Aminoflavone upregulates putative tumor suppressor miR-125b-2-3p to inhibit luminal A breast cancer stem cell-like properties

Metastatic breast Cancer is incurable and often due to breast Cancer stem cell (CSC)-mediated self-renewal. We previously determined that the Aryl Hydrocarbon Receptor (AhR) agonist aminoflavone (AF) inhibits the expression of the CSC biomarker α6-integrin (ITGA6) to disrupt the formation of luminal (hormone receptor-positive) mammospheres (3D breast Cancer spheroids). In this study, we performed miRNA-sequencing analysis of luminal A MCF-7 mammospheres treated with AF to gain further insight into the mechanism of AF-mediated anti-cancer and anti-breast CSC activity. AF significantly induced the expression of >70 MicroRNAs (miRNAs) including miR125b-2-3p, a predicted stemness gene regulator. AF-mediated miR125b-2-3p induction was validated in MCF-7 mammospheres and cells. miR125b-2-3p levels were low in breast Cancer tissues irrespective of subtype compared to normal breast tissues. While miR125b-2-3p levels were low in MCF-7 cells, they were much lower in AHR¹⁰⁰ cells (MCF-7 cells made unresponsive to AhR agonists). The miR125b-2-3p mimic decreased, while the antagomiR125b-2-3p increased the expression of stemness genes ITGA6 and SOX2 in MCF-7 cells. In MCF-7 mammospheres, the miR125b-2-3p mimic decreased only ITGA6 expression although the antagomiR125b-2-3p increased ITGA6, SOX2 and MYC expression. AntagomiR125b-2-3p reversed AF-mediated suppression of ITGA6. The miR125b-2-3p mimic decreased proliferation, migration, and mammosphere formation while the antagomiR125b-2-3p increased proliferation and mammosphere formation in MCF-7 cells. The miR125b-2-3p mimic also inhibited proliferation, mammosphere formation, and migration in AHR100 cells. AF induced AhR- and miR125b2-3p-dependent anti-proliferation, anti-migration, and mammosphere disruption in MCF-7 cells. Our findings suggest that miR125b-2-3p is a tumor suppressor and AF upregulates miR125b-2-3p to disrupt mammospheres via mechanisms that rely at least partially on AhR in luminal A breast Cancer cells.

aminoflavone; aryl hydrocarbon receptor; breast cancer; cancer stem cells; mammospheres; miR125b-2–3p; therapeutic targeting.