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Lysine 2-hydroxyisobutyrylation of NAT10 promotes cancer metastasis in an ac4C-dependent manner

Cell Research volume 33, pages 355–371 (2023)Cite this article

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Abstract

Posttranslational modifications add tremendous complexity to proteomes; however, gaps remain in knowledge regarding the function and regulatory mechanism of newly discovered lysine acylation modifications. Here, we compared a panel of non-histone lysine acylation patterns in metastasis models and clinical samples, and focused on 2-hydroxyisobutyrylation (Khib) due to its significant upregulation in cancer metastases. By the integration of systemic Khib proteome profiling in 20 paired primary esophageal tumor and metastatic tumor tissues with CRISPR/Cas9 functional screening, we identified N-acetyltransferase 10 (NAT10) as a substrate for Khib modification. We further showed that Khib modification at lysine 823 in NAT10 functionally contribute to metastasis. Mechanistically, NAT10 Khib modification enhances its interaction with deubiquitinase USP39, resulting in increased NAT10 protein stability. NAT10 in turn promotes metastasis by increasing NOTCH3 mRNA stability in an N4-acetylcytidine-dependent manner. Furthermore, we discovered a lead compound #7586-3507 that inhibited NAT10 Khib modification and showed efficacy in tumor models in vivo at a low concentration. Together, our findings bridge newly identified lysine acylation modifications with RNA modifications, thus providing novel insights into epigenetic regulation in human cancer. We propose that pharmacological inhibition of NAT10 K823 Khib modification constitutes a potential anti-metastasis strategy.

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Fig. 1: Comprehensive landscape of Khib in ESCC metastatic cell lines and tissue samples.
Fig. 2: Integration of proteomic analysis with CRISPR/Cas9 functional screening suggests that Khib modification of NAT10 at K823 is essential for cancer metastasis.
Fig. 3: KAT7 and SIRT7 are the writer and eraser for Khib modification of NAT10, respectively.
Fig. 4: Identification of deubiquitinase USP39 as an interacting protein of NAT10.
Fig. 5: Stabilization of Khib-modified NAT10 by USP39 leads to its upregulation in ESCC.
Fig. 6: NAT10 K823 is clinically and functionally important for ESCC metastasis.
Fig. 7: NAT10 catalyzes NOTCH3 mRNA ac4C modification to increase its stability.
Fig. 8: Pharmacological inhibition of NAT10 by small-molecule compounds offers a therapeutic option.

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Acknowledgements

This work was supported by the National Key R&D Program of China (2021YFC2501000, 2021YFC2501900), the National Natural Science Foundation of China (82273141, 82073196, 31961160727, 81973339, 82071372), Key Laboratory of Guangdong Higher Education Institutes (2021KSYS009), the Outstanding Scholar Program of Bioland Laboratory (2018GZR110102002), and the Science and Technology Program of Guangzhou (202007030012). We thank Profs. Didier Trono and Vladislav Verkhusha for the plasmids obtained from Addgene.

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  1. These authors contributed equally: Long Liao, Yan He, Shu-Jun Li, Xiao-Mei Yu, Zhi-Chao Liu.

Authors and Affiliations

  1. Key Laboratory of Biological Targeting Diagnosis, Therapy and Rehabilitation of Guangdong Higher Education Institutes, State Key Laboratory of Respiratory Disease, The Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China

    Long Liao, Yan He, Shu-Jun Li, Xiao-Mei Yu, Jing Yang, Guo-Geng Zhang, Chun-Miao Deng, Xian Wei, Yi-Dong Zhu, Tao-Yang Xu, Can-Can Zheng & Bin Li

  2. MOE Key Laboratory of Tumor Molecular Biology, National Engineering Research Center of Genetic Medicine, College of Life Science and Technology, Jinan University, Guangzhou, Guangdong, China

    Long Liao, Yan He, Shu-Jun Li, Xiao-Mei Yu, Jing Yang, Guo-Geng Zhang, Yi-Dong Zhu & Tao-Yang Xu

  3. Department of Thoracic Surgery, Shanghai Chest Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China

    Zhi-Chao Liu & Zhi-Gang Li

  4. Key Laboratory of CNS Regeneration, Ministry of Education, Guangdong Key Laboratory of Non-Human Primate Research, Guangdong-Hong Kong-Macau Institute of CNS Regeneration, Jinan University, Guangzhou, Guangdong, China

    Yi-Yao Liang & Ang Li

  5. Department of Thoracic Surgery, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, China

    Han Yang

  6. Department of Thoracic Surgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong, China

    Chao Cheng

  7. Guangdong Provincial Key Laboratory of Protein Modification and Degradation, State Key Laboratory of Respiratory Disease, and School of Basic Medical Sciences, Guangzhou Medical University, Guangzhou, Guangdong, China

    Jin-Bao Liu

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Contributions

B.L. conceived and designed the study; L.L., Y.H., S.J.L., X.M.Y., Z.C.L., Y.Y.L., J.Y., G.G.Z., C.M.D., X.W., Y.D.Z., T.Y.X. and C.C.Z. acquired, analyzed and interpreted the data; L.L., Y.H., S.J.L. and X.M.Y. performed statistical analysis and drafted the manuscript; H.Y., C.C., A.L., Z.G.L. and J.B.L. provided technical and/or material support and critically revised the manuscript for important intellectual content; B.L. supervised the study. All authors edited and approved the final version of the manuscript.

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Correspondence to Bin Li.

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The authors declare no competing interests.

Ethical approval

All animal experiments were approved by the Ethics Committee for Animal Experiments of Guangzhou Medical University (G2022-084), and mice were cared under standard conditions according to institutional guidelines. The human ESCC specimens were collected in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Shanghai Chest Hospital, Shanghai Jiao Tong University (No. KS(Y)22278). Informed consent was obtained from each participant.

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Liao, L., He, Y., Li, SJ. et al. Lysine 2-hydroxyisobutyrylation of NAT10 promotes cancer metastasis in an ac4C-dependent manner. Cell Res 33, 355–371 (2023). https://doi.org/10.1038/s41422-023-00793-4

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