SUMOylation of RNF146 results in Axin degradation and activation of Wnt/β-catenin signaling to promote the progression of hepatocellular carcinoma

SUMOylation of RNF146 results in Axin degradation and activation of Wnt/β-catenin signaling to promote the progression of hepatocellular carcinoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article SUMOylation of RNF146 results in Axin degradation and activation of Wnt/β-catenin signaling to promote the progression of hepatocellular carcinoma Dong Yin, Wenjia Li, Qingfang Han, Yuanxin Zhu, Yingshi Zhou, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2212462/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Apr, 2023 Read the published version in Oncogene → Version 1 posted 10 You are reading this latest preprint version Abstract Aberrant SUMOylation contributes to the progression of hepatocellular carcinoma (HCC), yet the molecular mechanisms have not been well elucidated. RNF146 is a key regulator of the Wnt/β-catenin signaling pathway, which is frequently hyperactivated in HCC. Here, it is identified that RNF146 can be modified by SUMO3. By mutating all lysines in RNF146, we found that K19, K61, K174 and K175 are the major sites for SUMOylation. UBC9/PIAS3/MMS21 and SENP1/2/6 mediated the conjugation and deconjugation of SUMO3, respectively. Furthermore, SUMOylation of RNF146 promoted its nuclear localization, while deSUMOylation induced its cytoplasmic localization. Importantly, SUMOylation promotes the association of RNF146 with Axin to accelerate the ubiquitination and degradation of Axin. Intriguingly, only UBC9/PIAS3 and SENP1 can act at K19/K175 in RNF146 and affect its role in regulating the stability of Axin. In addition, inhibiting RNF146 SUMOylation suppressed the progression of HCC both in vitro and in vivo. And, patients with higher expression of RNF146 and UBC9 have the worst prognosis. Taken together, we conclude that RNF146 SUMOylation at K19/K175 promotes its association with Axin and accelerates Axin degradation, thereby enhancing β-catenin signaling and contributing to cancer progression. Our findings reveal that RNF146 SUMOylation is a potential therapeutic target in HCC. Biological sciences/Cell biology/Post-translational modifications/Sumoylation Biological sciences/Cancer/Oncogenes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Full Text Additional Declarations There is NO conflict of interest to disclose. Supplementary Files supplymentarymaterials.pdf Cite Share Download PDF Status: Published Journal Publication published 07 Apr, 2023 Read the published version in Oncogene → Version 1 posted Editorial decision: revise 03 Jan, 2023 Review # 2 received at journal 21 Dec, 2022 Review # 1 received at journal 29 Nov, 2022 Reviewer # 2 agreed at journal 11 Nov, 2022 Reviewer # 1 agreed at journal 08 Nov, 2022 Reviewers invited by journal 07 Nov, 2022 Submission checks completed at journal 01 Nov, 2022 First submitted to journal 01 Nov, 2022 Unknown event 31 Oct, 2022 Editor assigned by journal 28 Oct, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2212462","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":150253252,"identity":"9894e3e4-f486-4031-a93a-ac5507124a13","order_by":0,"name":"Dong Yin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8ElEQVRIiWNgGAWjYDACZgYGyQ8VDAxs7HChBMJapCXOALUwE60FCCR42yB6idNicJz34A3Jedvk+ZgZmD/z/DnMwM+eY8DwcwceLYf5ki0Kt902bGNmYJPmbTvMINnzxoCx9ww+LTxmEpLbbjOCtDDzNhxmMLiRY8DM2EZAC++c2/ZtMIfZE6el4XZiGyjoeNiAtkgQ0CJ5mMfYWuLY7eQ2oDLJuW3pPBJnnhUc7MWjhe/8GcObH2pu285vbz784c0fazn+9uSND37i0aJwAM5kbGDiYWDgATEP4FANBvINSBzGH/iUjoJRMApGwYgFABGYST5YoxQpAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-1878-4849","institution":"Sun Yat-sen Memorial Hospital, Sun Yat-sen University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Dong","middleName":"","lastName":"Yin","suffix":""},{"id":150253255,"identity":"37e07652-b22a-4638-8515-f71a0d4504ec","order_by":1,"name":"Wenjia