Potent Inhibition of Breast Cancer by Bis-Indole-Derived Nuclear Receptor

Breast Cancer Research and Treatment (2019) 177:29–40 https://doi.org/10.1007/s10549-019-05279-9 PRECLINICAL STUDY Potent inhibition of breast cancer by bis‑indole‑derived nuclear receptor 4A1 (NR4A1) antagonists Erik Hedrick1,2 · Xi Li1 · Yating Cheng1,3 · Alexandra Lacey4 · Kumaravel Mohankumar1 · Mahsa Zarei1 · Stephen Safe1 Received: 30 January 2019 / Accepted: 13 May 2019 / Published online: 22 May 2019 © Springer Science+Business Media, LLC, part of Springer Nature 2019 Abstract Background Nuclear receptor 4A1 (NR4A1) is overexpressed in mammary tumors, and the methylene-substituted bis-indole derivative 1,1-bis(3′-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH) acts as an NR4A1 antagonist (inverse agonist) and inhibits NR4A1-regulated pro-oncogenic pathways/genes in breast and other cancer cells. Methods Buttressed analogs of DIM-C-pPhOH were synthesized by condensation of the substituted p-hydroxybenzaldehydes with indole. Breast cancer cell growth, survival, and migration assays were carried out by cell counting, Annexin V staining, and Boyden chamber assays, respectively. Changes in RNA and protein expression were determined by RT-PCR and western blots, respectively. Analysis of RNAseq results was carried out using Ingenuity Pathway Analysis, and in vivo potencies of NR4A1 antagonists were determined in athymic nude mice bearing MDA-MB-231 cells in an orthotopic model. Results Ingenuity Pathway analysis of common genes modulated by NR4A1 knockdown or treatment with DIM-C-pPhOH showed that changes in gene expression were consistent with the observed decreased functional responses, namely inhibition of growth and migration and increased apoptosis. DIM-C-pPhOH is rapidly metabolized and the efects and potencies of buttressed analogs of DIM-C-pPhOH which contain one or two substituents ortho to the hydroxyl groups were investigated using NR4A1-regulated gene/gene products as endpoints. The buttressed analogs were more potent than DIM-C-pPhOH in both in vitro assays and as inhibitors of mammary tumor growth. Moreover, using 1,1-bis(3′-indolyl)-1-(3-chloro-4-hydroxy- 5-methoxyphenyl)methane (DIM-C-pPhOh-3-Cl-5-OCH3) signifcant tumor growth inhibition was observed at doses as low as 2 mg/kg/d which was at least an order of magnitude more potent than DIM-C-pPhOH. Conclusions These buttressed analogs represent a more potent set of second generation NR4A1 antagonists as inhibitors of breast cancer. Keywords NR4A1 · bis-indole ligands · Antagonists · Anticancer activities Abbreviations C-DIMs Methylene-substituted DIMs DIM-C-pPhOH 1,1-bis(3′-indolyl)-1-(p- hydroxyphenyl)methane Electronic supplementary material The online version of this DIM-C-pPhOh- article (https ://doi.org/10.1007/s1054 9-019-05279 -9) contains 3-Cl-5-OCH 1,1-bis(3′-indolyl)-1- supplementary material, which is available to authorized users. 3 (3-chloro-4-hydroxy-5- * Stephen Safe methoxyphenyl)methane [email protected] DMSO Dimethyl sulfoxide EGFR Epidermal growth factor 1 Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, TX 77843, USA receptor NR4A1 Nuclear receptor 4A1 2 Present Address: Cleveland Clinic, Lerner Research Institute, Cleveland, OH 44195, USA SERMs Selective estrogen receptor modulators 3 Present Address: MD Anderson Cancer Center, Houston, TX 77030, USA 4 Present Address: Shell Oil, Houston, TX 77002, USA Vol.:(0123456789)1 3 30 Breast Cancer Research and Treatment (2019) 177:29–40 Background (3′- or 3′,5′-) to the hydroxyl groups since it has previously been reported that this substitution pattern hinders metab- The 48 members of the nuclear receptor (NR) superfamily olism (conjugation) of phenolic compounds [24]. The of transcription factors play essential roles in maintain cel- in vitro results clearly demonstrate the enhanced potency lular homeostasis and are also potential druggable targets of the buttressed analogs compared to DIM-C-pPhOH for many diseases including cancer [1, 2]. Pharmaceutical and the IC50 for breast tumor growth inhibition by one agents that directly target NRs are often selective receptor of the buttressed analog 1,1-bis(3′-indolyl)-1-(3-chloro- modulators which bind the receptor and exhibit tissue- 4-hydroxy-5-methoxyphenyl)methane (DIM-C-pPhOH-3- specifc receptor agonist or antagonist activities [2]. For Cl-5-OCH3) was approximately 2 mg/kg/d and greater than example, tamoxifen is a widely used selective estrogen 10-fold more potent than DIM-C-pPhOH. receptor modulator (SERM) for treating breast cancer and tamoxifen exhibits ER antagonist activity against breast cancer but is an ER agonist in the uterus [rev. in [3, 4]. Materials and methods Diferential expression of some NRs in cancer vs. non-cancer tissues has been observed and these receptors may also Cell lines, antibodies and chemicals be prognostic factors for drug-resistance, patient survival, and disease recurrence [5–11]. Muscat and co-workers MDA-MB-231 and