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 2

DDX3X was cleaved at the Asn124 site, which is located in the IDR, producing a relatively stable carboxyl-terminal fragment of DDX3X.

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DDX3X was cleaved at the Asn124 site, which is located in the IDR, produ...
(A) Immunoblots of Flag-tagged DDX3X, AEP, and β-actin in HEK293T cells cotransfected with AEP and DDX3X-WT or different point mutants of Asn (DDX3X-N45A/N124A/N155A/N159A). (B) Structure diagram of human AEP binding with DDX3X and the appropriate domains of DDX3X interacting with the AEP enzymatic center (human AEP pdb code: 4D3Z). (C) Sequence alignment of DDX3X amino acids among different species. (D) Percentage of AEP cleavage site–located regions, divided into the disordered region (IDR), ordered region (OR) and uncertain region. The regional discrimination of regional structure is based on literature retrieval combined with AlphaFold and Pondr-VSL2 prediction. (E) Cleavage-site view of the invariant chain chaperone, P53, synapsin I, amphiphysin, SET, and γ-adducin (sequence numbering following UniProt entries P04233, P04637, P17600, P49418, Q9EQU5, and Q9QYB5). (F) Cleavage-site view of DDX3X (sequence numbering following UniProt entries O00571). The IDR region is depicted in salmon. The side chains of Asn are shown as stick models. All panels were created within PyMOL with carbon depicted in green, oxygen in red, and nitrogen in blue. (G) The potential regulatory motif in the IDR region of DDX3X analysis by ELM. The red motifs included the potential modification site by the corresponding enzyme, and the green motifs included the potential combination site with the corresponding proteins. (H) A protein fragment stability assay was used to analyze the halflife of DDX3X and its cleavage fragment. Immunoblots of Flag-tagged DDX3X-FL, tDDX3X-N, tDDX3X-C and β-actin in HEK293T cells treated with 20 μg/mL cycloheximide (CHX) for the indicated time courses. (I) Degradation curves of DDX3X-FL, tDDX3X-N, and tDDX3X-C. Data were plotted as the mean ± SEM.