The Pyk2 FERM domain as a target to inhibit glioma migration

• Published in: Molecular cancer therapeutics 2009-06, Vol.8 (6), p.1505-1514
• Main Authors: Loftus, Joseph C, Yang, Zhongbo, Tran, Nhan L, Kloss, Jean, Viso, Carole, Berens, Michael E, Lipinski, Christopher A
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino Acid Substitution; Animals; Antibodies, Monoclonal - genetics; Antibodies, Monoclonal - metabolism; Antibodies, Monoclonal - pharmacology; Base Sequence; Binding Sites - genetics; Binding Sites - immunology; Cell Line; Cell Line, Tumor; Cell Movement - drug effects; Epitopes - chemistry; Epitopes - metabolism; Female; focal adhesion kinase; Focal Adhesion Kinase 2 - chemistry; Focal Adhesion Kinase 2 - genetics; Focal Adhesion Kinase 2 - metabolism; glioma; Glioma - drug therapy; Glioma - metabolism; Glioma - pathology; Humans; Immunoblotting; Immunoglobulin Variable Region - genetics; Immunoglobulin Variable Region - metabolism; invasion; Mice; Mice, Inbred BALB C; Mice, Nude; Models, Molecular; Molecular Sequence Data; Phosphorylation; Protein Binding; Protein Structure, Tertiary; Pyk2; single-chain Fv; Xenograft Model Antitumor Assays
• ISSN: 1535-7163; 1538-8514
• DOI: 10.1158/1535-7163.MCT-08-1055

The invasion of malignant glioma cells into the surrounding normal brain precludes effective clinical treatment. In this report, we investigated the role of the NH 2 -terminal FERM domain in the regulation of the promigratory function of Pyk2. We report that the substitution of residues that constitute a small cleft on the surface of the F3 module of the FERM domain do not significantly alter Pyk2 expression but result in the loss of Pyk2 phosphorylation. A monoclonal antibody, designated 12A10, specifically targeting the Pyk2 FERM domain was generated and recognizes an epitope located on the β5C-α1C surface of the F3 module of the FERM domain. Amino acid substitutions in the F3 module that resulted in the loss of Pyk2 phosphorylation also inhibited the binding of 12A10, suggesting that the 12A10 epitope overlaps a site that plays a role in Pyk2 activity. Conjugation of 12A10 to a membrane transport peptide led to intracellular accumulation and inhibition of glioma cell migration in a concentration-dependent manner. A single chain Fv fragment of 12A10 was stable when expressed in the intracellular environment, interacted directly with Pyk2, reduced Pyk2 phosphorylation, and inhibited glioma cell migration in vitro . Stable intracellular expression of the 12A10 scFv significantly extended survival in a glioma xenograft model. Together, these data substantiate a central role for the FERM domain in regulation of Pyk2 activity and identify the F3 module as a novel target to inhibit Pyk2 activity and inhibit glioma progression. [Mol Cancer Ther 2009;8(6):1505–14]