The deubiquitinase EIF3H promotes hepatocellular carcinoma progression by stabilizing OGT and inhibiting ferroptosis

Abstract

Hepatocellular carcinoma (HCC) is one of the most prevalent and lethal human malignancies, and with quite limited treatment alternatives. The proteasome is responsible for most of the protein degradation in eukaryotic cells and required for the maintenance of intracellular homeostasis. However, its potential role in HCC is largely unknown. In the current study, we identified eukaryotic translation initiation factor 3 subunit H (EIF3H), belonging to the JAB1/MPN/MOV34 (JAMM) superfamily, as a bona fide deubiquitylase of O-GlcNAc transferase (OGT) in HCC. We explored that EIF3H was positively associated with OGT in HCC and was related to the unfavorable prognosis. EIF3H could interact with, deubiquitylate, and stabilize OGT in a deubiquitylase-dependent manner. Specifically, EIF3H was associated with the GT domain of ERα via its JAB/MP domain, thus inhibiting the K48-linked ubiquitin chain on OGT. Besides, we demonstrated that the knockdown of EIF3H significantly reduced OGT protein expression, cell proliferation and invasion, and caused G1/S arrest of HCC. We also found that the deletion of EIF3H prompted ferroptosis in HCC cells. Finally, the effects of EIF3H depletion could be reversed by further OGT overexpression, implying that the OGT status is indispensable for EIF3H function in HCC carcinogenesis. In summary, our study described the oncogenic function of EIF3H and revealed an interesting post-translational mechanism between EIF3H, OGT, and ferroptosis in HCC. Targeting the EIF3H may be a promising approach in HCC. Video Abstract.

Keywords: Deubiquitination; EIF3H; Ferroptosis; Hepatocellular carcinoma; OGT.

Fig. 1

EIF3H depletion decreases O-GlcNac pathway activity and correlates with poor prognosis in hepatocellular carcinoma. A The siRNAs specific to each deubiquitinating enzyme (JAMMs family) were transfected into LM3 cells. After 48 h, cells were lysed and the O-GlcNac and OGT protein levels were analyzed by Western blot. B EIF3H depletion reduced OGT protein levels in both LM3 and HepG2 cells. C Genetic alternations of JAMMs family DUBs in HCC. The OncoPrint schematic was constructed in cBioPortal (TCGA dataset). D-E Prognostic analysis between EIF3H expression and OS (D) and RFS (E) in TCGA HCC patients. F Immunohistochemistry analysis of EIF3H and OGT expression in the Xiangya HCC cohort (n = 97), including normal liver tissue (n = 20) and HCC tumor tissues (n = 77). G Kaplan–Meier curve analysis of the EIF3H in Xiangya HCC cohort. H Correlations between EIF3H and OGT protein levels in Xiangya HCC cohort (Pearson correlation, R = 0.45). HCC, hepatocellular carcinoma; DUBs, deubiquitinating enzymes; OS, overall survival; RFS, relapse-free survival

