Inhibition of Aryl Hydrocarbon Receptor (AhR) Expression Disrupts Cell Proliferation and Alters Energy Metabolism and Fatty Acid Synthesis in Colon Cancer Cells

Abstract

The aryl hydrocarbon receptor (AhR) plays a wide range of physiological roles in cellular processes such as proliferation, migration or control of immune responses. Several studies have also indicated that AhR might contribute to the regulation of energy balance or cellular metabolism. We observed that the AhR is upregulated in tumor epithelial cells derived from colon cancer patients. Using wild-type and the corresponding AhR knockout (AhR KO) variants of human colon cancer cell lines HCT116 and HT-29, we analyzed possible role(s) of the AhR in cell proliferation and metabolism, with a focus on regulation of the synthesis of fatty acids (FAs). We observed a decreased proliferation rate in the AhR KO cells, which was accompanied with altered cell cycle progression, as well as a decreased ATP production. We also found reduced mRNA levels of key enzymes of the FA biosynthetic pathway in AhR KO colon cancer cells, in particular of stearoyl-CoA desaturase 1 (SCD1). The loss of AhR was also associated with reduced expression and/or activity of components of the PI3K/Akt pathway, which controls lipid metabolism, and other lipogenic transcriptional regulators, such as sterol regulatory element binding transcription factor 1 (SREBP1). Together, our data indicate that disruption of AhR activity in colon tumor cells may, likely in a cell-specific manner, limit their proliferation, which could be linked with a suppressive effect on their endogenous FA metabolism. More attention should be paid to potential mechanistic links between overexpressed AhR and colon tumor cell metabolism.

Keywords: AhR; Akt pathway; colon cancer cells; fatty acid synthesis; metabolism; proliferation.

Figure 1

AhR protein levels are increased in human colon cancer tissue. (A) A representative image of AhR staining in colon tumor; (B) a comparison of AhR staining between normal (left) and tumor (right) colon tissue. H-scores were calculated based on intensity and positivity values. Pictograms at the bottom of the panel document staining of all formalin-fixed paraffin-embedded tissue specimens (n = 22; grey, H-score 0–59; blue, H-score in quartiles up to 119, 179, 239 and 300); (C) H-score values comparing AhR staining in normal and tumor tissue. (D) AhR protein levels were determined by Western blotting in representative samples of isolated tumor and non-tumor colon epithelial cells (n = 10), using anti-human AhR rabbit monoclonal antibody, with β-actin being used as loading control. Representative Western blotting images are shown at the right. For original blots, see Supplementary File S1. Relative AhR levels determined by densitometry and normalized to -actin are shown at the left. * denotes a significant difference (p < 0.05) between AhR protein levels in tumor and non-tumor cells.

Figure 2

Loss of the AhR blocks induction of its target genes in HT-29 and HCT116 AhR KO cells. (A) Wild-type cells (WT), cells transfected with a CRISPR-Cas9 empty vector (gRNA) and AhR KO cell clones of HCT116 and HT-29 were used for detection of the AhR protein levels by Western blotting. For original blots, see Supplementary File S1. (B) Induction of AhR-target gene mRNAs after 24 h treatment with TCDD (10 nM) was determined by RT-qPCR. The data are shown as means + SD of at least three independent experiments. * and ** denote a significant difference (p < 0.05 and p < 0.01, respectively) between TCDD-treated and respective control cells. # and ## denote a significant difference (p < 0.05 and p < 0.01, respectively) between TCDD-treated AhR KO cells and the respective TCDD-treated wild-type cells.

Figure 3

Loss of AhR reduces proliferative rate of HCT116 and HT-29 cells. Cell numbers (A) and cell cycle distribution (B) of wild-type cells (WT) and AhR KO clones were determined 72 h after seeding by CyQUANT assay and flow cytometric analysis of cell cycle, respectively. The data are shown as means + SD of at least three independent experiments. ** denotes a significant difference (p < 0.01) between G0/G1 S-phase numbers of WT and AhR KO cells; ^## denotes a significant difference (p < 0.01) between S-phase numbers of WT and AhR KO cells; ^ denotes a significant difference (p < 0.05) between G2/M numbers of WT and AhR KO cells.

