Tissue factor pathway inhibitor-2 was repressed by CpG hypermethylation through inhibition of KLF6 binding in highly invasive breast cancer cells

Hongshen Guo,Hongwei Zhang,Duan Ma,Desheng Kong,Yifeng Lin,Juan Liu,Qiqun Tang,Nong Zhang,Yiming Li

doi:10.1186/1471-2199-8-110

Abstract

BackgroundTissue factor pathway inhibitor-2 (TFPI-2) is a matrix-associated Kunitz inhibitor that inhibits plasmin and trypsin-mediated activation of zymogen matrix metalloproteinases involved in tumor progression, invasion and metastasis. Here, we have investigated the mechanism of DNA methylation on the repression of TFPI-2 in breast cancer cell lines.ResultsWe found that both protein and mRNA of TFPI-2 could not be detected in highly invasive breast cancer cell line MDA-MB-435. To further investigate the mechanism of TFPI-2 repression in breast cancer cells, 1.5 Kb TFPI-2 promoter was cloned, and several genetic variations 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 deleted mutation. Scan mutation and informatics analysis identified a potential KLF6 binding site in TFPI-2 promoter. It was revealed, by bisulfite modified sequence, that the CpG island in TFPI-2 promoter region was hypermethylated in MDA-MB-435. Finally, using EMSA and ChIP assay, we demonstrated that the CpG methylation in the binding site of KLF-6 diminished the binding of KLF6 to TFPI-2 promoter.ConclusionIn this study, we found that the CpG islands in TFPI-2 promoter was hypermethylated in highly invasive breast cancer cell line, and DNA methylation in the entire promoter region caused TFPI-2 repression by inducing inactive chromatin structure and decreasing KLF6 binding to its DNA binding sequence.

Highlights

Tissue factor pathway inhibitor-2 (TFPI-2) is a matrix-associated Kunitz inhibitor that inhibits plasmin and trypsin-mediated activation of zymogen matrix metalloproteinases involved in tumor progression, invasion and metastasis
Expression of TFPI-2 in breast cancer cells Expression of TFPI-2 protein in human breast cancer cell lines with different metastasis potential was examined by western blotting
Hypermethylation of promoter CpG islands, which is frequently observed in breast cancer [18,19,20], is often associated with transcriptional silencing of the associated gene. We explored both genetic and epigenetic mechanisms controlling TFPI-2 expression in human breast cancer cells and the results indicated that TFPI-2 expression could be silenced by promoter hypermethylation by inducing inactive chromatin structure and decreasing KLF6 binding to its DNA binding sequence

Summary

Introduction

Tissue factor pathway inhibitor-2 (TFPI-2) is a matrix-associated Kunitz inhibitor that inhibits plasmin and trypsin-mediated activation of zymogen matrix metalloproteinases involved in tumor progression, invasion and metastasis. Human tissue factor pathway inhibitor-2 (TFPI-2) is a kunitz-type serine proteinase inhibitor synthesized and secreted into extrocelluar matrix (ECM) by endothelial cells, smooth muscle cells, fibroblasts, keratinocytes and urothelium [1,2]. It had already been reported that the expression of TFPI-2 was down regulated in several invasive tumor cell lines, including choriocarcinoma, glioma, prostate cancer, melanoma and fibrosarcoma, ectopic expression of this gene inhibits tumors growth and metastasis in vivo by regulating pericellular ECM remodeling and angiogenesis [5,6,7,8,9,10]. The mechanisms that alter/modify the expression of TFPI-2 gene in cancer cells are not well understood

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