A Noncompetitive Small Molecule Inhibitor of Estrogen-regulated Gene Expression and Breast Cancer Cell Growth That Enhances Proteasome-dependent Degradation of Estrogen Receptor α

Abstract

The mechanisms responsible for 17␤-estradiol (E 2 )-stimulated breast cancer growth and development of resistance to tamoxifen and other estrogen receptor ␣ (ER␣) antagonists are not fully understood. We describe a new tool for dissecting ER␣ action in breast cancer, p-fluoro-4-(1,2,3,6,-tetrahydro-1,3-dimethyl-2-oxo-6-thionpurin-8-ylthio) (TPSF), a potent small-molecule inhibitor of estrogen receptor ␣ that does not compete with estrogen for binding to ER␣. TPSF noncompetitively inhibits estrogen-dependent ER␣-mediated gene expression with little inhibition of transcriptional activity by NF-B or the androgen or glucocorticoid receptor. TPSF inhibits E 2 -ER␣-mediated induction of the proteinase inhibitor 9 gene, which is activated by ER␣ binding to estrogen response element DNA, and the cyclin D1 gene, which is induced by tethering ER␣ to other DNA-bound proteins. TPSF inhibits anchorage-dependent and anchorage-independent E 2 -ER␣-stimulated growth of MCF-7 cells but does not inhibit growth of ER-negative MDA-MB-231 breast cancer cells. TPSF also inhibits ER␣-dependent growth in three cellular models for tamoxifen resistance; that is, 4-hydroxytamoxifen-stimulated MCF7ER␣HA cells that overexpress ER␣, fully tamoxifen-resistant BT474 cells that have amplified HER-2 and AIB1, and partially tamoxifen-resistant ZR-75 cells. TPSF reduces ER␣ protein levels in MCF-7 cells and several other cell lines without altering ER␣ mRNA levels. The proteasome inhibitor MG132 abolished down-regulation of ER␣ by TPSF. Thus, TPSF affects receptor levels at least in part due to its ability to enhance proteasome-dependent degradation of ER␣. TPSF represents a novel class of ER inhibitor with significant clinical potential. Estrogen receptor ␣ (ER␣)3 is a well studied member of the steroid/nuclear receptor family of transcription regulators. ER␣ acts in the nucleus to regulate gene expression by binding to estrogen response elements (EREs) and related DNA sequences (1-4) and through association with transcription factors bound at SP1 and AP-1 DNA binding sites (4 -7). In response to high affinity estrogen binding, ER␣ dimerizes, binds to ERE DNAs, and undergoes a conformational change in the ligand binding domain that facilitates the recruitment of coactivators (8). Bound coactivators promote assembly of a multiprotein complex that enables chromatin remodeling and stabilization of an active transcription complex (9 -11). In contrast, antagonist-occupied ER␣ recruits corepressors (12).At detection, growth of most human breast cancers depends on 17␤-estradiol (E 2 ) binding to ER␣ (13-16). Treatment strategies that inhibit estrogen-dependent breast cancer include selective ER modulators such as tamoxifen, which binds in the ER␣ ligand binding pocket, and aromatase inhibitors, which block estrogen production. Nearly half of patients treated with aromatase inhibitors develop resistance (17). The long-term effectiveness of tamoxifen is limited by the development of resistance in nearly all patients with metastatic breast cancer and in ϳ4...

Small Molecule Inhibitors as Probes for Estrogen and Androgen Receptor Action

“…MCF-7 cells grown in medium containing E 2 formed large colonies. The addition of 10 M TPSF completely blocked formation of MCF-7 cell colonies (47). Thus, TPSF inhibits estrogen stimulation of both anchorage-dependent and anchorage-independent growth of breast cancer cells.…”

“…Because TPSF has very little or no effect on the levels of AR and GR, TPSF is highly selective for down-regulation of ER. The proteasome inhibitor MG132 abolished down-regulation of ER␣ by TPSF (47). Thus, TPSF influences receptor levels at least in part due to its ability to enhance proteasome-dependent degradation of ER␣.…”

“…Because 30 M TPSF had no effect on the NF-B-mediated induction of IL-8 mRNA in MCF-7 cells and because very high concentrations of TPSF were required to inhibit GR-and AR-mediated transcription, TPSF is a selective inhibitor of ER␣ (47).…”

Cell-Based Protein Stabilization Assays for the Detection of Interactions between Small-Molecule Inhibitors and BRD4

“…21 Another interesting option is to use such assays for the identification and characterization of protein destabilizers (e.g., estrogen receptor antagonists). 22 Finally, our assays may be applied to identify or characterize pharmacological chaperones, which have been described to correct enzyme deficiencies. 23 Our results revealed that protein stability, which is a fundamental cellular mechanism, can be directly influenced by small-molecule ligands.…”

Estrogen receptor α inhibitor activates the unfolded protein response, blocks protein synthesis, and induces tumor regression

“…Noncompetitive ERα inhibitors targeting this unmet therapeutic need, including DIBA, TPBM, TPSF, and LRH-1 inhibitors that reduce ERα levels, show limited specificity, require high concentrations (>5 μM), and usually have not advanced through preclinical development (9)(10)(11)(12). These noncompetitive ERα inhibitors and competitor antiestrogens are primarily cytostatic and act by preventing estrogen-ERα action; therefore, they are largely ineffective in therapy-resistant ERα containing cancer cells that no longer require estrogens and ERα for growth.…”