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 6

Pharmacological inhibition of USP9X attenuates the tumor-initiating ability of MES GSCs with high ALDH1A3 activity.

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Pharmacological inhibition of USP9X attenuates the tumor-initiating abil...
(A) MES 21 and 505 GSCs were cotransfected with His-ALDH1A3, HA-Ub, and Flag-USP9X in the absence or presence of 1 μM WP1130, and cell lysates were subjected to IP with His beads followed by IB with antibodies against HA and His. Cells were treated with 20 μM MG132 for 8 hours before harvesting. (B) IB analysis of ALDH1A3 in MES 21 and 505 GSCs treated with 1 μM WP1130 or vehicle with or without MG132. (C) MES 21 and 505 GSCs were treated with 1 μM WP1130 or vehicle for 24 hours, followed by 100 μg/ml CHX, harvested at the indicated times, and then subjected to IB with antibodies against ALDH1A3. SE, short exposure; LE, long exposure. (D) Quantification of FACS analysis for ALDH1 activity in MES 21 and 505 GSCs following treatment with 1 μM WP1130, 150 μM DEAB, or vehicle. (E) In vitro limiting dilution sphere-forming frequency of MES 21 and 505 GSCs after treatment with 1 μM WP1130 or vehicle. (F) IB analysis of the indicated proteins in MES 21 and 505 GSCs after treatment with 1 μM WP1130 or vehicle. (G) T2-weighted MRI images (left) and quantification of tumor volume (right) in mice bearing xenografts derived from MES 21 or 505 GSCs following treatment with 25 mg/kg WP1130 or vehicle. Red arrows indicate tumors. (H) H&E- and IHC-stained images of USP9X, ALDH1A3, and CD44 in mice intracranially implanted with MES 21 or 505 GSCs after treatment with WP1130 (25 mg/kg) or vehicle. Red arrows indicate tumors. Scale bars: 1 mm (G); 1 mm (H&E) and 100 μm (IHC) (H). Data are represented as mean ± SD of 3 independent experiments. ***P < 0.001, 1-way ANOVA with Dunnett’s post test (D); 2-tailed Student’s t test (G).