USP22 upregulates ZEB1-mediated VEGFA transcription in hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is a common solid tumor with high rate of recurrence and mortality. Anti-angiogenesis drugs have been used for the therapy of HCC. However, anti-angiogenic drug resistance commonly occurs during HCC treatment. Thus, identification of a novel VEGFA regulator would be better understanding for HCC progression and anti-angiogenic therapy resistance. Ubiquitin specific protease 22 (USP22) as a deubiquitinating enzyme, participates in a variety of biological processes in numerous tumors. While the molecular mechanism underlying the effects of USP22 on angiogenesis is still needed to be clarified. Here, our results demonstrated that USP22 acts as a co-activator of VEGFA transcription. Importantly, USP22 is involved in maintenance of ZEB1 stability via its deubiquitinase activity. USP22 was recruited to ZEB1-binding elements on the promoter of VEGFA, thereby altering histone H2Bub levels, to enhance ZEB1-mediated VEGFA transcription. USP22 depletion decreased cell proliferation, migration, Vascular Mimicry (VM) formation, and angiogenesis. Furthermore, we provided the evidence to show that knockdown of USP22 inhibited HCC growth in tumor-bearing nude mice. In addition, the expression of USP22 is positively correlated with that of ZEB1 in clinical HCC samples. Our findings suggest that USP22 participates in the promotion of HCC progression, if not all, at least partially via up-regulation of VEGFA transcription, providing a novel therapeutic target for anti-angiogenic drug resistance in HCC.

Fig. 1. USP22 is positively correlated with VEGFA in HCC.

A, B The mRNA or protein expression of USP22 is positively correlated with that of VEGFA. The data was downloaded from GEPIA (A) or CPTAC (B). C Evaluation of the indicated proteins in 35 clinical HCC samples (T) and the matched adjacent non-cancerous tissues (N) by western blotting. D, E The protein expression of USP22 or VEGFA in HCC samples is higher than that in the matched adjacent non-cancerous tissues. Data from (C) were quantified by densitometry, with GAPDH as the reference. F The protein expression of USP22 is positively correlated with VEGFA in 35 pairs of HCC samples and the matched adjacent noncancerous tissues. Pearson r indicates the degree of correlation. P < 0.05 is considered statistically significant.

Fig. 2. USP22 increases ZEB1-induced VEGFA transcription in HCC cells.

A–C The effects of USP22 on protein expression of VEGFA in HCCLM3 or Huh7 cells with GAPDH as the reference. D, E Evaluation of mRNA expression of VEGFA in USP22 knockdown PLC/PRF/5 or Huh7 cells. F Evaluation of mRNA expression of VEGFA in USP22 overexpressed PLC/PRF/5 or Huh7 cells. G USP22 enhances VEGFA transcription. HEK293 cells were transfected with PGL3-Basic or PGL3-VEGFA to evaluate the effect of USP22 on the transcription of VEGFA. Cells were lysed and assayed using the dual-luciferase reporter assay system. H USP22 and ZEB1 co-upregulate VEGFA transcription. HEK293 cells were transfected with PGL3-VEGFA together with the indicated expression plasmids. Cells were lysed and assayed using the dual-luciferase reporter assay system. I USP22 lost its regulatory effect on VEGFA protein expression when ZEB1 was knocked down. FLAG-USP22 or Vector was transfected with or without ZEB1 siRNA into Huh7 cells. J, K Evaluation of mRNA expression of three ZEB1 target gene in USP22 knockdown HCCLM3 or PLC/PRF/5 cells.

Fig. 3. USP22 interacts with ZEB1, and USP22/ZEB1 is recruited to the ZEB1-binding elements of VEGFA promoter region in HCC cell lines.

