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
Rosen, Eliot M
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 cellsShyam Nathan, MSDissertation Advisor: Eliot M. Rosen, MD, PhD.ABSTRACTThis dissertation examines the mechanism of action of the BRCA1-mimetic compound NSC35446.HCl and identifies a potential mechanism by which it is able to overcome anti-estrogen resistance in human breast carcinoma cell lines. Previous work has identified small molecule compounds that fit into a BRCA1-binding cavity within estrogen receptor-alpha (ERα), mimic the ability of BRCA1 to inhibit ERα activity (“BRCA1-mimetics”), and function differently from other anti-estrogens such as Tamoxifen and Fulvestrant. These BRCA1-mimetics inhibit ERα activity in both antiestrogen-sensitive and antiestrogen-resistant cells. One such compound, the hydrochloride salt of NSC35446 (“NSC35446.HCl”), also inhibited growth of antiestrogen-resistant LCC9 tumor xenografts. In this dissertation, I show that NSC35446.HCl inhibits proliferation of anti-estrogen resistant LCC9, T47DCO, MCF-7/RR, and LY2 cells but not of ERα-negative breast cancer cell lines (MDA-MB-231, MDA-MB-468, and HCC1806) and causes apoptosis in LCC9 and T47DCO cells but not in ERα-negative breast cancer cell lines. I identified IKKB, an upstream activator of NF-κB, as a BRCA1-mimetic-regulated gene, based on an RNA-seq analysis. I showed that NSC35446.HCl inhibited IKKB mRNA and protein expression in LCC9 cells. NSC35446.HCl also inhibited NF-κB transcriptional activity and expression of NF-κB target genes in ant-iestrogen resistant cell lines. In silico analysis of the IKKB (IKBKB) promoter identified nine estrogen response element (ERE) half-sites and one ERE-like full-site (which differs from the canonical ERE full-site by one nucleotide). Chromatin immunoprecipitation (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 strongly inhibited by compound NSC35446.HCl in LCC9 and T47DCO cells. These studies identified functional EREs within the IKKB promoter and identified the IKKB gene as a target of NSC35446.HCl. They further suggest that NF-κB, which has previously been linked to antiestrogen resistance, may be a target for NSC35446.HCl in reversing antiestrogen resistance.Index words: BRCA1-mimetic, estrogen receptor-alpha (ERα), antiestrogen resistant, IKKB, NF-κB
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