Li","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wenjia","middleName":"","lastName":"Li","suffix":""},{"id":150253257,"identity":"668cbd61-a15c-4883-89b9-480a7708406e","order_by":2,"name":"Qingfang Han","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qingfang","middleName":"","lastName":"Han","suffix":""},{"id":150253260,"identity":"3df33bf7-f55a-4262-b186-d3d309a605e5","order_by":3,"name":"Yuanxin Zhu","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yuanxin","middleName":"","lastName":"Zhu","suffix":""},{"id":150253262,"identity":"76f23b6b-5965-4d64-bb71-a4996ea4f35e","order_by":4,"name":"Yingshi Zhou","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yingshi","middleName":"","lastName":"Zhou","suffix":""},{"id":150253264,"identity":"ab140f5a-ed3c-41cd-8853-a907908be73c","order_by":5,"name":"Jingyuan Zhang","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jingyuan","middleName":"","lastName":"Zhang","suffix":""},{"id":150253265,"identity":"2496133f-ba70-40f0-a6db-02db686cbceb","order_by":6,"name":"Weijun Wu","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Weijun","middleName":"","lastName":"Wu","suffix":""},{"id":150253266,"identity":"f44208ea-bcb4-4996-bb88-746d4562b08f","order_by":7,"name":"Yu Li","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Li","suffix":""},{"id":150253267,"identity":"aeadc986-74f5-4dfc-a5b4-07e1ad10571e","order_by":8,"name":"Long Liu","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Long","middleName":"","lastName":"Liu","suffix":""},{"id":150253268,"identity":"b2dd51e2-b54b-4ba4-8c4e-66f48c4dc19f","order_by":9,"name":"Yuntan Qiu","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yuntan","middleName":"","lastName":"Qiu","suffix":""},{"id":150253269,"identity":"23bc8ecb-b31b-479a-a4ee-4c74f2f127c6","order_by":10,"name":"Kaishun Hu","email":"","orcid":"https://orcid.org/0000-0003-2157-6239","institution":"Sun Yat-Sen Memorial Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kaishun","middleName":"","lastName":"Hu","suffix":""}],"badges":[],"createdAt":"2022-10-28 09:52:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2212462/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2212462/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41388-023-02689-4","type":"published","date":"2023-04-07T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":28925960,"identity":"3b52404b-b648-499e-88d3-392a9ad17d19","added_by":"auto","created_at":"2022-11-10 23:10:50","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2834116,"visible":true,"origin":"","legend":"\u003cp\u003eRNF146 is SUMOylated by SUMO3 at lysine 19, lysine 61, and lysine 174/175. A HeLa cells stably expressing SFB-tagged RNF146 (SFP-RNF146) or the SFB-tagged vector were transfected with HA-SUMO1, HA-SUMO2 or HA-SUMO3 for 24 h. The harvested cells were lysed with NETN buffer and subjected to immunoprecipitation (IP) and Western blotting with indicated antibodies. B HeLa cells stably expressing SFB-RNF146 or SFB-tagged vector were transfected with HA-SUMO3 for 24 h, and the cell lysates were then subjected to IP using anti-HA beads and detected with the indicated antibodies. C HeLa cells stably overexpressing SFB-RNF146 were transfected with either the scrambled or UBC9 siRNA for 48 h and were then transfected HA-SUMO3 for 24 h. The cells were harvested and subjected to IP using anti-S beads prior to Western blot analysis. D HeLa cells stably expressing SFB-RNF146 or the SFB-tagged vector were transfected with HA-SUMO3 for 24 h and were then treated with 100 μM 2-D08 for 24 h. Cell lysates were then analysed as indicated. E HeLa cells were cotransfected with the indicated SFB-RNF146 plasmids and HA-SUMO3 for 24 h. Then, the cell lysates were subjected to IP using anti-S beads and Western blotting with the indicated antibodies. F Alignment of the SUMOylation sites at RNF146 in different species.