Effects Of Raf Kinase Inhibitor Protein On Breast Cancer Metastasis

Objective:Raf kinase inhibitor protein(RKIP) is a member of the phosphatid-ylethanolamine-binding protein(PEBP) family.It has various physiologies function(1-7). RKIP plays a pivotal modulatory role in several protein kinase signaling cascades. It has been shown to fit the criteria of being a metastasis suppressor gene in many kinds of malignant tumors. But its function not yet clarify completely in breast cancer.Our purposes is to determine whether RKIP is responsible for the metastasis suppression in breast cancer, whether it can be a biomarker in breast cancer, and whether RKIP can block breast cancer to transfer after regulating the level of RKIP.Methods Partâ… ,RKIP expression level assay:we investigated RKIP expression in different clinical specimens of breast tumors and their lymph node metastases use immunohistochemical approach,and then investigated RKIP expression in different breast cell lines MDA-MB-231,MDA-MB-435,HBL-100,T-47D,MCF-7with Real-time RT-PCR,Western Blot.Partâ…¡,The effect of RKIP on the malignant phenotypes of the breast cancer cells, we transfected the human Ss-RKIP, As-RKIP, pc3.1(+), pc3.1(-) cDNA into highly malignant breast cancer cell line MDA-MB-435 with FUGENE? HD Transfection Reagent.Clones of G418-resistant cells were selected. We evaluated the effect of RKIP in vitro on cell proliferation, soft-agar colony formation, cell adhesion, cell invasion, cell migration, morphological change in three-dimensional culture,and then measured the level of IL-8 in the culture supernatants. Flow cytometric analysis was performed to observe the effect of RKIP on cell cycle distribution.Partâ…¢,In vivo assay:Parental and transfectant clones were injected into the second mammary fat pads of athymic nude mice.After the mean tumor diameter reached 1.0-1.5 cm, tumors were surgically removed, and the wounds closed. Tumor tissues were measured and fixed paraffin embedding for histologic analysis.Four weeks later, mice were killed and examined for the presence of metastasis, assessment of angiogenesis and vascular invasion and the expression of RKIP.ResultsPartâ… ,RKIP expression was significantly higher in normal breast duct epithelia, hyperplastic breast tissue and ductal carcinoma in situ than that of invasive ductal breast carcinomas. The intensity of RKIP positive cells was significantly reduced in paired lymph node metastases than that of primary invasive ductal breast carcinomas. In five cell lines,RKIP expression is lower in metastatic cell lines:MDA-MB-435, MDA-MB-231,but higher in breast epithelial cell line HBL-100 and non-matastatic cell lines:T-47D,MCF-7.This distribution of RKIP down-regulation is statistically not significant but suggestive of a trend that breast metastatic cancer cells with reduced levels of RKIP expression.Partâ…¡, There were no statistically significant differences in in vitro proliferation or colony forming abilities between the control vector-transfected and sense RKIP vector-transfected cells or between the control vector-transfected and the antisense RKIP vector-transfected cells.High RKIP expression significantly suppressed the invasiveness, migration, adhesion and IL-8 secretion in vitro.Low RKIP expression significantly increased the invasiveness, migration, adhesion and IL-8 secretion in vitro. Furthermore, RKIP expression changed the shape of cells. Ss-RKIP cells became conglomeration.As-RKIP cells formed branches and linked each other to form tube-like structure.Partâ…¢, High RKIP expression significantly suppressed angiogenesis and decreased the number of lung metastases. In contrast,low RKIP expression significantly increased angiogenesis,and the number of lung metastases.There were no statistically significant differences in the volume of the tumors.ConclusionOur clinical data and in vivo/vitro assay show RKIP doesn't affect tumor growth,but it can suppress tumor metastasis.So we call RKIP a metastasis suppressor gene of breast cancer. It is suggesting that RKIP can be a prognostic biomarker to identify those patients with occult metastases.