Tumor Biology

, Volume 35, Issue 10, pp 10555–10569

The combination of a nuclear HMGB1-positive and HMGB2-negative expression is potentially associated with a shortened survival in patients with pancreatic ductal adenocarcinoma

  • Toru Takeda
  • Hiroto Izumi
  • Shohei Kitada
  • Hidetaka Uramoto
  • Takashi Tasaki
  • Li Zhi
  • Xin Guo
  • Yuichiro Kawatsu
  • Tomoko Kimura
  • Seichi Horie
  • Atsunori Nabeshima
  • Hirotsugu Noguchi
  • Ke-Yong Wang
  • Yasuyuki Sasaguri
  • Kimitoshi Kohno
  • Sohsuke Yamada
Research Article


High-mobility group box (HMGB) proteins are ubiquitous, abundant nuclear non-histone chromosomal proteins that play a critical role in binding to distorted DNA structures and subsequently regulating DNA transcription, replication, repair, and recombination. Both HMGB1 and HMGB2 exhibit a high expression in several human cancers and are closely associated with tumor progression and a poor prognosis. However, the expression patterns of these molecules in pancreatic ductal adenocarcinoma (PDAC) remain to be elucidated. As most cases of postoperative relapse of PDAC occur within the first 2 years, the clinical significance of accurate biomarkers is needed. Therefore, we investigated the correlation between the immunohistochemical HMGB1 and HMGB2 expression and the clinicopathological characteristics and prognosis using 62 paraffin-embedded tumor samples obtained from patients with surgically resected PDAC. The HMGB1/2 expression was considered to be positive when 10 % or more of the cancer cells showed positive nuclear, not merely cytoplasmic, staining. Consequently, the expression of HMGB1/2 was observed in 54 (87.1 %) and 31 (50.0 %) patients, respectively. Unexpectedly, a positive HMGB1 expression was found to have a significantly close relationship with a negative HMGB2 expression. The univariate and multivariate analyses demonstrated that the patients with a HMGB1+ and HMGB2− status had markedly lower disease-specific survival rates, especially within the first 2 years postoperatively, whereas those with a HMGB1+ status alone did not. Therefore, the combination of a HMGB1+ and HMGB2− expression potentially predicts a poor prognosis in patients with PDAC, and these new biomarkers may be useful parameters for clinical management in the early postoperative phase.


Pancreatic ductal adenocarcinoma (PDAC) HMGB1 HMGB2 Lymphatic permeation (LI) Prognosis 

Supplementary material

13277_2014_2328_MOESM1_ESM.pdf (127 kb)
Supplementary Figure 1The specificity of our original polyclonal antibodies for HMGB1 and HMGB2 was confirmed, with a larger amount of HMGB1/2 found to be localized in the nuclei of the PDAC cell lines. (A) Western blotting analyses of HMGB1/2 in the PDAC cell lines PANC-1 and MIA PaCa-2 divided into CF and NF. The majority of the HMGB1 and HMGB2 expression was localized in the NF rather than the CF in both of the cell lines. (B) Next, each cell lysate was diluted 1/2 sequentially. The same volume was subjected to SDS–PAGE, and Western blotting was performed with the indicated antibodies. The maximum amount of proteins was 100 μg. Sp-1 is a control for NF, whereas β-tubulin is a control for CF. Based on these data, approximately 75 % to 90 % of the HMGB1/2 expression was localized in the NF in each cell line. CF: cytoplasmic fraction. NF: nuclear fraction. (PDF 126 kb)
13277_2014_2328_MOESM2_ESM.pdf (46 kb)
Supplementary Figure 2Immunofluorescence confirmed the Western blotting data for the PDAC cell lines, showing a larger amount of HMGB1/2 localized in the nuclei of the PDAC cells. Immunofluorescence staining of the PANC-1 and MIA PaCa-2 cells also showed a specific expression of HMGB1 (green-stained) and HMGB2 (red-stained) in both the nuclei (blue-stained by Hoechst) and cytoplasm in the PDAC cell lines. Correspondingly, a weaker cytoplasmic expression of HMGB1/2 was detectable in both cell lines. (PDF 46 kb)
13277_2014_2328_MOESM3_ESM.pdf (2 mb)
Supplementary Figure 3The HMGB1+ and HMGB2− expression in the PDAC cells exhibited a potentially close relationship with a pathological LI, VI and/or PNI potential, manifesting as poorly differentiated characteristics associated with invasive/aggressive behavior. Representative pictures for H&E, EVG and the immunohistochemical analyses of HMGB1, HMGB2, D2-40 and S-100 protein in the areas of vascular (VI; Case No.27), lymphatic (LI; Case No.9) and perineural (PNI; Case No.9) invasion among the advanced PDAC components (Original magnification: ×100; inset: ×400). EVG, D2-40 and S-100 protein staining very clearly revealed elastic fibers in the vascular medial wall (VI(+)), with a lymphatic endothelium (LI(+)) and neuronal fibers (PNI(+)). Each inset provides a representative image of LI or PDAC nuclei and cytoplasm with a nuclear and cytoplasmic (HMGB1) or only cytoplasmic, not nuclear, (HMGB2) staining pattern, respectively, on high-power view. Bar = 100 μm (×100) or 20 μm (×400). H&E: hematoxylin and eosin. EVG: Elastica van Gieson. LI: lymphatic vessel invasion. VI: vascular invasion. PNI: perineural involvement. (PDF 2018 kb)