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2212462/v1/587f0683ea7476f83badae2f.jpg"},{"id":28925955,"identity":"8fea0e84-221a-4f60-bc1c-7ccdfc0204ff","added_by":"auto","created_at":"2022-11-10 23:10:50","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2054959,"visible":true,"origin":"","legend":"\u003cp\u003ePIAS3 and MMS21 are the dominant SUMO E3 ligases for RNF146. A HeLa cells stably expressing SFB-RNF146 were cotransfected with HA-SUMO3 and each plasmid expressing the indicated 3MYC-tagged PIAS family members for 24 h. The harvested cells were lysed with NETN buffer, and subjected to IP using anti-S beads and Western blotting. B HeLa cells stably expressing SFB-RNF146 were transiently cotransfected with HA-SUMO3 and either 3MYC- tagged PIAS3 or 3MYC- tagged MMS21 for 24h. Then, whole-cell lysates were subjected to IP using anti-MYC beads and detection with the indicated antibodies. C HeLa cells were lysed with RIPA buffer, and whole-cell lysates were subjected to co-IP using IgG or an anti-RNF146 antibody and analysed by Western blotting. 710 D SK-hep1 cells were cotransfected with mCherry-RNF146 and V5-turbo-PIAS3/MMS21 for 36h. After fixation, immunofluorescence staining was performed using the indicated antibody and DAPI; the scale bar indicates 10 μm. E, F HeLa cells stably overexpressing SFB-RNF146 were transfected with HA-SUMO3 for 24h prior to treatment with scrambled siRNA and either the PIAS3 (E) or MMS21 (F) siRNAs for another 48 h. The cells were harvested and subjected to IP using the indicated antibodies. G HeLa cells stably overexpressing SFB-RNF146 were transfected with scrambled ，PIAS3, MMS21 or PIAS3/MMS21 siRNAs for 48 h and lysed with NETN buffer, prior to IP and Western blot analysis with the indicated antibodies.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2212462/v1/487b58b975ffff1c5c00d0ad.jpg"},{"id":28925956,"identity":"06ca2013-5720-4484-99db-a05565274b95","added_by":"auto","created_at":"2022-11-10 23:10:50","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1959679,"visible":true,"origin":"","legend":"\u003cp\u003eSENP1/2/6 are the dominant deSUMOylases responsible for removing SUMOylation from RNF146. A HeLa cells stably expressing SFB-RNF146 were cotransfected with HA-SUMO3 and each plasmid expressing the indicated 3MYC-tagged SENP family members for 24 h. The harvested cells were lysed with NETN buffer and subjected to IP using anti-S beads and Western blotting using the indicated antibodies. B HeLa cells overexpressing SFB-RNF146 were transiently cotransfected with HA-SUMO3 and 3MYC-tagged SENP1/2/6 for 24 h, and whole-cell lysates were then subjected to IP using anti-MYC beads and detection with the indicated antibodies. C HeLa cell lysates were incubated with protein-G agarose beads conjugated to IgG or an anti-RNF146 antibody; the results of an IP assay are shown. D 293T and SK-hep1 cells were cotransfected with mCherry-RNF146 and V5-turbo-SENP1 for 36 h. Then, the cells were fixed and stained with the indicated antibodies and DAPI. The scale bar represents 10 μm. E SFB-RNF146 stably overexpressing HeLa cells were transfected with scrambled and siRNAs for SENP1 for 48 h, and then transfected with HA-SUMO3 for another 24 h. After being lysed with NETN buffer, cell lysates were analyzed by IP and Western blot using indicated antibodies. F HeLa cells stably expressing SFB-RNF146 were transfected 740 with scrambled, SENP1, SENP2, SENP6 or SENP1/SENP2/SENP6 siRNAs for 48 h and then transfected with HA-SUMO3 for another 24 h. Cell lysates were harvested and subjected to IP using anti-S beads prior to Western blot analysis.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2212462/v1/17b1aa81cdac481cc18cc6c1.jpg"},{"id":28926016,"identity":"ffce1518-d1ca-4e73-8cad-2cf69701cc72","added_by":"auto","created_at":"2022-11-10 23:18:50","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1393628,"visible":true,"origin":"","legend":"\u003cp\u003eSUMOylation of RNF146 at lysine 19 and lysine 175 promotes the interaction of RNF146 with Axin and the degradation of Axin. A Endogenous RNF146 was knocked out with sgRNA, and SFB-vector or the indicated SFB-RNF146 mutation plasmids were then transfected into SK-hep1 cells for 24 h. The expression of the indicated proteins was