BACKGROUND: Different microRNAs have been shown to have oncogenic and tumor‐suppressive functions in human cancers. Detection of their expression may lead to identifying novel markers for breast cancer.METHODS: The authors detected miR‐340 expression in 4 human breast cell lines and then focused on its role in regulation of tumor cell growth, migration, and invasion and target gene expression. They then analyzed miR‐340 expression in benign and cancerous breast tissue specimens.RESULTS: Endogenous miR‐340 expression was down‐regulated in the more aggressive breast cancer cell lines, which was confirmed in breast cancer tissue specimens by using quantitative real‐time polymerase chain reaction. Further studies showed that induction of miR‐340 expression was able to suppress tumor cell migration and invasion, whereas knockdown of miR‐340 expression induced breast cancer cell migration and invasion. At the gene level, the authors identified c‐Met as a direct miR‐340 target to mediate cell migration and invasion through regulation of MMP‐2 and MMP‐9 expression. Ex vivo, loss of miR‐340 expression was associated with lymph node metastasis, high tumor histological grade, clinical stage, and shorter overall survival of breast cancer as well as increased c‐Met expression in breast cancer tissue specimens.CONCLUSIONS: miR‐340 may play an important role in breast cancer progression, suggesting that miR‐340 should be further evaluated as a novel biomarker for breast cancer metastasis and prognosis, and potentially a therapeutic target. Cancer 2011. © 2011 American Cancer Society.

Although multidrug-resistance-associated protein-1 (MRP1) is a major contributor to multi-drug resistance (MDR), the regulatory mechanism of Mrp1 still remains unclear. Nrf2 is a transcription factor that regulates cellular defense response through antioxidant response elements (AREs) in normal tissues. Recently, Nrf2 has emerged as an important contributor to chemo-resistance in tumor tissues. In the present study, the role of Nrf2-ARE pathway on regulation of Mrp1 was investigated. Compared with H69 lung cancer cells, H69AR cells with MDR showed significantly higher Nrf2-ARE pathway activity and expression of Mrp1 as well. When Nrf2 was knocked down in H69AR cells, MRP1's expression decreased accordingly. Moreover, those H69AR cells with reduced Nrf2 level restored sensitivity to chemo-drugs. To explore how Nrf2-ARE pathway regulates Mrp1, the promoter of Mrp1 gene was searched, and two putative AREs—ARE1 and ARE2—were found. Using reporter gene and ChIP assay, both ARE1 and ARE2 showed response to and interaction with Nrf2. In 40 cases of cancer tissues, the expression of Nrf2 and MRP1 was measured by immunohistochemistry (IHC). As the quantitive data of IHC indicated, both Nrf2 and MRP1 showed significantly higher expression in tumor tissue than adjacent non-tumor tissue. And more important, the correlation analysis of the two genes proved that their expression was correlative. Taken together, theses data suggested that Nrf2-ARE pathway is required for the regulatory expression of Mrp1 and implicated Nrf2 as a new therapeutic target for MDR.

MicroRNAs (miRNAs) play an important role in the regulation of cell growth, differentiation, apoptosis, and carcinogenesis. Detection of their expression may lead to identifying novel markers for breast cancer.

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. 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. 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.

Activation of AKT pathway by Nrf2/PDGFA feedback loop contributes to HCC progression