Trabid inhibits hepatocellular carcinoma growth and metastasis by cleaving RNF8-induced K63 ubiquitination of Twist1

Abstract

TRAF-binding domain (Trabid), one of deubiquitination enzymes, was recently reported to activate Wnt/ β-catenin signaling pathway. However, the role of Trabid in tumors including hepatocellular carcinoma (HCC) and the underlying mechanisms controlling its activity remain poorly understood. Here, we report that Trabid is significantly downregulated in HCC tumor samples and cell lines compared with normal controls and that its expression level is negatively correlated with HCC pathological grading, recurrence, and metastasis. The reintroduction of Trabid expression in tumor cells significantly decreases HCC progression as well as pulmonary metastasis. The effect of Trabid on HCC development occurs at least partially through regulation of Twist1 activity. Mechanistically, Trabid forms a complex with Twist1 and specifically cleaves RNF8-induced K63-linked poly-ubiquitin chains from Twist1, which enhances the association of Twist1 with β-TrCP1 and allows for subsequent K48-linked ubiquitination of Twist1. Knockdown of Trabid increases K63-linked ubiquitination, but abrogates K48-linked ubiquitination and degradation of Twist1, thus enhancing HCC growth and metastasis. Interestingly, Twist1 negatively regulates the promoter activity of Trabid, indicating that a double-negative feedback loop exists. Our findings also identify an essential role for activation of Trabid by AKT-mediated phosphorylation at Ser78/Thr117 in negatively regulating Twist1 signaling, which further provides insights into the mechanisms by which Trabid regulates Twist1 ubiquitination. Our results reveal that Trabid is a previously unrecognized inhibitor of HCC progression and metastasis, which sheds light on new strategies for HCC treatment.

Fig. 1

Trabid is markedly decreased in HCC which predicts poor prognosis. a The levels of Trabid protein in 10 pairs of HCC and matched adjacent nontumorous tissues were measured using Western blot. b The quantitative polymerase chain reaction (qPCR) was used to measure the levels of Trabid mRNA in the above samples. RT-PCR analysis of Trabid expression in healthy control tissues, matched adjacent nontumorous tissues, and HCC tissues (c1). RT-PCR was used to measure Trabid expression in liver tissues from patients with different recurrence statuses (c2), or metastasis statuses (c3), or tumor volumes (c4), or disease stages (c5). c6 qPCR assays and WB of Trabid expression in normal hepatocytes, normal liver cell lines, and HCC cell lines. d Representative images of immunohistochemical (IHC) staining in tissue microarray-based IHC study of Trabid, and the percentage of patients with different expression of Trabid based on TNM stage. e, f Compared with patients with high Trabid levels in two cohorts, patients with low Trabid levels have a higher probability of recurrence and poorer overall survival

Fig. 2

Trabid significantly inhibits HCC growth and metastasis. a Western blot results demonstrate transfection of lentiviral vectors containing individual shRNA against Trabid (shRNA 1–4) (LV-sh-Trabid) induced significant downregulation of Trabid in HCC cells. ShTrabid-1# produced the best knock down effect on Trabid. b1 Western blot for Trabid in the indicated HCC cells transfected with Trabid or shTrabid or the shRNA-resistant expression construct, TrabidΔ, or their matched controls. The effects of Trabid overexpression (b2) or knockdown (b3) on HCC cell proliferation and foci formation. The effects of Trabid overexpression (c1) or knockdown (c2) on HCC cell foci formation. Representative images of the whole plate are shown in the right. d Indicated HCC cells were injected subcutaneously into six NOD/SCID mice. The growth of xenograft tumors was monitored at different time points. e The volume of MHCC97H orthotopic hepatic tumors was determined at various time points, and representative bioluminescence images or orthotopic tumors are shown

Fig. 3

Trabid inhibits HCC invasion and lung metastasis by inducing EMT. a The effects of Trabid expression on wound closure in indicated HCC cells. b The effects of Trabid expression on the invasion of HCC cells. Western blotting (c), immunofluorescence (d), and e real-time PCR were used to measure the levels of vimentin, fibronectin, E-cadherin, and ZO-1 expression in the indicated HCC cells. f, g An in vivo lung metastasis analysis. The indicated HCC cell lines were injected into the mice through the tail vein. The metastatic lung foci were counted and detected using bioluminescence imaging. g Representative images of mice lungs and H&E staining (n = 10/group) are shown. h Silencing Trabid prolonged overall survival of xenograft mice models bearing HCCs

Fig. 4

Downregulation of Twist1 is required for Trabid-induced inhibition of proliferation and EMT of HCC cells. a The relative mRNA and protein levels of EMT-related genes in indicated HCC cells were determined using qPCR. b Indicated cells were transfected with indicated plasmids for 24 h, and then cell lysates were probed with the indicated antibodies. c E-cadherin and vimentin in HCC were analyzed using IF staining. d The invasion assay in indicated HCC cells. e The luciferase-expressing HCC cell lines were injected by way of the tail vein into the NOD/SCID mice. Tumor metastasis was monitored by bioluminescence imaging. The incidence of lung metastases observed in the different experimental groups of NOD/SCID mice. Representative images showing lung and H&E staining of lung tissues from the different experimental groups. f The BrdU assay of HCC cells treated as indicated was determined at indicated times

