High AHR expression in breast tumors correlates with expression of genes from several signaling pathways namely inflammation and endogenous tryptophan metabolism

Abstract

Increasing epidemiological and animal experimental data provide substantial support for the role of aryl hydrocarbon receptor (AhR) in mammary tumorigenesis. The effects of AhR have been clearly demonstrated in rodent models of breast carcinogenesis and in several established human breast cancer cell lines following exposure to AhR ligands or AhR overexpression. However, relatively little is known about the role of AhR in human breast cancers. AhR has always been considered to be a regulator of toxic and carcinogenic responses to environmental contaminants such as TCDD (dioxin) and benzo[a]pyrene (BaP). The aim of this study was to identify the type of breast tumors (ERα-positive or ERα-negative) that express AHR and how AhR affects human tumorigenesis. The levels of AHR, AHR nuclear translocator (ARNT) and AHR repressor (AHRR) mRNA expression were analyzed in a cohort of 439 breast tumors, demonstrating a weak association between high AHR expression and age greater than fifty years and ERα-negative status, and HR-/ERBB2 breast cancer subtypes. AHRR mRNA expression was associated with metastasis-free survival, while AHR mRNA expression was not. Immunohistochemistry revealed the presence of AhR protein in both tumor cells (nucleus and/or cytoplasm) and the tumor microenvironment (including endothelial cells and lymphocytes). High AHR expression was correlated with high expression of several genes involved in signaling pathways related to inflammation (IL1B, IL6, TNF, IL8 and CXCR4), metabolism (IDO1 and TDO2 from the kynurenine pathway), invasion (MMP1, MMP2 and PLAU), and IGF signaling (IGF2R, IGF1R and TGFB1). Two well-known ligands for AHR (TCDD and BaP) induced mRNA expression of IL1B and IL6 in an ERα-negative breast tumor cell line. The breast cancer ER status likely influences AhR activity involved in these signaling pathways. The mechanisms involved in AhR activation and target gene expression in breast cancers are also discussed.

Fig 1. Survival curves of two groups of patients according to AHRR mRNA expression level in the cohort of 439 breast tumors.

AUC analysis was used to divide the population into two relevant AHRR expression subgroups.

Fig 2. mRNA expression levels of AHR, AHRR and ARNT, and of genes involved in inflammation, in MDA-MB-436 breast cancer cell line treated with two different AHR ligands.

MDA-MB-436 cells were cultivated in absence (CTL) or in presence of TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) (10^-9M) or BaP (benzo[a]pyrene) (10^-6M) for 16 h. Cells were then lysed and mRNA extracted. mRNA expression levels of AHR, AHRR and ARNT (A), and of IL1B, IL6, IL8 and CXCR4 (B) were determined by qRT-PCR. All experiments were performed in triplicate. Results were expressed as mean +/- s.e.m and normalized so that the mean of the control cells was 1. Three levels of statistical significance are distinguished: *p-value<0.05; **p-value<0.01; ***p-value<0.001.

Fig 3. Immunocytochemical staining for AhR in human breast tissue.

a, peritumoral “normal” tissue. b, tumor tissue. Note the intense staining in both nuclei and cytoplasm in b. G, epithelial glands; C, capillaries; T, tumor cells; S, intratumoral stroma. Original Magnification, x 20.

Fig 4. Immunocytochemical staining for AhR and CD4 in breast tumors.

a-b, moderate AhR-expressing tumor cells. Note positive AhR staining in the intratumoral stroma. c-d, immunostaining for AhR (c) or CD4 (d) in the same tumor sample. Immunostaining for both AhR and CD4 is observed in stromal cells in the intratumoral compartment. T, tumor cells; S, intratumoral stroma. Original Magnification, x 20.