Activation of the aryl-hydrocarbon receptor inhibits invasive and metastatic features of human breast cancer cells and promotes breast cancer cell differentiation

Abstract

The current statistics associated with breast cancer continue to show a relatively high recurrence rate together with a poor survival for aggressive metastatic disease. These findings reflect, in part, the pharmaceutical intractability of processes involved in the metastatic process and highlight the need to identify additional drug targets for the treatment of late-stage disease. In the current study, we report that ligand activation of the aryl-hydrocarbon receptor (AhR) inhibits multiple aspects of the metastatic process in a panel of breast cancer cell lines that represent the major breast cancer subtypes. Specifically, it was observed that treatment with exogenous AhR agonists significantly inhibited cell invasiveness and motility in the Boyden chamber assay and inhibited colony formation in soft agar regardless of estrogen receptor (ER), progesterone receptor, or human epidermal growth factor receptor 2 status. Knockdown of the AhR using small interfering RNA duplexes demonstrated that the inhibition of invasiveness was receptor dependent and that endogenous receptor activity was protective in each cell type examined. The inhibition of invasiveness and anchorage-independent growth correlated with the ability of exogenous AhR agonists to promote differentiation. Finally, exogenous AhR agonists were able to promote differentiation in a putative mammary cancer stem cell line. Cumulatively, these results suggest that the AhR plays an important role in mammary epithelial differentiation and, as such, represent a promising therapeutic target for a range of phenotypically distinct human breast cancers.

Figure 1

AhR is expressed in normal breast and in a range of human breast tumors. Tissues were derived from 48 patients, and RNA was isolated and provided as first-strand cDNAs on the TissueScan Breast Tissue pPCR Array Panel I (Origene Technologies; BCRT-01). qPCR was used to analyze hAhR expression in the 48 independent patient-derived tissue samples. Data were normalized to β-actin, and samples in each tissue type (Normal or stage 0–III) were averaged. Graphic data are depicted as relative fold expression over the average of values obtained using normal breast samples (set at 1). Error bars represent sd among independent samples of each tissue type. The distribution of the 48 patient samples represented as a function of breast tissue tumor grade was: normal breast: two samples; stage 0: five samples; stage I: 10 samples; stage II, 20 samples; stage III, 11 samples.

Figure 2

Dioxin responsiveness is a characteristic of both ER-negative and ER-positive breast cancer cells. A, CYP1A1, CXCR4, and MMP-9 in SKBR3 or MDA MB-231 ER-negative breast cancer cells after 24-h exposure to 10 nm TCDD. Cells were harvested for total RNA, which was used for qPCR. Tabular data are represented as fold induction over vehicle (Veh) (set at 1). B and C, Fold induction of CYP1A1, CXCR4, and MMP-9 in MCF-7 or ZR-75-1 ER-positive breast cancer cells. For panels A–C, data represent the average of triplicate amplification reactions for each condition in a representative experiment; n = 3 independent assays. *, P < 0.05 for comparison between Veh and TCDD. +, P < 0.05 for comparison between Veh and E₂. a, P < 0.05 for comparison between E₂ and E₂ + TCDD.

Figure 3

AhR agonists inhibit invasiveness of human breast cancer cells. A, Invasiveness of SKBR3 cells after 24-h exposure to vehicle (Veh) or multiple AhR ligands (TCDD, 10 nm; TCBDF, 10 nm; and DIM, 20 μm) and the AhR antagonist α-NF. Graphic data are represented as percent of Veh control (set at 100%). B, Invasiveness of MDA MB-231 cells after 24-h exposure to Veh or TCDD (10 nm). C, Invasiveness of MCF-7 cells after 24-h exposure to Veh or multiple AhR ligands (TCDD, 10 nm; TCBDF, 10 nm; and DIM, 20 μm) and the AhR antagonist α-NF in the presence and absence of 10 nm E₂. D, Invasiveness of ZR-75-1 cells after 24-h exposure to Veh or TCDD (10 nm) in the presence and absence of 10 nm E₂. For panels A–D, n = 3–4 independent assays, and bars are mean ± sd. *, P < 0.05 compared to vehicle.