A The endogenous interaction between USP22 and ZEB1 in HCCLM3 cells verified by Co-Immunoprecipitation. B Diagram of full-length (FL) and truncated mutants of USP22. C Exogenous USP22 or its truncated mutants interact with ZEB1 in HEK293 cells. D Identification of binding domains in USP22 for ZEB1 interaction. ZEB1 protein was synthesized by transcription and translation kit in vitro. Bound proteins were analyzed by western blot. GST and GST- USP22 deletion mutants were stained by Coomassie brilliant blue staining. *, position of GST and GST- USP22 deletion mutants. E The Immunofluorescence confocal experiments were used to identify the localizations of FLAG-USP22 (green) and ZEB1 (red) in HCCLM3 cells. HCCLM3 cell was transfected with FLAG-USP22 expression plasmid for 24 h before harvested. Nucleus was stained by DAPI (blue), scale bars, 15 μm. The colocation coefficient was analyzed and calculated by Image Pro Plus software. F Schematic diagram of the predicted ZEB1 binding sites on VEGFA promoter region. G USP22 were recruited to ZEB1-binding elements in VEGFA. H USP22 knockdown reduced the recruitment of ZEB1 and accumulation of levels of H2Bub on ZEB1-binding site at VEGFA-promoter I. The DNA fragments were amplified by qPCR with the primers indicated in Supplementary data (Table S3). I Effects of USP22 on the ubiquitination level of histone H2B near the five predicted ZEB1 binding sites. J Schematic diagram of PGL3-VEGFA mutant (ZEB1 binding site mutation) luciferase assay plasmid, CACCCG was replaced by AAAAAA. K USP22 failed to upregulate transcription of VEGFA without wild type ZEB1 binding site in dual luciferase assay. In histogram, the bars represent mean ± SD (n ≥ 3), P < 0.05 is considered statistically significant.

Fig. 4. USP22 maintains ZEB1 stability by triggering deubiquitination of ZEB1.

A, B USP22 deletion reduced ZEB1 protein expression while ectopic expression of USP22 upregulated that in HCC-derived cell lines in western blotting experiments. C USP22 had no obvious effect on mRNA expression of ZEB1 in qPCR experiments, the bars represent mean ± SD (n ≥ 3), P < 0.05 is considered statistically significant. D Western blotting analysis of ZEB1 expression in wild-type and USP22 knockdown Huh7 cells treated with MG132 (10 μM) for 8 h as indicated. E Depletion of USP22 decreases endogenous ZEB1 protein stability in HCCLM3 cells. Cells were treated with 100 μM cycloheximide (CHX) as indicated. F Gray values of ZEB1 were calculated and presented in a line chart with statistical analysis. G Ectopic expression of USP22 decreased the ubiquitination of ZEB1. Immunoprecipitation of ubiquitinated proteins from Huh7 cell extracts upon overexpression of USP22. Protein was harvested after MG132 (5 μM) treatment for 3 h and ubiquitinated ZEB1 species were detected by western blotting with anti-His. H Ubiquitination of ZEB1 was upregulated by USP22 deletion. HCCLM3 cells were transfected with USP22 siRNA. Cells were immunoprecipitated with ZEB1 and immunoblotted with anti-His. I The ubiquitination level of ZEB1 could not be reduced by deubiquitinase inactive mutant USP22(m). Cells were immunoprecipitated with ZEB1 and immunoblotted with anti-His. HEK293 cells were transfected with expression plasmid encoding ZEB1 and His-ubiquitin together with wild type USP22 or USP22(m). J–L Denaturing ubiquitination assays were performed to detect the effects of USP22 on ubiquitination levels and types of ZEB1. HCCLM3 cells were transfected with ZEB1 and FLAG-USP22 together with His-tagged ubiquitin (J). HEK293 cells were transfected with ZEB1 and FLAG-USP22 or USP22(m) together with HA-tagged ubiquitin mutants, including K0 (lysineless), K48-, K63-, and K27-linked ubiquitin as indicated (K, L). The cells were treated with MG132 (5 μM) before collected. The cell lysate was immunoprecipitated with anti-ZEB1 and immunoblotted with anti-His or anti-HA.

Fig. 5. USP22 promotes HCC-derived cell growth/invasion/Vascular Mimicry (VM) formation and angiogenesis.

