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

Abstract

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.

Keywords

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)

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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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