Fig. 5

Trabid inhibits Twist1 activity by cleaving RNF8-mediated Twist1 K63-linked ubiquitination. a1 HEK293T cells were co-transfected with indicated plasmids. Co-IP was performed with indicated antibody. a2 co-IP assays for endogenous Twist1 and Trabid from HCC cells. a3 GST pulldown assays. b1 An illustration to depict Trabid full-length (FL) and its deletion constructs. The interaction between Twist1 and Trabid domains is indicated by the number of plus signs (+), and detected via immunoblotting. b2 Schematic diagram of the functional domains of human Twist1 protein. And the Twist1 domain required for the interaction of Trabid was shown. c1 HCC cells were transfected with shRNA targeting Trabid for 72 h, and the lysates were analyzed to determine the K63 ubiquitination on Twist1. HEK293T cells were transfected with HA-tagged K63-only ubiquitin (c2) or K48-only ubiquitin (c3) and other indicated plasmids. Then all samples were immunoprecipitated with anti-Flag antibody, followed by probing. c4 In vitro K63-linked deubiquitination of Twist1 by Trabid. Purified recombinant Trabid or Trabid–C443A was incubated with K63 ubiquitinated Twist1 in vitro, and then probed with indicated antibody. d1 Trabid removes Twist1 K63 ubiquitination mediated by RNF8. HEK293 cells were cotransfected with indicated plasmids. Following Myc-Twist1 IP, K63 ubiquitination was detected. d2 HEK293T cells were transfected with shcontrol or Trabid shRNA and treated with 20 μM MG132. Then lysates were immunoprecipitated using an antibody against RNF8 (upper panel) or Twist1 (lower panel), and then immunoblotting was performed. d3 HEK293T cells were treated as indicated, and then cell lysates were immunoprecipitated and analyzed. e β-TrCP1 knockdown abolished Trabid-induced polyubiquitination of Twist1. HEK293T cells were treated as indicated, and then lysate was used in immunoprecipitation

Fig. 6

Twist1 negatively regulated Trabid expression in HCC. a The nucleotide sequences and positions of four putative Twist1 binding site in the Trabid proximal promoter are shown. Luciferase activity assays of the pGL3-Trabid promoter-reporter. b1 Serially truncated and mutation analysis b2 identified twist1-bindinf sites in the Trabid promoter. ChIP assay demonstrated the direct binding of Twist1 to the Trabid promoter in HCC cells (b3) and human HCC tissues (b4). Hepatocytes were separated from liver tissues of healthy controls (HC) and HCC patients. c ChIP assays in clinical HCC tumor samples (case #1 and case #2) and HCC cell lines. d Indicated HCC cells were transduced with or without Ad-Twist1. The levels of Twist1 and Trabid were determined using Western blot

Fig. 7

Trabid is an AKT substrate and this phosphorylation activates the DUB activity of Trabid. a Putative Akt phosphorylation sites predicted by Scansite within Trabid orthologs. b1 Electrospray ionization mass spectrometry (ESI-MS) analysis reveals the levels of S78 and Thr117 phosphorylation in Trabid from HEK293T cells. b2 Endogenous Trabid in Pten knockout mouse embryonic fibroblasts (MEFs) cells treated with or without AKT inhibitor were immunoprecipitated and probed with phospho-Ser/Thr antibody. b3 The phosphorylation of Trabid was found using a phospho-Ser/Thr antibody. b4 HEK293T cells were transfected with wild-type or mutant Trabid constructs, then phosphorylation of Trabid was detected using the phospho-Ser/Thr antibody. b5 The level of phosphorylated Trabid from HEK293T cells treated with or without λ-phosphatase. c1 Recombinant Trabid protein (1 μg) was mixed with or without active AKT (1 μg) in vitro, and Ub-AMC assay was performed. Relative fluorescence units (RFU). c2, c3 AKT activates Trabid in cells. Trabid was immunoprecipitated from HEK293T cells coexpressed with activated AKT (c2) or treated with 10 μM MK2206 (c3), and followed by Ub-AMC hydrolysis assay. c4 S78A/T117A mutation blocks Trabid activation by AKT. d Trabid^−/− MEF cells were virally transfected with indicated plasmids, and the K63-Ubiquitination of Twist1 was analyzed. e Trabid^−/− MEF cells were virally transfected with indicated plasmids with or without Myr-Akt, and the K63-Ubiquitination of Twist1 was analyzed. f Trabid^−/− HCC cells were virally transfected with indicated plasmids with or without 1 µM MK2206, and the K63-Ubiquitination of Twist1 was analyzed

Fig. 8

Clinical correlation analysis between Trabid, RNF8, and Twist1 in HCC patients. a Immunohistochemistry pictures of Trabid, RNF8, and Twist1 expressions in 20 representative HCC patients. Scale bar, 200 μm. b The percentage of patients with different expression of Trabid, RNF8, and Twist1 according to different clinical parameters. c The Kaplan–Meier analysis of OS of HCC patients as indicated. d Schematic model of the mechanism underlying Trabid mediated HCC growth and metastasis inhibition