Modulation of the Molecular Expression and Function of BCL‐2 Family Proteins by the Aryl Hydrocarbon Receptor in Breast Cancer Stem Cells of the MDA‐MB‐231

Breast cancer (BC) is a frequently occurring neoplasm in women worldwide and is a second major cause of cancer related deaths. It gains stemness by the formation of breast cancer stem cells (BCSCs) which are one of the tumor‐inducing populations of cells and cause BC recurrence. These cells possess cancer initiating properties and are hard to treat because of their self‐renewal characteristics. BCSCs survive through the maintenance of several cell signalling pathways among which aryl hydrocarbon receptor (AhR) has recently been evaluated. The AhR and Bcl‐2 family proteins are known to be expressed and play an important role in BC. However, the correlation between AhR and Bcl‐2 proteins, remains unclear. To test our hypothesis human epithelial MCF‐10A and breast cancer MDA‐MB‐231 cells were utilized as study model. The tumor spheroids of MDA‐MB‐231 cells were cultured in ultralow attachment flask provided with Stemflex medium. The cells were seeded in the cell culture plates for flow cytometry, RNA isolation, western blot analysis, Muse cell analysis and immunofluorescence, for each experiment. RNA and protein isolation were performed and the expression of AhR and Bcl‐2 family proteins in BC cells and BCSCs was determined through qRT‐PCR and western blot analyses. The functional studies of cells were performed using muse cell analyser and the cytoplasmic and nuclear localization of proteins were determined through immunofluorescence. The present study is conducted to explore the relationship between AhR and Bcl‐2 proteins in BCSCs development. The constitutive expression of AhR, CYP1B1 and anti‐apoptotic Bcl‐2 family proteins such as was found to be significantly higher in BCSCs (tumor spheroids) than non‐BCSCs differentiated cancer cells and MCF‐10A cells. To explore the role of AhR, we examined the effect of AhR antagonist, alpha‐Naphthoflavone (α‐NF), on the expression of BCL‐2 family proteins. Our results showed that chemical inhibition of AhR in MDA‐MB‐231 cells resulted in reduced expression of AhR, CYP1A1, and CYP1B1 which was associated with a proportional decrease in the mRNA and protein expression of BCL‐2 family; Bcl‐2, Mcl‐1 and bid. This inhibition also caused a significant reduction in CSCs (spheroid formation). The results from the immunofluorescence assay also indicated reduced expression of Bcl‐2 and AhR proteins upon treatment with AhR inhibitor, α‐NF. The reduced expression of AhR and Bcl‐2 family proteins consequently increased apoptosis (annexin V/PI) levels in MDA‐MB‐231 cells. The present study provides the first evidence of the crosstalk between AhR and Bcl‐2 proteins in MDA‐MB‐231 cells for the development of BCSCs. The results of this study are highly significant in the field of recognizing molecular targets for the treatment of BCSCs.