15-Lipoxygenase-1 suppression of colitis-associated colon cancer through inhibition of the IL-6/STAT3 signaling pathway

Abstract

The IL-6/signal transducer and activator of transcription 3 (STAT3) pathway is a critical signaling pathway for colitis-associated colorectal cancer (CAC). Peroxisome proliferator-activated receptor (PPAR)-δ, a lipid nuclear receptor, up-regulates IL-6. 15-Lipoxygenase-1 (15-LOX-1), which is crucial to production of lipid signaling mediators to terminate inflammation, down-regulates PPAR-δ. 15-LOX-1 effects on IL-6/STAT3 signaling and CAC tumorigenesis have not been determined. We report that intestinally targeted transgenic 15-LOX-1 expression in mice inhibited azoxymethane- and dextran sodium sulfate-induced CAC, IL-6 expression, STAT3 phosphorylation, and IL-6/STAT3 downstream target (Notch3 and MUC1) expression. 15-LOX-1 down-regulation was associated with IL-6 up-regulation in human colon cancer mucosa. Reexpression of 15-LOX-1 in human colon cancer cells suppressed IL-6 mRNA expression, STAT3 phosphorylation, IL-6 promoter activity, and PPAR-δ mRNA and protein expression. PPAR-δ overexpression in colonic epithelial cells promoted CAC tumorigenesis in mice and increased IL-6 expression and STAT3 phosphorylation, whereas concomitant 15-LOX-1 expression in colonic epithelial cells (15-LOX-1-PPAR-δ-Gut mice) suppressed these effects: the number of tumors per mouse (mean ± sem) was 4.22 ± 0.68 in wild-type littermates, 6.67 ± 0.83 in PPAR-δ-Gut mice (P = 0.026), and 2.25 ± 0.25 in 15-LOX-1-PPAR-δ-Gut mice (P = 0.0006). Identification of 15-LOX-1 suppression of PPAR-δ to inhibit IL-6/STAT3 signaling-driven CAC tumorigenesis provides mechanistic insights that can be used to molecularly target CAC.

Keywords: 15-LOX-1; CAC; IL-6 expression; PPAR-δ; STAT3 phosphorylation.

Figure 1.

Effects of 15-LOX-1 transgenic expression on colonic tumorigenesis induced by AOM and DSS. A–D) FVB/N (FVB) and C57BL/6 (B6) mice with (15-LOX-1-Gut) and without (WT) 15-LOX-1 transgenic expression were treated with AOM and DSS to induce colitis-associated colon tumors. Mice were then killed and examined for tumor formation. A, C) Scatter plots of tumor incidence. Horizontal bars represent means. B, D) Representative photographs of dissected colons of mice after treatment. Magnification ×10. E–G) Proliferative zone lengths in the small intestine and colon in the mice. E) Representative photographs of Ki-67 immunohistochemistry staining. F, G) Proliferative zone lengths (means ± sd). H) 15-LOX-1 mRNA relative expression levels measured by qRT-PCR in normal colonic epithelial cells and paired tumors of the15-LOX-1-Gut mice. I) 15-LOX-1 protein expression levels measured by Western blot in representative normal colonic epithelial cells and paired tumors of the 15-LOX-1-Gut mice. J) Densitometric analyses of the protein bands in I.

Figure 2.

15-LOX-1 transgenic expression inhibited IL-6/STAT3 signaling. A) Effects of 15-LOX-1 on IL-6 expression. FVB/N mice were treated with AOM and DSS to induce colitis-associated colon tumors. The mice were then killed, and crypts of normal and colonic tumor tissues were isolated. IL-6 mRNA expression was measured by qRT-PCR. Values are means ± sd. *P < 0.0001 compared with WT normal crypts. B–E) Effects of 15-LOX-1 on STAT3(705) phosphorylation and IL-6/STAT3 target gene expression. Crypt samples were obtained as described for A. B) Protein levels of p-STAT3(705) and total STAT3 were measured by Western blotting. C) Densitometric analyses of the protein bands in B. The data are presented as ratio of p-STAT3(705) to total STAT3. D, E) mRNA expression levels of the STAT3 target genes MUC1 and Notch3 measured by qRT-PCR. Values are means ± sd. *P < 0.01 compared with WT normal crypts. F, G) Effects of 15-LOX-1 on IL-6-induced p-STAT3(705) expression. F) Colonic crypts from FVB/N WT and 15-LOX-1-Gut^(+/+) mice were isolated and cultured with mouse recombinant IL-6 (50 ng/ml) for the indicated times. Protein levels of p-STAT3(705) and total STAT3 were measured by Western blotting. G) Densitometric analyses of the protein bands in F.

Figure 3.