determined by Western blotting. B Cytoplasmic and nuclear distribution of β-catenin and SFB-RNF146 in SK-hep1 cells stably expressing SFB-vector or SFB-RNF146. C Endogenous RNF146 was depleted in SK-hep1 cells. The cytoplasmic and nuclear distribution of β-catenin was analysed after transfection with SFB-vector or the indicated SFB-RNF146 plasmids. D SK-hep1 cells stably expressing SFB-RNF146 were treated with scrambled siRNA or siRNAs for PIAS3 and MMS21 for 72 h. Then, the cells were lysed with RIPA buffer, and the expression of Axin and β-catenin was analysed by Western blotting. E SK-hep1 cells stably expressing SFB-RNF146 were treated with scrambled siRNA or siRNAs for SENP1, SENP2 or SENP6 for 72 h. The expression of Axin and β-catenin were analysed by Western blotting. F SK-hep1 cells with depletion of endogenous RNF146 and stab expression of SFB-vector, wild-type SFB-RNF146 or mutant SFB-RNF146 plasmids were lysed with NETN buffer. Then, whole-cell lysates were subjected to IP using anti-S beads and analysed by Western blotting to evaluate the interaction between RNF146 and Axin. G SK-hep1 cells with depletion of endogenous RNF146 and stable expression of SFB-vector, wild-type SFB-RNF146 or mutant SFB-RNF146 plasmids were transfected with HA-Ub for 24 h. Cell lysates were subjected to co-IP using an anti-Axin antibody, and Western blotting was performed using the indicated antibodies.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2212462/v1/130088d75822bfe5a8496f52.jpg"},{"id":28925958,"identity":"4eecd7d4-8374-4dce-9124-3b73e6019d15","added_by":"auto","created_at":"2022-11-10 23:10:50","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2085461,"visible":true,"origin":"","legend":"\u003cp\u003eRNF146 SUMOylation activates β-catenin signaling and promotes the proliferation of HCC cells in vitro. A SFB-vector and SFB-RNF146 overexpression plasmids were stably transfected into SK-hep1 and HCC-LM3 cells. Axin and β-catenin signaling was analysed by Western blotting using the indicated antibodies. B RNF146 was knocked out with two sgRNAs in SK-hep1 and HCC-LM3 cells. Cell lysates were analysed by Western blotting using the indicated antibodies. C The proliferative ability of SK-hep1 and HCC-LM3 cells stably expressing SFB-vector or SFB-RNF146 was determined by the colony formation assay. n = 3 independent experiments. D SK-hep1 and HCC-LM3 cells with stable depletion of RNF146 were subjected to the colony formation assay. n = 3 independent experiments. E, F SK-hep1 or HCC-LM3 cells with stable overexpression (E) or depletion (F) of RNF146 were sequentially labelled with IdU and CldU for 20 min. Three independent experiments were performed, and the replication fork speed was calculated and analysed. G The SFB-vector, SFB-RNF146 wild-type and SFB-RNF146 mutant plasmids were stably expressed in SK-hep1 and HCC-LM3 cells in which endogenous RNF146 was depleted by sgRNA. Three independent experiments were performed, and the percentage of BrdU-positive cells was quantified and analysed. H The indicated stable cell lines were subjected to a colony formation assay. n = 3 independent experiments. I, J SK-hep1 and HCC-LM3 stable cell lines were used, and the DNA fiber assay was performed. Representative images are shown in (I). The replication fork speed was quantified and analysed as shown in (J). n = 3 independent experiments. n. s, not significant; *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2212462/v1/000212f26a20b8e717d4b31b.jpg"},{"id":28925962,"identity":"41e7e9ab-c58f-483b-b794-179e7fec5401","added_by":"auto","created_at":"2022-11-10 23:10:50","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":4973552,"visible":true,"origin":"","legend":"\u003cp\u003eAbolishing RNF146 SUMOylation inhibits HCC cell tumorigenesis. A SK-hep1 cells with depletion of endogenous RNF146 and stable expression of SFB-vector, SFB-RNF146-WT or SFB-RNF146-mutants were injected into the flanks of nude mice. Tumour growth was measured every 2 days. B, C Harvested xenografts were photographed (800 B) and weighed (C). D The expression of Axin and β-catenin in xenografts was analysed by Western blotting. E IHC staining of harvested xenografts using antibodies against Ki67, RNF146 and Axin. The scale bar indicates 100 μm. F, G SK-hep1 cells were injected into the flanks of nude mice. Six days after injection, the mice were intraperitoneally injected with 2-D08 (5 mg/kg) or vehicle (10% DMSO, 40% PEG300, 5% Tween 80, 45% saline) every 2 days. Tumour growth (F) and weight (G) were measured as indicated. n. s, not significant; *p \u0026lt; 0.05; ***p \u0026lt; 808 0.001.