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Transcriptional repression of ER through hMAPK dependent histone deacetylation by class I HDACs

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Abstract

Anti-estrogen therapies are not effective in ER− breast cancers, thus identifying mechanisms underlying lack of ER expression in ER− breast cancers is imperative. We have previously demonstrated that hyperactivation of MAPK (hMAPK) downstream of overexpressed EGFR or overexpression/amplification of Her2 represses ER protein and mRNA expression. Abrogation of hMAPK in ER− breast cancer cell lines and primary cultures causes re-expression of ER and restoration of anti-estrogen responses. This study was performed to identify mechanisms of hMAPK-induced transcriptional repression of ER. We found that ER promoter activity is significantly reduced in the presence of hMAPK signaling, yet did not identify specific promoter sequences responsible for this repression. We performed an epigenetic compound screen in an ER− breast cancer cell line that expresses hMAPK yet does not exhibit ER promoter hypermethylation. A number of HDAC inhibitors were identified and confirmed to modulate ER expression and estrogen signaling in multiple ER− cell lines and tumor samples lacking ER promoter methylation. siRNA-mediated knockdown of HDACs 1, 2, and 3 reversed the mRNA repression in multiple breast cancer cell lines and primary cultures and ER promoter-associated histone acetylation increased following MAPK inhibition. These data implicate histone deacetylation downstream of hMAPK in the observed ER mRNA repression associated with hMAPK. Importantly, histone deacetylation appears to be a common mechanism in the transcriptional repression of ER between ER− breast cancers with or without ER promoter hypermethylation.

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Acknowledgments

This work was supported by grants from the National Institutes of Health (NIH 1R01 CA113674 to DEA and NIH 1R01NS067289 to NA), the Bankhead Coley Foundation (BC-09BW-04-RC1 to DEA), and by the Sylvester Comprehensive Cancer Center at the University of Miami Miller School of Medicine (to DEA). We would like to thank the Center for Therapeutic Innovation at the University of Miami Miller School of Medicine for the use of their epigenetic compound library and laboratory equipment, and Laura Parsons and Joeli Brinkman for technical contributions. We would also like to thank Drs. Marc Lippman, Zafar Nawaz, and Kerry Burnstein of the University of Miami for thoughtful discussion.

Disclosures

The authors declare that they have no conflicts of interest.

Author information

Authors and Affiliations

  1. Sheila and David Fuente Graduate Program in Cancer Biology, University of Miami Miller School of Medicine, 1501 NW 10th Ave., Miami, FL, 33136, USA

    Amy Plotkin

  2. Department of Psychiatry & Behavioral Sciences, Center for Therapeutic Innovation, University of Miami Miller School of Medicine, 1501 NW 10th Ave., Miami, FL, 33136, USA

    Claude-Henry Volmar, Claes Wahlestedt & Nagi Ayad

  3. Department of Medicine, Braman Breast Cancer Institute at the Sylvester Comprehensive Cancer Center, University of Miami Miller School of Medicine, 1501 NW 10th Ave., Biomedical Research Building, Rm 716, Miami, FL, 33136, USA

    Dorraya El-Ashry

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  1. Amy Plotkin
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Correspondence to Dorraya El-Ashry.

Electronic supplementary material

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Online Resource 1

Graph shows ESR1 promoter methylation status in a number of primary tumor cell lines, SUM cell lines, and engineered hyperactive ERK1/2 MAP kinase MCF-7 cells analyzed by the Epitect Methyl-II PCR assay (Qiagen). MDA-MB-231 serves as a positive control for ESR1 promoter methylation while MCF-7 cells are the negative control. Red boxes identify those cell lines utilized for screening and validation studies. Supplementary material 1 (PDF 33 kb)

Online Resource 2

a Plasmid map of SABiosciences pGreenFire lentiviral reporter vector. 4 tandem EREs were inserted into the multiple cloning site prior to the minimal CMV promoter upstream of GFP and luciferase genes. In the absence of ER activation, there is no expression of GFP or luciferase. b 24 h treatment with ethanol vehicle, 10 nM estradiol, 100 nM faslodex, or estradiol + faslodex. Y-axis is relative luciferase units (RLU). **p < 0.001. c Graphic representation of luciferase expression in response to compound + estradiol treatment. Expression is shown as percent of baseline expression, as calculated from untreated wells of the compound plate. Three standard deviations from the mean gives a range of 71–129 % of baseline leading to a number of candidates providing a statistically significant increase in luciferase expression, the highest expressers being HDAC inhibitors. Supplementary material 2 (PDF 567 kb)

Online Resource 3

Compounds represented in the plate-based compound screen. Compounds include: HDAC inhibitors, HDAC stimulators, histone lysine deacetylase inhibitors, HIF1a inhibitor, histone demethylase, histone acetyltransferase inhibitor, sirtuin inhibitors (type III HDAC), etc. Supplementary material 3 (PDF 47 kb)

Online Resource 4

Feedback loop involving class I HDACs, ERK1/2, and DUSP5. We hypothesize that class I HDAC inhibition leading to increased DUSP5 expression leads to reduced ERK1/2 phosphorylation/activation, leading to further decreased class I HDAC activity. This reduced HDAC activity further increases both DUSP5 and ER expression, leading to further reduced ERK1/2 phosphorylation. Supplementary material 4 (PDF 546 kb)

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Plotkin, A., Volmar, CH., Wahlestedt, C. et al. Transcriptional repression of ER through hMAPK dependent histone deacetylation by class I HDACs. Breast Cancer Res Treat 147, 249–263 (2014). https://doi.org/10.1007/s10549-014-3093-5

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  • DOI: https://doi.org/10.1007/s10549-014-3093-5

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