2-Hydroxyisobutyrylation and Phosphorylation Crosstalk Guides Metastasis Prediction and Immunotherapy in Esophageal Squamous Cell Carcinoma

Abstract

Posttranslational modification (PTM) is pivotal in cancer progression. However, the mechanisms, biological function, and clinical significance underlying PTM crosstalk are unclear. Here, we performed systematic analyses of 2-hydroxyisobutyrylation (Khib), phosphoproteomic, proteomic, and transcriptomic profiles from 60 esophageal squamous cell carcinoma (ESCC) samples, comprising matched normal, primary tumor, and metastatic lymph node tissues. The integrative analysis identified 4499 proteins co-modified by Khib and phosphorylation on distinct residues. Interestingly, while the two PTMs target distinct motifs across proteins, they engage common motifs within individual proteins. Functionally, Khib and phosphorylation show prevalent positive crosstalk linked to metastatic progression. Mechanistically, Khib mediates this intra-protein crosstalk by recruiting kinases and potentiating phosphorylation events. Machine learning-based artificial intelligence (AI) analysis of secreted proteins with PTM crosstalk revealed an independent and highly effective plasma signature predicting lymph node metastasis. Moreover, molecular subtyping stratified ESCC into three groups, with immunotherapy-resistant Subtype 3 associated with the worst survival. While combined treatment of integrin inhibitor cilengitide and immunotherapy exhibited targeted efficacy. By deciphering the mechanistic basis of Khib-phosphorylation crosstalk, identifying a novel plasma biomarker for lymph node metastasis, and providing a therapeutic strategy to sensitize tumors to immunotherapy, this work collectively advances the precision diagnosis and treatment of ESCC.

Keywords

esophageal squamous cell carcinoma / immunotherapy combination / metastasis / plasma biomarker / PTM crosstalk

References

[1] A. C. Conibear, “Deciphering Protein Post-Translational Modifications Using Chemical Biology Tools,” Nature Reviews Chemistry 4, no. 12 (2020): 674–695.

[2] M. Leutert, S. W. Entwisle, and J. Villen, “Decoding Post-Translational Modification Crosstalk With Proteomics,” Molecular & Cellular Proteomics 20 (2021): 100129.

[3] A. S. Venne, L. Kollipara, and R. P. Zahedi, “The next Level of Complexity: Crosstalk of Posttranslational Modifications,” Proteomics 14, no. 4–5 (2014): 513–524.

[4] K. Y. Huang, T. Y. Lee, H. J. Kao, et al., “dbPTM in 2019: Exploring Disease Association and Cross-Talk of Post-Translational Modifications,” Nucleic Acids Research 47, no. D1 (2019): D298–D308.

[5] Y. Geffen, S. Anand, Y. Akiyama, et al., “Pan-Cancer Analysis of Post-Translational Modifications Reveals Shared Patterns of Protein Regulation,” Cell 186, no. 18 (2023): 3945–3967.e26.

[6] Y. Wang, J. Zhang, B. Li, and Q.-Y. He, “Advances of Proteomics in Novel PTM Discovery: Applications in Cancer Therapy,” Small Methods 3, no. 5 (2019): 1900041.

[7] X. P. Tan, Y. He, J. Yang, et al., “Blockade of NMT1 Enzymatic Activity Inhibits N-Myristoylation of VILIP3 Protein and Suppresses Liver Cancer Progression,” Signal Transduction and Targeted Therapy 8, no. 1 (2023): 14.

[8] Y. He, C. C. Zheng, J. Yang, et al., “Lysine Butyrylation of HSP90 Regulated by KAT8 and HDAC11 Confers Chemoresistance,” Cell Discovery 9, no. 1 (2023): 74.

[9] L. Dai, C. Peng, E. Montellier, et al., “Lysine 2-Hydroxyisobutyrylation Is a Widely Distributed Active Histone Mark,” Nature Chemical Biology 10, no. 5 (2014): 365–370.

[10] Y. Fu, J. Yu, F. Li, and S. Ge, “Oncometabolites Drive Tumorigenesis by Enhancing Protein Acylation: From Chromosomal Remodelling to Nonhistone Modification,” Journal of Experimental & Clinical Cancer Research 41, no. 1 (2022): 144.

[11] L. Liao, Y. He, S.-J. Li, et al., “Lysine 2-Hydroxyisobutyrylation of NAT10 Promotes Cancer Metastasis in an ac4C-Dependent Manner,” Cell Research 33, no. 5 (2023): 355–371.

