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. 2019 Apr 8;129(5):2043-2055.
doi: 10.1172/JCI126414.

USP9X deubiquitinates ALDH1A3 and maintains mesenchymal identity in glioblastoma stem cells

Zhengxin Chen  1   2 , Hong-Wei Wang  3 , Shuai Wang  4 , Ligang Fan  1 , Shuang Feng  1 , Xiaomin Cai  1 , Chenghao Peng  1 , Xiaoting Wu  5 , Jiacheng Lu  1 , Dan Chen  6 , Yuanyuan Chen  7 , Wenting Wu  8 , Daru Lu  9 , Ning Liu  1 , Yongping You  1   2 , Huibo Wang  1   2
Affiliations

USP9X deubiquitinates ALDH1A3 and maintains mesenchymal identity in glioblastoma stem cells

Zhengxin Chen et al. J Clin Invest. .

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.

Keywords: Brain cancer; Cell Biology; Oncology.

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Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. USP9X maintains ALDH1A3 stability.
(A) Four Flag-tagged DUBs (USP3, USP9X, USP22, and OTUD1) were expressed in HEK293T cells, and cell lysates were analyzed by IP with Flag beads followed by IB with antibodies against ALDH1A3 and Flag. (B) Increasing amounts of Flag-tagged USP9X (WT or C1566A mutant) were transfected into HEK293T cells, and cell lysates were analyzed by IB with antibody against ALDH1A3. (C) IB analysis of USP9X protein expression in ALDH1hi and ALDH1lo subpopulations isolated from MES 21 and 505 GSCs. (D) MES 21 and 505 GSCs transfected with 2 independent USP9X shRNA were treated with or without the proteasome inhibitor MG132 (20 μM, 8 hours), and then USP9X and ALDH1A3 were analyzed. (E) IB analysis of ALDH1A3 levels in MES 21 and 505 GSCs transduced with USP9X shRNA, together with either shRNA-resistant (sh-res) Flag-tagged USP9X WT or USP9X C1566A. (F) HEK293T cells were cotransfected with His-tagged ALDH1A3 and Flag-tagged USP9X WT or USP9X C1566A, treated with 100 μg/ml CHX, collected at the indicated times, and then subjected to IB with antibodies against His and Flag. Quantification of ALDH1A3 levels relative to β-actin is shown. (G and H) MES 21 (G) and 505 (H) GSCs stably expressing control shRNA or USP9X shRNA were treated with 100 μg/ml CHX, harvested at the indicated times, and then subjected to IB with antibodies against ALDH1A3 and USP9X. Quantification of ALDH1A3 levels relative to β-actin is shown. Data are represented as mean ± SD of 3 independent experiments. **P < 0.01, 1-way ANOVA with Dunnett’s post test (F); 2-tailed Student’s t test (G and H).
Figure 2
Figure 2. USP9X interacts with ALDH1A3.
(A) HEK293T cells were transfected with His-ALDH1A3 alone or in combination with Flag-tagged USP9X WT or USP9X C1566A, and cell lysates were analyzed by IP with Flag beads followed by IB with antibodies against His and Flag. (B) Cell lysates from MES 21 and 505 GSCs were analyzed by IP using antibodies against USP9X and ALDH1A3, then subjected to IB analysis. IgG was used as the isotype control. (C) Purified Flag-tagged USP9X WT or USP9X C1566A was incubated with GST or GST-ALDH1A3 coupled to glutathione-Sepharose beads. Proteins retained on Sepharose were then subjected to IB with indicated antibodies. Recombinant GST-ALDH1A3 was purified from bacteria and analyzed by SDS-PAGE and Coomassie blue staining. (D) Schematic representation of Flag-tagged full-length (FL) ZEB1, His-tagged FL ALDH1A3, and their various deletion mutants. (E) HEK293T cells were cotransfected with His-ALDH1A3 and Flag-tagged FL USP9X or its deletion mutants, and cell lysates were analyzed by IP with Flag beads followed by IB with antibodies against His and Flag. (F) HEK293T cells were cotransfected with Flag-USP9X and His-tagged FL ALDH1A3 or its deletion mutants, and cell lysates were analyzed by IP with His beads followed by IB with antibodies against Flag and His.
