title: Hypoxia integration in the serological proteome analysis unmasks tumor antigens and fosters the identification of anti-phospho-eEF2 antibodies as potential cancer biomarkers.
abstract: The expression by tumor cells of proteins with aberrant structure, expression or distribution accounts for the development of a humoral immune response. Autoantibodies (aAb) directed against tumor-associated antigens (TAA) may thus be particularly relevant for early detection of cancer. Serological proteome analysis (SERPA) aims to identify such circulating aAb through the immunoblotting of 2D-separated tumor cell proteins with cancer patient serum and the consecutive MS identification of proteins in reactive spots. This method has the advantage to use post-translationally modified proteins as a source of potential TAA. Here, we applied this strategy by using colorectal tumor cells pre-exposed to hypoxia in order to promote the expression of a pattern of TAA more likely to represent in vivo conditions. We used two human HCT116 and HT29 colorectal cancer cell lines exposed for 48 hours to 1% O2. Spots positive after immunoblotting of 2D-separated lysates of hypoxic cells with the sera of tumor-bearing mice, were collected and analysed by MS for protein identification. Among the hypoxia-specific immunogenic proteins, we identified a phosphorylated form of eukaryotic translation elongation factor 2 (phospho-Thr56 eEF2). We confirmed the increased phosphorylation of this protein in hypoxic colorectal tumor cells as well as in mouse tumors. Using a specific immunoassay, we could detect the presence of corresponding anti-phospho-Thr56 eEF2 aAb in the serum of tumor-bearing mice (vs healthy mice). We further documented that the detection of these aAb preceded the detection of a palpable tumor mass in mice and validated the presence of anti-phospho-Thr56 eEF2 aAb in the serum of patients with adenomatous polyps and colorectal carcinoma. In conclusion, this study validates a phosphorylated form of eEF2 as a new TAA and more generally, provides evidence that integrating hypoxia upstream of SERPA offers a more relevant repertoire of TAA able to unmask the presence of circulating aAb.
authors.affiliation: UCLouvain, Institut de Recherche Expérimentale et Clinique (IREC), Pole of Pharmacology and Therapeutics (FATH), Brussels, Belgium.
authors.forename: Marie
authors.initials: M
authors.lastname: Grandjean
authors.forename: Alexandra
authors.initials: A
authors.lastname: Sermeus
authors.forename: Samuel
authors.initials: S
authors.lastname: Branders
authors.forename: Florence
authors.initials: F
authors.lastname: Defresne
authors.forename: Marc
authors.initials: M
authors.lastname: Dieu
authors.forename: Pierre
authors.initials: P
authors.lastname: Dupont
authors.forename: Martine
authors.initials: M
authors.lastname: Raes
authors.forename: Mark
authors.initials: M
authors.lastname: De Ridder
authors.forename: Olivier
authors.initials: O
authors.lastname: Feron
mesh_terms: D000368:Aged; D000595:Amino Acid Sequence; D000818:Animals; D000951:Antigens, Neoplasm / Q000097:blood* / Q000276:immunology; D001323:Autoantibodies / Q000097:blood* / Q000276:immunology; D014408:Biomarkers, Tumor / Q000097:blood / Q000737:chemistry / Q000276:immunology*; D015687:Cell Hypoxia; D049109:Cell Proliferation; D015179:Colorectal Neoplasms / Q000097:blood / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D045325:HCT116 Cells; D019073:HT29 Cells; D006801:Humans; D007118:Immunoassay; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D020652:Peptide Elongation Factor 2 / Q000097:blood / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D010750:Phosphoproteins / Q000097:blood / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D010766:Phosphorylation; D040901:Proteomics; D012698:Serologic Tests; D013912:Threonine / Q000378:metabolism
chemical_list: D000951:Antigens, Neoplasm; D001323:Autoantibodies; D014408:Biomarkers, Tumor; D020652:Peptide Elongation Factor 2; D010750:Phosphoproteins; D013912:Threonine