Search

Background Lysophosphatidic acid (LPA), generated extracellularly by the action of autotaxin and phospholipase A2, functions through LPA receptors (LPARs) or sphingosine-1-phosphate receptors (S1PRs) to induce pro-fibrotic signaling in the lower respiratory tract of patients with idiopathic pulmonary fibrosis (IPF). We hypothesized that LPA induces changes in small airway epithelial (SAE) basal cells (BC) that create cross-talk between the BC and normal human lung fibroblasts (NHLF), enhancing myofibroblast formation. Methods To assess LPA-induced signaling, BC were treated with LPA for 2.5 min and cell lysates were analyzed by phosphokinase array and Western blot. To assess transcriptional changes, BC were treated with LPA for 3 h and harvested for collection and analysis of RNA by quantitative polymerase chain reaction (qPCR). To assess signaling protein production and function, BC were washed thoroughly after LPA treatment and incubated for 24 h before collection for protein analysis by ELISA or functional analysis by transfer of conditioned medium to NHLF cultures. Transcription, protein production, and proliferation of NHLF were assessed. Results LPA treatment induced signaling by cAMP response element-binding protein (CREB), extracellular signal-related kinases 1 and 2 (Erk1/2), and epithelial growth factor receptor (EGFR) resulting in elevated expression of connective tissue growth factor (CTGF), endothelin-1 (EDN1/ET-1 protein), and platelet derived growth factor B (PDGFB) at the mRNA and protein levels. The conditioned medium from LPA-treated BC induced NHLF proliferation and increased NHLF expression of collagen I (COL1A1), smooth muscle actin (ACTA2), and autotaxin (ENPP2) at the mRNA and protein levels. Increased autotaxin secretion from NHLF correlated with increased LPA in the NHLF culture medium. Inhibition of CREB signaling blocked LPA-induced changes in BC transcription and translation as well as the pro-fibrotic effects of the conditioned medium on NHLF. Conclusion Inhibition of CREB signaling may represent a novel target for alleviating the LPA-induced pro-fibrotic feedback loop between SAE BC and NHLF.

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. 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. 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. 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.

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 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

Context: Resistance to conventional antiestrogens is a major cause of treatment failure and, ultimately, death in breast cancer. Objective: The objective of the study was to identify small-molecule estrogen receptor (ER)-α antagonists that work differently from tamoxifen and other selective estrogen receptor modulators. Design: Based on in silico screening of a pharmacophore database using a computed model of the BRCA1-ER-α complex (with ER-α liganded to 17β-estradiol), we identified a candidate group of small-molecule compounds predicted to bind to a BRCA1-binding interface separate from the ligand-binding pocket and the coactivator binding site of ER-α. Among 40 candidate compounds, six inhibited estradiol-stimulated ER-α activity by at least 50% in breast carcinoma cells, with IC50 values ranging between 3 and 50 μM. These ER-α inhibitory compounds were further studied by molecular and cell biological techniques. Results: The compounds strongly inhibited ER-α activity at concentrations that yielded little or no nonspecific toxicity, but they produced only a modest inhibition of progesterone receptor activity. Importantly, the compounds blocked proliferation and inhibited ER-α activity about equally well in antiestrogen-sensitive and antiestrogen-resistant breast cancer cells. Representative compounds disrupted the interaction of BRCA1 and ER-α in the cultured cells and blocked the interaction of ER-α with the estrogen response element. However, the compounds had no effect on the total cellular ER-α levels. Conclusions: These findings suggest that we have identified a new class of ER-α antagonists that work differently from conventional antiestrogens (eg, tamoxifen and fulvestrant).