BRCA1-mimetic compound NSC35446.HCl inhibits IKKB expression by reducing estrogen receptor-α occupancy in the IKKB promoter and inhibits NF-κB activity in antiestrogen-resistant human breast cancer cells

Abstract

Purpose

We previously identified small molecules that fit into a BRCA1-binding pocket within estrogen receptor-alpha (ERα), mimic the ability of BRCA1 to inhibit ERα activity (“BRCA1-mimetics”), and overcome antiestrogen resistance. One such compound, the hydrochloride salt of NSC35446 (“NSC35446.HCl”), also inhibited the growth of antiestrogen-resistant LCC9 tumor xenografts. The purpose of this study was to investigate the down-stream effects of NSC35446.HCl and its mechanism of action.

Methods

Here, we studied antiestrogen-resistant (LCC9, T47DCO, MCF-7/RR, LY2), ERα-negative (MDA-MB-231, HCC1806, MDA-MB-468), and antiestrogen-sensitive (MCF-7) cell lines. Techniques utilized include RNA-seq, qRT-PCR, cell growth analysis, cell-cycle analysis, Western blotting, luciferase reporter assays, TUNEL assays, in silico analysis of the IKKB gene, and ChIP assays.

Results

SC35446.HCl inhibited proliferation and induced apoptosis in antiestrogen-resistant LCC9, T47DCO, MCF-7/RR, and LY2 cells but not in ERα-negative breast cancer cell lines. IKKB (IKKβ, IKBKB), an upstream activator of NF-κB, was identified as a BRCA1-mimetic-regulated gene based on an RNA-seq analysis. NSC35446.HCl inhibited IKKB, IKKA, and IKKG/NEMO mRNA and protein expression in LCC9 cells. NSC35446.HCl also inhibited NF-κB activity and expression of NF-κB target genes. In silico analysis of the IKKB promoter identified nine estrogen response element (ERE) half-sites and one ERE-like full-site. ChIP assays revealed that ERα was recruited to the ERE-like full-site and five of the nine half-sites and that ERα recruitment was inhibited by NSC35446.HCl in LCC9 and T47DCO cells.

Conclusions

These studies identify functional EREs in the IKKB promoter and identify IKKB as an ERα and NSC35446.HCl-regulated gene, and they suggest that NF-κB and IKKB, which were previously linked to antiestrogen resistance, are targets for NSC35446.HCl in reversing antiestrogen resistance.

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Abbreviations

BCL2:

B-cell leukemia 2

BCLXL:

BCL2-related protein, long isoform

BRCA1:

Breast cancer susceptibility gene 1

CASP:

Caspase

ChIP:

Chromatin immunoprecipitation

CIAP1:

Baculoviral IAP repeat-containing protein 1

CSS:

Charcoal-stripped serum

DMSO:

Dimethyl sulfoxide

E2:

17β-estradiol

ERα:

Estrogen receptor α

ERE:

Estrogen response element

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

IKKB (IKBKB):

I kappa B kinase-β

IRF1:

Interferon regulatory factor-1

NCI/DTP:

National Cancer Institute/Developmental Therapeutics Program)

NF-κB:

Nuclear factor-kappa B

PVDF:

Polyvinylidene difluoride

qRT-PCR:

Quantitative reverse transcriptase polymerase chain reaction

TdT:

Terminal deoxytransferase

TRAF2:

TNF receptor-associated factor 2

XBP1:

X-box binding protein-1

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Acknowledgments

The authors wish to thank Garret Graham for his in silico contribution to the paper, Karen Creswell in the Flow Cytometry Shared Resource at the Lombardi Comprehensive Cancer Center (LCCC), and David Goerlitz, Archana Kishore, and MD Islam of the Genomics and Epigenomics Shared Resource at the LCCC. We also thank Dr. James Li for assisting with the data analysis of the RNA-seq experiment. And we thank Drs. Priscilla A. Furth, Anna Riegel, Steven J. Metallo, and Robert Clarke (LCCC and Georgetown University) for their helpful advice during the performance of these studies and the preparation of this manuscript.

Funding

This research was supported, in part, by grants from Susan G. Komen for the Cure (KG110580), the United States Public Health Service (R01-CA150646), and the Living in Pink Foundation.

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Authors

Contributions

S Nathan and EM Rosen conceived and designed all experiments. S Nathan and Y Ma developed the methodology and performed all experiments; S Nathan and EM Rosen were responsible for data acquisition, analysis, and interpretation. S Nathan and EM Rosen wrote the manuscript. EM Rosen served as the principal investigator. YA Tomita designed compounds A7 and NSC35446, and ML Brown and E De Oliveira synthesized compound NSC35446.HCl.

Corresponding author

Correspondence to Eliot M. Rosen.

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Competing interests

Drs. Tomita, Brown, and Rosen have submitted a patent application based on the compounds described in this manuscript.

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Nathan, S., Ma, Y., Tomita, Y.A. et al. BRCA1-mimetic compound NSC35446.HCl inhibits IKKB expression by reducing estrogen receptor-α occupancy in the IKKB promoter and inhibits NF-κB activity in antiestrogen-resistant human breast cancer cells. Breast Cancer Res Treat 166, 681–693 (2017). https://doi.org/10.1007/s10549-017-4442-y

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Keywords

  • BRCA1-mimetic
  • Estrogen receptor-alpha (ER-alpha)
  • Antiestrogen resistant
  • IKKB
  • NF-kappaB
  • NSC35446.HCl
  • A7 (NSC31303)