SKBR3 human breast cancer cell lines [7] investigated the relative expression of NRs in breast were purchased from American Type Culture Collection tumors and normal breast and identifed 41 NRs in breast (Manassas, VA). MCF10A cells were kindly provided by cancers and 33 of these receptors exhibited diferential Dr. Weston Porter, Texas A&M University. Cells were expression in tumor versus non-tumor tissues. Twenty-six maintained 37 °C in the presence of 5% CO2 in Dulbecco’s NRs were higher in normal breast versus ER-positive and modifed Eagle’s medium/Ham’s F-12 medium with 10% ER-negative breast cancer and only seven NRs were more fetal bovine serum with antibiotic. β-Actin antibody and highly expressed in tumors. Two of the three NRs overex- Dulbecco’s Modifed Eagle’s Medium were purchased from pressed in both ER-positive and ER-negative breast tumors Sigma-Aldrich (St. Louis, MO). Sp1 antibody was purchased were members of the NR4A subfamily, namely NR4A1 from Millipore (Temecula, CA); bcl2, CHOP and epidermal (Nur77, TR3) and NR4A3 (Nor1) [7]. Subsequent stud- growth factor receptor (EGFR) antibodies were purchased ies on the expression, prognostic value, and functions of from Santa Cruz Biotech (Santa Cruz, CA). Cleaved cas- NR4A1 in breast cancer are contradictory and functional pase 3, cleaved poly ADP ribose polymerase (c-PARP), studies report that NR4A1 exhibits both pro- and anticar- phospho mTOR, mTOR, phospho S6RP, S6RP, phospho cinogenic activities [11–16]. 4EBP1, 4EBP1, XBP1-s, SERPINB5, GADD45α, and p21 Research in this laboratory has focused on a series antibodies were purchased from Cell Signaling Technologies of methylene-substituted bis-indole-derived compounds (Danvers, MA). TXNDC5 and IDH1 antibodies were pur- (C-DIMs) as NR4A1 ligands which exhibit tissue-specifc chased from Genetex (Irvine, CA). Apoptotic, Necrotic, and NR4A1 agonist or antagonist activities and are selective Healthy Cells Quantifcation Kit was purchased from Bio- NR4A1 modulators [8, 14–22]. In breast cancer and other tium (Hayward, CA). Cells were visualized under an EVOS solid tumors NR4A1, acts as a pro-oncogenic factor regu- f, fuorescence microscope, from Advanced Microscopy lating pathways/genes such as β1-integrin and TXNDC5 Group using a multiband flter set for FITC, rhodamine, and associated with cell proliferation, survival, and migra- DAPI. The C-DIM compounds were prepared as previously ′ tion/invasion and NR4A1 ligands typifed by 1,1-bis(3 - described by the condensation of indole with substituted p indolyl)-1-( -hydroxyphenyl)methane (DIM-C-pPhOH, benzaldehydes [9–11], and Supplemental Table S1 summa- CDIM8) act as antagonists [8, 14–20]. Previous studies in rizes the substituted benzaldehydes purchased from Sigma- breast, colon, kidney, pancreatic, and lung cancer cell lines Aldrich that were used to synthesize the buttressed analogs. and rhabdomyosarcoma cells show that both knockdown of NR4A1 (siNR4A1) or treatment with DIM-C-pPhOH decreased cell growth and migration, induced apoptosis, Cell proliferation assay and regulated expression of genes associated with these responses [8, 14–21]. Although DIM-C-pPhOH inhibits MDA-MB-231 and SKBR3 breast cancer cells and MCF10A 5 tumor growth in xenograft models (30 mg/kg/d) [8, 17, cells (1.0 × 10 per well) were plated in 12-well plates and 21], this compound is rapidly metabolized [23]. There- allowed to attach for 24 h, and cells were treated with vari- fore, we synthesized several buttressed analogs of DIM- ous C-DIM analogs (dimethyl sulfoxide, DMSO, as empty C-pPhOH containing at least one or two substituents ortho vehicle) for 18–24 h and efects on cell proliferation were determined as described [14]. 1 3 Breast Cancer Research and Treatment (2019) 177:29–40 31 Annexin V staining Ham’s F-12 medium in six-well plates. Cells were allowed to attach for 24 h after they were seeded and subsequently MDA-MB-231 and SKBR3 breast cancer cells(1.0 × 105 per treated with varying concentrations of C-DIM analogs well) were seeded in two-well Nunc Lab-Tek chambered for 24 h and western blots were determined as described B#1.0 Borosilicate coverglass slides from Thermo Scien- [14–16]. tifc (Waltham, MA) and were allowed to attach for 24 h and treatment-related efects on Annexin V staining were TNBC orthotopic xenograft model determined as described [14]. Female BALB/c nude mice (6–8 weeks old) were obtained Boyden chamber assay (Charles River Laboratory, Wilmington, MA) and maintained under specific pathogen-free conditions, housed MDA-MB-231 and SKBR3 breast cancer cells (3.0 × 105 per in isolated vented cages, and allowed to acclimate for well) were seeded in Dulbecco’s modifed Eagle’s medium/ 1 week with standard chow diet. The animals were housed Ham’s F-12 medium supplemented with 2.5% charcoal- at Texas A&M University Laboratory Animal Resources stripped fetal bovine serum and were allowed to attach for and Research facility in accordance with the standards of 24 h.

Load more