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Epigenetic silencing of the tumor suppressor klotho in human breast cancer

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Abstract

Klotho is a single pass transmembrane protein, associated with premature aging. We identified tumor suppressor activities for klotho, associated with reduced expression in breast cancer. We now aimed to analyze klotho expression in early stages of breast tumorigenesis and elucidate mechanisms leading to klotho silencing in breast tumors. We studied klotho expression, using immunohistochemistry, and found high klotho expression in all normal and mild hyperplasia samples, whereas reduced expression was associated with moderate and atypical ductal hyperplasia. Promoter methylation and histone deacetylation were studied as possible mechanisms for klotho silencing. Using bisulfite sequencing, and methylation-specific PCR, we identified KLOTHO promoter methylation in five breast cancer cell lines and in hyperplastic MCF-12A cells, but not in the non-tumorous mammary cell line HB2. Importantly, methylation status inversely correlated with klotho mRNA levels, and treatment of breast caner cells with 5-aza-2-deoxycytidine elevated klotho expression by up to 150-fold. KLOTHO promoter methylation was detected in 8/23 of breast cancer samples but not in normal breast samples. Chromatin immunoprecipitation revealed that in HB2 KLOTHO promoter was enriched with AcH3K9; however, in breast cancer cells, H3K9 was deacetylated, and treatment with the histone deacetylase inhibitor suberoylanilide bishydroxamide (SAHA) restored H3K9 acetylation. Taken together, these data indicate loss of klotho expression as an early event in breast cancer development, and suggest a role for DNA methylation and histone deacetylation in klotho silencing. Klotho expression and methylation may, therefore, serve as early markers for breast tumorigenesis.

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Acknowledgments

This research was supported by The Riva and Joel Koschitzky Fund for Breast Cancer Research at the Sheba Medical Center; the Chief Scientist Office of the Ministry of Health, Israel (grant no. 4055_3 to IW); the Israel Cancer Association Research Grant; The Israel Science Foundation (to I.W.); the “Talpiut” Sheba Career Development Award; and the Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.

Conflicts of interest

The authors declare no conflicts of interest.

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Authors and Affiliations

  1. The Institute of Oncology, Chaim Sheba Medical Center, 52621, Ramat-Gan, Israel

    Tami Rubinek, Michal Shulman, Shira Israeli, Adi Zundelevich, Einav Nili Gal-Yam, Bella Kaufman & Ido Wolf

  2. Tel Aviv University, Tel Aviv, Israel

    Ella Evron & Ido Wolf

  3. Department of Anatomic Pathology, Cedars-Sinai Medical Center, Los Angeles, CA, USA

    Shikha Bose

  4. Department of Oncology, Assaf Harofeh Medical Center, Zerifin, Israel

    Ayelet Avraham & Ella Evron

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  1. Tami Rubinek
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  2. Michal Shulman
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Correspondence to Tami Rubinek.

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Rubinek, T., Shulman, M., Israeli, S. et al. Epigenetic silencing of the tumor suppressor klotho in human breast cancer. Breast Cancer Res Treat 133, 649–657 (2012). https://doi.org/10.1007/s10549-011-1824-4

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  • DOI: https://doi.org/10.1007/s10549-011-1824-4

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