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.

An Optimized Fluorescence-Based Bidimensional Immunoproteomic Approach for Accurate Screening of Autoantibodies

“…Whereas CyDye labeling was initially described as mainly affecting the low molecular weight proteins, we observed a more heterogeneous distortion of apparent molecular weight, likely related to the lysine distribution in the amino acid sequence of proteins. This mass shift was recently observed in another study using a similar labeling design [ 68 ]. However, the restricted focusing on a single antigenic target could not be expected to resolve this question.…”

“…This approach uses an internal standard in order to match and normalize the protein profiles. A similar approach was used in a recently published study [ 68 ]. In our study we took advantage of the internal standard and transposed it as a repeated proteomic map on each immunoblot in order to guarantee the matching of the antigenic maps.…”

Eukaryotic elongation factor 2 kinase confers tolerance to stress conditions in cancer cells

“…In vivo and in vitro studies indicated that eEF2K is a novel therapeutic target for cancer. Moreover, clinical studies of patients showed that eEF2K may serve as a biomarker for prognosis and personalized cancer treatment (White-Gilbertson et al 2009;Grandjean et al 2013;Sun et al 2013). Future studies are necessary to further investigate the mechanism and clinical application of this kinase.…”

Progress in mass spectrometry-based proteomic research of tumor hypoxia

“…These UPR proteins could be applicable as novel biomarker proteins for DTC detection in cancer patients. A few years later, in 2013, Grandjean et al (56) identified a potential colon cancer biomarker through their research of the human colorectal carcinoma HCT116 and HT29 cell lines, through which the phosphorylated form of translation elongation 2 was verified as a new tumor-associated antigen.…”