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Hypoxia integration in the serological proteome analysis unmasks tumor antigens and fosters the identification of anti-phospho-eEF2 antibodies as potential cancer biomarkers.

Grandjean M, Sermeus A, Branders S, Defresne F, Dieu M, Dupont P, Raes M, De Ridder M, Feron O - PLoS ONE (2013)

Bottom Line: Autoantibodies (aAb) directed against tumor-associated antigens (TAA) may thus be particularly relevant for early detection of cancer.Among the hypoxia-specific immunogenic proteins, we identified a phosphorylated form of eukaryotic translation elongation factor 2 (phospho-Thr56 eEF2).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.

View Article: PubMed Central - PubMed

Affiliation: UCLouvain, Institut de Recherche Expérimentale et Clinique (IREC), Pole of Pharmacology and Therapeutics (FATH), Brussels, Belgium.

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.

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Validation of hypoxia-induced phosphorylations of eEF2 in colorectal cancer cells.A. Representative eEF2 and phospho-Thr56 eEF2 immunoblotting of HCT116 and HT29 cultured for 48 hours under hypoxia (Hx) or maintained in normoxia (Nx). B. Normalized expression of phospho-Thr56 eEF2 in normoxic vs hypoxic HCT116 and HT29 cells; n = 3, **p<0.01 C. Representative phospho-Thr56 eEF2 immunostaining of sections of HCT116 tumors in the absence (top) or the presence (bottom) of phosphatase lambda; note the complete disappearance of the phosphorylated form of eEF2 upon treatment with the phosphatase.
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pone-0076508-g005: Validation of hypoxia-induced phosphorylations of eEF2 in colorectal cancer cells.A. Representative eEF2 and phospho-Thr56 eEF2 immunoblotting of HCT116 and HT29 cultured for 48 hours under hypoxia (Hx) or maintained in normoxia (Nx). B. Normalized expression of phospho-Thr56 eEF2 in normoxic vs hypoxic HCT116 and HT29 cells; n = 3, **p<0.01 C. Representative phospho-Thr56 eEF2 immunostaining of sections of HCT116 tumors in the absence (top) or the presence (bottom) of phosphatase lambda; note the complete disappearance of the phosphorylated form of eEF2 upon treatment with the phosphatase.

Mentions: Since the phosphorylation of eEF2 is known to occur in response to hypoxia (leading to eEF2 inactivation and the arrest of protein translation), we examined the extent of eEF2 phosphorylation on Thr56 previously described as the first and main residue modified by a phosphate group within the eEF2 sequence [31]. Re-probing the 2D membrane used for SERPA confirmed that the spot recognized by the serum of tumor-bearing mice was also positively stained by a commercial anti-phospho-Thr56 eEF2 antibody (Figure 4B, lower panel). We also confirmed in conventional Western blotting experiments that eEF2 phosphorylation on Thr56 was significantly increased in hypoxic HCT116 cells (p<0.01) and that a similar trend was observed in HT29 cells (Figures 5A and 5B). Also, the injection of HCT116 cells into mice led to the development of a tumor with a robust staining of phospho-Thr56 eEF2 confirming the occurrence of this post-translational modification in vivo (Figure 5C, top panel). The treatment of tumor sections with phosphatase lambda completely abrogated the staining obtained with a commercial anti-phospho-eEF2 antibody (Figure 5C, bottom panel).


Hypoxia integration in the serological proteome analysis unmasks tumor antigens and fosters the identification of anti-phospho-eEF2 antibodies as potential cancer biomarkers.

Grandjean M, Sermeus A, Branders S, Defresne F, Dieu M, Dupont P, Raes M, De Ridder M, Feron O - PLoS ONE (2013)

Validation of hypoxia-induced phosphorylations of eEF2 in colorectal cancer cells.A. Representative eEF2 and phospho-Thr56 eEF2 immunoblotting of HCT116 and HT29 cultured for 48 hours under hypoxia (Hx) or maintained in normoxia (Nx). B. Normalized expression of phospho-Thr56 eEF2 in normoxic vs hypoxic HCT116 and HT29 cells; n = 3, **p<0.01 C. Representative phospho-Thr56 eEF2 immunostaining of sections of HCT116 tumors in the absence (top) or the presence (bottom) of phosphatase lambda; note the complete disappearance of the phosphorylated form of eEF2 upon treatment with the phosphatase.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3794947&req=5

pone-0076508-g005: Validation of hypoxia-induced phosphorylations of eEF2 in colorectal cancer cells.A. Representative eEF2 and phospho-Thr56 eEF2 immunoblotting of HCT116 and HT29 cultured for 48 hours under hypoxia (Hx) or maintained in normoxia (Nx). B. Normalized expression of phospho-Thr56 eEF2 in normoxic vs hypoxic HCT116 and HT29 cells; n = 3, **p<0.01 C. Representative phospho-Thr56 eEF2 immunostaining of sections of HCT116 tumors in the absence (top) or the presence (bottom) of phosphatase lambda; note the complete disappearance of the phosphorylated form of eEF2 upon treatment with the phosphatase.
Mentions: Since the phosphorylation of eEF2 is known to occur in response to hypoxia (leading to eEF2 inactivation and the arrest of protein translation), we examined the extent of eEF2 phosphorylation on Thr56 previously described as the first and main residue modified by a phosphate group within the eEF2 sequence [31]. Re-probing the 2D membrane used for SERPA confirmed that the spot recognized by the serum of tumor-bearing mice was also positively stained by a commercial anti-phospho-Thr56 eEF2 antibody (Figure 4B, lower panel). We also confirmed in conventional Western blotting experiments that eEF2 phosphorylation on Thr56 was significantly increased in hypoxic HCT116 cells (p<0.01) and that a similar trend was observed in HT29 cells (Figures 5A and 5B). Also, the injection of HCT116 cells into mice led to the development of a tumor with a robust staining of phospho-Thr56 eEF2 confirming the occurrence of this post-translational modification in vivo (Figure 5C, top panel). The treatment of tumor sections with phosphatase lambda completely abrogated the staining obtained with a commercial anti-phospho-eEF2 antibody (Figure 5C, bottom panel).

Bottom Line: Autoantibodies (aAb) directed against tumor-associated antigens (TAA) may thus be particularly relevant for early detection of cancer.Among the hypoxia-specific immunogenic proteins, we identified a phosphorylated form of eukaryotic translation elongation factor 2 (phospho-Thr56 eEF2).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.

View Article: PubMed Central - PubMed

Affiliation: UCLouvain, Institut de Recherche Expérimentale et Clinique (IREC), Pole of Pharmacology and Therapeutics (FATH), Brussels, Belgium.

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.

Show MeSH
Related in: MedlinePlus