Aryl hydrocarbon receptor-microRNA-212/132 axis in human breast cancer suppresses metastasis by targeting SOX4

Hanieh Molecular Cancer (2015) 14:172 DOI 10.1186/s12943-015-0443-9 RESEARCH Open Access Aryl hydrocarbon receptor-microRNA-212/ 132 axis in human breast cancer suppresses metastasis by targeting SOX4 Hamza Hanieh Abstract Background: MicroRNAs (miRNAs) are a class of short non-coding RNAs that pave a new avenue for understanding immune responses and cancer progression. Although the miRNAs are involved in breast cancer development, their axis with the transcription factors that show therapeutic potential in breast cancer is largely unknown. Previous studies showed anti-metastatic roles of agonist-activated aryl hydrocarbon receptor (Ahr) in various breast cancer cell lines. Recently, we demonstrated that agonist-activated Ahr induced a highly conserved miRNA cluster, named miR-212/132, in murine cellular immune compartment. Therefore, current study was performed to examine if this miRNA cluster mediates the anti-metastatic properties of Ahr agonists. Methods: The expression of miR-212/132 cluster and coding genes were examined by real-time PCR, and the protein levels were detected by western blot. The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3,3′-diindolylmethane (DIM) were used to activate Ahr in MDA-MB-231 and T47D breast cancer cells. Chromatin immunoprecipitation (ChIP) assay was used to identify the binding site(s) for Ahr on miR-212/132 promoter. For prediction of potentially target gene of the miRNA cluster, bioinformatics analysis was carried out, and to test targeting, luciferase activity was quantified. Besides, biological effects of Ahr-miR-212/132 axis were examined in vitro by cell migration, expansion and invasion, and examined in vivo by orthotopic model of spontaneous metastasis. Results: The miR-212/132 cluster was transcriptionally activated in MDA-MB-231 and T47D cells by TCDD and DIM, and this activation was regulated by Ahr. A reciprocal correlation was identified between Ahr agonists-induced miR-212/132 and the pro-metastatic SRY-related HMG-box4 (SOX4), and a new specific binding sites for miR-212/132 were identified on the untranslated region (3′UTR) of SOX4. Interestingly, miR-212/132 over-expression showed direct anti-migration, anti-expansion and anti-invasion properties, and an inhibition of the miRNA cluster mitigated the anti-invasive properties of TCDD and DIM. Further in vivo studies demonstrated that the Ahr-miR-212/132-SOX4 module was induced by Ahr activation. Conclusion: Taken together, the findings provide the first evidences of the synergistic anti-metastatic properties of miR-212/132 cluster through suppression of SOX4. Also, current study suggest a new miRNA-based mechanism elucidating the anti-metastatic properties of Ahr agonists, suggesting possibility of using miR-212/132 to control metastasis in breast cancer patients. Keywords: miR-212/132, Aryl hydrocarbon receptor, Breast cancer, Metastasis Correspondence: Laboratory of Physiology, Biological Sciences Department, College of Science, King Faisal University, Faisal Bin Fahd road, Hofuf 31982 Ahsaa, Saudi Arabia © 2015 Hanieh. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Hanieh Molecular Cancer (2015) 14:172 Introduction Breast cancer is the most common cause of cancerassociated deaths amongst women in developed and developing countries [1]. Metastasis, the spread of a tumor to distant organs, accounts for 90 % of breast cancer patients’ mortality [2]. Important advances have been achieved to understand the complicated process of metastasis, and various molecules have shown promising anti-metastatic properties [3–5]. However, detailed mechanisms remain to be defined. The microRNAs (miRNAs) are small non-coding RNAs of ∼ 22 nt that regulate gene expression at the posttranscriptional level. These molecules add a new dimension for understanding cancer progression. An increasing paradigm has clearly shown that miRNAs are involved in breast cancer metastasis. For example, miR-10b promotes breast cancer cell invasion and metastasis by targeting syndecan-1 (SDC1) in MDA-MB-231 and MCF-7 cells [6]. The invasion of MDA-MB-231 and BT-20 cells is diminished by over-expression of c-Met-targeting miR-335 [7]. Furthermore, miR-135 and miR-203 reduce tumor growth and metastasis of MD-MB-231 cells to the bones by targeting the runt-related transcription factor 2 (Runx2) [8]. MiR-212 and miR-132 are tandem miRNAs at the same location on chromosome 17 in humans, called miR-212/ 132 cluster, and they share the same seed sequence and the transcriptional regulatory elements. Extensive studies have revealed important roles of this miRNA cluster in the different body systems, which may suggest potential therapeutic strategy. For example, miR-212/132 cluster is involved in mammary gland development [9, 10], neuronal differentiation and cognitive processes [11–13], cardiac hypertrophy and cardiomyocyte autophagy [14], autoimmune inflammation [15], vasodilatory function and angiogenic responses [16]. In breast cancer, miR-132 suppresses cell proliferation, invasion, migration and metastasis of different breast cancer cells through direct suppression of hematological and neurological expressed 1 (HN1) [17]. Over-expression of this miRNA suppresses proliferation and colony formation of MDA-MB-231 and MCF-7 [18]. Moreover, miR-132 causes expression changes of genes involved in metabolism, DNA damage and cell motility in immortalized fibroblasts co-cultured with epithelial columnar cell hyperplasia (CCH) cells [19]. Although the role of miR-212 has been investigated in different cancer types [20, 21], it has never been investigated alone or in a combination with miR-132 in breast cancer. The aryl hydrocarbon receptor (Ahr) is an environmentally responsive transcription factor activated by structurally diverse agonists see [22]. It is demonstrated that the Ahractive omeprazole decreases invasion and metastasis in estrogen receptor (ER)-negative breast cancer cell lines by Page 2 of 13 down-regulation of matrix metalloproteinase-9 (MMP-9) and C-X-C chemokine receptor 4 (CXCR4) [23]. Activation of Ahr by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 6-methyl-1,3,-trichlorodibenzofuran (MCDF) suppresses metastasis of ER-negative breast cancer cells to the lungs [24, 25]. Zhang and colleagues [25] suggest that both TCDD and MCDF induce miR-335 targeting the prometastatic mediator SRY-related HMG-box4 (SOX4). However, no more studies were performed to provide (...truncated)