High Mobility Group Box 1 Protein Is Methylated and Transported to Cytoplasm in Clear Cell Renal Cell Carcinoma

  • Wu, Fei (Department of Urology, Provincial Hospital Affiliated to Shandong University) ;
  • Zhao, Zuo-Hui (Department of Urology, Provincial Hospital Affiliated to Shandong University) ;
  • Ding, Sen-Tai (Department of Urology, Provincial Hospital Affiliated to Shandong University) ;
  • Wu, Hai-Hu (Department of Urology, Provincial Hospital Affiliated to Shandong University) ;
  • Lu, Jia-Ju (Department of Urology, Provincial Hospital Affiliated to Shandong University)
  • Published : 2013.10.30


Background: The high mobility group box 1 (HMGB1) protein is a widespread nuclear protein present in most cell types. It typically locates in the nucleus and functions as a nuclear cofactor in transcription regulation. However, HMGB1 can also localize in the cytoplasm and be released into extracellular matrix, where it plays critical roles in carcinogenesis and inflammation. However, it remains elusive whether HMGB1 is relocated to cytoplasm in clear cell renal cell carcinoma (ccRCC). Methods: Nuclear and cytoplasmic proteins were extracted by different protocols from 20 ccRCC samples and corresponding adjacent renal tissues. Western blotting and immunohistochemistry were used to identify the expression of HMGB1 in ccRCC. To elucidate the potential mechanism of HMGB1 cytoplasmic translocation, HMGB1 proteins were enriched by immunoprecipitation and analyzed by mass spectrometry (MS). Results: The HMGB1 protein was overexpressed and partially localized in cytoplasm in ccRCC samples (12/20, 60%, p<0.05). Immunohistochemistry results indicated that ccRCC of high nuclear grade possess more HMGB1 relocation than those with low grade (p<0.05). Methylation of HMGB1 at lysine 112 in ccRCC was detected by MS. Bioinformatics analysis showed that post-translational modification might affect the binding ability to DNA and mediate its translocation. Conclusion: Relocation of HMGB1 to cytoplasm was confirmed in ccRCC. Methylation of HMGB1 at lysine 112 might the redistribution of this cofactor protein.


