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The E3 ubiquitin ligase MG53 inhibits hepatocellular carcinoma by targeting RAC1 signaling.

ABSTRACT: Ras-related C3 botulinum toxin substrate 1 (RAC1) overexpressiosn and hyperactivation are correlated with aggressive growth and other malignant characteristics in a wide variety of cancers including hepatocellular carcinoma (HCC). However, the regulatory mechanism of RAC1 expression and activation in HCC is not fully understood. Here, we demonstrated that E3 ubiquitin ligase MG53 (also known as tripartite motif 72, TRIM72) acted as a direct inhibitor of RAC1, and it catalyzed the ubiquitination of RAC1 and further inhibited RAC1 activity in HCC cells. Mechanistically, MG53 directly bound with RAC1 through its coiled-coil domain and suppressed RAC1 activity by catalyzing the Lys48 (K48)-linked polyubiquitination of RAC1 at Lys5 residue in HCC cells. We further demonstrated that MG53 significantly suppressed the malignant behaviors of HCC cells and enhanced the chemosensitivity of HCC cells to sorafenib treatment by inhibiting RAC1-MAPK signaling axis. In summary, we identified MG53 as a novel RAC1 inhibitor and tumor suppressor in HCC, and it suppressed HCC progression by inducing K48-linked polyubiquitination of RAC1 and further inhibiting the RAC1-MAPK signaling. Altogether, our investigation provided a new therapeutic strategy for RAC1 overactivated tumors by modulating MG53.

SUBMITTER: Ma X

PROVIDER: S-EPMC9300626 | biostudies-literature | 2022 Jul

REPOSITORIES: biostudies-literature

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The E3 ubiquitin ligase MG53 inhibits hepatocellular carcinoma by targeting RAC1 signaling.

Ma Xiaomin X Ma Xiaoxiao X Zhu Lihui L Zhao Yunxue Y Chen Mengmeng M Li Tao T Lin Yueke Y Ma Dapeng D Sun Caiyu C Han Lihui L

Oncogenesis 20220720 1

Ras-related C3 botulinum toxin substrate 1 (RAC1) overexpressiosn and hyperactivation are correlated with aggressive growth and other malignant characteristics in a wide variety of cancers including hepatocellular carcinoma (HCC). However, the regulatory mechanism of RAC1 expression and activation in HCC is not fully understood. Here, we demonstrated that E3 ubiquitin ligase MG53 (also known as tripartite motif 72, TRIM72) acted as a direct inhibitor of RAC1, and it catalyzed the ubiquitination ...[more]

PMID: 35858925

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Project description:BackgroundAberrant activation of β-catenin and Yes-associated protein (YAP) signaling pathways has been associated with hepatocellular carcinoma (HCC) progression. The LIM domain protein Ajuba regulates β-catenin and YAP signaling and is implicated in tumorigenesis. However, roles and mechanism of Ajuba expression in HCC cells remain unclear. The E3 ligase Hakai has been shown to interact with other Ajuba family members and whether Hakai interacts and regulates Ajuba is unknown.MethodsHCC cell lines stably depleted of Ajuba or Hakai were established using lentiviruses expressing shRNAs against Ajuba or Hakai. The effects of Ajuba on HCC cells were determined by a number of cell-based analyses including anchorage-independent growth, three dimension cultures and trans-well invasion assay. In vivo tumor growth was determined in a xenograft model and Ajuba expression in tumor sections was examined by immunohistochemistry. Co-immunoprecipitation, confocal microscopy and immunoblot assay were used to examine the expression and interaction between Ajuba and Hakai.ResultsDepletion of Ajuba in HCC cells significantly enhanced anchorage-independent growth, invasion, the formation of spheroids and tumor growth in a xenograft model, suggesting a tumor suppressor function for Ajuba in HCC. Mechanistically, Ajuba depletion triggered E-cadherin loss and β-catenin translocation with increased Cyclin D1 levels. In addition, depletion of Ajuba upregulated the levels of YAP and its target gene CYR61. Furthermore, siRNA-mediated knockdown of either β-catenin or YAP attenuated the pro-tumor effects by Ajuba depletion on HCC cells. Notably, Ajuba stability in HCC cells was regulated by Hakai, an E3 ligase for E-cadherin. Hakai interacted with Ajuba via its HYB domain and induced Ajuba neddylation, which was antagonized by the neddylation inhibitor, MLN4924, but not MG132. We further show that overexpression of Hakai in HCC cells markedly increased anchorage-independent growth, spheroid-formation ability and tumor growth in xenografts whereas Hakai depletion resulted in these opposite effects, indicating an oncogenic role for Hakai in HCC. Hakai also induced β-catenin translocation with increased levels of Cyclin D1.ConclusionsOur data suggest a role for Ajuba and Hakai in HCC, and uncover the mechanism underlying the regulation of Ajuba stability.