15-LOX-1 inhibited IL-6 up-regulation (A) and STAT3(705) phosphorylation (B) in colitis-associated colorectal tumorigenesis. 15-LOX-1-Gut and WT littermate mice were treated with AOM and DSS to induce colitis-associated colon tumors (T). IL-6 and p-STAT3(705) protein expression levels were measured by immunohistochemistry staining. N, nonmalignant tissue.

Figure 4.

15-LOX-1 inhibited IL-6 up-regulation and STAT3(705) phosphorylation in human colon cancer cells. A, B) IL-6 and 15-LOX-1 expression levels measured by qRT-PCR in paired human normal and colorectal tumor tissue samples. Values shown are means ± sd of triplicate measurements for each patient. C–H) 15-LOX-1 inhibited IL-6 up-regulation and STAT3(705) phosphorylation in colorectal cancer cells. The cells were infected with either modified Ad-hTert-15-LOX-1 (Ad-15-LOX-1) or modified Ad-hTert-luciferase (Ad-luciferase) at a ratio of 200 adenovirus particles per cell for HCT116 cells and 400 adenovirus particles per cell for LoVo cells. Cells were harvested 48 hours after infection for measurement of IL-6 mRNA expression by qRT-PCR (C, D) and 72 hours after infection for measurement of p-STAT3(705) protein expression by Western blotting (E, F). G, H) Densitometric analyses of the protein bands in E and F. The data are presented as ratio of p-STAT3(705) to total STAT3. I–L) Effects of 15-LOX-1 protein expression and enzymatic activity on IL-6/STAT3 signaling. Caco-2 cells stably transfected with either nontargeted shRNA (control shRNA) or 15-LOX-1 shRNA were cultured for 14 days after confluence to induce differentiation and then harvested and analyzed for 15-LOX-1 mRNA expression by qRT-PCR (I) and protein expression by Western blotting (J). Control shRNA and 15-LOX-1 shRNA cells were cultured as described for I for 14 days and then treated with caffeic acid (2.2 µM), 13-S-HODE (13.5 µM), or solvent control as indicated with or without IL-6 (50 ng/ml) for 40 min before cell harvesting for protein expression analyses by Western blotting (K). L) Densitometric analyses of the protein bands in J. M, N) 15-LOX-1 inhibited IL-6 transcription in colorectal cancer cells. HCT116 and LoVo cells were transfected with either Ad-15-LOX-1 or Ad-GFP combined with the IL-6 promoter luciferase vector. O–T) 15-LOX-1 inhibited PPAR-δ mRNA and protein expression. HCT116 and LoVo cells were treated as described for C–H. O, P) PPAR-δ mRNA expression was measured by qRT-PCR 48 hours after infection. Q, R) PPAR-δ protein expression was measured by Western blotting 72 hours after infection. S, T) Densitometric analyses of the protein bands in Q and R.

Figure 5.

Effects of 15-LOX-1 on PPAR-δ-mediated promotion of CAC tumorigenesis and IL-6/STAT3 signaling. A, B) Indicated genetic strains of mice were treated with AOM and DSS to induce CAC tumors. The mice were then killed and examined for tumor formation. A) Scatter plot of tumor incidence. B) Representative photographs taken at ×10 magnification. C–G) 15-LOX-1 decreased PPAR-δ expression in murine colonic crypts. Mice were treated as described for A and B, and crypts of normal colonic tissues were isolated. 15-LOX-1 and PPAR-δ mRNA relative expression levels were measured by qRT-PCR (C, D), and protein levels were measured by Western blotting (E). F, G) Densitometric analyses of the protein bands in E. H–K) Effects of 15-LOX-1 on PPAR-δ-mediated activation of IL-6/STAT3 signaling and target gene expression. Mice were treated as described for A and B. Colonic crypt samples were obtained as for C–G. H, K) IL-6 and Notch3 mRNA relative expression levels were measured by qRT-PCR. I) Protein expression levels of p-STAT3(705) and total STAT3 were measured by Western blotting. J) Densitometric analyses of the protein bands in I. L–N) Effects of PPAR-δ expression and activation on IL-6/STAT3 signaling. WT and PPAR-δ-Gut (PPAR-δ+) mice were fed a control diet or a GW501516 (50 mg/kg) or GSK3787 (50 mg/kg) diet for 2 weeks and then treated with 1.2% DSS for 1 wk. The mice were then killed, and colon crypts were isolated. L) IL-6 mRNA relative expression levels were measured by qRT-PCR. M) Protein expression levels of p-STAT3(705) and total STAT3 were measured by Western blotting. N) Densitometric analyses of the protein bands in M.

Figure 6.

Conceptual model of the effects of 15-LOX-1 on the PPAR-δ–IL-6/STAT3 signaling pathway in relation to colorectal tumorigenesis.