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Genomic screening for genes upregulated by demethylation revealed novel targets of epigenetic silencing in breast cancer

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Abstract

Breast cancer arises through the accumulation of multiple genetic alterations and epigenetic changes such as methylation, which silences gene expression in a variety of cancers. In the present study, we applied genomic screening to identify genes upregulated by the demethylating agent 5-aza-2′-deoxycytidine (DAC) in a human breast cancer cell line (MCF7). We identified 288 genes upregulated and 29 genes downregulated more than fivefold after treatment with DAC, and gene ontology analyses revealed the genes to be involved in immune responses, apoptosis, and cell differentiation. In addition, real-time PCR analysis of ten genes silenced in MCF7 cells confirmed that they are upregulated by DAC, while bisulfite-pyrosequencing analysis confirmed that nine of those genes were silenced by methylation. We also found that treating MCF7 cells with DAC restored induction of DFNA5 by p53, as well as by two other p53 family genes, p63γ and p73β. Introduction of NTN4 into MCF7 cells suppressed cell growth, indicating that NTN4 has tumor suppressive activity. In primary breast cancers, we detected cancer-specific methylation of NTN4, PGP9.5, and DKK3, suggesting that methylation of these genes could be useful markers for diagnosis of breast cancer. Thus, DNA methylation appears to be a common event in breast cancer, and the genes silenced by methylation could be useful targets for both diagnosis and therapy.

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Acknowledgments

The authors thank Dr. William F. Goldman for editing the manuscript. This study was supported in part by Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science, and Technology (K.I., T.T., and M.T.), Grants-in-Aid for Scientific Research (S) from Japan Society for Promotion of Science (K.I.), a Grant-in-Aid for the Third-term Comprehensive 10-year Strategy for Cancer Control, and Grant-in-Aid for Cancer Research from the Ministry of Health, Labor, and Welfare, Japan (M.T.).

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Authors and Affiliations

  1. Department of Molecular Biology, Cancer Research Institute, Sapporo Medical University, Sapporo, 060-8556, Japan

    Tomoko Fujikane, Minoru Toyota, Hiromu Suzuki, Reo Maruyama, Mutsumi Ohe-Toyota, Yasushi Sasaki & Takashi Tokino

  2. First Department of Surgery, Sapporo Medical University, Sapporo, 060-8556, Japan

    Tomoko Fujikane, Noriko Nishikawa, Toshihiko Nishidate, Tousei Ohmura & Koichi Hirata

  3. First Department of Internal Medicine, Sapporo Medical University, Sapporo, 060-8556, Japan

    Minoru Toyota, Hiromu Suzuki & Reo Maruyama

  4. Department of Biochemistry, Sapporo Medical University, South 1, West 17, Chuo-ku, Sapporo, 060-8556, Japan

    Minoru Toyota, Masami Ashida & Masahiro Kai

  5. Department of Public Health, Sapporo Medical University, Sapporo, 060-8556, Japan

    Masanori Nojima

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  1. Tomoko Fujikane
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Correspondence to Minoru Toyota.

Additional information

Tomoko Fujikane, Noriko Nishikawa, Minoru Toyota, Hiromu Suzuki contributed equally to this work.

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Fujikane, T., Nishikawa, N., Toyota, M. et al. Genomic screening for genes upregulated by demethylation revealed novel targets of epigenetic silencing in breast cancer. Breast Cancer Res Treat 122, 699–710 (2010). https://doi.org/10.1007/s10549-009-0600-1

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