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. 2023 Sep 4;13(14):4919-4935.
doi: 10.7150/thno.85662. eCollection 2023.

E3 ubiquitin ligase TRIM21 targets TIF1γ to regulate β-catenin signaling in glioblastoma

YanLan Li  1   2 , Lingbo Bao  1 , Hong Zheng  3 , Mingying Geng  1 , TianYi Chen  1 , Xiaoyan Dai  1 , He Xiao  1 , Lujie Yang  1 , Chengyi Mao  4 , Yuan Qiu  5 , Yu Xu  1 , Dong Wang  1 , Meng Xia Li  1 , Qian Chen  1
Affiliations

E3 ubiquitin ligase TRIM21 targets TIF1γ to regulate β-catenin signaling in glioblastoma

YanLan Li et al. Theranostics. .

Abstract

Background: Elucidation of the mechanism of ubiquitation has led to novel ways to treat glioblastoma (GBM). A tripartite motif (TRIM) protein mediates a reversible, stringent ubiquitation which is closely related to glioma malignancy. This study intends to screen the most vital and abnormal regulating component of the tripartite motif protein and to explore its underlying mechanisms. Methods: TRIM21 is identified as an important oncogene that accelerates the progression of glioma cell through database in a multidimensional way and this is confirmed in human samples and cells. Tandem Mass Tags (TMT) and MS analysis are performed to discover the substrates of TRIM21.The underlying mechanisms are further investigated by CO-IP, luciferase reporter assays and gain and loss of function assays. In vivo treatment with siRNA is applied to evaluate the therapeutic significance of TRIM21. Result: We screened a panel of TRIM proteins and identified TRIM21, a E3 ubiquitin-protein ligase and autoantigen, as well as a prognostic biomarker for GBM. Functionally, high expression of wild-type TRIM21 accelerates tumor progression in vitro and in vivo, whereas TRIM21 mutants, including one with a critical RING-finger deletion, do not. Mechanistically, TRIM21 stimulates K63-linked ubiquitination and subcellular translocation of active β-catenin from the cytoplasm to the nucleus. Moreover, TRIM21 forms a complex with the β-catenin upstream regulator, TIF1γ, in the nucleus and accelerated its degradation by inducing K48-linked ubiquitination at K5 site, consequently increasing further nuclear β-catenin presence. Endogenous TRIM21 levels are found to be inversely correlated with TIF1γ but positively correlated with β-catenin in glioma tissue microarray experiments. Furthermore, direct injection of TRIM21 small interfering RNA (siRNA) into U87 cell-derived tumors (in vivo treatment with siRNA) is proved to inhibit tumor growth in nude mice. Conclusion: This work suggests that TRIM21/TIF1γ/β-catenin axis is involved in the progression of human GBM. TRIM21 is a promising therapeutic and prognostic biomarker for glioma with hyperactive β-catenin.