[12] S. J. Li, Z. R. Liang, Z. C. Liu, et al., “LIMK1 as a Novel Kinase of Beta-Catenin Promotes Esophageal Cancer Metastasis by Cooperating With CDK5,” Advanced Science 12, no. 29 (2025): e03223.

[13] K. Qiao, L. Wu, L. Yang, et al., “Ubiquitin-Centered Post-Translational Modification Crosstalk Orchestrates Tumor Immunity and Immunotherapy Response,” Experimental Hematology & Oncology 15, no. 1 (2026): 22.

[14] W. Wang, Y. Li, L. Tang, et al., “Cross-Talk Between BCKDK-Mediated Phosphorylation and STUB1-Dependent Ubiquitination Degradation of BCAT1 Promotes GBM Progression,” Cancer Letters 591 (2024): 216849.

[15] N. Deboever, C. M. Jones, K. Yamashita, J. A. Ajani, and W. L. Hofstetter, “Advances in Diagnosis and Management of Cancer of the Esophagus,” BMJ 385 (2024): e074962.

[16] H. Ji, C. Hu, X. Yang, et al., “Lymph Node Metastasis in Cancer Progression: Molecular Mechanisms, Clinical Significance and Therapeutic Interventions,” Signal Transduction and Targeted Therapy 8, no. 1 (2023): 367.

[17] X. Huang, Q. Wang, W. Xu, et al., “Machine Learning to Predict Lymph Node Metastasis in T1 Esophageal Squamous Cell Carcinoma: A Multicenter Study,” International Journal of Surgery 110, no. 12 (2024): 7852–7859.

[18] N. Wu, J. Cai, J. Jiang, et al., “Biomarkers of Lymph Node Metastasis in Esophageal Cancer,” Frontiers in Immunology 15 (2024): 1457612.

[19] J. Yates and E. M. Van Allen, “New Horizons at the Interface of Artificial Intelligence and Translational Cancer Research,” Cancer Cell 43, no. 4 (2025): 708–727.

[20] M. Liu, H. An, Y. Zhang, et al., “Molecular Analysis of Chinese Oesophageal Squamous Cell Carcinoma Identifies Novel Subtypes Associated With Distinct Clinical Outcomes,” EBioMedicine 57 (2020): 102831.

[21] F. Wang, Z. Yan, J. Lv, et al., “Gene Expression Profiling Reveals Distinct Molecular Subtypes of Esophageal Squamous Cell Carcinoma in Asian Populations,” Neoplasia 21, no. 6 (2019): 571–581.

[22] Y. Li, B. Yang, Y. Ma, et al., “Phosphoproteomics Reveals Therapeutic Targets of Esophageal Squamous Cell Carcinoma,” Signal Transduction and Targeted Therapy 6, no. 1 (2021): 381.

[23] W. Liu, L. Xie, Y. H. He, et al., “Large-Scale and High-Resolution Mass Spectrometry-Based Proteomics Profiling Defines Molecular Subtypes of Esophageal Cancer for Therapeutic Targeting,” Nature Communications 12, no. 1 (2021): 4961.

[24] D. Zhao, Y. Guo, H. Wei, et al., “Multi-Omics Characterization of Esophageal Squamous Cell Carcinoma Identifies Molecular Subtypes and Therapeutic Targets,” JCI Insight 9, no. 10 (2024): e171916.

[25] Y. M. Yang, P. Hong, W. W. Xu, Q. Y. He, and B. Li, “Advances in Targeted Therapy for Esophageal Cancer,” Signal Transduction and Targeted Therapy 5, no. 1 (2020): 229.

[26] T. A. Yap, E. E. Parkes, W. Peng, J. T. Moyers, M. A. Curran, and H. A. Tawbi, “Development of Immunotherapy Combination Strategies in Cancer,” Cancer Discovery 11, no. 6 (2021): 1368–1397.

[27] Y. He, M. M. Sun, G. G. Zhang, et al., “Targeting PI3K/Akt Signal Transduction for Cancer Therapy,” Signal Transduction and Targeted Therapy 6, no. 1 (2021): 425.

[28] N. A. Kuburich, P. den Hollander, M. Castaneda, et al., “Stabilizing Vimentin Phosphorylation Inhibits Stem-Like Cell Properties and Metastasis of Hybrid Epithelial/Mesenchymal Carcinomas,” Cell Reports 42, no. 12 (2023): 113470.

