MG and MDA-MB-231 cells (Flagged-tDDX3X-C OE) with or without hypoxia and nutrient deprivation stimulus for 4 hours. Data were plotted as the mean ± SEM. Statistical analysis was performed using 1-way ANOVA followed by Tukey’s multiple comparisons test (H), paired t test (I), and unpaired t test (J). *P < 0.05, **P < 0.01, *** P < 0.001, ****P < 0.0001. Data shown are representative of 3 independent experiments.