Abstract 3602: Novel BRCA1 mimetic 35446HCl inhibits growth of anti estrogen resistant breast cancer cells through the NF-κB pathway
Authors: Nathan Shyam , Rosen Eliot M. 
Published: July 2017

Nearly a third of breast cancer patients develop resistance to antiestrogen treatments although the estrogen receptor ER-α continues to affect cell survival and proliferation. Thus there is a need for new interventions to inhibit ER-α function. Based on high-resolution mapping, we proposed interaction sites on both BRCA1 and ER-α in a three-dimensional model of BRCA1. Utilizing this model, we identified a class of compounds that fits into a predicted BRCA1-binding cavity within ER-α, mimics the ability of BRCA1 to repress ER-α activity, and functions differently than other antiestrogens. Previous publication has shown that the 1st generation BRCA1 mimetic compound (A7) reduced proliferation and inhibited ER-α activity in antiestrogen-sensitive and resistant breast cancer cells with negligible non-specific toxicity. More importantly we have shown that the 2nd generation compounds (35446, 4631) partially restored sensitivity to Tam/ICI-resistant cells (LCC9), caused dissociation of ER-α from a model estrogen response element (ERE) in MCF-7 cells, and inhibited growth of LCC9 tumor xenografts at nontoxic concentrations utilizing compound 35446-HCl (35446 prepared as a hydrochloride salt). The focus of this study is the mechanism of action of 35446-HCl compound’s inhibition of ER-α, which includes inhibition of a major signaling pathway and mode of cell death. Eight genes identified from RNA-seq in LCC9 cells treated with BRCA1 mimetic A7 were verified by qPCR using 35446-HCl. One verified gene is the NF-κB activator IKKB, which is linked to therapy resistance. Treatment with 35446-HCl inhibited IKKB expression and proliferation in LCC9 cells compared to untreated Control at 48 and 72 hrs (p&lt;0.0001). Transcriptional activity from the p65 promoter as measured with a luciferase assay was decreased in the presence of BRCA1 mimetic treatment (p&lt;0.0001). Expression of downstream targets of NF-κB, including genes known to inhibit apoptosis, was reduced (p value ranging from &lt; 0.05- 0.001). Apoptosis due to 35446-HCl treatment was verified by caspase-7 cleavage and increased TUNEL staining at both 48 and 72 hrs. In silico analysis of the IKBKB promoter within 10kb upstream of the transcription start site identified one putative full ERE and five putative half ERE sites. Of these, 35446-HCl inhibited the interaction of ER-α with the full ERE (p&lt;0.0001) and five half ERE (p value ranging from &lt; 0.05- 0.001) sites upstream of the IKKB start site in LCC9 cells as well as Tam resistant/ER-α mutated T47Dco cells. These results identify a mechanism of a novel compound that can overcome antiestrogen resistance.

Citation Format: Shyam Nathan, Eliot M. Rosen. Novel BRCA1 mimetic 35446HCl inhibits growth of anti estrogen resistant breast cancer cells through the NF-κB pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3602. doi:10.1158/1538-7445.AM2017-3602