Monoclonal antibody to cytokeratin VKIALEVEIATY sequence motif reduces plasminogen activation in breast tumour cells

Cytokeratins (CKs) are the main structural proteins of epithelial cells. Although they mainly form cytoplasmic structures, they are also localized at the plasma membrane or secreted from the cells. Some CKs are over-expressed in tumour cells and are used as diagnostic and prognostic biomarkers. A stable hybridoma cell line producing anti-cytokeratin monoclonal antibody (anti-CK MAb) was prepared after immunizing mice with breast cancer MCF-7 cell lysate. As shown by 2D electrophoresis, immunoblotting and mass spectroscopy, the monoclonal antibody recognizes an epitope on CK1, CK2, CK8, CK10 and CK18 in MCF-7 cells. To identify the binding site of the antibody three peptides of 12 amino acids were synthesized, each overlapping a 27 amino acid consensus sequence of the recognized CKs. Anti-CK MAb expressed high affinity for a dodecapeptide with the sequence VKIALEVEIATY, localized in the CK α-helical B2 domain, as shown by ELISA and surface plasmon resonance. Treatment of MCF-7 cells by anti-CK MAb impaired plasminogen activation and consequently invasiveness of the cells. Our results show that, besides their use in diagnosis, anti-cytokeratin antibodies could be used in therapy of invasive breast cancer.