Keywords: TIF1γ; TRIM21; TRIMs family; ubiquitination; β-catenin.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
TRIM21 is highly expressed in GBM samples and correlates with poor prognosis. (A) Venn diagram showing the number of changed proteins from TRIMs family identified in TCGA-datasets in GBM relative to normal brain tissue samples (Blue) and the prognosis difference using Kaplan-Meier curves in GBM from TCGA-database (Yellow) and Rembrandt-Database (Green) and overlapping proteins in the datasets. (B) TRIM21 and TRIM48 were identified. (C) Western blotting of TRIM21 and TRIM48 protein in 5 paired surgically removed glioma (T) and the corresponding adjacent normal tissues (N). (D) The mRNA of TRIM21 in glioma in different grades. (E-F) Representative IHC staining images (E) and score (F) of TRIM21 in WHO grade I-IV glioma tissue microarrays and normal brain tissues (NBT). Scale bar, 100 μm. (G-H) Kaplan-Meier curves showing the relationship between the expression of TRIM21 and the overall survival rate (G) and Disease-free survival rate (H) of patients with glioma (n = 120). (I) Heatmap showing the distribution of clinical features and genetic characteristics and TRIM21 expressions in glioma specimens from TCGA-LGGGBM database (n = 330).
Figure 2
Figure 2
TRIM21 facilitated invasion and growth of GBM via its RING domain in vivo and in vitro. (A) Expression of TRIM21 protein in GBM cells (GBM-1 and U87-MG) transfected with Scramble or shTRIM21. (B) The growth curve of GBM cells GBM cells (GBM-1 and U87-MG) transfected with Scramble or shTRIM21. (C) Representative images (Left panel) and number (Right panel) of U87-MG cells transfected with Scramble or shTRIM21. Scar bars, 50μm. (D) Western blot analysis of TRIM21 in GBM cells (U87-MG and U251-MG) transfected with Mock, TRIM21-FL, TRIM21-△RING plasmid. (E) Representative images (Left panel) and number (Right panel) of Mock, TRIM21-FL, TRIM21-△RING GBM cells (U87-MG and U251-MG) in transwell assay. (F-G) Summary graph indicates the motilities (F) and Brdu-positive immunofluorescence staining (G) of GBM cells transfected with Mock, TRIM21-FL, TRIM21-△RING plasmid. (H-I) Bioluminescent images (H) and the quantification (I) of tumors in mice implanted with Mock-, TRIM21-FL-, or TRIM21-△RING- U87-MG cells. The bioluminescent images were obtained at day 7 and 14 after injection. (J) Survival curves of tumor-bearing mice implanted with indicated cells. (K) Representative IHC staining of the tumor tissues were performed with anti-Ki-67 antibodies. Scar bars, 100μm
Figure 3
Figure 3
TRIM21-mediated WNT/β-catenin through K63 polyubiquitination of β-catenin. (A) GSEA of positive regulation of WNT signaling pathway score in human GBMs from the GSE108474 (n = 110). (B) Gene oncology enrichment analysis of identified Differential Expression Analysis (DEG) by TMT from U87-MG. (C) Western blot analysis to evaluate β-catenin and C-myc in lysates prepared from mock, TRIM21-FL and TRIM21-△RING-GBM (U87-MG and U251-MG) cells. (D) Western blot analysis to evaluate β-catenin and C-myc in lysates prepared from TRIM21 knock down. (E) Immunofluorescence (left) and quantitative analysis (right) of β-catenin nuclear translocation induced by overexpression of TRIM21. Scale bar,20 µm. (F-G) Overexpression (F) and silencing (G) of TRIM21 enhanced and abolished the nuclear translocation of β-catenin, respectively. β-catenin protein in the cytoplasm and nucleus were assayed in U87-MG cells with up- or down- of TRIM21 by a nuclear extraction assay. (H) Immunoprecipitation was performed with U87-MG-OETRIM21-Flag (Left panel) and HEK293-T (Right panel) lysates using Flag or β-catenin antibodies, and the precipitants were measured by western blotting with the indicated antibodies. (I) The Armadillo domain of β-catenin mediates the interaction of this protein with TRIM21 (Top panel), Schematic illustration of β-catenin. Bottom panel, β-Catenin