USP19 regulates DNA methylation damage repair and confers temozolomide resistance through MGMT stabilization

Abstract

Objective: To elucidate the relationship between USP19 and O(6)-methylguanine-DNA methyltransferase (MGMT) after temozolomide treatment in glioblastoma (GBM) patients with chemotherapy resistance.

Methods: Screening the deubiquitinase pannel and identifying the deubiquitinase directly interacts with and deubiquitination MGMT. Deubiquitination assay to confirm USP19 deubiquitinates MGMT. The colony formation and tumor growth study in xenograft assess USP19 affects the GBM sensitive to TMZ was performed by T98G, LN18, U251, and U87 cell lines. Immunohistochemistry staining and survival analysis were performed to explore how USP19 is correlated to MGMT in GBM clinical management.

Results: USP19 removes the ubiquitination of MGMT to facilitate the DNA methylation damage repair. Depletion of USP19 results in the glioblastoma cell sensitivity to temozolomide, which can be rescued by overexpressing MGMT. USP19 is overexpressed in glioblastoma patient samples, which positively correlates with the level of MGMT protein and poor prognosis in these patients.

Conclusion: The regulation of MGMT ubiquitination by USP19 plays a critical role in DNA methylation damage repair and GBM patients' temozolomide chemotherapy response.

Keywords: MGMT; deubiquitination; glioblastoma; temozolomide resistence; usp19.

FIGURE 1

USP19 regulates MGMT protein level. (A) Deubiquitinases (DUBs) were transfected in cells. After 48 h, cells were lysed, and Western blot was performed with the indicated antibodies. (B) Cells stably expressing USP19 shRNAs were treated with vehicle or MG132 for 4 h, and then lysed. Western blot was performed with the indicated antibodies. (C, D) Cells stably expressing control or USP19 shRNAs were harvested for check the mRNAs were extracted from the cells and subjected to qPCR. Error bars represent ± S.E.M. from three independent experiments. ***p < 0.001. Statistical analyses were performed with the Student's t‐test. (E, F) Cells stably expressing control shRNA, USP19 shRNA, and USP19 shRNA together with shRNA‐resistant USP19 were treated with cycloheximide (1.0 μg/mL), and harvested at the indicated times. Figure E shows immunoblots of USP19 and MGMT. Figure F shows the quantification of the MGMT level relative to GAPDH.

FIGURE 2

USP19 interacts with MGMT. (A) MGMT interacts with USP19 in cells. Cells were transfected with FLAG‐MGMT. FLAG‐MGMT was purified on anti‐FLAG‐agarose, and then coprecipitating endogenous USP19 was detected by anti‐USP19 antibody. (B) Endogenous MGMT coprecipitates with endogenous USP19. Cell lysates were subjected to immunoprecipitation with control IgG. The immunoprecipitates were then blotted with the indicated antibodies. (C) HCT116 cells transfected with FLAG‐tagged USP19 WT, USP19 1‐596(CS), USP19 USP, USP19 USP+TMD mutant were lysed, and then cell lysates were subjected to immunoprecipitation with anti‐FLAG‐agarose. The immunoprecipitates were then blotted with the indicated antibodies. (D) A schematic illustration of the USP19 constructs used in the study.

FIGURE 3

USP19 deubiquitinates MGMT. (A) USP19 deubiquitinates MGMT in cells. Cells stably expressing control or USP19 shRNAs and control or FLAG‐MGMT were transfected with His‐Ub, and then were treated with MG132 for 4 h before harvested. Covalently modified proteins purified on NiNTI‐agarose under denatured conditions. Ubiquitinated MGMT was detected by anti‐FLAG antibody. (B) Cells transfected with indicated constructs were treated with MG132 for 4 h before harvested. Covalently modified proteins were purified on NiNTI‐agarose under denatured conditions and then blotted with indicated antibodies. (C) Deubiquitination of MGMT in vitro by USP19. Ubiquitinated MGMT was incubated with purified USP19WT or USP19CS in vitro and then blotted with indicated antibodies.

FIGURE 4

USP19 regulates temozolomide chemotherapy resistance via MGMT. (A) Different brain tumor cells were harvested and lysed, and western blot was performed with the indicated antibodies. (B) T98G, (C) LN18, (D) U87, (E) U251 cells stably expressing indicated constructs were treated with the indicated concentration of temozolomide. Cell survival was performed by colony formation. (F, G) T98G cells stably expressing indicated constructs were subcutaneously injected into the flank of nude mice. Animals were randomized into four groups (n = 5) and treated with temozolomide (50 mg/kg) by oral. Tumor volumes were measured by caliper every 3 days, and solid tumor weight was measured at sacrifice. (H) Images of temozolomide‐treated tumor.

FIGURE 5

USP19‐deficient glioblastoma sensitizes to temozolomide in vivo. USP19 expression is correlated to MGMT in clinical glioblastoma sample. (A, B) Representative images of immunohistochemistry staining of USP19 and MGMT in glioblastoma. Scar bars, 50 μm. (C) Quantification of USP19 and MGMT protein levels in glioblastoma. Statistical analyses were performed with Chi‐squared test. R, Pearson correlation coefficient. (D) Survival analysis of glioblastoma patients was performed. As per data from cbioportal database, patients were categorized into with and without alteration of USP19.