High Expression of AhR and Environmental Pollution as AhR-Linked Ligands Impact on Oncogenic Signaling Pathways in Western Patients with Gastric Cancer-A Pilot Study

Abstract

The vast majority of gastric cancer (GC) cases are adenocarcinomas including intestinal and diffuse GC. The incidence of diffuse GC, often associated with poor overall survival, has constantly increased in Western countries. Epidemiological studies have reported increased mortality from GC after occupational exposure to pro-carcinogens that are metabolically activated by cytochrome P450 enzymes through aryl hydrocarbon receptor (AhR). However, little is known about the role of AhR and environmental AhR ligands in diffuse GC as compared to intestinal GC in Western patients. In a cohort of 29, we demonstrated a significant increase in AhR protein and mRNA expression levels in GCs independently of their subtypes and clinical parameters. AhR and RHOA mRNA expression were correlated in diffuse GC. Further, our study aimed to characterize in GC how AhR and the AhR-related genes cytochrome P450 1A1 (CYP1A1) and P450 1B1 (CYP1B1) affect the mRNA expression of a panel of genes involved in cancer development and progression. In diffuse GC, CYP1A1 expression correlated with genes involved in IGF signaling, epithelial-mesenchymal transition (Vimentin), and migration (MMP2). Using the poorly differentiated KATO III epithelial cell line, two well-known AhR pollutant ligands, namely 2-3-7-8 tetrachlorodibenzo-p-dioxin (TCDD) and benzo[a]pyrene (BaP), strongly increased the expression of CYP1A1 and Interleukin1β (IL1B), and to a lesser extend UGT1, NQO1, and AhR Repressor (AhRR). Moreover, the increased expression of CYP1B1 was seen in diffuse GC, and IHC staining indicated that CYP1B1 is mainly expressed in stromal cells. TCDD treatment increased CYP1B1 expression in KATO III cells, although at lower levels as compared to CYP1A1. In intestinal GC, CYP1B1 expression is inversely correlated with several cancer-related genes such as IDO1, a gene involved in the early steps of tryptophan metabolism that contributes to the endogenous AhR ligand kynurenine expression. Altogether, our data provide evidence for a major role of AhR in GC, as an environmental xenobiotic receptor, through different mechanisms and pathways in diffuse and intestinal GC. Our results support the continued efforts to clarify the identity of exogenous AhR ligands in diffuse GC in order to define new therapeutic strategies.

Keywords: 2,3,7,8-tetrachlorodibenzeno-p-dioxin (TCDD or dioxin); aryl hydrocarbon receptor (AhR); benzo[a]pyrene (BaP); diffuse gastric subtype; gastric cancers; intestinal gastric subtype; tryptophan metabolism; xenobiotic metabolism.

Figure 1

Immunohistochemical staining of AhR and CYP1B1 in peritumoral and diffuse GCs. AhR in peritumoral gastric tissue (A); weak cytoplasmic and/or nuclear staining were observed in glandular tissue and stroma. In tumoral tissue (B,D), strong AhR immunostaining is observed in most cells, both epithelial and stromal compartments. CYP1B1 (C) and AhR (D) immunostaining are shown on the same tumor (diffuse GC). CYP1B1 was mainly observed in the stromal compartment in diffuse GC (C). Original magnification ×20. Bar scale, 500 μm.

Figure 2

mRNA expression levels of AhR and AhR-related genes in KATO III and AGS gastric cells upon treatment with either TCDD or BaP. (a) The KATO III cells were cultivated in the absence (Ctrl) or presence of either TCDD (dioxin) 30 nM or BaP (10 µM) for 16 h. The cells were incubated with (gray column) or without (black column) CHH223191 (10 μM). The expression of the indicated genes was determined by qRT-PCR. All the experiments were performed in triplicate. The results are expressed as means +/− S.E.M and normalized so that the mean of the control cells was 1. * p value < 0.005, ** p value < 0.01; *** p value < 0.001; **** p value < 0.0001. (b) The KATO III cells were cultivated in the presence or absence of dioxin at the indicated concentrations. The expression levels of CYP1A1 and CYP1B1 were determined by qRT-PCR in the same experiment. The results were expressed as in (a). (c) The AGS cells were cultivated in the absence (Ctrl in black) or presence of (dioxin) (0.01–10 nM, in gray). The expression levels of the indicated genes were determined by qRT-PCR in the same experiment. All the experiments were performed in triplicate. The results were expressed as in (a).

Figure 3

AhR role in cancer biology; environmental compounds at the crossroads of toxicity and several signaling pathways.