The Differential Selectivity of Aryl Hydrocarbon Receptor (AHR) Agonists towards AHR-Dependent Suppression of Mammosphere Formation and Gene Transcription in Human Breast Cancer Cells

Abstract

We had previously reported that treatment with the aryl hydrocarbon receptor (AHR) agonist β-naphthoflavone (βNF) suppressed mammosphere formation derived from cancer stem cells in human breast cancer MCF-7 cells (Cancer Lett., 317, 2012, Zhao et al.). Here, using several AHR agonists, we have investigated the association of this suppression with the classical ability to induce AHR-mediated gene transcription in the xenobiotic response element (XRE). The mammosphere formation assays were performed using wild-type and AHR-knockout MCF-7 cells in the presence of AHR agonists including 3-methylcholanthrene (3MC), benzo[a]pyrene (BaP), 7,12-dimethylbenz[a]anthracene (DMBA), 6-formylindolo[3,2-b]carbazole (FICZ), indirubin, indole-3-carbinol (I3C), indole-3-acetic acid (IAA), and kynurenine (KYN), followed by the XRE-reporter gene assays of the agonists. We showed that treatments with 3MC, BaP, and DMBA strongly suppressed mammosphere formation of the stem cells in an AHR-dependent manner, while other agonists showed weaker suppression. In reporter gene assays, the strength or duration of AHR/XRE-mediated gene transcription was found to be dependent on the agonist. Although strong transcriptional activation was observed with 3MC, FICZ, indirubin, I3C, IAA, or KYN after 6 h of treatment, only weak activation was seen with BaP or DMBA. While transcriptional activation was sustained or increased at 24 h with 3MC, BaP, or DMBA, appreciable reduction was observed with the other agonists. In conclusions, the results demonstrated that the suppressive effects of AHR agonists on mammosphere formation do not necessarily correlate with their abilities to induce AHR-mediated gene transcription. Hence, different AHR functions may be differentially induced in an agonist-dependent manner.

Aryl Hydrocarbon Receptor Directly Regulates VTCN1 Gene Expression in MCF-7 Cells

“…We reported that ligand-activated AhR suppressed tumorsphere (mammosphere) formation in MCF-7 cells. [2][3][4][5] On the other hand, we also found that AhR promoted cell migration and mammosphere formation in human epidermal growth factor receptor 2 overexpressing breast cancer cell lines. 6,7) In addition, AhR is involved in doxorubicin resistance in MDA-MB-231 cells.…”

Possible Involvement of the Upregulation of ΔNp63 Expression Mediated by HER2-Activated Aryl Hydrocarbon Receptor in Mammosphere Maintenance

“…Agonist-mediated AHR activation represses mammosphere formation and reduces the ALDH-high population, suggesting the repressive effects of AHR on breast CSCs [ 47 , 48 , 49 , 50 ]. Moreover, we have previously reported the suppressive effect of AHR (depending on the agonist used) on mammosphere formation in BC cells [ 22 , 51 ]. These results suggest that AHR has both pro- and anti-tumorigenic activities in cancers and that an agonist can determine its activity.…”

“…The mammosphere formation assay was performed as previously described [ 22 ]. Cells were seeded into 96-well ultra-low adherent plates at 1000 cells/well in MammoCult™ Medium (StemCell Technologies, Vancouver, Canada) and cultured for 7 days.…”

“…KpnI- and XhoI-digested fragments were inserted into the pGL4.24[luc2P/minP] vector (Promega). The pCMV-3Tag-6 plasmid containing the AHR-coding sequence (pCMV-3Tag-6 AHR) has been reported previously [ 22 ]. The pCMV-3Tag-6 ARNT plasmid was constructed by inserting amplified human ARNT cDNA into pCMV-3Tag-6 (Stratagene, Los Angeles, CA, USA) at the EcoRV/XhoI restriction sites.…”

“…The mammospheres were observed under a light microscope with 2× magnification (scale bar is 2000 μm) and 20× magnification (scale bar is 200 μm). The percentage of cells contributing to mammosphere formation was calculated as the number of spheres divided by the original number of seeded cells [ 22 ]. Each column represents the mean of six wells.…”