Project description:BackgroundTumor metastasis is a major factor for poor prognosis of hepatocellular carcinoma (HCC), but the relationship between ubiquitination and metastasis need to be studied more systematically. We analyzed the ubiquitinome of HCC in this study to have a more comprehensive insight into human HCC metastasis.MethodsThe protein ubiquitination levels in 15 HCC specimens with vascular invasion and 15 without vascular invasion were detected by ubiquitinome. Proteins with significantly different ubiquitination levels between HCCs with and without vascular invasion were used to predict E3 ubiquitin ligases associated with tumor metastasis. The topological network of protein substrates and corresponding E3 ubiquitin ligases was constructed to identify the key E3 ubiquitin ligase. Besides, the growth, migration and invasion ability of LM3 and HUH7 hepatoma cell lines with and without SYVN1 expression interference were measured by cell proliferation assay, subcutaneous tumor assay, umphal vein endothelium tube formation assay, transwell migration and invasion assays. Finally, the interacting proteins of SYVN1 were screened and verified by protein interaction omics, immunofluorescence, and immunoprecipitation. Ubiquitin levels of related protein substrates in LM3 and HUH7 cells were compared in negative control, SYVN1 knockdown, and SYVN1 overexpression groups.ResultsIn this study, our whole-cell proteomic dataset and ubiquitinomic dataset contained approximately 5600 proteins and 12,000 ubiquitinated sites. We discovered increased ubiquitinated sites with shorter ubiquitin chains during the progression of HCC metastasis. In addition, proteomic and ubiquitinomic analyses revealed that high expression of E3 ubiquitin-protein ligase SYVN1 is related with tumor metastasis. Furthermore, we found that SYVN1 interacted with heat shock protein 90 (HSP90) and impacted the ubiquitination of eukaryotic elongation factor 2 kinase (EEF2K).ConclusionsThe ubiquitination profiles of HCC with and without vascular invasion were significantly different. SYVN1 was the most important E3 ubiquitin-protein ligase responsible for this phenomenon, and it was related with tumor metastasis and growth. Therefore, SYVN1 might be a potential therapeutic target for HCC.

Project description:Stroke is one of the leading causes of morbidity and mortality worldwide. Inflammation, oxidative stress, apoptosis, and excitotoxicity contribute to neuronal death during ischemic stroke; however, the mechanisms underlying these complicated pathophysiological processes remain to be fully elucidated. Here, we found that the expression of tumor necrosis factor receptor-associated factor 6 (TRAF6) was markedly increased after cerebral ischemia/reperfusion (I/R) in mice. TRAF6 ablation in male mice decreased the infarct volume and neurological deficit scores and decreased proinflammatory signaling, oxidative stress, and neuronal death after cerebral I/R, whereas transgenic overexpression of TRAF6 in male mice exhibited the opposite effects. Mechanistically, we demonstrated that TRAF6 induced Rac1 activation and consequently promoted I/R injury by directly binding and ubiquitinating Rac1. Either functionally mutating the TRAF6 ubiquitination site on Rac1 or inactivating Rac1 with a specific inhibitor reversed the deleterious effects of TRAF6 overexpression during I/R injury. In conclusion, our study demonstrated that TRAF6 is a key promoter of ischemic signaling cascades and neuronal death after cerebral I/R injury. Therefore, the TRAF6/Rac1 pathway might be a promising target to attenuate cerebral I/R injury.SIGNIFICANCE STATEMENT Stroke is one of the most severe and devastating neurological diseases globally. The complicated pathophysiological processes restrict the translation of potential therapeutic targets into medicine. Further elucidating the molecular mechanisms underlying cerebral ischemia/reperfusion injury may open a new window for pharmacological interventions to promote recovery from stroke. Our study revealed that ischemia-induced tumor necrosis factor receptor-associated factor 6 (TRAF6) upregulation binds and ubiquitinates Rac1 directly, which promotes neuron death through neuroinflammation and neuro-oxidative signals. Therefore, precisely targeting the TRAF6-Rac1 axis may provide a novel therapeutic strategy for stroke recovery.

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The E3 ubiquitin ligase MG53 inhibits hepatocellular carcinoma by targeting RAC1 signaling.

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The E3 ubiquitin ligase MG53 inhibits hepatocellular carcinoma by targeting RAC1 signaling.

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