Figure 4

AhR deficiency alters ATP production rate in HCT116 and HT-29 cells. (A) Real-time ATP production rate was measured by SeaHorse XFp, and the contribution of mitochondria-produced ATP (mito-ATP) and glycolysis-produced ATP (glyco-ATP) to total ATP levels was determined. The data are shown as means + SD of three independent experiments. * and ** denote a significant difference (p < 0.05 and p < 0.01, respectively) between AhR KO and wild-type cells. (B) Glucose uptake was measured by colorimetric assay determining the concentration of glucose in cell culture medium collected from cells after 48 h of cultivation. The effect of AhR KO was compared with that of incubation of cells with the AhR antagonist CH-223191 (10 μM).

Figure 5

Loss of the AhR reduces expression of FA synthesis genes and their regulators, alters calculated SCD1 activities and modulates Akt protein levels/phosphorylation. The levels of mRNAs encoded by genes involved in FA synthesis or control of lipid metabolism 48 h after their seeding (A,B). The data are shown as means + SD of at least three independent experiments. * and ** denote a significant difference (p < 0.05 and p < 0.01, respectively) between AhR KO and wild-type cells. The full line indicates mRNA level in wild-type cells. (C) The levels of palmitoleic (C16:1), palmitic (C16:0), oleic (C18:1) and stearic (C18:0) fatty acids in wild-type (WT) and AhR KO cell lines were determined by GC/MS, and ratios of C16:1/C16:0 and C18:1/18:0 were calculated. The data are shown as means + SD of three independent experiments. * and ** denote a significant difference (p < 0.05 and p < 0.01, respectively) between AhR KO and wild-type (WT) cells. (D) Western blotting analysis of levels of total Akt and phosphorylated Akt (pAkt; Ser473) with or without treatment by insulin (ins; 100 nM) for 1 h. Representative Western blot images are shown at the left; results of densitometry analysis, normalized to β-actin, are shown at the right. For original blots, see Supplementary File S1. The data are shown as means + SD of at least three independent experiments. * denotes a significant difference (p < 0.05) between AhR KO and wild-type (WT) cells.

Figure 6

The AhR antagonist CH-223191 interferes with proliferation, Akt pathway activity and SCD1 expression in colon carcinoma cells. (A) Cells were treated with CH-223191 (10 μM), 24 h after their seeding, and cell numbers were determined after 48 h treatment. The data are shown as means + SD of at least three independent experiments. * denotes a significant difference (p < 0.05) between CH-223191-treated and control cells. (B,C) HCT116 cells were pre-treated with CH-223191 (10 μM) for 48 h and then with insulin (100 nM) for 1 h. The levels of phosphorylated Akt (pAkt; Ser473) and total Akt were determined by Western blotting, with β-actin being used as loading control. Representative Western blot images are shown in (B). For original blots, see Supplementary File S1; the results of densitometry analysis, normalized to β-actin are shown in (C). The data are shown as means + SD of at least three independent experiments. * and ** denote a significant difference (p < 0.05 and p < 0.01, respectively) between insulin-treated and the respective control cells. # denotes a significant difference (p < 0.05) between control wild-type cells treated with insulin and insulin treated AhR KO cells or insulin-treated wild-type cells pre-treated with CH-223191. (D) SCD1 mRNA levels were determined in control HT-29 and HCT116 cells, and in cells treated with CH-223191 (10 μM) for 24 h, by RT-qPCR. The data are shown as means + SD of at least three independent experiments. * denotes a significant difference (p < 0.05) between control and CH-223191-treated cells. (E) Expression of FA synthesis/transport genes in WT (line) and AhR KO differentiated HepaRG cells (black bars). The data are shown as means + SD of at least three independent experiments. * denotes a significant difference (p < 0.05) between WT and AhR KO cells.