Expression Of Tissue Factor Pathway Inhibitor-2 In Different Stage Of Breast Tumor And Its Mechanism Of Down-Regulation

Tissue factor pathway inhibitor-2(TFPI-2),also known as placenta protein-5 (PP5)or matrix associated serine protease inhibitor(MSPI),is a protease inhibitor with three Kunitz domains and belongs to the superfamily of serine protease inhibitor. It plays a major role in ECM degradation during development,tumor cell invasion and metastasis,wound healing and angiogenesis.To observe the expression of TFPI-2 in different stages of breast tumor,we cooperated with Zhongshan Hospital and collected 83 breast tumor samples of in-patients,from Aug.2004 to Aug.2006,21 were benign and 61 were breast cancer tumor and we grouped the malignant by pTNM stage.Detection of TFPI-2 by immunohistochemistry revealed that the expression of TFPI-2 was down regulated in breast cancer and one way-ANOVA analysis showed that expression of TFPI-2 was significantly higher in benign than that in malignant and TFPI-2 in pTNMIV was significantly lower than that of other malignant groups(p<0.05).Then we applied Spearman correlation methods to analyze the relationship between TFPI-2 expression and pTNM staging,the results indicated that expression of TFPI-2 inversely correlated with the malignancy of breast cancer(r_s0.01=-0.41,p<0.01).We found that both protein and mRNA level of TFPI-2 could not be detected by western-blot and real-time PCR in highly invasive breast cancer cell MDA-MB-435, while in the low invasive breast cancer cell MCF-7 and T47D the expression of TFPI-2 was slightly decreased compared with normal human umbilical vein cell (HUVEC).Then we chose the highly invasive MDA-MB-435 cell and the low invasive MCF-7 for further investigating the silence mechanism of TFPI-2 during the development of breast cancer.The TFPI-2 expression vector pcDNA3-TFPI-2 and control vector pcDNA3 were transfected into MDA-MB-435 cells,and the stable overexpressing TFPI-2 clones were obtained.The transfected cells,namely pcDNA3-TFPI-2 and pcDNA3-mock,were injected subcutaneously in athymic nude mice.The growth of xenograft tumor was not fully suppressed by overexpression of TFPI-2,that is although the growth rate was decreased and final weight was smaller in TFPI-2 expression group,but the difference was not significant(p=0.324).However,the invasion of xenograft tumor to surrounding connective tissue was significantly suppressed.To further investigate the mechanism of TFPI-2 silence in breast cancer cell,a 1.5 Kb TFPI-2 promoter was cloned,and several genetic mutations were detected,but the promoter luciferase activities were not affected by the point mutation in the promoter region and the phenomena was further supported by delete mutation.It was revealed by delete mutation that the core promoter of TFPI-2 lies in -144 to+1 region. This region was densely methylated in MDA-MB-435,but no CpG methylation was detected in MCF-7.Methylation of promoter -144 in vitro significantly suppressed the promoter activity compared with nonmethylated promoter -144.Treating MDA-MB-435 cell with DNA methyltransferases(DNMT)inhibitor restored the expression TFPI-2 in dosage dependent manner.Scan mutation and informatics analysis identified the potential KLF6 binding site in TFPI-2 promoter and the CpG dinucleotides in the core sequence of KLF6 binding site was methylated in MDA-MB-435 cell revealed by bisulfite modified sequence. Finally,using Electrophoretic mobility shift assay(EMSA)and Chromatin immunoprecipitation(ChIP)assay,we demonstrated that CpG methylation blocked the binding of KLF6 to TFPI-2 promoter,diminished the trans-activation of KLF6 on TFPI-2 promoter.Our data suggested that the more malignancy the lower expression of TFPI-2 gene,and this phenomena might correlated with the growth,invasion and metastasis of breast cancer.Silence of TFPI-2 in highly invasive breast cancer MDA-MB-435 correlated with DNA methylation in TFPI-2 promoter,especially CpG methylation in KLF6 binding site;this abnormal hypermethylation of CpG dinucleotides affected the binding of transcription factor and might be the main mechanism of TFPI-2 silence in highly invasive breast cancer cell MDA-MB-435.Our data provide useful clues for elucidating the genesis and development mechanism of breast cancer.We will further investigate the relationship of histone modification and promoter methylation on the expression of TFPI-2.