BRCA1-mimetic compound NSC35446.HCl inhits IKKB expression by reducing estrogen receptor alpha occupancy in the IKKB promoter and inhibts NF-?B activity in anti-estrogen resitant human breast cells

We previously identified small molecules that fit into a BRCA1-binding pocket within estrogen receptor-alpha (ER?), mimic the ability of BRCA1 to inhibit ER? activity (“BRCA1-mimetics”), and overcome antiestrogen resistance. One such compound, the hydrochloride salt of NSC35446 (“NSC35446.HCl”), also inhibited 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?-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: NSC35446.HCl inhibited proliferation and induced apoptosis in antiestrogen resistant LCC9, T47DCO, MCF-7/RR, and LY2 cells but not in ER?-negative breast cancer cell lines. IKKB (IKKß, IKBKB), an upstream activator of NF-?B, was identified as a BRCA1-mimetic-regulated gene, based on an RNA-seq analysis; and NSC35446.HCl inhibited IKKB mRNA and protein expression in LCC9 cells. NSC35446.HCl also inhibited NF-?B activity and expression of NF-?B 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 ER? was recruited to the ERE-like full-site and five of the nine half-sites and that ER? 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 NSC35446.HCl-regulated gene; and they suggest that NF-?B and IKKB, which were previously linked to antiestrogen resistance, are targets for NSC35446.HCl in reversing antiestrogen resistance. Overall design: Examination of mRNA profiles of BRCA1 mimetic treated LCC9 cells: 4 samples Total: 2 Vehicle(DMSO) Control and 2 BRCA1 mimetic (A7) treated LCC9 cells