Construction And Expression Of CXCR7Targeting Antagonists

BackgroundBreast cancer is the most malignant tumor of women with with highest morbidity and mortality. Cancer Journal for Clinicians magazine released statistics show that in2011an estimated230,480new cases of invasive breast cancer were expected to be diagnosed in women in the U.S, along with57,650new cases of non-invasive (in situ) breast cancer in2011. It accounts for30%in the new cases of malignant tumors of women. Breast cancer is also the leading cause of death for the female patient in developing country or district.Breast cancer possesses the characteristics of high incidence and aggressive, slow progression, easily to metastasize, and is difficult to be detected in early diagnosis. Therefore, the research of mechanism of occurrence, development and metastasis of breast cancer is very important. Breast cancer is a kind of tumor that highly dependent on blood vessel. Various events such as changes in the tumor microenvironment, local tumor microvascular generation and the cells invade the extracellular matrix and basement membrane in the primary tumor is closely related with chemokines and their receptors.Chemokine, also named as chemotactic hormone or chemical hormone, is a kind of cytokines that secreted by different types of cells with low relative molecular mass of8～12kD, which mainly function in regulation of immune cells chemotaxis movement. Chemokine receptor is a kind of membrane proteins belonging to the seven transmembrane G protein coupled receptor family that can specifically bind with chemokines. The binding of chemokine and its receptor can trigger or regulate relevant signal transduction and involved in various physiological and pathological processes.Stromal cell derived factor1(stromal cell-derived factor1, SDF-1), also named CXCL12, is a protein of CXC chemokines that produced by marrow stromal cells, including SDF-1α and SDF-1β SDF-1is the only chemokines that can combine with and activate the receptor CXCR4. SDF-1is widely expressed by various cells and tissues including immune cells, lung, brain, heart, kidney etc. Research have shown that the many cells and tissues of malignant tumors can synthesize and secrete CXCR4the receptor of SDF-1including breast cancer. SDF-1plays key roles in regulating B lymphopoiesis, the formation of hemopoietic stem cell, and lymphocytes migration. SDF-1also induces the hemopoietic stem cell moved from liver into the bone marrow in embryogenesis. There are abnormal development in heart and brain in early embryo of the SDF-1deficient mice.SDF-1mediates the actin aggregate and initiates the downstream signaling pathways by combining with CXCR4. It results in MEK/ERK phosphorylation and increased intracellular free calcium concentration. Besides, SDF-1participates in the formation of tumor migration and metastasis environment and tumor growth by inducing pseudopodia formation, regulating the expression and the activity of adhesion factors in tumor cells, thereby controlling the tumor cells growth, survival, metastasis and tumor angiogenesis.CXCR7is another newly found receptor of SDF-1whose ability to combine with SDF-1is stronger than CXCR4. Due to the lack of typical sequence of G protein coupled receptor, the binding of SDF-1and CXCR7does not cause cell calcium current change and cell migration, nor activation of intracellular MAP kinase or PI1kinase PKB signaling pathways. Ulrike Naumann and others found that CXCR7can remove SDF-1(CXCL12) andI-TAC(CXCL11) and reduce their concentrations. In zebrafish and mammalian cells, CXCR7was found to recycle constantly between plasma membrane and cytoplasm regardless of the existence of the ligand.or not. There is a view that, the MAP kinase is activated via β-arrestin instead of canonical activation of G protein signaling pathways after SDF-1binding to the decoy receptor CXCR7. The migration of vascular smooth muscle cells which express endogenous CXCR7can be significantly weakened by CXCR7antagonist or β-arrestin siRNA. Thus, β-arrestin mediates the signaling pathway of CXCR7, and CXCR7is an independent β-arrestin receptor.Although the specific funtion of CXCR7is not clear at present, there is no doubt that it plays an important role in immunity, tumor progression and angiogenesis. CXCR7can promote cancer cell survival. The wild type breast cancer cells MDA-MB-435s with lower expression of CXCR7are hard to survive in medium containing low concentration of serum (1%FBS), however, the cells transfected with CXCR7overexpression plasmid can grow into the logarithmic phase. Wang and others found that CXCR7is highly expressed in prostate cancer. The result of high density organic dye micro series showed that the expression of CXCR7on invasive tumors is more than others. The expression of CXCR7is positively correlated with cancer cells adhesion, invasion, proliferation and survival. CXCR7influence the expression of several molecules involved in tumor invasion, such as CD44, calcium binding (CDH11), IL-8, vascular endothelial growth factor, and TGF-beta. Specificly blocking of CXCR7is expected to become a new treatment strategy due to the multiple functions of CXCR7. Some small molecule inhibitors such as CCX733, CCX266, siRNA and blocking antibodies have been used in in vivo and in vitro experiments. In spite of this, on the whole, the mechanisms of CXCR7expression and regulation, and the signal pathway mediated by CXCR7remain unclarified and need further in-depth study.We have previously constructed a competitive antagonist of CXCR4, named SDF-1/54R, by gene reconstruction of SDF-1. It has been proved that SDF-1/54R could eliminate CXCR4on cell surface by induction of internalization of CXCR4quickly with no influence on its downstream signaling pathways, showing good anti-cancer effect. However, the SDF-1/54R protein was easily to be expressed in the way of inclusion