AEP-cleaved DDX3X induces alternative RNA splicing events to mediate cancer cell adaptation in harsh microenvironments
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
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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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 7

ARRB-Δexon 13 proteoform is critical for AEP/tDDX3X-C-mediated sustained tumor growth.

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ARRB-Δexon 13 proteoform is critical for AEP/tDDX3X-C-mediated sustained...
(A) Representative bright field images of GBO no. 1 and the corresponding IF of Ki-67 were used to detect the viability of GBOs. Scale bar: 200 μm. (B) Histogram of the diameter of GBOs. (C) Ratio of Ki-67 in GBOs. (D and E) Histogram of the number and diameter of colonies in soft agar colony formation assays of U87-MG (D) and MDA-MB-231 (E) cells in the NC, AEP KD, AEP KD/tDDX3X-C res, AEP KD/ARRB1-WT res, and AEP KD/ARRB1-Δexon 13 res groups. The number and average diameter of cell colonies (μm) were counted and measured by ImageJ. (F) Protein binding profile of ARRB1-Δexon 13 detected by IP-MS. (G) Pyruvate content in the corresponding groups of GBOs. (H) Preferential binding proteins of ARRB1-Δexon 13 associated with glycolysis. (I) Co-IP assays of ARRB1-Δexon 13, ARRB1-WT, and ENO1. Data were plotted as the mean ± SEM, and statistical analysis was performed using 1-way ANOVA followed by Šidák’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data shown are representative of 3 independent experiments.