Site-Specific O-Glycosylation in Oncofetal Fibronectin IIICS Domain Creates Cancer Stage-Specific Biomarkers

Despite the pathological importance of oncofetal fibronectin isoforms associated with cancer cell progression and metastasis, our understanding of the structural and molecular details that occurred in this abundant and ubiquitous extracellular matrix component remains largely unknown. Here, we communicate that site-specific O-glycosylation in the oncofetal fibronectin creates cancer stage-specific serum biomarkers for hepatocellular carcinoma (HCC) by quantitative MS-based glycoproteomic approach. Selective reaction monitoring (SRM) using a structure-defined synthetic glycopeptide library enabled absolute quantitation of the targeted label-free tryptic fragments bearing cancer-relevant O-glycans derived from the type III homology connective segment (IIICS) domain of the oncofetal fibronectin (onfFN) in the sera of HCC patients and healthy controls. We found site-specific O-glycoform alteration from T/sialyl T antigens to Tn/sialyl Tn antigens at the consecutive threonine residues ²¹⁵⁵Thr-Thr-Ala²¹⁵⁷ within this fragment during cancer progression. Surprisingly, this dynamic glycoform alteration is observed specifically in the patient sera diagnosed newly as stage 2-4 groups not in the normal control and stage 1 patient groups. Our results provide compelling evidence that site-specific glycoform changes observed in the onfFN IIICS domain during the tumor proliferation elaborate new class cancer stage-relevant "dynamic epitopes" as highly potential cancer diagnostic and therapeutic targets.