  1. 1.
    Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin. 2005;55(2):74–108.CrossRefPubMedGoogle Scholar
  2. 2.
    Li Z, Yamada S, Inenaga S, et al. Polypeptide N-acetylgalactosaminyltransferase 6 expression in pancreatic cancer is an independent prognostic factor indicating better overall survival. Br J Cancer. 2011;104(12):1882–9.PubMedCentralCrossRefPubMedGoogle Scholar
  3. 3.
    Cleary SP, Gryfe R, Guindi M, et al. Prognostic factors in resected pancreatic adenocarcinoma: analysis of actual 5-year survivors. J Am Coll Surg. 2004;198(5):722–31.CrossRefPubMedGoogle Scholar
  4. 4.
    Katz MH, Hwang R, Fleming JB, Evans DB. Tumor-node-metastasis staging of pancreatic adenocarcinoma. CA Cancer J Clin. 2008;58(2):111–25.CrossRefPubMedGoogle Scholar
  5. 5.
    Stathis A, Moore MJ. Advanced pancreatic carcinoma: current treatment and future challenges. Nat Rev Clin Oncol. 2010;7(3):163–72.CrossRefPubMedGoogle Scholar
  6. 6.
    Sharma S, Kelly TK, Jones PA. Epigenetics in cancer. Carcinogenesis. 2010;31(1):27–36.PubMedCentralCrossRefPubMedGoogle Scholar
  7. 7.
    Nalls D, Tang SN, Rodova M, Srivastava RK, Shankar S. Targeting epigenetic regulation of miR-34a for treatment of pancreatic cancer by inhibition of pancreatic cancer stem cells. PLoS One. 2011;6(8):e24099.PubMedCentralCrossRefPubMedGoogle Scholar
  8. 8.
    Thomas JO. HMG1 and 2: architectural DNA-binding proteins. Biochem Soc Trans. 2001;29(Pt4):395–401.CrossRefPubMedGoogle Scholar
  9. 9.
    Pallier C, Scaffidi P, Chopineau-Proust S, Agresti A, Nordmann P, Bianchi ME, et al. Association of chromatin proteins high mobility group box (HMGB) 1 and HMGB2 with mitotic chromosomes. Mol Biol Cell. 2003;14(8):3414–26.PubMedCentralCrossRefPubMedGoogle Scholar
  10. 10.
    Ronfani L, Ferraguti M, Croci L, Ovitt CE, Schöler HR, Consalez GG, et al. Reduced fertility and spermatogenesis defects in mice lacking chromosomal protein Hmgb2. Development. 2001;128(8):1265–73.PubMedGoogle Scholar
  11. 11.
    Krynetskaia NF, Phadke MS, Jadhav SH, Krynetskiy EY. Chromatin-associated proteins HMGB1/2 and PDIA3 trigger cellular response to chemotherapy-induced DNA damage. Mol Cancer Ther. 2009;8(4):864–72.PubMedCentralCrossRefPubMedGoogle Scholar
  12. 12.
    Kostova N, Zlateva S, Ugrinova I, Pasheva E. The expression of HMGB1 protein and its receptor RAGE in human malignant tumors. Mol Cell Biochem. 2010;337(1–2):251–8.CrossRefPubMedGoogle Scholar
  13. 13.
    Sharma A, Ray R, Rajeswari MR. Overexpression of high mobility group (HMG) B1 and B2 proteins directly correlates with the progression of squamous cell carcinoma in skin. Cancer Invest. 2008;26(8):843–51.CrossRefPubMedGoogle Scholar
  14. 14.
    Gnanasekar M, Thirugnanam S, Ramaswamy K. Short hairpin RNA (shRNA) constructs targeting high mobility group box-1 (HMGB1) expression leads to inhibition of prostate cancer cell survival and apoptosis. Int J Oncol. 2009;34(2):425–31.PubMedGoogle Scholar
  15. 15.
    Yao X, Zhao G, Yang H, Hong X, Bie L, Liu G. Overexpression of high-mobility group box 1 correlates with tumor progression and poor prognosis in human colorectal carcinoma. J Cancer Res Clin Oncol. 2010;136(5):677–84.CrossRefPubMedGoogle Scholar
  16. 16.
    Song B, Song WG, Li ZJ, Xu ZF, Wang XW, Wang CX, et al. Effect of HMGB1 silencing on cell proliferation, invasion and apoptosis of MGC-803 gastric cancer cells. Cell Biochem Funct. 2012;30(1):11–7.CrossRefPubMedGoogle Scholar
  17. 17.
    Jiao Y, Wang HC, Fan SJ. Growth suppression and radiosensitivity increase by HMGB1 in breast cancer. Acta Pharmacol Sin. 2007;28(12):1957–67.CrossRefPubMedGoogle Scholar
  18. 18.
    Wang W, Jiang H, Zhu H, Zhang H, Gong J, Zhang L, et al. Overexpression of high mobility group box 1 and 2 is associated with the progression and angiogenesis of human bladder carcinoma. Oncol Lett. 2013;5(3):884–8.PubMedCentralPubMedGoogle Scholar
  19. 19.
