The aryl hydrocarbon receptor functions as a tumor suppressor of liver carcinogenesis

Abstract

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the biological and toxic effects of its xenobiotic ligands. Previous cell culture studies have shown that, in addition to controlling the xenobiotic detoxification response, AHR activation leads to G0-G1 arrest, diminished capacity for DNA replication, and inhibition of cell proliferation. In fact, recent work from our own and from other laboratories suggests that AHR may function as a tumor suppressor gene that becomes silenced during the process of tumor formation. To test this hypothesis and determine whether the mouse Ahr gene acts as a tumor suppressor gene in vivo, we have examined the role of Ahr ablation in liver tumorigenesis induced by the genotoxic chemical diethylnitrosamine (DEN), a hepatic carcinogen that is not an AHR ligand. In mice given a single i.p. injection of DEN, AHR antagonized liver tumor formation and growth by regulating cell proliferation, inflammatory cytokine expression, and DNA damage, parameters which were significantly elevated in the livers of control and, more so, of DEN-exposed Ahr-/- mice. Ahr-/- hepatocytes also showed significantly higher numbers of 4N cells, increased expression of proliferative markers, and repression of tumor suppressor genes. These data support the concept that in its basal state in the absence of a xenobiotic ligand, the Ahr gene functions as a tumor suppressor gene, and that its silencing may be associated with cancer progression.

Figure 1. AHR-deficient DEN-induced tumors show increased DNA proliferation, DNA damage and apoptosis

(A) Four-micron-thick sections from formalin-fixed, paraffin-embedded left liver lobe of control and DEN-treated Ahr^+/+ and Ahr^−/− male mice were processed 30 – 35 weeks after i.p. inoculation for histological analysis by H&E staining, and (B), BrdU incorporation, and γH2A.X and TUNEL staining by immunohistochemistry. Corresponding micrographs from a single representative animal from each genotype are shown. Micrographs from at least 3 mice in each group were recorded blind by counting at least 400 hepatocytes per section chosen from several random fields of each micrograph. Open and closed bars represent means ± SEM of controls and DEN-treated mice, respectively. (*) denotes significantly different (p<0.05) from untreated controls of the same genotype; (+) denotes significantly different from untreated controls of different genotypes. (^) denotes significantly different between DEN-treated mice of different genotypes.

Figure 2. Aberrant ploidy in Ahr^−/− hepatocytes from DEN-treated male mice

Nuclei were isolated from hepatocytes from control livers and from tumor and tumor-adjacent normal tissue of DEN-treated Ahr^+/+ and Ahr^−/− male mice (n=3 – 5) and their DNA content was analyzed by flow cytometry. (*) Significantly different (p<0.05) between genotypes.

Figure 3. Differential expression of pro-inflammatory cytokines Il6 and Tnfα in livers of control and DEN-treated Ahr^+/+ and Ahr^−/− male mice

Il6 and Tnfα mRNA levels were quantified at 3 weeks (A) and 30 – 35 weeks (B) following control or DEN injection. Parallel determinations of the protein levels of the two cytokines were done at 3 weeks (C) and 30 – 35 weeks (D). Open and closed bars represent mean ± SEM of controls and DEN-treated mice, respectively. The symbols denoting significance are as detailed in the legend to Figure 1.

Figure 4. Analysis of the expression of pro-proliferative and pro-apoptotic markers in Ahr^+/+ and Ahr^−/− mice

RNA extracted from hepatocytes of control livers and of tumor and tumor-adjacent normal tissue of DEN-treated Ahr^+/+ and Ahr^−/− male mice (n=3 – 5) was analyzed by real time RT-PCR for the accumulation of mRNA from the genes shown on the abscissa. The ordinate denotes the log₂ of the ratio of the means ± SEM expression of those genes in the indicated Ahr^−/− tissues (control, tumor, or tumor-adjacent normal) relative to their expression in the corresponding tissues of Ahr^+/+ mice. The numerical expression value was determined from the change in threshold cycle number (ΔC_t) of each mRNA relative to β-actin used as the normalization factor.