A Knockdown of USP22 inhibited HCCLM3 proliferation. The absorbance of HCCLM3 cells carrying shCtrl or shUSP22 added MTS reagent in indicated time was measured at 490 nm. B, C Effects of USP22 on the cell growth in HCCLM3 cells were photographed in the colony formation assay. VEGFA-165 stands for recombinant human VEGFA protein. D The morphology of HCCLM3 cells changed with shUSP22 was photographed under microscope. E The left panel shows tube formation pictures of the HCCLM3 cells. Scale bars, 200 μm. The right panel is a quantification plot of the number of tubes in each visual field in the left. F, G Effect of USP22 on cell migration and invasion in Huh7 cells. Cells were planted in the transwell chamber coated with Matrigel (G) or without Matrigel (F) for 24 h. Scale bars, 250 μm. H ELISA assay was used to verify the effect of USP22 on VEGFA secretion from Huh7 cells. I Tube formation of HUVEC cells were photographed under microscope. Conditioned medium collected from shCtrl or shUSP22 Huh7 cells were applied to culture HUVEC cells on the Matrigel for 24 h. J Colony formation experiments were used to determine the effect of USP22 on the cell growth of HUVEC cells. Scale bars, 250 μm. Histogram data were shown as mean ± SD, P < 0.05 is considered statistically significant.

Fig. 6. USP22 promotes HCC progression in mice.

A Pictures showed all the xenograft tumors formed by HCCLM3 cells in different groups. VEGFA-165 stands for recombinant human protein VEGFA-165. B The image closely up displays the tumors in situ, with yellow arrows indicating blood vessels forming on the tumor surface. C Growth curves drawn by tumor volume of xenograft tumors in the USP22 knockdown group and the control group at indicated time. D Tumor weight of xenografts in indicated groups were shown. E Protein levels of USP22, VEGFA and ZEB1 in every xenograft tumor were detected by western blotting. GAPDH was used as a control. F The protein expression of ZEB1 from (E) was quantified by densitometry, with GAPDH as the reference. P < 0.05 is considered statistically significant. G Pictures showed all the xenograft tumors formed by HCCLM3 cells in different groups. H Growth curves drawn by tumor volume of xenograft tumors in the USP22 overexpressing, USP22 overexpressing with ZEB1 deletion, and the control group at indicated time. I Tumor weight of xenografts in indicated groups were shown. J Protein levels of USP22, and ZEB1 in every xenograft tumor were detected by western blotting. GAPDH was used as a control.

Fig. 7. Knockdown of USP22 reduced VM formation in xenograft tumors.

A Expression of USP22, ZEB1 and VEGFA in xenograft tumors (Fig. 6A) were detected by IHC assays; CD31/PAS double staining was used to evaluate the appearance of vasculogenic mimicry (PAS + CD31-) and vessels (PAS + and CD31 + ) in the shUSP22 and shCtrl groups. VEGFA-165 stands for recombinant human protein VEGFA-165. Scale bars, 50μm. Average optical density (AOD) was used to evaluate the relative expression levels of USP22, ZEB1 and VEGFA. Counts of channels per sections was used to evaluate the effect of USP22 on the formation of vascular mimicry and angiogenesis within tumors. B The representative images of expression of USP22 and ZEB1 in immunohistochemical staining of human HCC pathological sections. The expression of specific marker from the HCC pathological data showed that, the tumor with high USP22 and ZEB1 expression on the left, AFP (+), Ki67(30%); the tumor with low USP22 and ZEB1 expression on the right, AFP (+), Ki67 (15%). Scale bars, 200 μm.

Fig. 8. Schematic diagram illustrating the function of USP22 on modulation of ZEB1-induced VEGFA transcription and the role of USP22 in promotion of HCC progression.

USP22 maintains ZEB1 stability by triggering deubiquitination of ZEB1. USP22/ZEB1 being recruited to the ZEB1 binding elements of VEGFA promoter region, inducing the ubiquitination of histone H2B, enhances the transcription of VEGFA.This effect ultimately leads to increased expression and secretion of VEGFA, which may accelerate HCC progression.