Figure 3
Figure 3. USP9X deubiquitinates ALDH1A3.
(A) HEK293T cells or NHAs were cotransfected with His-ALDH1A3, HA-ubiquitin (HA-Ub), and Flag-tagged USP9X WT or USP9X C1566A, 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) MES 21 and 505 GSCs were cotransfected with the indicated siRNA and HA-Ub, and cell lysates were subjected to IP with ALDH1A3 antibody, followed by IB with antibodies against HA and ALDH1A3. Cells were treated with 20 μM MG132 for 8 hours before harvesting. (C) Unubiquitylated or ubiquitylated His-ALDH1A3 was incubated with GST-USP9X WT or GST-USP9X C1566A coupled to glutathione-Sepharose beads. His-ALDH1A3 was subjected to IP with His beads followed by IB with antibodies against HA and His. Recombinant GST-USP9X or GST-USP9X C1566A was analyzed by SDS-PAGE and Coomassie blue staining. (D) MES 21 and 505 GSCs were cotransfected with His-ALDH1A3, Flag-USP9X, and the indicated HA-Ub Lys0, Lys48-only, or Lys63-only plasmids, and then the ALDH1A3 ubiquitylation linkage was analyzed. (E) MES 21 and 505 GSCs transfected with Ub WT or Ub Lys48R were cultured for 72 hours in the presence of control siRNA or USP9X siRNA. Cell lysates were analyzed by IB using antibodies against ALDH1A3 and USP9X.
Figure 4
Figure 4. High USP9X expression predicts enrichment of ALDH1A3hi MES GSCs with potent tumorigenic capability.
(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.
Figure 5
Figure 5. Ablation of USP9X expression impairs the self-renewal, tumorigenicity, and radio/chemoresistance of MES GSCs.
(A and B) Primary neurosphere formation was assessed in MES 21 and 505 GSCs transduced with shCtrl or shUSP9X, reconstituted with vector control or ALDH1A3. Representative images are shown (A). Neurosphere formation efficiency (spheres/cells plated) was quantified. (B) Data are represented as means ± SD of 3 independent experiments. ***P < 0.001, 2-tailed Student’s t test. (C) In vitro limiting dilution sphere-forming frequency of MES 21 and 505 GSCs transduced with shCtrl or shUSP9X, reconstituted with vector control or ALDH1A3. Stem cell frequencies were estimated as the ratio 1/x with the upper and lower 95% confidence intervals, where 1 = stem cell and x = all cells. (D) IB analysis of USP9X, ALDH1A3, CD44, C/EBPβ, TAZ, p-STAT3, STAT3, VEGF-A, and c-MET levels in MES 21 and 505 GSCs expressing shCtrl or shUSP9X, reconstituted with vector control or ALDH1A3. (E) H&E-stained brain sections from mice intracranially implanted with MES 21 or 505 GSCs with indicated modifications. Red arrows indicate tumors. (F) Kaplan-Meier survival curves of mice intracranially injected with MES 21 or 505 GSCs with indicated modifications (n = 8). ****P < 0.0001, log-rank (Mantel-Cox) test. Scale bars: 500 μm (A); 1 mm (E).
Figure 6
Figure 6. Pharmacological inhibition of USP9X attenuates the tumor-initiating ability of MES GSCs with high ALDH1A3 activity.
(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).
Figure 7
Figure 7. USP9X shows a positive correlation with ALDH1A3 protein levels and is associated with poor survival of ALDH1A3hi MES GBMs.
(A) IHC staining of 138 human GBM specimens for USP9X and ALDH1A3. Representative consecutive sections from 2 specimens are shown. Scale bars: 50 μm. (B) Kaplan-Meier curves showing overall survival (upper panels) and progression-free survival (lower panels) of GBM patients divided based on USP9X expression in ALDH1A3hi MES tumors or OLIG2hi PN tumors (P values by log-rank test).
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