Screening A Phage Display Library For A Novel FGF8B-binding Peptide With Anti-tumor Effect On Prostate Cancer

Objective: Various experiments have shown increased expression of FGF8b in prostate cancerbut none or little in normal prostates. So we isolate a novel FGF8b-binding peptide by screeninga phage display library with FGF8b, and investigate the therapeutic potential and mechanism ofthe lead peptide in prostate cancer, thereby providing the base for developing FGF8b antagonists.Methods: Using FGF8b as the target, Ph.D.-7phagy display library was biopanned for3roundsto screen phage clones. The affinity and specificity of the phage clones were assessed by ELISA.DNA sequencing and identity predicting were applied to analyze the positive clones to select thelead peptide homologousing to FGFR3c D2. PC-3and HUVEC cells were chosen as target cellsfor further investigation. Cell viability was measured by MTT. Cell cycle progression wasdetermined by propidium iodide staining and flow cytometry. Western blotting was carried out todetect the expression of Cyclin D1, Proliferating Cell Nuclear Antigen (PCNA) and theactivation of Erk1/2, Akt, p38, JNK, FGF receptor substrate-2(FRS2), Signal Transducers andActivators of Transcription5(STAT5) phosphorylation. HUVEC migration was determined bymonolayer wound healing assay.Results: After3rounds biopanning, seven candidate peptides were isolated from twelve positiveclones. The peptide P12(HSQAAVP) corresponding to one of these candidates showed highhomology to the immunoglobulin-like (Ig-like) domain II(D2) of high-affinity FGF8b receptor(FGFR3c), contained3identical amino acids (AVP) to the authentic FGFR3D2sequence aa163–169(LLAVPAA) directly participating in ligand binding, carried the same charges as itscorresponding motif, suggesting that P12may have a greater potential to interrupt FGF8bbinding to its receptors than other identified heptapeptides do. Functional analysis indicated thatsynthetic P12peptides mediate significant inhibition of FGF8b-induced cell proliferation, arrestcell cycle at the G0/G1phase via suppression of Cyclin D1and PCNA, and blockade of theactivations of Erk1/2and Akt cascades in both prostate cancer cells and vascular endothelialcells. Moreover, P12significantly inhibited FGF8b-stimulatd HUVEC migration via blockade ofthe activations of p38and JNK cascades, and reduced FGF8b-FGFR-FRS2-induced STAT5phosphorylation, which involve in angiogenesis.Conclusion: We successfully isolated an FGF8b-binding peptide P12with high affinity and specificity from a phage display heptapeptide library, which provides an effective FGF8b/FGFRantagonist, and may have potential application for the treatment of a variety of cancers includingprostate cancer characterized by abnormal high expression level of FGF8b. Meanwhile, the studywould offer the basis for further researching the peptide antagonists of FGF8b.