    Takeuchi T, Sakazume K, Tonooka A, Zaitsu M, Takeshima Y, Mikami K, et al. Cytosolic HMGB1 expression in human renal clear cell cancer indicates higher pathological T classifications and tumor grades. Urol J. 2013;10(3):960–5.PubMedGoogle Scholar
  20. 20.
    Pardo M, García A, Thomas B, Piñeiro A, Akoulitchev A, Dwek RA, et al. The characterization of the invasion phenotype of uveal melanoma tumour cells shows the presence of MUC18 and HMG-1 metastasis markers and leads to the identification of DJ-1 as a potential serum biomarker. Int J Cancer. 2006;119(5):1014–22.CrossRefPubMedGoogle Scholar
  21. 21.
    Mita AC, Mita MM, Nawrocki ST, Giles FJ. Survivin: key regulator of mitosis and apoptosis and novel target for cancer therapeutics. Clin Cancer Res. 2008;14(16):5000–5.CrossRefPubMedGoogle Scholar
  22. 22.
    Müller S, Ronfani L, Bianchi ME. Regulated expression and subcellular localization of HMGB1, a chromatin protein with a cytokine function. J Intern Med. 2004;255(3):332–43.CrossRefPubMedGoogle Scholar
  23. 23.
    Naglova H, Bucova M. HMGB1 and its physiological and pathological roles. Bratisl Lek Listy. 2012;113(3):163–71.PubMedGoogle Scholar
  24. 24.
    Sobin LH, Gospodarowicz MK, Wittekind Ch. TNM classification of malignant tumours 7th edition. Wiley-Blackwell; 2009.Google Scholar
  25. 25.
    Bosman F, Carneiro F, Hruban RH, Theise N, editors. Ductal adenocarcinoma of the pancreas. In: Hruban RH, Boffetta P, Hiraoka N, lacobuzio-Donahue C, Kato Y, Kern SE, Klimstra DS, Kloppel G, Marita A, Offerhaus GJA, Pitman MB, editors. World Health Organization (WHO) classification of tumours of the digestive system. IARC, Lyon: Springer; 2010, pp. 281–91.Google Scholar
  26. 26.
    Ise T, Nagatani G, Imamura T, et al. Transcription factor Y-box binding protein 1 binds preferentially to cisplatin-modified DNA and interacts with proliferating cell nuclear antigen. Cancer Res. 1999;59(2):342–6.PubMedGoogle Scholar
  27. 27.
    Wakasugi T, Izumi H, Uchiumi T, et al. ZNF143 interacts with p73 and is involved in cisplatin resistance through the transcriptional regulation of DNA repair genes. Oncogene. 2007;26(36):5194–203.CrossRefPubMedGoogle Scholar
  28. 28.
    Izumi H, Wakasugi T, Shimajiri S, et al. Role of ZNF143 in tumor growth through transcriptional regulation of DNA replication and cell-cycle-associated genes. Cancer Sci. 2010;101(12):2538–45.CrossRefPubMedGoogle Scholar
  29. 29.
    Kitada S, Yamada S, Kuma A, et al. Polypeptide N-acetylgalactosaminyl transferase 3 independently predicts high-grade tumours and poor prognosis in patients with renal cell carcinomas. Br J Cancer. 2013;109(2):472–81.PubMedCentralCrossRefPubMedGoogle Scholar
  30. 30.
    Wu Y, Yamada S, Izumi H, et al. Strong YB-1 expression is associated with liver metastasis progression and predicts shorter disease-free survival in advanced gastric cancer. J Surg Oncol. 2012;105(7):724–30.CrossRefPubMedGoogle Scholar
  31. 31.
    Kawatsu Y, Kitada S, Uramoto H, et al. The combination of strong expression of ZNF143 and high MIB-1 labelling index independently predicts shorter disease-specific survival in lung adenocarcinoma. Br J Cancer. 2014, in print.Google Scholar
  32. 32.
    Hanley JA. Receiver operating characteristic (ROC) methodology: the state of the art. Crit Rev Diagn Imaging. 1989;29(3):307–35.PubMedGoogle Scholar
  33. 33.
    Kanda Y. Investigation of the freely available easy-to-use software ‘EZR’ for medical statistics. Bone Marrow Transplant. 2013;48(3):452–8.PubMedCentralCrossRefPubMedGoogle Scholar
  34. 34.
    Conroy T, Desseigne F, Ychou M, et al. FOLFIRINOX versus gemcitabine for metastatic pancreatic cancer. N Engl J Med. 2011;364(19):1817–25.CrossRefPubMedGoogle Scholar
  35. 35.
    Chang BP, Wang DS, Xing JW, Yang SH, Chu Q, Yu SY. miR-200c inhibits metastasis of breast cancer cells by targeting HMGB1. J Huazhong Univ Sci Technolog Med Sci. 2014;34(2):201–6.CrossRefPubMedGoogle Scholar
  36. 36.
    Earl J, Yan L, Vitone LJ, et al. Evaluation of the 4q32-34 locus in European familial pancreatic cancer. Cancer Epidemiol Biomarkers Prev. 2006;15(10):1948–55.CrossRefPubMedGoogle Scholar