HMGB1;methylation;RCC;renal cancer;transcription regulation;cytoplasmic location


  1. Bianchi ME, Beltrame M, Paonessa G (1989). Specific recognition of cruciform DNA by nuclear protein HMG1. Science, 243, 1056-9.
  2. Alao JP, Gamble SC, Stavropoulou AV, et al (2006). The cyclin D1 proto-oncogene is sequestered in the cytoplasm of mammalian cancer cell lines. Mol Cancer, 5, 7.
  3. Apetoh L, Ghiringhelli F, Tesniere A, et al (2007). Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy. Nat Med, 13, 1050-9.
  4. Assenberg R, Webb M, Connolly E, et al (2008). A critical role in structure-specific DNA binding for the acetylatable lysine residues in HMGB1. Biochem J, 411, 553-61.
  5. Banerjee S, Kundu TK (2003). The acidic C-terminal domain and A-box of HMGB-1 regulates p53-mediated transcription. Nucleic Acids Res, 31, 3236-47.
  6. Bonaldi T, Langst G, Strohner R, et al (2002). The DNA chaperone HMGB1 facilitates ACF/CHRAC-dependent nucleosome sliding. EMBO J, 21, 6865-73.
  7. Bonaldi T, Talamo F, Scaffidi P, et al (2003). Monocytic cells hyperacetylate chromatin protein HMGB1 to redirect it towards secretion. EMBO J, 22, 5551-60.
  8. Campana L, Bosurgi L, Rovere-Querini P (2008). HMGB1: a two-headed signal regulating tumor progression and immunity. Curr Opin Immunol, 20, 518-23.
  9. Carneiro V C, de Moraes Maciel R, de Abreu da Silva IC, et al (2009). The extracellular release of Schistosoma mansoni HMGB1 nuclear protein is mediated by acetylation. Biochem Biophys Res Commun, 390, 1245-9.
  10. Chandrasekharan MB, Huang F, Sun ZW (2010). Histone H2B ubiquitination and beyond: Regulation of nucleosome stability, chromatin dynamics and the trans-histone H3 methylation. Epigenetics, 5, 460-8.
  11. Curtin J F, Liu N, Candolfi M, et al (2009). HMGB1 mediates endogenous TLR2 activation and brain tumor regression. PLoS Med, 6, e10.
  12. de Abreu da Silva IC, Carneiro VC, Maciel Rde M, et al (2011). CK2 phosphorylation of Schistosoma mansoni HMGB1 protein regulates its cellular traffic and secretion but not its DNA transactions. PLoS One, 6, e23572.
  13. Ellerman JE, Brown CK, de Vera M, et al (2007). Masquerader: high mobility group box-1 and cancer. Clin Cancer Res, 13, 2836-48.
  14. Evankovich J, Cho SW, Zhang R, et al (2010). High mobility group box 1 release from hepatocytes during ischemia and reperfusion injury is mediated by decreased histone deacetylase activity. J Biol Chem, 285, 39888-97.
  15. Gauley J and Pisetsky DS (2009). The translocation of HMGB1 during cell activation and cell death. Autoimmunity, 42, 299-301.
  16. Goodwin GH, Johns EW (1977). The isolation and purification of the high mobility group (HMG) nonhistone chromosomal proteins. Methods Cell Biol, 16, 257-67.
  17. Gupta K, Miller JD, Li JZ, et al (2008). Epidemiologic and socioeconomic burden of metastatic renal cell carcinoma (mRCC): a literature review. Cancer Treat Rev, 34, 193-205.
  18. Hanahan D, Weinberg RA (2000). The hallmarks of cancer. Cell, 100, 57-70.
  19. Jung Y and Lippard S J (2003). Nature of full-length HMGB1 binding to cisplatin-modified DNA. Biochemistry, 42, 2664-71.
  20. Hoppe G, Talcott KE, Bhattacharya SK, et al (2006). Molecular basis for the redox control of nuclear transport of the structural chromatin protein Hmgb1. Exp Cell Res, 312, 3526-38.
  21. Ito I, Fukazawa J, Yoshida M (2007). Post-translational methylation of high mobility group box 1 (HMGB1) causes its cytoplasmic localization in neutrophils. J Biol Chem, 282, 16336-44.
  22. Jiao Y, Wang HC, Fan S J (2007). Growth suppression and radiosensitivity increase by HMGB1 in breast cancer. Acta Pharmacol Sin, 28, 1957-67.
  23. Kang HJ, Lee H, Choi HJ, et al (2009). Non-histone nuclear factor HMGB1 is phosphorylated and secreted in colon cancers. Lab Invest, 89, 948-59.
  24. Knapp S, Muller S, Digilio G, et al (2004). The long acidic tail of high mobility group box 1 (HMGB1) protein forms an extended and flexible structure that interacts with specific residues within and between the HMG boxes. Biochemistry, 43, 11992-7.
  25. Kovacs G, Akhtar M, Beckwith BJ, et al (1997). The Heidelberg classification of renal cell tumours. J Pathol, 183, 131-3.<131::AID-PATH931>3.0.CO;2-G
  26. Krynetski EY, Krynetskaia NF, Bianchi ME, Evans WE (2003). A nuclear protein complex containing high mobility group proteins B1 and B2, heat shock cognate protein 70, ERp60, and glyceraldehyde-3-phosphate dehydrogenase is involved in the cytotoxic response to DNA modified by incorporation of anticancer nucleoside analogues. Cancer Res, 63, 100-6.
  27. Lee H, Park M, Shin N, et al (2012). High mobility group box-1 is phosphorylated by protein kinase C zeta and secreted in colon cancer cells. Biochem Biophys Res Commun, 424, 321-6.
  28. Lotze MT, DeMarco RA (2003). Dealing with death: HMGB1 as a novel target for cancer therapy. Curr Opin Investig Drugs, 4, 1405-9.
  29. Lee KB, Thomas JO (2000). The effect of the acidic tail on the DNA-binding properties of the HMG1,2 class of proteins: insights from tail switching and tail removal. J Mol Biol, 304, 135-49.
  30. Li J, Kokkola R, Tabibzadeh S, et al (2003). Structural basis for the proinflammatory cytokine activity of high mobility group box 1. Mol Med, 9, 37-45.
  31. Lin L, Zhong K, Sun Z, et al (2012). Receptor for advanced glycation end products (RAGE) partially mediates HMGB1-ERKs activation in clear cell renal cell carcinoma. J Cancer Res Clin Oncol, 138, 11-22.