[29] C. Zhu, F. Fan, C. Y. Li, Y. Xiong, and X. Liu, “Caspase-3 Promotes Oncogene-Induced Malignant Transformation via EndoG-Dependent Src-STAT3 Phosphorylation,” Cell Death & Disease 15, no. 7 (2024): 486.

[30] H. N. Cuesta-Hernandez, J. Contreras, P. Soriano-Maldonado, et al., “An Allosteric Switch Between the Activation Loop and a C-Terminal Palindromic Phospho-Motif Controls c-Src Function,” Nature Communications 14, no. 1 (2023): 6548.

[31] H. Zhao, W. Chen, J. Wu, L. Wang, and W. Mao, “Clinical Significance of Preoperative Serum Tumor Markers in Esophageal Squamous Cell Carcinoma,” Journal of Cancer Research and Therapeutics 10S supplement, (2014): C179–C185.

[32] M. A. Gillette, S. Satpathy, S. Cao, et al., “Proteogenomic Characterization Reveals Therapeutic Vulnerabilities in Lung Adenocarcinoma,” Cell 182, no. 1 (2020): 200–225.e35.

[33] L. Cao, C. Huang, D. Cui Zhou, et al., “Proteogenomic Characterization of Pancreatic Ductal Adenocarcinoma,” Cell 184, no. 19 (2021): 5031–5052.e26.

[34] Y. Qu, J. Feng, X. Wu, et al., “A Proteogenomic Analysis of Clear Cell Renal Cell Carcinoma in a Chinese Population,” Nature Communications 13, no. 1 (2022): 2052.

[35] Y. Li, Y. Wang, G. Ren, et al., “Characterization of Novel Mouse Esophageal Squamous Cell Carcinoma Cell Lines and Their Utility as Preclinical Models,” Cancer Letters 616 (2025): 217600.

[36] M. Liu, J. Yang, B. Xu, and X. Zhang, “Tumor Metastasis: Mechanistic Insights and Therapeutic Interventions,” MedComm 2, no. 4 (2021): 587–617.

[37] P. Luo, J. Duan, Q. Chen, et al., “PTK2 Promotes Uveal Melanoma Metastasis by Activating Epithelial-to-Mesenchymal Transition,” MedComm—Oncology 2, no. 3 (2023): e44.

[38] X. Pei, Z. Liu, L. Tang, et al., “Single-Cell Multi-Omic and Spatial Profiling of Esophageal Squamous Cell Carcinoma Reveals the Immunosuppressive Role of GPR116⁺ Pericytes in Cancer Metastasis,” Nature Genetics 57, no. 10 (2025): 2494–2508.

[39] P. Peng, S. Qin, L. Li, et al., “Epigenetic Remodeling Under Oxidative Stress: Mechanisms Driving Tumor Metastasis,” MedComm—Oncology 3, no. 4 (2024): e70000.

[40] X. Shi, X. Wang, W. Yao, et al., “Mechanism Insights and Therapeutic Intervention of Tumor Metastasis: Latest Developments and Perspectives,” Signal Transduction and Targeted Therapy 9, no. 1 (2024): 192.

[41] W. W. Xu, C. C. Zheng, Y. N. Huang, et al., “Synephrine Hydrochloride Suppresses Esophageal Cancer Tumor Growth and Metastatic Potential Through Inhibition of Galectin-3-AKT/ERK Signaling,” Journal of Agricultural and Food Chemistry 66, no. 35 (2018): 9248–9258.

[42] Y. J. Zhan, C. M. Deng, L. Tang, et al., “NAT10 Increases Lysosomal Acidification to Promote Esophageal Cancer Metastasis via ac4C Acetylation of ATP6V0E1 mRNA,” Advanced Science 12, no. 31 (2025): e02931.

[43] C. Zheng, X. Yu, T. Xu, et al., “KCTD4 Interacts With CLIC1 to Disrupt Calcium Homeostasis and Promote Metastasis in Esophageal Cancer,” Acta Pharmaceutica Sinica B 13, no. 10 (2023): 4217–4233.

[44] H. J. Xie, M. J. Jiang, K. Jiang, et al., “Communication Between Cancer Cell Subtypes by Exosomes Contributes to Nasopharyngeal Carcinoma Metastasis and Poor Prognosis,” Precision Clinical Medicine 7, no. 3 (2024): pbae018.

