The Regulatory Role And Mechanisms Of Centromere Protein U(CENP-U) In Breast Cancer Cell Proliferation And Progression

Background: CENP-U(centromere protein U,CENP-U), an important resident centromerebinding protein, is one of the important elements composing CCAN(constitutively centromere-associated network) in kinetochore lining, which participates in assemble of the middle mature kinetochore and takes an important role in the process of cellular mitosis and regulates cell-cycle status by recruitment of function-specific proteins.Recent studies indicated the expression of CENP-U was closely related to malignancy. Our research aims to demonstrate the effect and mechanism of CENP-U in breast cancer cells proliferation and metastasis in vivo, in vitro and clinical data, which would enrich the recognition of CENP-U function to provide new target for the breast cancer research in initiation and development.Methods:1. The expression of CENP-U was detected in Ductal carcinoma in situ(DCIS) tissue, invasive ductal breast cancer tissue and adjacent paired tissues by immunohistochemistry. Besides, we also compared CENP-U expression between the metastasis group and the non- metastasis group. Then, we analyzed whether CENP-U was associated with the prognosis of patients with invasive breast cancer.2. To detect CENP-U protein expression in normal mammary epithelial cells and in human breast cancer cell lines, Western-blotting and immunofluorescence(IF) was performed.3. We knocked down CENP-U by RNA interference and transfected CENP-U over-expression plasmids to cells. Then we chose soft agar colony formation assay and IF to compare the effect of CENP-U in the above cell lines.4. With fluorescence activated cell sorter(FACS), we analyzed whether Knockdown or over-expression CENP-U has any effect on cell cycle and the percentage of apoptotic cells. To explore the relationship between CENP-U and PI3K/AKT/NF-κB pathways, we tested several proteins by Western-blotting under the condition of knocking down or over-expressing CENP-U.5. Nude mouse transplantation tumor model was constructed to detect the effect of CENP-U on breast cancer in vivo.6. In order to find whether CENP-U has any effect on breast cancer metastasis, wound healing and transwell migration assays were performed. Besides, several proteins associated with EMT were detected by Western-blotting under the condition of knocking down or over-expressing CENP-U.7. We examined the expression of CENP-U in breast cancer stem cells(SCs) and non-stem cells(NSCs) by Western-blotting and the portion of ALDHhigh cells in sh CENPU/ p CDH-CENPU cells by flow cytometry.Results: 1. CENP-U was overexpressed in breast cancer. Positive ratio of CENP-U was seemed to be higher in the patients with metastasis. The Kaplan-Meier analysis showed that the DFS and OS in CENP-U negative invasive breast cancer patients was higher than that in the positive patients.2. The expression of CENP-U was lowest in MCF-10 A cell line and was highest in MDA-MB-231 cell line. Moreover, in the MDR breast cancer cells, the amount of CENP-U was significantly increased.3. The ability of proliferation and colony formation of sh CENP-U cells was significantly supressed compared with sh Controls, while the portion of apoptotic cells and G2/M cells were higher than which in sh CENP-U cells. PI3K-p110α, S6, NF-κB p65, AKT Ser473 and S6 Ser235/236 were detected lower in sh CENP-U cells.4. The ability of proliferation and colony formation of p CDH-CENP-U cells was increased compared with the p CDH-Control cells. And overexpression CENP-U induced cells into mitosis(G2/M). PI3K-p110α and NF-κB p65 were upregulated in p CDH-CENP-U cells. Also the expression of AKT Ser473 and S6 Ser235/236 were detected higher in p CDH-CENP-U cells.5. In vivo studies, nude mouse transplantation tumor model was constructed. When we injected 231-sh CENP-U into the nude mouse, we found the ability of tumorigenicity was obviously weaken compared with the sh Control cells.Otherwise, the tumor grew rapidly when injecting T47D-CENP-U cells compared with the T47D-Control cells.6. The results of wound healing and transwell migration assays revealed that the migration and invasive ability of 231-sh CENP-U cells was suppressed compared with the 231-sh Control cells. E-cadherin and β-catenin were increased, while Vimentin and N-cadherin were decreased. T47D-CENP-U cells were more invasive and migratory. Mesenchymal markers were upregulated and epithelial markers were downregulated.7. Compared with NSCs, the expression of CENP-U was significantly downregulated in SCs. We evaluated the portion of ALDHhigh cells in both p CDH-CENP-U and sh CENP-U cell lines. The results showed that there was no relationship between the portion of ALDHhigh cells and the expression level of CENP-U.Conclusion: 1. It was confirmed that the expression of CENP-U protein was related to tumorigenesis and development of breast cancer, and CENP-U may be used to predict DFS/OS of patients with breast cancer as prognostic marker.2. CENP-U may act as an oncogene in breast cancer, which affects the proliferation of breast cancer in vivo and in vitro through regulating the cell cycle, inhibiting cell apoptosis and changing the activity of PI3K/AKT/NF-κB pathway.3. Over-expression CENP-U might induce the EMT of breast cancer and improve the invasive ability. While slience CENP-U might inhibit the EMT.4. The expression of CENP-U was lower in breast cancer stem cells and the amount of CENP-U seemed to have no relation with breast cancer stem cells.