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2212462/v1/93e361a7966bcae89dde7c30.jpg"},{"id":28925963,"identity":"2cdd853c-4b28-4ab7-b2f9-a4bc1e81546a","added_by":"auto","created_at":"2022-11-10 23:10:51","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3306034,"visible":true,"origin":"","legend":"\u003cp\u003eThe clinical significance of RNF146 SUMOylation/Axin/β-catenin axis in human HCC tissues. A, B The expression of RNF146, UBC9, PIAS3 and SENP1 in HCC tumor and adjacent specimens (n = 12) was analyzed by Western blotting using indicated antibodies (A), and their expression were quantified and analysed as shown in (B). C Representative IHC images of RNF146, Axin, PIAS3 and SENP1 staining using HCC tissues. Scale bar indicates 100 μm. D The association between Axin expression and UBC9, RNF146, PIAS3 or SENP1 (n = 99). The percentage of positive staining and p value based on Pearson's χ2 test and Pearson's correlations are shown in the tables. E UBC9 expression in HCC (n = 369) and non-tumour tissues (n = 50) analyzed by the GEPIA2 web tool. F The correlation between the expression pattern of RNF146 and UBC9 with the overall survival of HCC patients was analyzed using data from TCGA. The high and low grouping of RNF146 and UBC9 was based on the median of the gene expression. n. s, not significant; *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2212462/v1/d8a665d55beebe592fa24f70.jpg"},{"id":28925961,"identity":"c0d38a8a-71e6-4b09-9256-658c83c77eda","added_by":"auto","created_at":"2022-11-10 23:10:50","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":88535,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic model of the SUMO-RNF146-Axin pathway.\u003c/p\u003e\n\u003cp\u003ePIAS3-mediated SUMOylation of RNF146 promotes its translocation from the\u003c/p\u003e\n\u003cp\u003ecytoplasm to the nucleus, while deSUMOylation of RNF146 is catalysed by\u003c/p\u003e\n\u003cp\u003eSENP1, facilitating its nuclear export. SUMOylation 830 of RNF146 promotes its\u003c/p\u003e\n\u003cp\u003eassociation with Axin and accelerates Axin ubiquitination and degradation,\u003c/p\u003e\n\u003cp\u003ethereby activating β-catenin signaling and resulting in HCC progression.\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2212462/v1/01925df7e7da2481b8aef6da.jpg"},{"id":35473977,"identity":"2fb3e069-0409-46ed-a942-e2c3fa31e556","added_by":"auto","created_at":"2023-04-08 07:09:51","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1497567,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2212462/v1_covered.pdf"},{"id":28926017,"identity":"2c70019e-6c3a-40dd-a3a9-4a7631184674","added_by":"auto","created_at":"2022-11-10 23:19:17","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1492076,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2212462/v1_covered.pdf"},{"id":28926015,"identity":"cf695a9a-849d-4b8c-b84f-dea2f7e3f884","added_by":"auto","created_at":"2022-11-10 23:18:50","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1406731,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"supplymentarymaterials.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2212462/v1/0c0a3cebb064fce5234b56dc.