Aminoflavone

More than 40% of patients with luminal breast cancer treated with endocrine therapy agent tamoxifen demonstrate resistance. Emerging evidence suggests tumor initiating cells (TICs) and aberrant activation of Src and Akt signaling drive tamoxifen resistance and relapse. We previously demonstrated that aryl hydrocarbon receptor ligand aminoflavone (AF) inhibits the expression of TIC gene α6‐integrin and disrupts mammospheres derived from tamoxifen‐sensitive breast cancer cells. In the current study, we hypothesize that tamoxifen‐resistant (TamR) cells exhibit higher levels of α6‐integrin than tamoxifen‐sensitive cells and that AF inhibits the growth of TamR cells by suppressing α6‐integrin–Src–Akt signaling. In support of our hypothesis, TamR cells and associated mammospheres were found to exhibit elevated α6‐integrin expression compared with their tamoxifen‐sensitive counterparts. Furthermore, tumor sections from patients who relapsed on tamoxifen showed enhanced α6‐integrin expression. Gene expression profiling from the TCGA database further revealed that basal‐like breast cancer samples, known to be largely unresponsive to tamoxifen, demonstrated higher α6‐integrin levels than luminal breast cancer samples. Importantly, AF reduced TamR cell viability and disrupted TamR mammospheres while concomitantly reducing α6‐integrin messenger RNA and protein levels. In addition, AF and small interfering RNA against α6‐integrin blocked tamoxifen‐stimulated proliferation of TamR MCF‐7 cells and further sensitized these cells to tamoxifen. Moreover, AF reduced Src and Akt signaling activation in TamR MCF‐7 cells. Our findings suggest elevated α6‐integrin expression is associated with tamoxifen resistance and AF suppresses α6‐integrin–Src–Akt signaling activation to confer activity against TamR breast cancer.

We have previously reported the synthesis and breast cancer selectivity of (Z)-2-(3,4-dichlorophenyl)-3-(1H-pyrrol-2-yl)acrylonitrile (ANI-7) in cancer cell lines. To further evaluate the selectivity of ANI-7, we have expanded upon the initial cell line panel to now include the breast cancer cell lines (MCF7, MCF7/VP16, BT474, T47D, ZR-75-1, SKBR3, MDA-MB-468, BT20, MDA-MB-231); normal breast cells (MCF-10A); and cell lines derived from colon (HT29), ovarian (A2780), lung (H460), skin (A431), neuronal (BE2C), glial (U87, SJG2), and pancreatic (MIA) cancers. We now show that ANI-7 is up to 263-fold more potent at inhibiting the growth of breast cancer cell lines (MCF7, MCF7/VP16, BT474, T47D, ZR-75-1, SKBR3, MDA-MB-468) than normal breast cells (MCF-10A) or cell lines derived from other tumor types. Measures of growth inhibition, cell cycle analysis, morphologic assessment, Western blotting, receptor binding, gene expression, small interfering RNA technology, reporter activity, and enzyme inhibition assays were exploited to define the mechanism of action of ANI-7. In this work, we report that ANI-7 mediates its effects via the activation of the aryl hydrocarbon receptor (AhR) pathway and the subsequent induction of CYP1-metabolizing mono-oxygenases. The metabolic conversion of ANI-7 induces DNA damage, checkpoint activation, S-phase cell cycle arrest, and cell death in sensitive breast cancer cell lines. Basal expression of AhR, the AhR nuclear translocator, and the CYP1 family members do not predict for sensitivity; however, inherent expression of the phase II-metabolizing enzyme sulfur transferase 1A1 does. For the first time, we identify (Z)-2-(3,4-dichlorophenyl)-3-(1H-pyrrol-2-yl)acrylonitrile as a new AhR ligand.