Identification Of The Receptor Of The Peptide BRBP1 Which Targets To Human Brain Metastatic Breast Cancer Cells

Breast cancer is the most common cancer in women with the increasing incidence rate every year which has become a serious threat to women's health. Breast cancer brain metastasis is the leading reason of disability and death. About 10-30% of patients with disseminated breast cancer develop CNS metastasis, generally with a median survival of 1 year, despite the standard treatment including whole brain radiation therapy (WBRT), stereotactic radiosurgery (SRS), and surgery. Therefore, it is critical to understand the molecular mechanisms underlying brain metastatic processes and to identify relevant targets.A novel peptide, BRBP1 (Patent number:ZL201210538671.4) was identified using an in vitro selection of a phage-displayed peptide library against human brain-seeking breast carcinoma cells (231-BR cells) previously in our lab. In vivo and in vitro studies showed that BRBP1 preferentially bound to the 231-BR cells. Based on BRBPl peptide, we designed near-infrared fluorescence probe which could be used for imaging of 231-BR cells in the tumor-bearing mice. We also constructed a targeting compound peptide based on BRBP1 which inhibited the formation of both large and micro-metastases which 231-BR cells induced. Not only that, BRBPl peptide was able to specifically bind to human breast cancer axillary lymph node tissues ex vivo. Thus identification of the receptor of the peptide BRBP1 is of great importance to deeply understand the function and application value. Our study includes the following several parts:1. Expression and the binding specificity of BRBPl-Fc peptibody targeting to brain metastasis breast cancer cellsBRBP1 sequence with EcoR I, Nco I enzymes was synthesized and subcloned into pFUSE-hIgG2-Fc2 vector. After sequencing validation, the plasmid was transfected into 293 T cell line. The expression of the recombinant plasmid was proved by Western blotting. The specific binding was observed by Enzyme Linked Immunosorbent Assay (ELISA), immunofluorescence and flow cytometry method. This part laid the foundation for the identification of BRBP1 peptide receptor.2. Identification of the specific receptor for BRBP1 peptide by co-immunoprecipitationThere are many methods to identify receptor at present and we take co-immunoprecipitation as a research approach. Through incubation of BRBP1-Fc peptibody with Protein G magnetic bead, BRBP1-BIOTIN peptide with streptavidin magnetosphere, we captured the receptor of the peptide BRBP1 on 231-BR cells membrane. By silver staining and mass spectrum, we acquired dozens of candidate receptor. Annexin A2, the common protein of twice mass spectrum result, was first analyzed. It was confirmed that the expression of Annexin A2 on 231-BR, MDA-MB-231, MCF-7 cell surface was coincident with desired receptor distribution by immunofluorescence experiment. Furthermore, we detected if there have been ligand-receptor relationship between BRBP1 and Annexin A2 by small interference RNA (si-RNA) and co-immunoprecipitation methods. Unfortunately we did not get a confirmed answer and subsequent study was needed. Additionally we obtained three candidate receptor molecules (SND1, S100A8, AP2B1) through bioinformatics software which required post-proved.3. Screening of BRBP1 peptide receptor based on bioinformatics toolsMatthew D. Dun and his colleagues have made a comprehensive, systematic, comprehensive, cutting-edge analysis on the difference of protein, mRNA and miRNA expression between brain metastases breast cancer cells 231-BR and parental cells MDA-MB-231. Riesa M. group determined gene expression patterns in parental breast cancer cells MDA-MB-231 and its lung-metastasis (LMD-231), bone-metastasis (BMD-231), adrenal-metastasis (ADMD-231) and brain metastasis (231-BR) variants. Through database retrieve and quantitative real time PCR, we find Moesin was up-regulated on 231-BR cells compared with MEA-MB-231 cells, MCF-7cells. Subsequently it was proved that the expression of Moesin on 231-BR, MDA-MB-231, MCF-7 cell surface was coincident with desired receptor distribution by western blotting, immunofluorescence, flow cytometry experiment. Similarly we detected the relationship between BRBP1 and Moesin by si-RNA technology. Until now we have initially identified the relationship between BRBP1 and Moesin by interference experiment.In conclusion, the core content of this research is to identify the receptor of the peptide BRBP1 targeting to brain metastases breast cancer cell line 231-BR. We have adopted co-immunityprecipitation-mass spectrometry analysis, database screening methods, and preliminarily confirmed that there was the interaction between BRBP1 and Moesin. In addition, other candidate molecules acquired by mass spectrometry need further research.