body, tedious denaturation and refolding processes are needed to regain its biological activity. Except for this, low yield of the recombinant activated protein is another frustrating aspect.Base on the newly found receptor CXCR7of SDF-1and our previous study results of SDF-1/54R, we infer that SDF-1/54R could inhibit the proliferation and metastas of breast cancer by competitive inhibition, which is a mutant of SDF-1and CXCR7specific antagonist. In order to prove our hypothesis, we decided to use the SDF-1/54R/pET-30a+plasmid as the template to amplify the SDF-1/54R gene by PCR, and insert the gene into pET-41a+with GST label after double enzyme digestion. Then transform the correct recombinant pasmid into E.coli and obtain the soluble SDF-1/54R fusion protein after IPTG induction. The dialyzed recombinant protein was subjected to Enterokinase enzyme digestion and secondary purification to yield SDF-1/54R proteins for further research. Moreover, the breast cancer cell lines which overexpress CXCR7is selected by flow cytometry, and test the proliferation and metastas function mediate by CXCR7with this cell model. This study will lay an indispensable foundation for developing SDF-1/54R into a new polypeptides anti-cancer drug which targeting CXCR7. ObjectivesChange the prokaryotic expression vectors of pET-30a+into pET-41a+to yield soluble SDF-1/54R protein. And test the affect of SDF-1/54R on SDF-1/CXCR7mediated proliferation and metastasis of breast cancer cells.Methods1. Screening of breast cancer cells that overexpress CXCR7by flow cytometryA variety of breast cancer cell lines, including MCF-7, MDA-MB231were cultured in complete culture media. Then the cells were subjected to trypsin digestion when in good condition, collected and washed with PBS. The cells were suspended in200μl of PBS and divided into two s. One were added20μl PE-labeled mouse anti-human CXCR7monoclonal antibody, mix and incubated on ice for30min, another portion were added the same volume of the same type control antibodies. Then the cells were washed2times with PBS30min later and resuspended with300μl of PBS. The expression of CXCR7was analyzed by detecting the fluorescence intensity of the cell with flow cytometry.2.The construction and expression of SDF-1/54RIn order to obtain soluble SDF-1/54R protein, we designed the primers for PCR amplification using SDF-1/54R/pET-30a+as the template. Then oriented the PCR product into prokaryotic expression vector pET-41a+, transformed into E.coli BL21(DE3) and induced by chemical inducers IPTG. The expression of SDF-1/54R was tested by SDS-PAGE. Abundantly expression of SDF-1/54R was conducted after optimizing the expression conditions such as IPTG concentration, induction temperature and time.The soluble fusion protein was purified with GST affinity chromatography system, and eluted with glutathione elustion buffer, then collected and tested by SDS-PAGE. The purified fusion protein was subjected to dialysis for further subsequent enterokinase digestion. After optimizing the enzyme reaction condition, suchas reaction time, temperature and concentration in the pilot experiment, the N-terminal GST-tag of the recombinant fusion protein were digested according to Novagen recombinant the enterokinase manual. The digestion products were subjected to secondary separation and purification using GST affinity chromatography system. The SDF-1/54R protein was collected and tested by SDS-PAGE.3.The activity test of SDF-1/54RThe activity of SDF-1/54R was tested using in vitro experimental cell model. First, the induction of CXCR7internalization of SDF-1/54R was analyzed by flow cytometry using MCF-7cells which was proved to highly express CXCR7. The proliferation inhibition effect of SDF-1/54R on MCF-7cells was tested by MTT assay. Metastas effection of SDF-1/54R on MCF-7cells induces by SDF-1was assessed in transwell chemotaxis assay.Results1. Screening breast cancer cells that highly expressed CXCR7by flow cytometryTo detect expression of CXCR7on a variety of breast cancer cells membrane was detected by flow cytometry. It was found that CXCR7expression level is high in MCF-7and MDA-MB-231. The expression percentage were68.64%and66.13%in MCF-7and MDA-MB-231respectively2. Construction and expression of SDF-1/54R/pET-41a+The sequencing results show that the expression vector SDF-1/54R/pET-41a+was successfully constructed. The fusion protein SDF-1/54R was successfully expressed and in E. coli BL21(DE3).The recombinant fusion protein SDF-1/54R was abundantly expressed under most appropriate condition. After the route optimization of purification and dialysis, digestion and related technological process, the SDF-1/54R protein was obtained finally.3. The SDF-1/54R activity detectionThe result of flow cytometry shows that SDF-1/54R could significantly reduced the CXCR7in the membrane surface. MTT result shows that SDF-1/54R could inhibit the growth of breast cancer cell line MCF-7significantly within48hours. Transwell chemotaxis assay result shows that SDF-1/54R could inhibit the transmembrane of MCF-7induce by SDF-1/CXCR7.Conclusions1. Breast cancer cells we tested express CXCR7including MCF-7, MDA-MB-231, etc. Among which, the expression percentage of CXCR7on MCF-7was up to69.99%2. We successfully construct prokaryotic expression system which could express soluble fusion protein SDF-1/54R, setting a necessary basis for obtaining enough amount of active SDF-1/54R. The biological activaty, GST-removed protein SDF-1/54R was easily to obtain by optimizing purification, digestion conditions.3. SDF-1/54R showed the function of inducing internalization and elimination of CXCR7on MCF-7cell surface, and inhibiting the growth and metastas of tumor cell which express CXCR7. The results indicate that SDF-1/54R is a promising specific antagonist of CXCR7. The research has laid a good foundation for the functional studies of in vivo.