Fig. 2

EIF3H associates with OGT and enhances its stability. A The immunofluorescence assay presented that EIF3H and OGT at least partially colocalized in LM3 cells. B Co-IP assay showed a correlation between endogenous EIF3H and OGT in LM3 cells. C-D EIF3H and OGT structure domain and deletion mutants were applied in the study. E The JAM/MP domain of EIF3H interacted with OGT. HEK293 cells were transfected with 2 µg Myc-OGT and GFP-EIF3H full-length or mutants. Cells were harvested with NP-40 lysis buffer for 24 h. Co-IP was performed using a GFP antibody and the possible interacted EIF3H domains were detected by Myc antibody. F OGT interacted with EIF3H through its GT domain. HEK293 cells were transfected with 2 µg GFP-EIF3H together with Myc-OGT full-length or mutants., Cells were harvested with NP-40 lysis buffer After 24 h. Co-IP was performed using Myc antibody. The possible interacted OGT domains were detected by GFP antibody. G In the presence of the proteasome inhibitor MG132, the knockdown of EIF3H did not further reduce the OGT protein expression level. LM3 cells were transfected with siControl or siEIF3H. After 48 h, cells were treated with 10 µM MG132/vehicle for 6 h, cell lysates were prepared for Western Blot analysis. H LM3 cells were transfected with EIF3H (wild type or mutant DDQ/AAA) together with EIF3H siRNA. The OGT protein levels were detected by western blot assay. I Silencing EIF3H decreases OGT half-life in LM3 cells. LM3 cells were transfected with siControl or siEIF3H. After 48 h, cells were treated with 100 µM cycloheximide/vehicle for indicated times. Cell lysates were prepared for western blot analysis. J The mutant EIF3H^DDQ/AAA did not increase OGT half-life in HEK293 cells. HEK293 cells were transfected with Myc-tag, Myc-EIF3H or Myc-EIF3H^DDQ/AAA plasmids. After 24 h, cells were treated with 100 µM cycloheximide/vehicle for indicated times. Cell lysates were prepared for Western blot assay

Fig. 3

EIF3H deubiquitylates OGT. A OGT was immunoprecipitated with anti-OGT and immunoblotted with anti-HA. LM3 cells transfected with the indicated siEIF3H were treated with MG132 for 6 h before collection. B Immunoblotting to detect the ubiquitination of OGT in HEK293 cells co-transfected with Flag-OGT, HA-Ubiquitin and GFP-EIF3H (wild type or DDQ/AAA). C EIF3H removed the ubiquitin chain of OGT in a dose-dependent manner. D-E K48 or K63 Ub was co-transfected with Flag-OGT and GFP-EIF3H into HEK293 cells. After treatment with 10 μM MG132 for 6 h, cell lysates were subjected to ubiquitination assay and the ubiquitination level of OGT was detected by HA antibody

Fig. 4

EIF3H depletion inhibits HCC proliferation and migration. A EIF3H repression inhibited cell proliferation in HCC cells. B Knockdown of EIF3H-induced G1 cell cycle arrest in HCC cells. C Silence of EIF3H decreased clone formation capability of HCC cells. D-E Representative images of EdU assays of LM3 and Hep3B cells. F Transwell invasion assay of HCC cells. G-H EIF3H depletion inhibits tumor growth in vivo. LM3 cells were stably transfected with lentivirus carrying a scrambled shRNA or EIF3H shRNA. 1 × 10⁶ LM3 cells were injected into each mouse (n = 6). Tumor sizes were measured every five days until the end of the experiment. ***P value < 0.001

Fig. 5

EIF3H regulates ferroptosis in HCC cells. A-B Cell viability assay showing the response of LM3 (A) and Hep3B (B) cell lines to different concentrations of Erastin with or without EIF3H depletion. C-D Colony formation showing the response of LM3 (C) and Hep3B (D) cell lines to Erastin with or without EIF3H depletion. E–F Representative images illustrating LM3 (E) and Hep3B (F) cells response to Erastin (20 μM) ± Ferrostatin (1 μM) with or without EIF3H knockdown. I-K Levels of lipid ROS (I, J), GSH (K), and ferrous iron (L) in HCC cells with or without EIF3H depletion. ***P value < 0.001

Fig. 6

Increased OGT expression reverses the effect of EIF3H depletion. A Cell proliferation assay of LM3. B Colony formation of LM3. C Representative images of EdU assay of LM3. D Transwell invasion assay of LM3. E Cell Viability analysis showing the response of EIF3H-knockdown LM3 cells to different concentrations of Erastin with or without OGT overexpression. F Representative images presenting EIF3H- silenced LM3 cells response to Erastin (20 μM) with or without OGT overexpression. G-I Levels of lipid ROS (G), GSH (H), and ferrous iron (I) in EIF3H-depleted LM3 cells with or without OGT overexpression. ***P value < 0.001