Interleukin-6 mediated upregulation of CYP1B1 and CYP2E1 in colorectal cancer involves DNA methylation, miR27b and STAT3

Abstract

Background: The pro-inflammatory cytokine interleukin-6 (IL6) promotes colorectal cancer (CRC) development. It is also known to regulate cytochrome P450 (CYP450) enzymes, which are involved in CRC tumour initiation and promotion via activation of chemical carcinogens. Here, IL6 regulation of CYP450 expression was investigated in CRC.

Methods: The effect of IL6 on CYP 1A1, 1B1 and 2E1 expression was determined in vitro using CRC cell lines HCT116 and SW480, and CYP450 expression was determined by immunohistochemistry in CRC tissues previously shown to have increased levels of IL6.

Results: In mechanistic studies, IL6 treatment significantly induced CYP1B1 and CYP2E1, but not CYP1A1, gene expression in HCT116 and SW480 cells. CYP2E1 expression regulation occurred via a transcriptional mechanism involving STAT3. For CYP1B1 regulation, IL6 downregulated the CYP1B1-targeting microRNA miR27b through a mechanism involving DNA methylation. In clinical samples, the expression of CYP1B1 and CYP2E1, but not CYP1A1, was significantly increased in malignant tissue overexpressing IL6 compared with matched adjacent normal tissue.

Conclusions: Colonic inflammation with the presence of IL6 associated with neoplastic tissue can alter metabolic competency of epithelial cells by manipulating CYP2E1 and CYP1B1 expression through transcriptional and epigenetic mechanisms. This can lead to increased activation of dietary carcinogens and DNA damage, thus promoting colorectal carcinogenesis.

Figure 1

IL6 expression is increased in epithelial cells and stroma of colorectal tumour samples. Data are adapted from our previous publication (Maihofner et al, 2003) and are shown as fold change of immunohistochemistry score relative to control. Significant differences from normal tissue were calculated using Wilcoxon's signed-rank test (*P<0.05, ***P<0.001).

Figure 2

IL6 effect on CYP450 gene expression. HCT116 and SW480 cells were treated with 0, 100 and 1000 pg ml⁻¹ IL6 for 24 and 48 h. CYP1A1 (A), CYP1B1 (B) and CYP2E1 (C) expression was measured by RT-qPCR. Data were normalised to expression of GAPDH housekeeping gene and are shown relative to control. Significance was calculated using one-way ANOVA with a Dunnett post-test comparing treated groups with vehicle control and linear trend analysis (*P<0.05, **P<0.01, ***P<0.001). Data are presented as a mean of at least three biological replicates. Error bars represent the s.e.m.

Figure 3

MiRNA involvement in IL6-mediated regulation of CYP1B1 and CYP2E1 gene expression. (A–D) HCT116 and SW480 cells were treated with 0 and 1000 pg ml⁻¹ IL6 for 24 and 48 h. MiR378 (A) and miR27b (C) expression was measured by RT-qPCR. Fold-change expression of miR378 was correlated with fold-change expression of CYP2E1 (B) and fold-change expression of miR27b with fold-change CYP1B1 expression (D). Data were normalised to expression of U6 RNA and are shown relative to control. Significance was calculated using Student's t-test; Pearson's product-moment correlation coefficient test was used for correlation analysis (*P<0.05, **P<0.01). Data are presented as a mean of at least three biological replicates. Error bars represent the s.e.m.

Figure 4

STAT3 involvement in IL6-mediated regulation of CYP1B1 and CYP2E1 gene expression. (A) Potential STAT3 binding sites in the CYP2E1 promoter region (1000 bp upstream of the CYP2E1 start site) predicted using TFSEARCH ver1.3 (Heinemeyer et al, 1998) (B, C) HCT116 and SW480 were treated with 1000 pg ml⁻¹ IL6 or a combination of IL6 and 25 μM STAT3 inhibitor VII 5,15-diphenylporphyrin for 24 h (B) and 48 h (C). CYP2E1 (B) and CYP1B1 (C) expression was measured by RT-qPCR. Data were normalised to expression of GAPDH housekeeping gene and are shown relative to control. (D) SW480 cells were treated with 1000 pg ml⁻¹ IL6 or a combination of IL6 and 25 μM STAT3 inhibitor VII 5,15-diphenylporphyrin for 60 min before being fixed and STAT3 binding to predicted sites in the CYP2E1 promoter region was measured by ChIP-qPCR using an anti-STAT3 antibody. Data are presented as a mean of at least three biological replicates for gene expression and STAT3 ChIP experiments and error bars represent the s.e.m. Significance was calculated using two-way ANOVA with a Bonferroni post-test comparing treated groups with vehicle control (**P<0.01). ChIP experiments were repeated using IgG antibody to control for non-specific binding and performed in duplicate.

Figure 5

IL6-induced downregulation of miR27b is mediated by DNA methylation. HCT116 and SW480 cells were treated with 1000 pg ml⁻¹ IL6, 4 μM 5-aza-2′-deoxycytidine or a combination of IL6 and 5-aza-2′-deoxycytidine for 24 and 48 h. (A) MiR27b expression was measured by RT-qPCR. Data were normalised to expression of U6 RNA and are shown relative to control. (B) CYP1B1 expression was measured by RT-qPCR. Data were normalised to expression of GAPDH housekeeping gene and are shown relative to control. (C) Fold-change expression of miR27b was correlated with fold-change expression of CYP1B1. Significance was calculated using Student's t-test and one-way ANOVA with a Dunnett post-test comparing treated groups with vehicle control; Pearson's product-moment correlation coefficient test was used for correlation analysis (*P<0.05, **P<0.01). Data are presented as a mean of at least three biological replicates. Error bars represent the s.e.m.

Figure 6

Immunohistochemical localisation of CYP450s in normal and adjacent malignant colonic epithelia. The presence of the immunoreactive protein is indicated by brown staining. Normal (A) and tumour (B) tissues treated with pre-immune serum as primary antibody (negative control); normal (C) and tumour (D) tissues treated with anti-CYP1A1 primary antibody; normal (E) and tumour (F) tissues treated with anti-CYP1B1 primary antibody; normal (G) and tumour (H) tissues treated with anti-CYP2E1 primary antibody. (I) Expression of CYP1B1 in non-malignant colonic epithelial cells lying adjacent to tumour cells. (J) CYP2E1 expression in plasma cells. All sections are shown at × 200 or × 400 (inset) magnification. (K) Expression of CYP450s in matched normal and adjacent malignant colonic epithelia from 40 patients. Significant differences from normal tissue were calculated using Wilcoxon's signed-rank test (**P<0.01, ***P<0.001).

Figure 7

Potential mechanism of IL6-mediated induction of CYP1B1 and CYP2E1 mRNA expression in colorectal cancer cells. IL6 secreted by stromal cells binds to the soluble IL6 receptor (sIL6R) in the stroma, which interacts with transmembrane protein gp130 at the cellular surface resulting in activation of JAK/STAT3 and PI3K/AKT pathways. Activated STAT3 forms a homodimer, translocates to the nucleus and binds to the CYP2E1 promoter region, thus inducing its transcription. Activated AKT phosphorylates DNMT1 leading to its nuclear translocation. Once in the nucleus, DNMT1 induces DNA methylation at a CpG island located near miR27b, thus preventing its expression. Lower expression of miR27b results in less binding to the CYP1B1 mRNA leading to less degradation and increased CYP1B1 mRNA expression.