A cell-permeable peptide corresponding to the calmodulin-binding domain of Grb7 inhibits proliferation and migration of A431 cells

Abstract

[Introduction]: Grb7 (growth factor receptor bound 7) is a mammalian adaptor protein that transduces signals from tyrosine kinase receptors. This protein is overexpressed, together with ErbB2, in different human carcinomas. Grb7 is implicated in cell migration, contributing to the invasiveness and metastatic capacity of tumour cells. Also, Grb7 controls cell proliferation and tumour-associated angiogenesis. Our group demonstrated that calmodulin (CaM) binds to the proximal region of the pleckstrin homology (PH) domain of human Grb7 in a Ca2+-dependent manner, comprising the sequence 243RKLWKRFFCFLRRS256. A deletion mutant of Grb7, lacking its CaM-binding domain (CaM-BD), impairs the adhesion and migratory capacity of transfected cells. Moreover, the expression of this deletion mutant in rat C6 glioblastoma cells strongly inhibited its proliferation, the growth of brain tumours derived from stereotaxically implanted tumour cells, and tumour-associated angiogenesis. Therefore, the CaM-BD of Grb7 could be a potential target to inhibit tumour cells invasiveness and metastasis development. [Materials and Methods]: Human carcinoma epidermoid A431 cells were used in all the experiments. Custom-made synthesis of peptides (>99% purity) with the amino acid sequence RKLWKRFFCFLRRS, and a derivative with a myristoyl group at its N-terminus (Myr-RKLWKRFFCFLRRS) to facilitate cell internalization, were manufactured by Wuxi Nordisk Biotech Ltd. (China). Cell migration was measured by artificial wound healing assays and time-lapse microscopy, and cell proliferation was measured by Crystal Violet staining. The assays were done in the absence and presence of 10 nM EGF, and 20-50 µg/ml of the different peptides, or 10-50 µM W-7 and W-12 (CaM antagonists). [Results]: The addition of EGF first induced a 12-18 h lag phase in which the migration of A431 cells was arrested, and a significant retraction of the border of the artificial wound was observed. Thereafter, the migration rate of the cells increased several folds as compared to the rate of migration of cells in the absence of EGF. The RKLWKRFFCFLRRS peptide inhibited the migration of A431 cells both in the absence and presence of EGF. This inhibitory effect was more evident using the Myr-RKLWKRFFCFLRRS peptide. The high affinity CaM antagonist W-7, and in lesser extent its low affinity analogue W-12, incremented the lag phase in which the migration of the cells was arrested upon addition of EGF and decreased its subsequent migration rate. As previously demonstrated, EGF induced a significant inhibition of cell proliferation in A431 cells, and both the RKLWKRFFCFLRRS and Myr-RKLWKRFFCFLRRS peptides further inhibited the proliferative response. [Conclusion]: The mechanism of action of the peptides under study could be due to the capacity to sequester CaM and/or to the prevention of Grb7 dimerization, possibilities that are under study. These peptides, upon selected delivery to tumour cells, could have potential therapeutic effects against cancer and metastasis development.