The Role Of Protein 4.1N In Breast Cancer Metastasis

BackgroundBreast cancer is one of the most common malignancy in women and still represents a great health concern in China. Although the diagnosis and combined treatment, mainly the surgery and the radiotherapy & chemotherapy, of breast cancer have made great progresses, distant metastasis is still the main factor affecting the prognosis of patients. The recovery rate of non-metastatic breast cancer patients has been reported to be 98%, but can dramatically dicrease when the metastasis especially the internal organs and brain metastasis of the patients occur, with the 5-year survival rate being only 27%. So it is important to investegate the genes related to breast cancer genesis and identify the molecular mechanisms of tumor development and metastasis for bettering the clinical breast cancer treatment.Protein 4.1R, the member of the protein 4.1 gene superfamily, was first identified in blood which plays an important role in maintaining mechanical stability of red cell membrane. After that,3 homologous proteins,4.1G,4.1N and 4.1B, were also identified from mammalian cells which share the common homology with 4.1R and all contain three conserved domains:FERM domain (Four.1 protein, Ezrin, Radixin, Moesin), SABD (spectrin-actin-binding domain) and CTD (C-terminal domain). Previous study about protein 4.1 has showed the important function of these proteins in maintaining mechanical stability through co-localization bentween spectin and actin, and also promoting the interaction of spectrin-actin with transmembrane protein. With the identification of protein 4.1 family function in regulating transmembrane protein, more evidence suggest these proteins could have something to do with cell proliferation and motality. It was found that protein 4.1B was low-expressed in lung cancer, meningeoma, breast cancer and prostate cancer, and also found that protein 4.1R and 4.1G are involved in the genesis of brain tunor. Theses clinical evidence suggested the potential role in cancer development.As a cytoskeletal component, it is belived that 4.1s proteins play an important role in maintaining cell-cell and cell-matrix interactions, as well as the organization of the cytoskeleton. The facts that tumor genesis and metastasis process involved in the cell-cell and cell-matrix interaction changes, so 4.1s protein may have some potential roles in tumor metastasis. Previous studies have shown that 4.1B was related to metastasis of sarcoma cell lines and prostate cancer, but the roles of the other 4.1 members in tumor metastasis, especially 4.1N in breast cancer, have not been investigated. Based on these review, the study on relationship between protein 4.1N and breast cancer metastasis, and its functions in tumor development and metastasis were merit further investigated.Objective1. The expression and localization of protein4.1 family members (4.1R/G/N/B) in breast cancer cell lines with different metastatic capability were investigated. The relevance between expression and location of protein 4.1 s and metastatic of breast cancer was analized, followed by screening the 4.1s member which is closely related to breast cancer metastasis.2. The target protien 4.1 that closely related to breast cancer metastasis was expressed in cell line after transfection for the further functional mechanism sduty. 3. The role of screened 4.1 member in breast cancer metastasis was identified by comparision the biological characteristics before and after transfection in vitro.MethodsImmunolbloting and Immunolfluorescence methods were employed to detect the expression and subcellular location of 4.1R/G/N/B in breast cancer cell lines with different metastatic capability, MCF-7 (low metastatic), T-47D (middle metastatic) and MDA-MB-231 (high metastatic), by specific 4.1s antibodies. Protein was screened which is closely related to breast cancer metastasis. Then a plasmid fusion expressing this protein was constructed with EGFP and set up a stable transfected cell line. Finaly, the roles of the protein in breast cancer metastasis were identified by comparing the biological characteristics of breast cancer cells before and after transfection, cell proliferation assay, cell adhesion assay, cell migration and invasion assay.Results 1. Western blot and immunolfluorescence assay showed that 4.1R/B/G were all expressed in MCF-7, T-47D and MDA-MB-231 cells, but the subcellular location was different. In the MCF-7 and T-47D cells 4.1B protein was mainly located in the cell-cell junctions, In the highly metastatic MDA-MB-231 cells it was lacated in the nucleus, but 4.1R/G existed in the cytoplasm. Protein 4.1N was expressed in MCF-7 which was significantly higher than that in T-47D cells (P<005), but no expression was found in MDA-MB-231 cells. This result suggested that 4.1N may be closely related to breast cancer metastasis.2. IB and IF result showed that a stable cell line EGFP-4.1N/MDA-MB-231 was established which will serve as a cell model for the further research on 4.1N in breast cancer metastasis.3. Cell proliferation of MDA-MB-231 transfected with recombinant plasmid pEGFP-4.1N was significantly inhibited (P<0.05) compared to MDA-MB-231 which was transfected with empty vector and non-transfected controls. The adhesion of cells to matrix, cell migration and invasion were also significantly decreased (P<0.001).Conclusion1. Decreased expression of 4.1N protein is closely related to breast cancer metastasis.2. Study on the in vitro biological behavior showed that 4.1N can negatively regulate breast cancer cell proliferation and migration, which could become the potential target for cancer bio-therapy and drug development.