Screening Of Peptides Targeting FOXM1-DBD From Random Ph.D.-C7C Library And Preliminary Investigation On The Anti-Tumor Effects And Mechanisms Of 9R-CP29L Peptide

FOXM1 is a transcription factor of the mammalian Forkhead box（Fox）protein superfamily,which is overexpressed in most tumor cells including hepatocellular carcinoma,breast cancer,and lung cancer.Its high expression is closely associated with a variety of cancer phenotypes,including overproliferation,migration,invasion,anti-apoptosis,drug resistance,and angiogenesis,and therefore,FOXM1 is a very promising target for cancer therapy.In this study,the recombinantly expressed FOXM1-DBD protein was used as the target,and the peptides with high affinity to FOXM1 was screened by phage display technology.The anti-cancer activity and mechanism of the screened peptides were verified by in vitro cellular and molecular experiments.In this study,FOXM1-DBD protein was firstly expressed by inducing the recombinant E.coli strains containing the recombinant FOXM1-DBD gene,and the protein with high purity and concentration of 1.66 mg/m L was obtained after purification.Then,phage random peptide library of Ph.D.-C7C was used to screen against the FOXM1-DBD protein.The recovery rate of phage was gradually increased after four rounds of biopanning,and the phage monoclones from the third and fourth rounds were randomly piked up and sequenced.Phage ELISA and phage titer assay were performed to identify the affinity and specificity of different phages against to the target,and finally we obtained four peptides:CP13,CP18,CP29,FCP20 with high affinity and specificity.After synthesizing the four peptides with 9R membrane penetrating peptide,the anti-tumor activity of the four peptides（9R-CP13L、9R-CP18L、9R-CP29L、9R-FCP20L）were verified in four human tumor cells（human hepatoma Hep G2 cells,human high metastatic hepatocellular carcinoma cells HCCLM3,human non-small cell lung cancer cells A549,human triple negative breast cancer cells MDA-MB-231）.CCK8 results showed that the peptide 9R-CP29L exerted the highest inhibitory effect on tumor cells and the best effect on HCCLM3 cells among all the four peptides.30μM and 50μM of 9R-CP29L treatment for 48h achieved 96.2（±0.52）%and 99.1（±0.09）%inhibition rates on HCCLM3cells,respectively.Hence,9R-CP29L was selected as the optimal peptide for subsequent study.The strong inhibitory effects of 9R-CP29L on the viability of HCCLM3 and MDA-MB-231 cells were confirmed by CCK8 aasay.The IC₅₀ values of 9R-CP29L on HCCLM3 cells at12h,24h and 48h were 11.95±0.92μM,9.03±1.83μM and 9.26±0.69μM,respectively,and the IC₅₀ on MDA-MB-231 cells at three time periods were 25.59±5.49μM,11.11±0.52μM and 7.37±0.10μM,respectively.Subsequently,clone formation,AO-EB double fluorescent dye staining,Annexin V-FITC/PI apoptosis,Transwell,and cell cycle assays were performed with HCCLM3,and 9R-CP29L was found to significantly inhibit the proliferation and migration,induced apoptosis and arrest the cell cycle in G2 phase in HCCLM3 cells.Molecular docking confirmed the interactions of the peptide CP29L to FOXM1-DBD.CP29L can form hydrogen bonds with Asn283,His287,Asn288 and His292 in FOXM1-DBD,also the interaction of Pi-Cation and attractive charge interaction with Arg236,as well as the Pi-Pi T-shaped interaction with His287.The treatment of 9R-CP29L decreased the m RNA expression of FOXM1 and downstream target genes MMP2 and CDC25B,and increased the expression of pro-apoptotic genes BAX and CASPASE3,and significantly decreased the expression of FOXM1 and C-MYC protein.Differential gene expression analysis by RNA sequencing revealed that several genes closely related to cancer development were significantly down-regulated,such as EPHA10,EPHA3,DHFR,etc.Multiple tumor suppressor genes were upregulated,such as BAX,FOXO3,DKK1,etc.Differential gene signaling pathway enrichment analysis showed that some pathways closely related to cancer such as TNF signaling pathway,FOXO signaling pathway,AMPK signaling pathway,MAPK signaling pathway were significantly enriched.In summary,we obtained a peptide 9R-CP29L with strong anti-tumor activity by screening phage display peptide library of Ph.D.-C7C.9R-CP29L inhibits tumor cell proliferation and migration,promotes apoptosis and arrest cell cycle by regulating FOXM1and downstream genes and pathways.This study lays an important foundation for the development of drugs targeting FOXM1 protein for cancer therapy.