Tissue factor pathway inhibitor-2 was repressed by CpG hypermethylation through inhibition of KLF6 binding in highly invasive breast cancer cells.
BACKGROUND
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. Here, we have investigated the mechanism of DNA methylation on the repression of TFPI-2 in breast cancer cell lines.


RESULTS
We 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.


CONCLUSIONS
In 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.
Key Laboratory of Molecular Medicine, Ministry of Education, Yixueyuan Road 138#, Shanghai Medical College, Fudan University, Shanghai 200032, China. eagooselj@yahoo.com.cn
Hongshen
H
Guo
Yifeng
Y
Lin
Hongwei
H
Zhang
Juan
J
Liu
Nong
N
Zhang
Yiming
Y
Li
Desheng
D
Kong
Qiqun
Q
Tang
Duan
D
Ma
D001483:Base Sequence; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D045744:Cell Line, Tumor; D018899:CpG Islands / Q000235:genetics*; D019175:DNA Methylation; D015536:Down-Regulation; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D000075723:Kruppel-Like Factor 6; D051741:Kruppel-Like Transcription Factors / Q000037:antagonists & inhibitors* / Q000378:metabolism; D008969:Molecular Sequence Data; D009361:Neoplasm Invasiveness; D009362:Neoplasm Metastasis; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation / Q000235:genetics
D006023:Glycoproteins; C105465:KLF6 protein, human; D000075723:Kruppel-Like Factor 6; D051741:Kruppel-Like Transcription Factors; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; C021240:tissue-factor-pathway inhibitor 2