Figure 4

AhR expression and activation is associated with protection against breast cancer cell invasiveness. qPCR analysis of endogenous AhR expression. SKBR3 cells (panel A) and MDA MB-231 cells (panel B) were transfected either a nontargeting siRNA duplex (Control si) or one of two independent AhR siRNAs (AhR-si-1, AhR-si-2). After 24 h, cells were treated with vehicle (Veh) or TCDD (10 nm) for 24 h. Cells were harvested 48 h after transfection for total RNA, which was used for qPCR. Graphic data are represented as fold induction over vehicle (set at 1). Data points represent the average of triplicate amplification reactions for each condition in a representative experiment (n = 3 independent assays). Invasiveness of SKBR3 cells (panel C) and MDA MB-231 cells (panel D) in the modified Boyden chamber invasion assay. Before assay, cells were transfected with siRNAs for 48 h and administered Veh or TCDD (10 nm) for 24 h. Graphic data are represented as percent of Veh control (set at 100%). For panels C and D, a representative experiment (n = 3 independent assays) is shown. In panels A–D, *, P < 0.05 for comparison between Control si and AhR-si-1 and Control si and AhR-si-2 for each treatment (Veh or TCDD).

Figure 5

AhR agonists inhibit colonization of breast cancer cells. Results from the soft-agar colony formation assay. Anchorage-independent growth of SKBR3 cells (A) and MDA MB-231 cells (B) after continuous exposure to multiple AhR ligands for 21 d (TCDD, 10 nm; TCBDF, 10 nm; and DIM, 20 μm) and the AhR antagonist α-NF (10 μm). Cells were plated in six-well plates in soft agar containing AhR ligand and overlaid with media containing ligand. Graphic data are represented as percent of Vehicle (Veh) control (set at 100%). For A and B, n = 5 independent assays for TCDD and TCDF; n = 3 independent assays for DIM and α-NF. Anchorage-independent growth of MCF-7 cells (C) or ZR-75-1 cells (D) after continuous exposure to multiple AhR ligands as described above, in the presence or absence of E₂ (10 nm) (for C and D, n = 3 independent assays). For A–D, bars are mean ± sd. *, P < 0.01. All independent assays contained triplicate wells for each treatment condition.

Figure 6

Activation of AhR induces markers of breast epithelial cell differentiation. A, SKBR3 cells were seeded in six-well plates and treated with vehicle (Veh), TCDD (10 nm), TCDBF (10 nm), or 9-cisRA (1 μm) for 48 h. Cells were imaged by light microscopy. SKBR3 cells (B) and MCF-7 cells (C) were seeded in six-well plates and treated for 48 h as described above. Cells were fixed, stained with Oil Red O, counterstained with hematoxylin, and imaged by light microscopy. Red granules represent cellular lipid deposits. For A–C, a representative experiment (n =3 independent experiments each) is shown in each case. D, SKBR3, MDA MB-231, MCF-7, and ZR-75-1 cells were treated for 48 h with Veh (lanes 1, 4, 7, and 10), 10 nm TCDD (lanes 2, 5, 8, and 11), 500 nm SAHA (lane 3), or 1 μm 9-cisRA (lanes 6, 9, and 12). Cells were lysed and whole-cell extracts were prepared and used for SDS-PAGE and Western blotting using a rabbit antihuman antibody to MFG1. A mouse antihuman β-actin antibody was used as a control for total protein between different samples. Shown is a representative experiments (n = 4 independent experiments). E, LA7 cells were seeded in four-well chamber slides and treated with TCDD, TCDBF, or 9-cisRA as described above for 48 h. Cells were fixed, stained with Oil Red O, counterstained with hematoxylin, and imaged by light microscopy.