deletion mutants were co-expressed with HA-TRIM21 in 293T cells. the cells were subjected to IP with a HA antibody followed by immunoblotting (IB) with FLAG and HA antibodies. (J) In vivo ubiquitination assays performed in HEK 293T cells with mock, TRIM21, TRIM21-△RING plasmid, TRIM21-C16A, which then were transfected as indicated. Cells were treated with MG132 (10 mM for 4 h) and prior to lysis and then subjected to anti-β-catenin IP followed by anti-UB with immunoblot analysis. (K) His- Ub (WT, K6R, K27R, K29R, K33R, K48R, K63R) mutants were expressed with or without HA-TRIM21 in HEK293T cells. (L) HEK293T cells were transfected with Flag-β-catenin and HA-TRIM21 together with wild-type (WT) ubiquitin with K48O and K63O respectively. Cells were treated with MG132 (10 mM for 4 h) and prior to lysis and then subjected to anti-β-catenin IP followed by anti-His with immunoblot analysis.
Figure 4
Figure 4
TRIM21-mediated degradation of TIF1γ. (A) Volcano plot of the protein abundance changes in response to OETRIM21. Average protein expression ratio of 4 replicates (log 2 transformed) between TRIM21 overexpression and mock U87-MG cells was plotted against p-value by t-test (-log 10 transformed). Cutoffs of p = 0.05 and 2.0 -fold change were marked, respectively. The figure below shows the total number of proteins identified as well as the number of up- and down-regulated proteins. (B) MS analysis identified TIF1γ as the key interacting protein of TRIM21. TRIM21 was immunoprecipitated from U87-MG expressing HA-TRIM21 using anti-HA-conjugated beads. TIF1γ was identified through MS analysis by peptides covering the TIF1γ protein sequence. representative detected peptide (QIDLVDNYFVK) of TIF1γ was shown. (C) Co-IP of TRIM21 with TIF1γ in U87 cells (left panel) and U251-cells (Right panel). (D) Co-IP of TIF1γ with TRIM21 in U87 cells (left panel) and U251-cells (Right panel). (E) Full-length TRIM21 and a series of TRIM21 mutants with deletion (△) of various domains (top panel). 293 T cells were co-transfected with Flag-TIF1γ and WT HA-TRIM21 or their truncation mutants for 48 h. CO-IP assay was performed. (F) Full-length TIF1γ and a series of TIF1γ mutants with deletion (△) of various domains (top panel). 293 T cells were co-transfected with Flag-TIF1γ or their truncation mutants and WT HA-TRIM21 CO-IP assay was performed. (G) In vivo ubiquitination assays performed in U87-Flag-TIF1γ cells transfected with indicated plasmid. Cells were treated with MG132 (10 mM for 4 h) and prior to lysis and then subjected to anti-Flag IP followed by anti-UB with immunoblot analysis. (H-I) TRIM21 is a K48-linkage-specificubiquitin ligase for TIF1γ. HEK293T cells were transfected with Flag-TIF1γ and HA-TRIM21 together with wild-type (WT) ubiquitin (H), with each of the ubiquitin mutants UbK48O, Ub K63O (H), Ub K48R or Ub K63R (I). (J) HEK293-T cells were cotransfected with the indicated plasmids for 48 h and then treated with 10 mM MG132. The polyubiquitination levels of Flag-TIF1γ and Flag-TIF1γ-K5R; Flag-TIF1γ-K1115R mutant protein were analyzed. (K) Left panel: U87-MG cells were transfected with TRIM21-TIF1γ-WT or TRIM21-TIF1γ-K5R plasmid for 24 h, and then treated with cycloheximide (CHX, 20 μg/mL) or the indicated times before harvesting. Cells lysates were analyzed by immunoblotting.
Figure 5
Figure 5
TRIM21 / TIF1γ axis is involved in β-catenin stability and tumor progression. (A) Immunoblotting of TRIM21, TIF1γ and β-catenin in GBM cells (U87-MG and U251-MG) overexpressing TRIM21, TIF1γ and TRIM21 + TIF1γ. (B) Immunofluorescence scanning of β-catenin (Red), TIF1γ(Green), TRIM21(White) in GBM cells overexpressing TRIM21 and/or TIF1γ; Scar bars, 50μm. (C) In vivo ubiquitination assays performed in U87-MG cells transfected with indicated plasmid. Cells were treated with MG132 (10 mM for 4 h) and prior to lysis and then subjected to anti-β-catenin IP followed by anti-UB with immunoblot