1521-0111/93/2/168–177$25.00 https://doi.org/10.1124/mol.117.109827 MOLECULAR PHARMACOLOGY Mol Pharmacol 93:168–177, February 2018 Copyright ª 2018 by The American Society for Pharmacology and Experimental Therapeutics (Z)-2-(3,4-Dichlorophenyl)-3-(1H-Pyrrol-2-yl)Acrylonitrile Exhibits Selective Antitumor Activity in Breast Cancer Cell Lines via the Aryl Hydrocarbon Receptor Pathway s Jayne Gilbert, Geoffry N. De Iuliis, Mark Tarleton, Adam McCluskey, and Jennette A. Sakoff Experimental Therapeutics Group, Department of Medical Oncology, Calvary Mater Newcastle Hospital, Waratah, New South Wales, Australia (J.G., J.A.S.); and Priority Research Centre for Reproductive Science, Faculty of Science (G.N.D.I.), and Chemistry, School of Environmental and Life Sciences, Faculty of Science (M.T., A.M., J.A.S.), University of Newcastle, Callaghan, New South Wales, Australia Received July 4, 2017; accepted December 6, 2017 ABSTRACT We have previously reported the synthesis and breast cancer selectivity of ( Z)-2-(3,4-dichlorophenyl)-3-(1H-pyrrol-2-yl)- acrylonitrile (ANI-7) in cancer cell lines. To further evaluate the selectivity of ANI-7, we have expanded upon the initial cell line panel to now include the breast cancer cell lines (MCF7, MCF7/VP16, BT474, T47D, ZR-75-1, SKBR3, MDA-MB-468, BT20, MDA-MB-231); normal breast cells (MCF-10A); and cell lines derived from colon (HT29), ovarian (A2780), lung (H460), skin (A431), neuronal (BE2C), glial (U87, SJG2), and pancreatic (MIA) cancers. We now show that ANI-7 is up to 263-fold more potent at inhibiting the growth of breast cancer cell lines (MCF7, MCF7/VP16, BT474, T47D, ZR-75-1, SKBR3, MDA-MB-468) than normal breast cells (MCF-10A) or cell lines derived from other tumor types. Measures of growth inhibition, cell cycle analysis, morphologic assessment, Western blotting, receptor binding, gene expression, small interfering RNA technology, reporter activity, and enzyme inhibition assays were exploited to define the mechanism of action of ANI-7. In this work, we report that ANI-7 mediates its effects via the activation of the aryl hydrocarbon receptor (AhR) pathway and the subsequent induction of CYP1-metabolizing mono-oxygenases. The metabolic conversion of ANI-7 induces DNA damage, checkpoint activation, S-phase cell cycle arrest, and cell death in sensitive breast cancer cell lines. Basal expression of AhR, the AhR nuclear translocator, and the CYP1 family members do not predict for sensitivity; however, inherent expression of the phase II–metabolizing enzyme sulfur transferase 1A1 does. For the first time, we identify (Z)-2-(3,4-dichlorophenyl)-3-(1H-pyrrol-2-yl)- acrylonitrile as a new AhR ligand. Introduction Breast cancer is the most common cancer in women both in the developed and less developed world, and the incidence is on the rise. Early-stage breast cancer treatments include surgery and radiotherapy, whereas chemotherapy, hormonal, and tar- geted therapies are considered for more aggressive tumors. Tamoxifen and anastrozole are standard treatment of hormone- sensitive tumors; however, drug resistance is often induced and tumor selectivity is poor (Ma et al., 2015). Herceptin selectively targets the human epidermal growth factor receptor (HER2); however, 70% of HER2-positive patients fail to respond to treatment, with resistance developing rapidly. Herceptin also induces significant cardiac dysfunction in 2%–7% of patients. Even fewer options are available for triple-negative tumors that are receptor negative for estrogen (ER), progesterone, and HER2. Triple-negative breast cancers are vastly heterogeneous, hindering targeted therapy development (Sharma, 2016). Crit- ically and irrespective of the hormonal status, 33% of patients with initial breast cancer experience recurrence or metastasis, whereas 5% of new breast cancer patients present with metastases at diagnosis. The 5-year survival for these advanced breast cancer patients is only 25%, and, despite all efforts, metastatic breast cancer remains incurable (Steeg, 2016). The need to identify better therapies and translate these discoveries into the clinic has never been greater. We have previously reported the synthesis of (Z)-2-(3,4- dichlorophenyl)-3-(1H-pyrrol-2-yl)acrylonitrile (ANI-7, Fig. 1A) and identified it as a potent and selective inhibitor of cell growth in MCF-7 breast cancer cells (Tarleton et al., 2011), while having minimal to no effect on the growth of normal nontumor- derived breast cells or cells derived from other tumor types, including colon, ovarian, lung, skin, neuronal, glial, and pancreatic. Spurred on by this discovery, we set out to in- vestigate the breast cancer selectivity of ANI-7 and to identify its mode of action using standard cell-based technologies. In This work was supported by grants from Calvary Mater Newcastle Hospital Granting Scheme, Hunter Medical Research Institute, and Hunter Cancer Research Alliance, NSW, Australia. https://doi.org/10.1124/mol.117.109827. s This article has supplemental material available at molpharm. aspetjournals.org. ABBREVIATIONS: AhR, aryl hydrocarbon receptor; ANI-7, (Z)-2-(3,4-dichlorophenyl)-3-(1H-pyrrol-2-yl)acrylonitrile; ARNT, AhR nuclear translocator; BSA, bovine serum albumin; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethylsulfoxide; EGFR, epidermal growth factor receptor; ER, estrogen receptor; HER2, human epidermal growth factor receptor; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; P450, cytochrome p450; PB, phosphate buffer; siRNA, small interfering RNA; SULT, sulfur transferase; XRE, xenobiotic response element. 168 http://molpharm.aspetjournals.org/content/suppl/2017/12/21/mol.117.109827.DC1 Supplemental material to this article can be found at: at ASPET Journals on May 30, 2020 molpharm.aspetjournals.org Downloaded from