[45] Y. W. Cheng, F. M. Zeng, D. J. Li, et al., “P300/CBP-Associated Factor (PCAF)-Mediated Acetylation of Fascin at Lysine 471 Inhibits Its Actin-Bundling Activity and Tumor Metastasis in Esophageal Cancer,” Cancer Communications 41, no. 12 (2021): 1398–1416.

[46] Z. Liu, J. Chen, Y. Ren, et al., “Multi-Stage Mechanisms of Tumor Metastasis and Therapeutic Strategies,” Signal Transduction and Targeted Therapy 9, no. 1 (2024): 270.

[47] A. M. Holder, A. Dedeilia, K. Sierra-Davidson, et al., “Defining Clinically Useful Biomarkers of Immune Checkpoint Inhibitors in Solid Tumours,” Nature Reviews Cancer 24, no. 7 (2024): 498–512.

[48] C. Zheng, X. Yu, Y. Liang, et al., “Targeting PFKL With Penfluridol Inhibits Glycolysis and Suppresses Esophageal Cancer Tumorigenesis in an AMPK/FOXO3a/BIM-Dependent Manner,” Acta Pharmaceutica Sinica B 12, no. 3 (2022): 1271–1287.

[49] P. Sharma, S. Goswami, D. Raychaudhuri, et al., “Immune Checkpoint Therapy—Current Perspectives and Future Directions,” Cell 186, no. 8 (2023): 1652–1669.

[50] P. Sharma, B. A. Siddiqui, S. Anandhan, et al., “The Next Decade of Immune Checkpoint Therapy,” Cancer Discovery 11, no. 4 (2021): 838–857.

[51] Y. Huang, S. Ren, L. Ding, et al., “TP53-Specific Mutations Serve as a Potential Biomarker for Homologous Recombination Deficiency in Breast Cancer: A Clinical Next-Generation Sequencing Study,” Precision Clinical Medicine 7, no. 2 (2024): pbae009.

[52] D. Wang, D. Sun, J. Tu, et al., “The Recurrence or Metastasis Related Gene Predicts the Prognosis of Extremity and Trunk Soft Tissue Sarcoma,” Precision Clinical Medicine 8, no. 4 (2025): pbaf027.

[53] F. Ji, M. Zhou, H. Zhu, et al., “Integrative Proteomic Analysis of Multiple Posttranslational Modifications in Inflammatory Response,” Genomics, Proteomics & Bioinformatics 20, no. 1 (2022): 163–176.

[54] W. Zhu, X. Jiang, H. Sun, et al., “Global Lysine Acetylation and 2-Hydroxyisobutyrylation Profiling Reveals the Metabolism Conversion Mechanism in Giardia lamblia,” Molecular & Cellular Proteomics 20 (2021): 100043.

[55] Q. Zhao, S. Zhou, W. Lou, H. Qian, and Z. Xu, “Crosstalk Between O-GlcNAcylation and Phosphorylation in Metabolism: Regulation and Mechanism,” Cell Death and Differentiation 32, no. 7 (2025): 1181–1199.

[56] D. De Martino and J. J. Bravo-Cordero, “Collagens in Cancer: Structural Regulators and Guardians of Cancer Progression,” Cancer Research 83, no. 9 (2023): 1386–1392. PMID: 36638361.

[57] S. Xu, H. Xu, W. Wang, et al., “The Role of Collagen in Cancer: From Bench to Bedside,” Journal of Translational Medicine 17, no. 1 (2019): 309.

[58] R. J. Slack, S. J. F. Macdonald, J. A. Roper, R. G. Jenkins, and R. J. D. Hatley, “Emerging Therapeutic Opportunities for Integrin Inhibitors,” Nature Reviews Drug Discovery 21, no. 1 (2022): 60–78.

[59] A. Bagati, S. Kumar, P. Jiang, et al., “Integrin αvβ6–TGFβ–SOX4 Pathway Drives Immune Evasion in Triple-Negative Breast Cancer,” Cancer Cell 39, no. 1 (2021): 54–67.e9.

[60] A. Vannini, V. Leoni, C. Barboni, et al., “αvβ3-Integrin Regulates PD-L1 Expression and Is Involved in Cancer Immune Evasion,” Proceedings of the National Academy of Sciences of the United States of America 116, no. 40 (2019): 20141–20150.

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