Novel Aryl Hydrocarbon Receptor Agonist Suppresses Migration and Invasion of Breast Cancer Cells

RESEARCH ARTICLE Novel Aryl Hydrocarbon Receptor Agonist Suppresses Migration and Invasion of Breast Cancer Cells Hamza Hanieh1*, Omar Mohafez2,3, Villianur Ibrahim Hairul-Islam1,4, Abdullah Alzahrani1, Mohammad Bani Ismail1, Krishnaraj Thirugnanasambantham4 1 Biological Sciences Department, College of Science, King Faisal University, Hofuf Ahsaa, Saudi Arabia, 2 Biomedical Department, College of Clinical Pharmacy, Hofuf Ahsaa, Saudi Arabia, 3 Biochemistry Department, College of Pharmacy Al-Azhar University Assiut, Egypt, 4 Pondicherry Centre For Biological Sciences, Jawahar Nagar, Pondicherry, India * a11111 Abstract Background OPEN ACCESS Citation: Hanieh H, Mohafez O, Hairul-Islam VI, Alzahrani A, Bani Ismail M, Thirugnanasambantham K (2016) Novel Aryl Hydrocarbon Receptor Agonist Suppresses Migration and Invasion of Breast Cancer Cells. PLoS ONE 11(12): e0167650. doi:10.1371/journal. pone.0167650 Editor: Makoto Makishima, Nihon University School of Medicine, JAPAN Received: October 8, 2016 Accepted: November 17, 2016 Published: December 1, 2016 Copyright: © 2016 Hanieh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting data files. Funding: This work was funded by the Deanship of Scientific Research, King Faisal University, Hofuf, Saudi Arabia (Grants 160030 to HH and 160036 to AA). Competing Interests: All authors declare that they have no conflicts of interest. Despite the remarkable progress to fight against breast cancer, metastasis remains the dominant cause of treatment failure and recurrence. Therefore, control of invasiveness potential of breast cancer cells is crucial. Accumulating evidences suggest Aryl hydrocarbon receptor (Ahr), a helix-loop-helix transcription factor, as a promising target to control migration and invasion in breast cancer cells. Thus, an Ahr-based exploration was performed to identify a new Ahr agonist with inhibitory potentials on cancer cell motility. Methods For prediction of potential interactions between Ahr and candidate molecules, bioinformatics analysis was carried out. The interaction of the selected ligand with Ahr and its effects on migration and invasion were examined in vitro using the MDA-MB-231 and T47D cell lines. The silencing RNAs were transfected into cells by electroporation. Expressions of microRNAs (miRNAs) and coding genes were quantified by real-time PCR, and the protein levels were detected by western blot. Results The in silico and in vitro results identified Flavipin as a novel Ahr agonist. It induces formation of Ahr/Ahr nuclear translocator (Arnt) heterodimer to promote the expression of cytochrome P450 family 1 subfamily A member 1 (Cyp1a1). Migration and invasion of MDA-MB231 and T47D cells were inhibited with Flavipin treatment in an Ahr-dependent fashion. Interestingly, Flavipin suppressed the pro-metastatic factor SRY-related HMG-box4 (Sox4) by inducing miR-212/132 cluster. Moreover, Flavipin inhibited growth and adhesion of both cell lines by suppressing gene expressions of B-cell lymphoma 2 (Bcl2) and integrinα4 (ITGA4). PLOS ONE | DOI:10.1371/journal.pone.0167650 December 1, 2016 1 / 16 Flavipin Inhibits Breast Cancer in Ahr-Dependent Fashion Conclusion Taken together, the results introduce Flavipin as a novel Ahr agonist, and provide first evidences on its inhibitory effects on cancer cell motility, suggesting Flavipin as a candidate to control cell invasiveness in breast cancer patients. Introduction Breast cancer is a dominant cause of cancer-related death among cancer patients worldwide [1]. Despite the significant progress to fight against malignancy, the invasiveness and metastasis of breast cancer contribute to the high mortality rate [2]. In addition, the toxicity of the available drugs is a persistent problem in effective chemotherapies [3]. Therefore, development of drugs that show effective anticancer properties with less toxic effects is a priority [4, 5]. Recent decades have witnessed constant emergence of candidate molecules that show effective inhibition of cell motility in breast cancer [6–8]. However, there is a need for safe molecules of natural origin. The Aryl hydrocarbon receptor (Ahr) is a heterodimeric protein belongs to the helix-loophelix/Per-Arnt-Sim (bHLH/PAS) family of transcription factors. It is activated by wide range of natural and synthetic molecules. Upon ligation, Ahr heterodimerizes with the Ahr nuclear translocator (Arnt) to modulate cell functions through downstream genes such as cytochrome P450 (Cyp) [9, 10]. Recent paradigm of promising anti-breast cancer candidates has revealed that Ahr mediates their therapeutic potential. For example, (5S,7S)-7-methyl-3-(3-(trifluoromethyl)phenyl)-5,6,7,8-tetrahydrocinnolin-5-ol (NK150460) suppresses growth of different estrogen receptor (ER)-negative and ER-positive breast cancer cell lines via the molecular cascade of Ahr/Arnt [11]. The Ahr mediates the inhibitory effects of 2-(4-hydroxy-3-methoxyphenyl)-benzothiazole (YL-109) on growth and invasion of MDA-MB-231 through upregulation of Hsp70-interacting protein (CHIP) [12]. Furthermore, the icaritin derivative of prenylflavone inhibits cell growth of MCF-7 cells by destabilization of ERα in an Ahr-dependent fashion [13]. Due to the therapeutic potential role of Ahr against breast cancer, research efforts have been directed towards identification of new Ahr ligands. Importantly, Ahr has emerged as a potential therapeutic strategy to control motility of breast cancer cells including migration, invasiveness and metastasis. For example, omeprazole-activated Ahr based down-regulation of matrix metalloproteinase-9 (MMP-9) and C-X-C chemokine receptor 4 (CXCR4) suppresses invasion and metastasis of MDA-MB-231 cells [14]. The 3,30 -diindolylmethane (DIM) suppresses invasion of MCF-7 and T47D by inhibition of phosphorylation of type III epidermal growth factor receptor mutation (EGFRvIII) [15]. Previously, we found that DIM and the synthetic toxin 3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) suppress migration, invasion and metastasis of MDA-MB-231 and T47D in an Ahrdependent fashion [16]. These suppressive effects of Ahr agonists on cell motility were attributed mainly to the microRNA (miR)-212/132 cluster that targets the pro-metastatic SRYrelated HMG-box4 (Sox4). In lines, TCDD induces Sox4-targeting miR-335 to inhibit the motility of MDA-MB-231 [17]. Flavonoids are a wide range of natural compounds that have been intensively studied for their anticancer properties [18, 19]. Interestingly, some flavonoid compounds exert their effects through activation of Ahr [20, 21]. Unlike most of the known flavonoids, few studies have been performed to study the biological significance of Flavipin [22, 23] (...truncated)