Proteomics-based identification of DEAD-box protein 48 as a novel autoantigen, a prospective serum marker for pancreatic cancer

https://doi.org/10.1016/j.bbrc.2005.02.181Get rights and content

Abstract

Patients with cancer frequently develop autoantibodies, and the identification of panels of tumor autoantigens may have utility in early cancer diagnosis and immunotherapy. This study aims to exploit the autoantibody repertoire in pancreatic cancer and identify the possible serum marker for pancreatic cancer. Sera from 55 newly diagnosed patients with pancreatic cancer and 52 healthy controls were analyzed for antibody-based reactivity against Hep-2, a human larynx epithelioma cancer cell line, with one-dimensional immunoblot assay. From this analysis, we observed a prominent band with a molecular weight of 47 kDa in 63.64% (35/55) patients, while in only 1.9% normal group (1/52). Using immunoblot analysis after two-dimensional electrophoresis combined with liquid chromatography–electrospray ionization tandem mass spectrometry, this target antigen was identified as DEAD-box protein 48 (DDX48). BLAST analysis showed that it was highly similar to eukaryotic initiation factor 4A and might play a role in pre-mRNA processing. An enzyme-linked immunosorbent assay was performed using recombinant, purified DDX48 as an antigen to detect anti-DDX48 autoantibodies in sera. Reactivity was observed in 20 of 60 (33.33%) pancreatic cancer patients, 3 of 30 (10.00%) colorectal cancer patients, 2 of 30 (6.67%) gastric cancer patients, 2 of 30 (6.67%) hepatocellular cancer patients, while none of the 20 chronic pancreatitis patients, 30 lung cancer patients, and 60 normal individuals. Together, these results demonstrate that the detection of autoantibodies to DDX48 may have clinical utility for the improved diagnosis of pancreatic cancer.

Section snippets

Materials and methods

Sera and cells. Sera were obtained at the time of diagnosis from 60 patients with pancreatic cancer, after informed consent was given. Sera from 60 healthy individuals and from 140 patients with other diseases (including 30 with colorectal cancer, 30 with gastric cancer, 30 with hepatocellular cancer, 30 with lung cancer, and 20 with chronic pancreatitis) were used as controls. Hep-2 cells were grown in RPMI1640 (Gibco) supplemented with 10% fetal bovine serum.

Sample preparation. Cells were

Reactivity of sera from pancreatic cancer patients with DDX48

Hep-2 cell proteins were separated by SDS–PAGE and then transferred onto nitrocellulose membranes. Sera from 55 patients with pancreatic cancer and from 52 healthy donors were screened individually for the presence of autoantibodies. The results revealed that 87.27% pancreatic patient sera contained autoantibodies recognizing Hep-2 proteins in the 20–170 kDa range, and most of them showed more than one positive bands, while only 8 out of 52 (15.38%) normal individuals observed positive

Discussion

Pancreatic cancer presents many challenges to clinicians and researchers searching for more effective ways to combat its often-devastating effects. Among the central challenges of this disease is the identification of markers for improved diagnosis.

In this study, we first employed 1-D immunoblot assay to screen the repertoire of autoantibodies in pancreatic cancer patients, and then implemented a proteomic approach which involves 2-D gel electrophoresis, immunoblot analysis, and mass

References (24)

  • K. Holzmann et al.

    A human common nuclear matrix protein homologous to eukaryotic translation initiation factor 4A

    Biochem. Biophys. Res. Commun.

    (2000)
  • E.J. Marina et al.

    The molecular biology of pancreatic cancer

  • G. Covini et al.

    Diversity of antinuclear antibody response in hepatocellular carcinoma

    J. Hepatol.

    (1997)
  • F. Gautier et al.

    Identification of an apoptotic cleavage product of BARD1 as an autoantigen: a potential factor in the antitumoral response mediated by apoptotic bodies

    Cancer Res.

    (2000)
  • F.M. Brichory et al.

    An immune response manifested by the common occurrence of annexin I and II autoantibodies and high circulating levels of IL-6 in lung cancer

    Proc. Natl. Acad. Sci. USA

    (2001)
  • J. Raedle et al.

    p53 autoantibodies in patients with pancreatitis and pancreatic carcinoma

    Pancreas

    (1996)
  • S.H. Hong et al.

    An autoantibody-mediated immune response to calreticulin isoforms in pancreatic cancer

    Cancer Res.

    (2004)
  • Y. Hamanaka et al.

    Circulating anti-MUC1 IgG antibodies as a favorable prognostic factor for pancreatic cancer

    Int. J. Cancer

    (2003)
  • H. Maacke et al.

    Autoantibodies in sera of pancreatic cancer patients identify recombination factor Rad51 as a tumour-associated antigen

    J. Cancer Res. Clin. Oncol.

    (2002)
  • K.N. Syrigos et al.

    Autoantibodies against insulin and beta-islet cells in pancreatic adenocarcinoma: a possible explanation for diabetes mellitus

    Int. J. Cancer

    (1996)
  • M.F. Fernandez et al.

    Autoantibodies to Annexin XI-A and other autoantigens in the diagnosis of breast cancer

    Cancer Res.

    (2004)
  • J.H. Kim et al.

    Identification of epithelial cell adhesion molecule autoantibody in patients with ovarian cancer

    Clin. Cancer Res.

    (2003)
  • Cited by (59)

    • Identification of SEC61β and its autoantibody as biomarkers for colorectal cancer

      2011, Clinica Chimica Acta
      Citation Excerpt :

      Although factors resulting in the production of autoantibodies in malignancy are not completely understood, autoantibodies have been used as reporters that identify abnormal cellular processes during tumorigenesis [9]. In addition, the antibody response to a particular tumor-associated antigen is generally undetectable in healthy individuals [10–13] and in various non-malignant diseases (benign colonic adenoma, familial adenomatous polyposis, Peutz–Jeghers syndrome, gastritis, alcoholic cirrhosis, and chronic viral hepatitis) [14]. Therefore, the use of autoantibodies as serological markers for cancer detection is feasible.

    View all citing articles on Scopus
    View full text