Deubiquitinase USP9X deubiquitinates β-catenin and promotes high grade glioma cell growth

Abstract

β-catenin is a crucial signal transduction molecule in the Wnt/β-catenin signal pathway, and increased β-catenin expression has consistently been found in high grade gliomas. However, the mechanisms responsible for β-catenin overexpression have remained elusive.Here we show that the deubiquitinase USP9X stabilizes β-catenin and thereby promotes high grade glioma cell growth. USP9X binds β-catenin and removes the Lys 48-linked polyubiquitin chains that normally mark β-catenin for proteasomal degradation. Increased USP9X expression correlates with increased β-catenin protein in high grade glioma tissues. Moreover, patients with high grade glioma overexpressing USP9X have a poor prognosis. Knockdown of USP9X suppresses cell proliferation, inhibits G1/S phase conversion, and induces apoptosis in U251 and A172 cells. Interestingly, c-Myc and cyclinD1, which are important downstream target genes in the Wnt/β-catenin signal pathway, also show decreased expression in cells with siRNA-mediated down-regulation of USP9X. Down-regulation of USP9X also consistently inhibits the tumorigenicity of primary glioma cells in vivo.In summary, these results indicate that USP9X stabilizes β-catenin and activates Wnt/β-catenin signal pathway to promote glioma cell proliferation and survival. USP9X could also potentially be a novel therapeutic target for high grade gliomas.

Keywords: USP9X; deubiquitination; high grade gliomas; prognosis; β-catenin.

Figure 1. USP9X interacted with β-catenin

a. Kaplan–Meier curves of high grade glioma patients with negative- versus positive-expression of USP9X in 54 high grade glioma patients. b. Co-localization of USP9X and β-catenin in U251 and A172 cells was detected by immunofluorescence. c. Western blot analysis of vector-induced overexpression of β-catenin in HEK293T cells after treatment with pCMV-β-catenin plasmid. d and e. HEK293T cells treated with pCMV-β-catenin plasmid for 48 h were used for endogenous USP9X and β-catenin immunoprecipitation. WCL, whole cell lysate.

Figure 2. USP9X inhibition retarded WNT/β-catenin signal pathway

a and b. Western blot analysis of USP9X, β-catenin. c-Myc and cyclinD1 levels after USP9X siRNAs were transfected into U251 and A172 cells, respectively for 72 h. c. MG-132 recovered β-catenin protein expression after USP9X knock down. USP9X si-2 was transfected into U251 cells for 72 hours and treated with or without 10mM MG-132 for 4 h, cells were harvested and lysed for western blot. d. Staining of β-catenin protein in U251 and A172 cells treated by USP9X si-2.

Figure 3. USP9X knockdown caused increase of K48-ubiquitinated β-catenin

a. USP9X siRNA-2 was transfected into U251 cells, and after another 48 h, cells were treated with MG132 for 4 h before harvest. β-catenin was immunoprecipitated and immunoblotted with β-catenin antibodies or anti-ubiquitin antibodies. b. USP9X siRNA-2 was transfected into U251 cells along with Myc-UBK0 plasmid, and after another 48 h, cells were treated with MG132 for 4 h before harvest. Myc-UBK0 was immunoprecipitated and immunoblotted with β-catenin antibodies. c. USP9X siRNA-2 was transfected into U251 cells, and after another 48 h, cells were treated with MG132 for 4 h before harvest. β-catenin was immunoprecipitated and immunoblotted with K48-linkages specific ubiquitin antibody. d. The efficiency of USP9X shRNA was examined using anti-USP9X antibody. e. USP9X siRNA-2 was transfected into U251 cells, and after another 48 h, cells were stimulated with Wnt3a for 3 h and examined with indicated antibody.

Figure 4. USP9X specific inhibitor WP1130 treatment increased of K48-ubiquitinated β-catenin

a. U251 cells were treated with 1μM WP1130 for 24h before harvest. β-catenin was immunoprecipitated and immunoblotted with β-catenin antibody or anti-ubiquitin antibody. b. U251 cells were treated with 1μM WP1130 for 24h before harvest. β-catenin was immunoprecipitated and immunoblotted with β-catenin antibody or K48-linkages specific ubiquitin antibody. c. USP9X siRNA-2 was transfected into U251 cells along with Myc-UBK0 plasmid, and after another 48h, cells were treated with MG132 for 4 h before harvest. β-catenin was immunoprecipitated and immunoblotted with Myc-Tag antibody.

Figure 5. Cell proliferation decreased after USP9X knockdown

a and b. Cell proliferation curves of U251 and A172 cells after USP9X knockdown. c and d. USP9X siRNAs were transfected into U251 and A172 cells for 72 h, cells were harvested and lysed for western blot to detect caspase 3 and caspase 8.

Figure 6. Representative immunohistochemical staining for USP9X, β-catenin, c-Myc and cyclinD1 in high grade glioma tissues

a. Positive USP9X, β-catenin, c-Myc, cyclinD1 expression in patient a (Original magnification, ×400). b. Negative USP9X, β-catenin, c-Myc, cyclinD1 expression in patient b (Original magnification, ×400).

Figure 7. USP9X knockdown decreased tumorigenesis of glioma cells in vivo

a and b. Growth of GBM1and GBM2 xenograft tumors expressing USP9X-shRNA or NC-shRNA. Control, GBM1 and GBM2 cells.