The Regulatory Role And Mechanisms Of Gametogenetin Binding Protein(GGNBP2) In Breast Cancer Cell Proliferation And Progression

Background:In silico analyses of 693 genes encoded in human chromosome 17q12-q23 78 region revealed a gene ~6.3 MB upstream of BRCA1 called gametogenetin binding protein 2(GGNBP2),which has a single C2H2 zinc finger and a consensus 52 Lxx LL nuclear receptor binding motif。Recent studies indicated the expression of GGNBP2 might be related to malignancy. Our research aims to demonstrate the effect and mechanism of GGNBP2 in breast cancer cells proliferation and metastasis in vivo and in vitro, which would enrich the recognition of GGNBP2 function to provide new target for the breast cancer research in initiation and development.Methods:1. To examine GGNBP2 expression in nine human tissues, we performed multiple tissue expression array analyses. A cancer expression array containing 50pair-wise human normal and breast tumor tissues were hybridized with radiolabeled probes to examine GGNBP2 expression in breast cancer samples.2. The Wnt1 transgenic mice are known to spontaneously develop mammary gland tumors. We performed semi-quantitative RT-PCR analyses both normal mammary gland tissue and cancer cells.3. To address the potential anti-breast tumor activity of GGNBP2 in vitro, we 350 expressed exogenous His-tagged human GGNBP2 in T47 D, MCF-7, MCF-10 A and MCF-10 F cell. Semi-quantitative RT-PCR analysis confirmed the expression of exogenous GGNBP2 in these stably transfected cells. we examined the effect of overexpression of exogenous 354 GGNBP2 on cell proliferation by 3-D culture and E2 irrtation.4. To test if GGNBP2 has a role in regulating cell migration, we performed wound-healing assays. To further confirm that overexpression of GGNBP2 can inhibit cell migration, we performed cell migration assays using Matrigel chemotactic chambers. To examine if GGNBP2 can inhibit anchorage-independent cell growth in T47 D cells, we performed soft-agar experiments.5. We performed an in vitro protein-protein interaction assay using in vitro transcribed/translated His-GGNBP2, ERα and ERβ.6. We transfected pc DNA3-His C control vector and pc DNA3-GGNBP2 expression vector along with an ERE reporter plasmid into T47 D cells and performed luciferase reporter assays. We performed Western blot analysis to examine CCND1 and TFF1 expression in stable control T47D-His C cells and T47D-GGNBP2.7. Nude mouse transplantation tumor model was constructed to detect the effect of CENP-U on breast cancer in vivo.Results:1. Quantitative analyses by normalizing to the housekeeping gene GAPDH showed that GGNBP2 expression was detected in all nine tissues examined with the highest levels in normal breast tissues and the lowest levels in normal kidney tissues. Quantitative analyses by normalizing to GAPDH showed that GGNBP2 expression levels were dramatically reduced in 45 of 50 breast tumor samples.Reduced GGNBP2 expression was also observed in metastatic cancer samples of the same patients.2. The Wnt1 transgenic mice are known to spontaneously develop mammary gland tumors. RT-PCR revealed that GGNBP2 m RNA levels were dramatically reduced in the mammary tumors from Wnt1 transgenic mice.3. The number of cell of GGNBP2-overexpressing ERα positive T47 D and MCF-7cells but not ERα negative MCF-10 A and MCF-10 F cells were significantly reduced after 4 days of culture, when compared to corresponding empty vector transfected(His C) cells.4. The proliferation of T47 D cell stably overexpressing-GGNBP2 and T47D-His C control cells were compared with or without E2. The results show that stable expression of exogenous GGNBP2 significantly repressed T47 D cell proliferation and also rendered the cells unresponsive to E2-induced cell proliferation. The cell number of T47D-GGNBP2 masses in 3-D culture was significantly reduced after 4 days of culture.5. Parental T47 D and T47D-His C cells almost completely migrated into the empty spaces. In contrast, only a few cells migrated to the empty space. There was a significant reduction of invaded cells in T47D-GGNBP2 when compared to either parental T47 D or T47D-His C control cells. The total number of colonies in T47D-GGNBP2 cells was significantly less than those in both parental T47 D and T47D-His C cells.6. ERα but not ERβ was pulled down by His-tagged GGNBP2. More ERαwas pulled down by His-tagged GGNBP2 in the presence of 1 μM E2 than in the absence of E2.7. E2-stimulated activation of the ERE-reporter was observed in T47 D cells transfected with His C control vector plasmid. However, transfection of pc DNA3-GGNBP2 inhibited E2-induced ERE activation by >50%.8. GGNBP2 over-expression substantially reduced tumor progression in xenograft mice relative to parental T47 D cell controls.Conclusion:1. Reduced GGNBP2 expression in human and mouse breast cancer samples It was confirmed that the expression of GGNBP2 was related to tumorigenesis and development of breast cancer.2. GGNBP2 inhibited the tumorigenic potential of ERα positive T47 D cells in vitro and in vivo.3. GGNBP2 functions as an ERα corepressor to influence the expression of its target genes, such as CCND1 and TFF1, and ultimately to control breast epithelial cell proliferation.