  32. Lotze M T and Tracey K J (2005). High-mobility group box 1 protein (HMGB1): nuclear weapon in the immune arsenal. Nat Rev Immunol, 5, 331-42.
  33. Luo Y, Chihara Y, Fujimoto K, et al (2012). High mobility group box 1 released from necrotic cells enhances regrowth and metastasis of cancer cells that have survived chemotherapy. Eur J Cancer, 49, 741-51.
  34. Motzer RJ, Hutson TE, Tomczak P, et al (2007). Sunitinib versus interferon alfa in metastatic renal-cell carcinoma. N Engl J Med, 356, 115-24.
  35. Na S, Bandeira N, Paek E (2012). Fast multi-blind modification search through tandem mass spectrometry. Mol Cell Proteomics, 11, M111 010199.
  36. Pasheva EA, Pashev IG, Favre A (1998). Preferential binding of high mobility group 1 protein to UV-damaged DNA. Role of the COOH-terminal domain. J Biol Chem, 273, 24730-6.
  37. Peters R (1986). Fluorescence microphotolysis to measure nucleocytoplasmic transport and intracellular mobility. Biochim Biophys Acta, 864, 305-59.
  38. Read CM, Cary PD, Crane-Robinson C, et al (1993). Solution structure of a DNA-binding domain from HMG1. Nucleic Acids Res, 21, 3427-36.
  39. Pettersen EF, Goddard TD, Huang CC, et al (2004). UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem, 25, 1605-12.
  40. Pil P M, Lippard S J (1992). Specific binding of chromosomal protein HMG1 to DNA damaged by the anticancer drug cisplatin. Science, 256, 234-7.
  41. Ramstein J, Locker D, Bianchi ME, Leng M (1999). Domain-domain interactions in high mobility group 1 protein (HMG1). Eur J Biochem, 260, 692-700.
  42. Rini BI, Campbell SC, Escudier B (2009). Renal cell carcinoma. Lancet, 373, 1119-32.
  43. Sabio G, Arthur JS, Kuma Y, et al (2005). p38gamma regulates the localisation of SAP97 in the cytoskeleton by modulating its interaction with GKAP. EMBO J, 24, 1134-45.
  44. Scaffidi P, Misteli T, Bianchi ME (2002). Release of chromatin protein HMGB1 by necrotic cells triggers inflammation. Nature, 418, 191-5.
  45. Sessa L, Bianchi ME (2007). The evolution of High Mobility Group Box (HMGB) chromatin proteins in multicellular animals. Gene, 387, 133-40.
  46. Stott K, Watson M, Howe FS, et al (2010). Tail-mediated collapse of HMGB1 is dynamic and occurs via differential binding of the acidic tail to the A and B domains. J Mol Biol, 403, 706-22.
  47. Stros M, Stokrova J, Thomas J O (1994). DNA looping by the HMG-box domains of HMG1 and modulation of DNA binding by the acidic C-terminal domain. Nucleic Acids Res, 22, 1044-51.
  48. Tang D, Kang R, Zeh HJ, 3rd, Lotze MT (2010). High-mobility group box 1 and cancer. Biochim Biophys Acta, 1799, 131-40.
  49. Stros M, Ozaki T, Bacikova A, et al (2002). HMGB1 and HMGB2 cell-specifically down-regulate the p53- and p73- dependent sequence-specific transactivation from the human Bax gene promoter. J Biol Chem, 277, 7157-64.
  50. Taguchi A, Blood DC, del Toro G, et al (2000). Blockade of RAGE-amphoterin signalling suppresses tumour growth and metastases. Nature, 405, 354-60.
  51. Tan F, Lu L, Cai Y, et al (2008). Proteomic analysis of ubiquitinated proteins in normal hepatocyte cell line Chang liver cells. Proteomics, 8, 2885-96.
  52. Teo SH, Grasser KD, Thomas JO (1995). Differences in the DNA-binding properties of the HMG-box domains of HMG1 and the sex-determining factor SRY. Eur J Biochem, 230, 943-50.
  53. Tian J, Avalos AM, Mao SY, et al (2007). Toll-like receptor 9-dependent activation by DNA-containing immune complexes is mediated by HMGB1 and RAGE. Nat Immunol, 8, 487-96.
  54. Tsung A, Klune JR, Zhang X, et al (2007). HMGB1 release induced by liver ischemia involves Toll-like receptor 4 dependent reactive oxygen species production and calciummediated signaling. J Exp Med, 204, 2913-23.
  55. Wang H, Bloom O, Zhang M, et al (1999). HMG-1 as a late mediator of endotoxin lethality in mice. Science, 285, 248-51.
  56. Webb M, Thomas JO (1999). Structure-specific binding of the two tandem HMG boxes of HMG1 to four-way junction DNA is mediated by the A domain. J Mol Biol, 294, 373-87.
  57. Weir HM, Kraulis PJ, Hill CS, et al (1993). Structure of the HMG box motif in the B-domain of HMG1. EMBO J, 12, 1311-9.
  58. Yang H, Antoine DJ, Andersson U, and Tracey KJ (2013). The many faces of HMGB1: molecular structure-functional activity in inflammation, apoptosis, and chemotaxis. J Leukoc Biol, 93, 865-73.
  59. Youn JH, Shin JS (2006). Nucleocytoplasmic shuttling of HMGB1 is regulated by phosphorylation that redirects it toward secretion. J Immunol, 177, 7889-97.

Cited by

  1. Expression of High Mobility Group Box - B1 (HMGB-1) and Matrix Metalloproteinase-9 (MMP-9) in Non-small Cell Lung Cancer (NSCLC) vol.15, pp.12, 2014,
  2. Antibodies against High Mobility Group Box protein-1 (HMGB1) versus other anti-nuclear antibody fine-specificities and disease activity in systemic lupus erythematosus vol.17, pp.1, 2015,
  3. Label-free quantitative proteomic analysis reveals potential biomarkers and pathways in renal cell carcinoma vol.36, pp.2, 2015,
  4. HMGB1 promotes HCC progression partly by downregulating p21 via ERK/c-Myc pathway and upregulating MMP-2 vol.37, pp.4, 2016,
  5. Serum HMGB1 concentrations at 4 weeks is a useful predictor of extreme poor prognosis for advanced hepatocellular carcinoma treated with sorafenib and hepatic arterial infusion chemotherapy vol.53, pp.1, 2018,
  6. The Role of HMGB1 Signaling Pathway in the Development and Progression of Hepatocellular Carcinoma: A Review vol.16, pp.9, 2015,