Studies On Breast Cancer Treatment Mediated By ScFv1C9 Specific For FGF-1

The fibroblast growth factor 1, a member of FGF family, plays important role inproliferation of different kinds of cells and angiogenesis. However, studies have recentlyshown that FGF-1 is overexpressed in the early stages of several kinds of cancer, whichsuggest that FGF-1 is involved in the tumorigenesis and metastasis. In this study, slices ofnormal breast tissues, fibroadenomas tissues and breast ductal carcinoma tissues weresubjected to FGF-1 immunohistochemistry staining. The results indicated that FGF-1 wasoverexpressed in breast ductal carcinoma tissues, which suggest that FGF-1 might be acandidate for breast cancer immunotargeting. Thus, the variable regions of the light chain (VL)and heavy chain (VH) were cloned from an FGF-1 specific hybridoma and assembled intosingle chain variable fragment (scFv1C9) with a flexible linker which expected to be aneutralizing reagent of FGF-1.The cell lysates from HA-FGF-1, GFP overexpressing and HA-FGF-1, GFP-scFv1C9overexpressing MCF-7 cells were performed for immunoprecipitation assay. TheGFP-scFv1C9 was found to precipitate with HA-FGF-1. The results showed that therecombinant scFv1C9 retained enough affinity to bind with the antigen in vitro. To analyse theneutralizing function of scFv1C9, the scFv1C9 gene was stably expressed in MCF-7 cellmediated by lentivirus. Moreover, the cells expressing scFv1C9 had lower ability to inducetumor formation comparing with control cells.To investigate the mechanism that scFv1C9 neutralized the FGF-1, GFP orGFP-scFv1C9 overexpressed MCF-7 cells were stained with anti-FGF-1 antibody. In thecontrol cells overexpressing GFP, endogenous FGF-1 localized in the cytoplasm and nucleus.However, in GFP-scFv1C9 overespressing cells, endogenous FGF-1 was primarily detected inthe cytoplasmic compartment and co-localized with GFP-scFv1C9. These results indicatedthat overexpressed scFv1C9 targeted FGF-1 and blocked the nuclear import of FGF-1. Inaddition, we also analyzed the effect of scFv1C9 on the secretion of FGF-1 to the medium.Quantitative ELISA results indicated that scFv1C9 overexpression did not block theextracellular secretion of FGF-1. Accordingly scFv1C9 did not affect the receptor-dependentpathways.To explore what would happen upon blocking the nuclear translocation of FGF-1 inMCF-7 cells, the cell cycle distribution was analyzed by flow cytometry. GFP overexpressingcells had the following distribution of the cell cycle phases: 57-58% in G1, 25-31% in S and11-17% in G2/M phases, which was characteristic for proliferative cells. ContraryGFP-scFv1C9 overexpressing cells distributed with 73-82% in G1, 9-16% in S and 6-11% in G2/M which presented a senescent-like phenotype correlated with cell cycle arrest in G1phase, consequently, the cell proliferation index decreased significantly.In order to analyze the mechanisms of G1 arrest induced by scFv1C9 expression, wefirstly analyzed the expression of FGF-1 in scFv1C9 overexpressed cells. The RT-PCR resultsshowed that scFv1C9 did not affect the expression of FGF-1. Protein p21, an inhibitor of cellcycle, is an executant of cell cycle arrest. Therefore, we investigated the expression level ofp21 in scFv1C9 overexpressing cells. As expected, the p21 expression was up-regulated afterthe expression of scFv1C9. Moreover, p21 up-regulation could be attenuated byoverexpressing FGF-1 in MCF-7 cells. Together these findings showed that overexpressedscFv1C9 targeted FGF-1 and prevented the nuclear translocation of FGF-1, resulting inblocking of the intracrine pathway of FGF-1 which induced G1 arrest by p21 up-regulation inMCF-7 cells.To study the therapeutic use of scFv1C9 in vivo, the scFv1C9 gene was introduced intosolid tumor by electroporation. The conditions of electroporation is 100V/50ms/8pulses. Thetreatment was repeated five times. The body weight and tumor volume were measured onetime a week. The curve of body weight indicated that the weight was increased gradually,which showed the state of mice was healthy and the data was reliable. The weight and volumeof the tumor of the scFv1C9 group were markedly degraded comparing with the PBS andGFP group. The tumors were cut into slides and subjected for immunohistochemistry stainingof Ki-67 and CD31. The results showed that the Ki-67 index and MVD of the scFv1C9 groupwas reduced comparing with the PBS and GFP group. These results suggested that scFv1C9was an effective reagent for the treatment of breast cancer.In summary, it was demonstrated that scFv1C9 blocked the intracrine pathway of FGF-1by preventing its translocation, which regulated the expression of p21, resulting in the G0/G1areest. Further, the scFv1C9 gene was deliveried into solid tumor by in vivo electroporation.The results showed scFv1C9 exhibited the significant inhibition of MCF-7 transplanted solidtumor. These work has provided important foundation for the development of reagents forbreast cancer treatment.