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"SUMOylation of RNF146 results in Axin degradation and activation of Wnt/β-catenin signaling to promote the progression of hepatocellular carcinoma","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"oncogene","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"onc","sideBox":"Learn more about [Oncogene](http://www.nature.com/onc/)","snPcode":"41388","submissionUrl":"https://mts-onc.nature.com/cgi-bin/main.plex","title":"Oncogene","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-2212462/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2212462/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Aberrant SUMOylation contributes to the progression of hepatocellular carcinoma (HCC), yet the molecular mechanisms have not been well elucidated. RNF146 is a key regulator of the Wnt/β-catenin signaling pathway, which is frequently hyperactivated in HCC. Here, it is identified that RNF146 can be modified by SUMO3. By mutating all lysines in RNF146, we found that K19, K61, K174 and K175 are the major sites for SUMOylation. UBC9/PIAS3/MMS21 and SENP1/2/6 mediated the conjugation and deconjugation of SUMO3, respectively. Furthermore, SUMOylation of RNF146 promoted its nuclear localization, while deSUMOylation induced its cytoplasmic localization. Importantly, SUMOylation promotes the association of RNF146 with Axin to accelerate the ubiquitination and degradation of Axin. Intriguingly, only UBC9/PIAS3 and SENP1 can act at K19/K175 in RNF146 and affect its role in regulating the stability of Axin. In addition, inhibiting RNF146 SUMOylation suppressed the progression of HCC both in vitro and in vivo. And, patients with higher expression of RNF146 and UBC9 have the worst prognosis. Taken together, we conclude that RNF146 SUMOylation at K19/K175 promotes its association with Axin and accelerates Axin degradation, thereby enhancing β-catenin signaling and contributing to cancer progression. Our findings reveal that RNF146 SUMOylation is a potential therapeutic target in HCC.","manuscriptTitle":"SUMOylation of RNF146 results in Axin degradation and activation of Wnt/β-catenin signaling to promote the progression of hepatocellular carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-11-10 23:10:45","doi":"10.21203/rs.3.rs-2212462/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2023-01-03T14:38:08+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2022-12-22T04:24:48+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2022-11-29T08:06:57+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2022-11-11T20:07:50+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2022-11-08T08:27:27+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2022-11-07T22:00:48+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-11-01T11:07:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"Oncogene","date":"2022-11-01T05:06:43+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2022-10-31T15:15:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-10-28T09:49:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"oncogene","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"onc","sideBox":"Learn more about [Oncogene](http://www.nature.com/onc/)","snPcode":"41388","submissionUrl":"https://mts-onc.nature.com/cgi-bin/main.plex","title":"Oncogene","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d9c7cb93-dba2-4720-9864-a6cc9a7cea69","owner":[],"postedDate":"November 10th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":16793761,"name":"Biological sciences/Cell biology/Post-translational modifications/Sumoylation"},{"id":16793762,"name":"Biological sciences/Cancer/Oncogenes"}],"tags":[],"updatedAt":"2023-04-08T07:09:35+00:00","versionOfRecord":{"articleIdentity":"rs-2212462","link":"https://doi.org/10.1038/s41388-023-02689-4","journal":{"identity":"oncogene","isVorOnly":false,"title":"Oncogene"},"publishedOn":"2023-04-07 04:00:00","publishedOnDateReadable":"April 7th, 2023"},"versionCreatedAt":"2022-11-10 23:10:45","video":"","vorDoi":"10.1038/s41388-023-02689-4","vorDoiUrl":"https://doi.org/10.1038/s41388-023-02689-4","workflowStages":[]},"version":"v1","identity":"rs-2212462","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2212462","identity":"rs-2212462","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}