Copyright information

© International Society of Oncology and BioMarkers (ISOBM) 2014

Authors and Affiliations

  • Toru Takeda
    • 1
  • Hiroto Izumi
    • 2
  • Shohei Kitada
    • 3
    • 4
  • Hidetaka Uramoto
    • 5
  • Takashi Tasaki
    • 3
  • Li Zhi
    • 3
    • 6
  • Xin Guo
    • 3
  • Yuichiro Kawatsu
    • 1
  • Tomoko Kimura
    • 1
  • Seichi Horie
    • 1
  • Atsunori Nabeshima
    • 3
  • Hirotsugu Noguchi
    • 3
  • Ke-Yong Wang
    • 3
    • 7
  • Yasuyuki Sasaguri
    • 3
    • 8
  • Kimitoshi Kohno
    • 9
  • Sohsuke Yamada
    • 3
  1. 1.Department of Health Policy and Management, Institute of Industrial Ecological SciencesUniversity of Occupational and Environmental HealthKitakyushuJapan
  2. 2.Department of Occupational Pneumology, School of MedicineUniversity of Occupational and Environmental HealthKitakyushuJapan
  3. 3.Department of Pathology and Cell Biology, School of MedicineUniversity of Occupational and Environmental HealthKitakyushuJapan
  4. 4.Department of Urology, School of MedicineUniversity of Occupational and Environmental HealthKitakyushuJapan
  5. 5.Department of Second Surgery, School of MedicineUniversity of Occupational and Environmental HealthKitakyushuJapan
  6. 6.Department of Medical OncologyThe First Hospital of China Medical UniversityShenyangPeople’s Republic of China
  7. 7.Department of Bio-information Research Center, School of MedicineUniversity of Occupational and Environmental HealthKitakyushuJapan
  8. 8.Laboratory of PathologyFukuoka Wajiro HospitalFukuokaJapan
  9. 9.The President Laboratory, School of MedicineUniversity of Occupational and Environmental HealthKitakyushuJapan

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