BRCA1-mimetic compound NSC35446.HCl inhibits IKKB expression by reducing estrogen receptor-[alpha] occupancy in the IKKB promoter and inhibits NF-[kappa]B activity in antiestrogen-resistant human breast cancer cells

• Published in: Breast cancer research and treatment Vol. 166; no. 3; p. 681
• Main Authors: Nathan, Shyam, Ma, Yongxian, Tomita, York A, De Oliveira, Eliseu, Brown, Milton L, Rosen, Eliot M
• Format: Journal Article
• Language: English
• Published: Springer 01.12.2017
• Subjects: Analysis; Breast cancer; Estrogens; Phenols (Class of compounds); RNA
• ISSN: 0167-6806; 1573-7217
• DOI: 10.1007/s10549-017-4442-y

Purpose We previously identified small molecules that fit into a BRCA1-binding pocket within estrogen receptor-alpha (ER[alpha]), mimic the ability of BRCA1 to inhibit ER[alpha] 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), ER[alpha]-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 ER[alpha]-negative breast cancer cell lines. IKKB (IKK[beta], IKBKB), an upstream activator of NF-[kappa]B, 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-[kappa]B activity and expression of NF-[kappa]B target genes. Insilico analysis of the IKKB promoter identified nine estrogen response element (ERE) half-sites and one ERE-like full-site. ChIP assays revealed that ER[alpha] was recruited to the ERE-like full-site and five of the nine half-sites and that ER[alpha] 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 ER[alpha] and NSC35446.HCl-regulated gene, and they suggest that NF-[kappa]B and IKKB, which were previously linked to antiestrogen resistance, are targets for NSC35446.HCl in reversing antiestrogen resistance.