USP9X deubiquitinates ALDH1A3 and maintains mesenchymal identity in glioblastoma stem cells
Zhengxin Chen, Hong-Wei Wang, Shuai Wang, Ligang Fan, Shuang Feng, Xiaomin Cai, Chenghao Peng, Xiaoting Wu, Jiacheng Lu, Dan Chen, Yuanyuan Chen, Wenting Wu, Daru Lu, Ning Liu, Yongping You, Huibo Wang
Zhengxin Chen, Hong-Wei Wang, Shuai Wang, Ligang Fan, Shuang Feng, Xiaomin Cai, Chenghao Peng, Xiaoting Wu, Jiacheng Lu, Dan Chen, Yuanyuan Chen, Wenting Wu, Daru Lu, Ning Liu, Yongping You, Huibo Wang
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Research Article Cell biology Oncology

USP9X deubiquitinates ALDH1A3 and maintains mesenchymal identity in glioblastoma stem cells

Abstract

The mesenchymal (MES) subtype of glioblastoma (GBM) stem cells (GSCs) represents a subpopulation of cancer cells that are notorious for their highly aggressive nature and resistance to conventional therapy. Aldehyde dehydrogenase 1A3 (ALDH1A3) has been recently suggested as a key determinant for the maintenance of MES features of GSCs. However, the mechanisms underpinning aberrant ALDH1A3 expression remain elusive. Here, we identified ubiquitin-specific protease 9X (USP9X) as a bona fide deubiquitinase of ALDH1A3 in MES GSCs. USP9X interacted with, depolyubiquitylated, and stabilized ALDH1A3. Moreover, we showed that FACS-sorted USP9Xhi cells were enriched for MES GSCs with high ALDH1A3 activity and potent tumorigenic capacity. Depletion of USP9X markedly downregulated ALDH1A3, resulting in a loss of self-renewal and tumorigenic capacity of MES GSCs, which could be largely rescued by ectopic expression of ALDH1A3. Furthermore, we demonstrated that the USP9X inhibitor WP1130 induced ALDH1A3 degradation and showed marked therapeutic efficacy in MES GSC–derived orthotopic xenograft models. Additionally, USP9X strongly correlated with ALDH1A3 expression in primary human GBM samples and had a prognostic value for patients with the MES subgroup. Collectively, our findings unveil USP9X as a key deubiquitinase for ALDH1A3 protein stabilization and a potential target for GSC-directed therapy.

Authors

Zhengxin Chen, Hong-Wei Wang, Shuai Wang, Ligang Fan, Shuang Feng, Xiaomin Cai, Chenghao Peng, Xiaoting Wu, Jiacheng Lu, Dan Chen, Yuanyuan Chen, Wenting Wu, Daru Lu, Ning Liu, Yongping You, Huibo Wang

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Figure 4

High USP9X expression predicts enrichment of ALDH1A3hi MES GSCs with potent tumorigenic capability.

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High USP9X expression predicts enrichment of ALDH1A3hi MES GSCs with pot...
(A) Confocal images showing colocalization of USP9X (red) and ALDH1A3 (green) in MES 21 GSCs. Nuclei were counterstained with DAPI (blue). (B) FACS sorting of USP9Xhi or USP9Xlo fractions isolated from MES 21 and 505 GSCs. (C) IB analysis of USP9X and ALDH1A3 levels in FACS-sorted USP9Xhi and USP9Xlo subpopulations. (D) Quantification of FACS analysis for ALDH1 activity in USP9Xhi or USP9Xlo subpopulations. DEAB was used to inhibit ALDH1 activity, serving as a negative control. (E) Representative bioluminescent images of intracranial GBM xenografts derived from FACS-sorted USP9Xhi or USP9Xlo subpopulations. Quantification of bioluminescent images is shown on the right. Colored scale bars represent photons/s/cm2/steradian. (F) H&E staining images and IHC images of USP9X and ALDH1A3 are shown in consecutive brain sections from mice implanted with 5 × 104 USP9Xhi or USP9Xlo subpopulations. Red arrows indicate tumors. Scale bars: 25 μm (A); 1 mm (H&E staining) and 100 μm (IHC staining) (F). Data are represented as mean ± SD of 3 independent experiments. **P < 0.01; ***P < 0.001, 2-tailed Student’s t test.