A novel peptide inhibitor blocks invadopodia-mediated breast cancer metastasis

The non-receptor tyrosine kinase Pyk2 is highly expressed in breast cancer, where it mediates invadopodia formation and function and consequent breast cancer invasiveness via interaction with the actin-nucleation promoting factor cortactin. Here, we designed a cell-permeable peptide inhibitor that contains the second proline-rich region (PRR2) sequence of Pyk2, which binds to the SH3 domain of cortactin and inhibits the interaction between Pyk2 and cortactin in invadopodia. The PRR2-Pyk2 peptide blocks spontaneous lung metastasis in immune-competent mice by inhibiting cortactin tyrosine phosphorylation and actin polymerization-mediated maturation and activation of invadopodia, leading to reduced MMP-dependent tumor cell invasiveness. The native structure of the Pyk2-PRR2: cortactin-SH3 complex was determined using nuclear magnetic resonance (NMR), revealing an extended class II interaction surface spanning the canonical binding groove and a second hydrophobic surface which significantly contributes to ligand affinity. Using structure-guided design, we created a mutant peptide lacking critical residues involved in binding that failed to inhibit invadopodia maturation and function and consequent metastatic dissemination in mice. Further transcriptomics analysis revealed the downstream molecular and signaling mechanisms of metastasis inhibition by our Pyk2-derived peptide. Our findings shed light on the specific molecular interactions between Pyk2 and cortactin and on the downstream mechanisms of their inhibition and may lead to the development of a novel strategy for preventing metastatic dissemination of primary breast tumors that are predicted to be highly metastatic at the time of diagnosis, and of secondary tumors that have already spread to other parts of the body.