Primary breast cancer biomarkers based on glycosylation and extracellular vesicles detected from human serum

Background Breast cancer is a very common cancer that can be severe if not discovered early. The current tools to detect breast cancer need improvement. Cancer has a universal tendency to affect glycosylation. The glycosylation of circulating extracellular vesicle-associated glycoproteins, and mucins may offer targets for detection methods and have been only explored in a limited capacity. Aim Our aim was to develop an approach to detect the aberrant glycosylation of mucins and extracellular vesicle-associated glycoproteins from human sera using fluorescent nanoparticles, and preliminarily evaluate this approach for the differential diagnosis of breast cancer. Methods and results The assay involved immobilizing glycosylated antigens using monoclonal antibodies and then probing their glycosylation by using lectins and glycan-specific antibodies coated on Eu+3-doped nanoparticles. Detection of mucin 1 and mucin 16 glycosylation with wheat germ agglutinin, and detection of the extracellular vesicle-associated CD63 were found to have better diagnostic ability for localized breast cancer than the conventional assays for mucin 1 and mucin 16 based tumor markers when the receiver operating characteristics were compared. Conclusions These results indicate that successful differential diagnosis of primary breast cancer may be aided by detecting cancer-associated glycosylation of mucin 1 and mucin 16, and total concentration of CD63, in human serum.</p

The PSI domain of the MET oncogene encodes a functional disulfide isomerase essential for the maturation of the receptor precursor

The tyrosine kinase receptor encoded by the MET oncogene has been extensively studied. Surprisingly, one extracellular domain, PSI, evolutionary conserved between plexins, semaphorins, and integrins, has no established function. The MET PSI sequence contains two CXXC motifs, usually found in protein disulfide isomerases (PDI). Using a scrambled oxidized RNAse enzymatic activity assay in vitro, we show, for the first time, that the MET extracellular domain displays disulfide isomerase activity, abolished by PSI domain antibodies. PSI domain deletion or mutations of CXXC sites to AXXA or SXXS result in a significant impairment of the cleavage of the MET 175 kDa precursor protein, abolishing the maturation of alpha and beta chains, of, respectively, 50 kDa and 145 kDa, disulfide-linked. The uncleaved precursor is stuck in the Golgi apparatus and, interestingly, is constitutively phosphorylated. However, no signal transduction is observed as measured by AKT and MAPK phosphorylation. Consequently, biological responses to the MET ligand-hepatocyte growth factor (HGF)-such as growth and epithelial to mesenchymal transition, are hampered. These data show that the MET PSI domain is functional and is required for the maturation, surface expression, and biological functions of the MET oncogenic protein

Early diagnosis of breast cancer : detection of altered glycosylation in human haptoglobin

Breast cancer (BrCa) is the most common cancer in women worldwide, with 2.1 million of new cases occurring yearly, and the fifth most common cause of death in women. The five-year survival rate for early diagnosed BrCa is 80-90%, while it drops to 24-22% for cancers diagnosed at an advanced stage. Cancer biomarkers are an important target in research to achieve early diagnosis and improve the quality of cancer treatment. The target biomarker in this research is haptoglobin (HPT), a glycoprotein that has two subunits with four N-glycosylation sites which can form oligomers so that the final possible glycovariants are numerous. In the presence of cancer, it is known that alterations occur in the glycan structures, making glycovariants important potential biomarkers for the detection of cancer from bodily fluids, since current diagnostic methods do not achieve a good level of sensitvity in the detection of cancer and its stages of development. Six relevant pools from a BrCa cohort of 152 samples (which included cancerous and benign samples) and eight healthy samples were analyzed using an immobilized anti-HPT antibody and Eu+3-doped fluorescent nanoparticles, coated with glycan-binding proteins, which detected the glycan structures of HPT. HPT was used as the target antigen for the optimization of the assay, to achieve the best possible analytical performance. Finally, the samples were individually tested to assess the clinical performance of the assay, in comparison with an assay performed with the established conventional BrCa biomarker CA15-3. One out of four glycan-binding proteins that showed potential (RCA) was selected for evaluating the clinical performance of the assay. Statistical evaluation of the assay performance in individual samples screening exhibited an overall poor analytical sensitivity of the assay, and case samples had lower signals compared to the other samples’ groups. Further studies on the other glycan binders are needed to evaluate the potential of HPT glycovariants as possible BrCa biomarkers, in order to achieve a better assay resolution and, consequently, a better discrimination between the pools