Abstract
BackgroundY-box binding protein-1 (YB-1) is the prototypic member of the cold shock protein family that fulfills numerous cellular functions. In the nucleus YB-1 protein orchestrates transcription of proliferation-related genes, whereas in the cytoplasm it associates with mRNA and directs translation. In human tumor entities, such as breast, lung and prostate cancer, cellular YB-1 expression indicates poor clinical outcome, suggesting that YB-1 is an attractive marker to predict patients' prognosis and, potentially, is suitable to individualize treatment protocols. Given these predictive qualities of YB-1 detection we sought to establish a highly specific monoclonal antibody (Mab) for diagnostic testing and its characterization towards outcome prediction (relapse-free and overall survival).MethodsHybridoma cell generation was carried out with recombinant YB-1 protein as immunogen and Mab characterization was performed using immunoblotting and ELISA with recombinant and tagged YB-1 proteins, as well as immunohistochemistry of healthy and breast cancer specimens. Breast tumor tissue array staining results were analyzed for correlations with receptor expression and outcome parameters.ResultsYB-1-specific Mab F-E2G5 associates with conformational binding epitopes mapping to two domains within the N-terminal half of the protein and detects nuclear YB-1 protein by immunohistochemistry in paraffin-embedded breast cancer tissues. Prognostic evaluation of Mab F-E2G5 was performed by immunohistochemistry of a human breast cancer tissue microarray comprising 179 invasive breast cancers, 8 ductal carcinoma in situ and 37 normal breast tissue samples. Nuclear YB-1 detection in human breast cancer cells was associated with poor overall survival (p = 0.0046). We observed a close correlation between nuclear YB-1 detection and absence of progesterone receptor expression (p = 0.002), indicating that nuclear YB-1 detection marks a specific subgroup of breast cancer. Likely due to limitation of sample size Cox regression models failed to demonstrate significance for nuclear YB-1 detection as independent prognostic marker.ConclusionMonoclonal YB-1 antibody F-E2G5 should be of great value for prospective studies to validate YB-1 as a novel biomarker suitable to optimize breast cancer treatment.
Highlights
Y-box binding protein-1 (YB-1) is the prototypic member of the cold shock protein family that fulfills numerous cellular functions
Bargou et al [7] reported that nuclear localization of YB-1 was associated with P-glycoprotein expression in human primary breast cancers, other studies have shown a co-expression of YB-1 and P-glycoprotein in osteo- and synovial sarcoma as well as breast, ovarian and prostate cancer [3,5,811]
Characterization of monoclonal YB-1-specific antibody FE2G5 In a first approach the F-E2G5 antibody was examined for its substrate specificity by western blot analysis using cell extracts from HEK293 cells expressing green fluorescent protein (GFP), YB-1-GFP or YB-1(21-147)-GFP proteins
Summary
Y-box binding protein-1 (YB-1) is the prototypic member of the cold shock protein family that fulfills numerous cellular functions. In human tumor entities, such as breast, lung and prostate cancer, cellular YB1 expression indicates poor clinical outcome, suggesting that YB-1 is an attractive marker to predict patients' prognosis and, potentially, is suitable to individualize treatment protocols. Given these predictive qualities of YB-1 detection we sought to establish a highly specific monoclonal antibody (Mab) for diagnostic testing and its characterization towards outcome prediction (relapse-free and overall survival). Following its original cloning as binding activity of the EGF receptor promoter [25], Dunn et al recently reported that YB-1 is a strong transstimulator of EGF receptor expression in breast cancer cells [26]
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