analysis. (D) Immunoblotting of TRIM21, TIF1γ in U87-MG-siTIF1γ cells Overexpressing TIF1γ, TIF1γ-K5R, TRIM21+ TIF1γ, TIF1γ-K5R+ TRIM21. (E) The invasive number of Mock, OETRIM21, OETIF1γ and OETRIM21+TIF1γ GBM Cells (U87-MG and U251-MG). (F) The number of Brdu-positive immunofluorescence staining for the indicated GBM cells. (G-H) Bioluminescence (G) and Quantification (H) of tumors in NOD-SCID mice implanted with Mock, TRIM21, TIF1γ and TIF1γ+ TRIM21-U87-MG cells. (I) Survival curves of tumor-bearing mice implanted with Mock, OETRIM21, OETIF1γ and OETIF1γ+TRIM21 U87-MG cells. (J) Representative IHC image of the tumor tissues were performed with anti-β-catenin and anti-Ki-67 antibodies. Scar bars, 50μm.
Figure 6
Figure 6
Arginine 443 in TRIM21 is critical for mediating the ubiquitination and degradation of TIF1γ. (A) The R443W mutation of TRIM21 was identified from TCGA GBM database (http://www.cbioportal.org). (B) U87-MG cells were transfected with HA-tagged TRIM21 and its mutants R443W (HA-TRIM21, HA-TRIM21R443W respectively) Cell lysates were immunoprecipitated with anti-HA antibodies. (C) U87-MG cells were transfected with HA-tagged TRIM21, its mutants HA-TRIM21-R443W and/or Flag-TIF1γ respectively, cells were immunoprecipitated with anti-TIF1γ. (D) Western blot analysis to evaluate TIF1γ, β-catenin, C-myc and cyclin D1 in lysates prepared from Mock, TRIM21 and TRIM21-R443W-GBM (U87-MG) cells. (E) In vivo ubiquitination assays performed in U87-MG transfected with mock; HA-tagged TRIM21, its mutants HA-TRIM21-R443W. Cells were treated with MG132 (10 mM for 4 h) and prior to lysis and then subjected to anti-Flag IP followed by anti-HIS with immunoblot analysis. (F) Immunoblotting (Left panel) and Quantitation (Right panel) of TIF1γ in GBM cell (Mock, TRIM21, TRIM21R443W) treated with CHX (100 mg/ml) and MG132 for different times. (G) Brdu-positive immunofluorescence staining images (Upper panel) and number (down panel) of GBM cells transfected with (-Mock, -TRIM21, -TRIM21R443W) Scar bars, 100μm. (H) Representative images (Upper panel) and number (down panel) of invaded U87-MG cells and U251-MG transfected with (-Mock, -TRIM21, -TRIM21R443W) Scar bars, 100μm.
Figure 7
Figure 7
Correlation of TRIM21, TIF1γ and β-catenin in Glioma. (A-D) Representative image of TRIM21, TIF1γ and β-catenin expression in IDH1-WT and IDH1-MT glioma examined by IHC. Scare bars, 100 μm. (E-F) Kaplan-Meier plots of the OS (E) rates and DFS (F) rates in human glioma specimens in the groups with high and low expression of TIF1γ. (G) IHC staining of 120 human glioma specimens was performed with the indicated antibodies. Representative images from the staining of TRIM21, TIF1γ and β-catenin were shown scar bars, 100μm. (H) χ2 analysis showing the percentage of TIF1γ-high and TIF1γ-low in TRIM21-high group and TRIM21-low group. (I) χ2 analysis showing the percentage of β-catenin-high and β-catenin-low in TRIM21-high group and TRIM21-low group. (J) χ2 analysis showing the percentage of β-catenin-high and β-catenin-low in TIF1γ-high group and TIF1γ-low group. (K) Kaplan-Meier plots of the overall survival rates and Disease-free survival rates in human glioma specimens in the groups with TRIM21(+) β‐catenin (+), TRIM21(-) β‐catenin (-), and others.
Figure 8
Figure 8
Inhibition of xenograft tumor proliferation by in vivo treatment with siRNA. (A-C) U87-MG cells (1 × 106 cells) were subcutaneously implanted into nude mice. After 10 days, control or TRIM21 siRNA was injected into the developed xenograft tumors (n = 5). 10 days after siRNA injection, the nude mice were killed. Dashed lines show the outline of xenograft tumors in representative three mice (Left picture), and extirpated xenograft tumors are shown (Right picture) (A). The volumes (B) and weights (C) of five pair of tumors were measured. (D) Sections were stained with TRIM21 Scar bars, 50 μm.
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