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Inhibition of histone demethylase, LSD2 (KDM1B), attenuates DNA methylation and increases sensitivity to DNMT inhibitor-induced apoptosis in breast cancer cells

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Abstract

Increasing evidence suggests that dysfunction of histone lysine demethylase is associated with abnormal chromatin remodeling and gene silencing, contributing to breast tumorigenesis. In silico analysis shows that the newly identified histone demethylase lysine-specific demethylase 2 is highly expressed in breast cancer, especially in invasive tumors. However, it is currently unknown how LSD2 regulates chromatin remodeling and gene expression regulation in breast cancer. Using short hairpin RNA, we stably knocked down LSD2 (LSD2-KD) in MDA-MB-231 breast cancer cells. LSD2-KD led to accumulation of H3K4me1/2 without changing methylation levels of other key histone lysine residues, suggesting that LSD2 acts as a bona fide H3K4 demethylase in breast cancer cells. LSD2-KD resulted in decreased colony formation and attenuated global DNA methylation in MDA-MB-231 cells. Additionally, treatment with the DNMT inhibitor, 5-aza-deoxycytidine (DAC), synergistically increased mRNA expression of aberrantly silenced genes important in breast cancer development, including PR, RARβ, ERα, SFRP1, SFRP2, and E-cadherin in LSD2-KD cells. Furthermore, LSD2-KD cells are more susceptible to cell death than scramble controls, and combined treatment with tranylcypromine, an LSD2 inhibitor, and DAC resulted in synergistic growth inhibition of breast cancer cells. DNMT inhibition by DAC in LSD2-KD cells led to internucleosomal DNA fragmentation, enhanced PARP cleavage and increased sub-G1 apoptotic cell population. These results demonstrate an important role for LSD2 in regulation of DNA methylation and gene silencing in breast cancer, and suggest that inhibition of LSD2 in combination with DNA methyltransferase inhibition represents a novel approach for epigenetic therapy of breast cancer.

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Abbreviations

LSD:

Lysine-specific demethylase

KD:

Knockdown

HDAC:

Histone deacetylase

DAC:

Decitabine

TCP:

Tranylcypromine

DNMT:

DNA methyltransferase

PGR (PR):

Progesterone receptor

ESR1 (ER):

Estrogen receptor

SFRP:

Secreted frizzled related protein

RAR:

Retinoic acid receptor

CDH1:

E-cadherin

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Acknowledgments

Partially supported by Breast Cancer Research Foundation, Samuel Winters Foundation, and Competitive Medical Research Fund of UPMC. These studies used the UPCI Genomics Core Facility supported by P30CA047904.

Conflicts of interest

The authors declare no competing interests.

Ethical standard

All experiments comply with the current laws of the United States of America.

Author information

Authors and Affiliations

  1. UPMC Cancer Research Pavilion, University of Pittsburgh Cancer Institute, Suite 500, 5150 Centre Ave, Pittsburgh, PA, 15232, USA

    Tiffany A. Katz, Shauna N. Vasilatos, Emily Harrington, Steffi Oesterreich, Nancy E. Davidson & Yi Huang

  2. Department of Pharmacology & Chemical Biology, University of Pittsburgh, Pittsburgh, PA, USA

    Tiffany A. Katz, Emily Harrington, Steffi Oesterreich, Nancy E. Davidson & Yi Huang

  3. Women’s Cancer Research Center, University of Pittsburgh, Pittsburgh, PA, USA

    Tiffany A. Katz, Shauna N. Vasilatos, Emily Harrington, Steffi Oesterreich, Nancy E. Davidson & Yi Huang

  4. Magee Womens Research Institute, Room 406, 204 Craft Ave, Pittsburgh, PA, 15213, USA

    Yi Huang

Authors
  1. Tiffany A. Katz
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  2. Shauna N. Vasilatos
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  3. Emily Harrington
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  4. Steffi Oesterreich
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  5. Nancy E. Davidson
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Corresponding authors

Correspondence to Nancy E. Davidson or Yi Huang.

Electronic supplementary material

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Supplemental Fig. 1

LSD2 gene expression is increased in breast cancer. Three data sets from the oncomine.org database are presented, as well as LSD2 OncoPrint data in cBioPortal.org. (PPTX 8122 kb)

Supplemental Fig. 2

Increased sensitivity in LSD2-KD cells is specific for DAC. Dose responses for 4-OH-tamoxifen, carboplatin, and lapatinib after 120 h and for paclitaxel, ABT-888, and doxorubicin after 96 h were assessed by crystal violet assay. Each point represents the mean of 3 experiments ± SEM (t test, * = p < 0.05) (PPTX 209 kb)

Supplementary material 3 (DOC 45 kb)

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Katz, T.A., Vasilatos, S.N., Harrington, E. et al. Inhibition of histone demethylase, LSD2 (KDM1B), attenuates DNA methylation and increases sensitivity to DNMT inhibitor-induced apoptosis in breast cancer cells. Breast Cancer Res Treat 146, 99–108 (2014). https://doi.org/10.1007/s10549-014-3012-9

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  • DOI: https://doi.org/10.1007/s10549-014-3012-9

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