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Development of JNK2-selective peptide inhibitors that inhibit breast cancer cell migration.

ABSTRACT: Despite their lack of selectivity toward c-Jun N-terminal kinase (JNK) isoforms, peptides derived from the JIP (JNK Interacting Protein) scaffolds linked to the cell-penetrating peptide TAT are widely used to investigate JNK-mediated signaling events. To engineer an isoform-selective peptide inhibitor, several JIP-based peptide sequences were designed and tested. A JIP sequence connected through a flexible linker to either the N-terminus of an inverted TAT sequence (JIP(10)-?-TAT(i)) or to a poly arginine sequence (JIP(10)-?-R(9)) enabled the potent inhibition of JNK2 (IC(50) ? 90 nM) and exhibited 10-fold selectivity for JNK2 over JNK1 and JNK3. Examination of both peptides in HEK293 cells revealed a potent ability to inhibit the induction of both JNK activation and c-Jun phosphorylation in cells treated with anisomycin. Notably, Western blot analysis indicates that only a fraction of total JNK must be activated to elicit robust c-Jun phosphorylation. To examine the potential of each peptide to selectively modulate JNK2 signaling in vivo, their ability to inhibit the migration of Polyoma Middle-T Antigen Mammary Tumor (PyVMT) cells was assessed. PyVMTjnk2-/- cells exhibit a lower migration potential compared to PyVMTjnk2+/+ cells, and this migration potential is restored through the overexpression of GFP-JNK2?. Both JIP(10)-?-TAT(i) and JIP(10)-?-R(9) inhibit the migration of PyVMTjnk2+/+ cells and PyVMTjnk2-/- cells expressing GFP-JNK2?. However, neither peptide inhibits the migration of PyVMTjnk2-/- cells. A control form of JIP(10)-?-TAT(i) containing a single leucine to arginine mutation lacks ability to inhibit JNK2 in vitro cell-free and cell-based assays and does not inhibit the migration of PyVMTjnk2+/+ cells. Together, these data suggest that JIP(10)-?-TAT(i) and JIP(10)-?-R(9) inhibit the migration of PyVMT cells through the selective inhibition of JNK2. Finally, the mechanism of inhibition of a D-retro-inverso JIP peptide, previously reported to inhibit JNK, was examined and found to inhibit p38MAPK? in an in vitro cell-free assay with little propensity to inhibit JNK isoforms.

PROVIDER: S-EPMC3401522 | BioStudies |

REPOSITORIES: biostudies

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