IDO1 metabolites activate β-catenin signaling to promote cancer cell proliferation and colon tumorigenesis in mice

Abstract

Background & aims: Indoleamine 2,3 dioxygenase-1 (IDO1) catabolizes tryptophan along the kynurenine pathway. Although IDO1 is expressed in inflamed and neoplastic epithelial cells of the colon, its role in colon tumorigenesis is not well understood. We used genetic and pharmacologic approaches to manipulate IDO1 activity in mice with colitis-associated cancer and human colon cancer cell lines.

Methods: C57Bl6 wild-type (control), IDO1-/-, Rag1-/-, and Rag1/IDO1 double-knockout mice were exposed to azoxymethane and dextran sodium sulfate to induce colitis and tumorigenesis. Colitis severity was assessed by measurements of disease activity, cytokine levels, and histologic analysis. In vitro experiments were conducted using HCT 116 and HT-29 human colon cancer cells. 1-methyl tryptophan and small interfering RNA were used to inhibit IDO1. Kynurenine pathway metabolites were used to simulate IDO1 activity.

Results: C57Bl6 mice given pharmacologic inhibitors of IDO1 and IDO1-/- mice had lower tumor burdens and reduced proliferation in the neoplastic epithelium after administration of dextran sodium sulfate and azoxymethane than control mice. These reductions also were observed in Rag1/IDO1 double-knockout mice compared with Rag1-/- mice (which lack mature adaptive immunity). In human colon cancer cells, blockade of IDO1 activity reduced nuclear and activated β-catenin, transcription of its target genes (cyclin D1 and Axin2), and, ultimately, proliferation. Exogenous administration of IDO1 pathway metabolites kynurenine and quinolinic acid led to activation of β-catenin and proliferation of human colon cancer cells, and increased tumor growth in mice.

Conclusions: IDO1, which catabolizes tryptophan, promotes colitis-associated tumorigenesis in mice, independent of its ability to limit T-cell-mediated immune surveillance. The epithelial cell-autonomous survival advantage provided by IDO1 to colon epithelial cells indicate its potential as a therapeutic target.

Keywords: 1-mT; 1-methyl tryptophan; AOM; APC; CAC; CRC; DSS; DbKO; GCN; IBD; IDO; IFN; Metabolism; Mouse Model; Ulcerative Colitis; antigen-presenting cell; azoxymethane; colitis-associated cancer; colorectal cancer; dextran sodium sulfate; double gene knockout mouse for indoleamine 2,3 dioxygenase 1 and Rag 1; general control nonrepressed; indoleamine 2,3 dioxygenase; inflammatory bowel disease; interferon.

Figure 1

IDO1 expression is increased in AOM/DSS model tumors. Tumor and adjacent colitis tissue was compared to colon tissue from untreated (control) mice. A) mRNA levels of IDO1 and IDO1-inducing cytokines are shown (n=6 mice). B) Representative IDO1 protein expression in same comparator groups (numerical fold increase over control shown below IDO1 band). C) H&E (left) and immunofluorescence images of OCT-frozen fixed tissues (25x and 400x original) highlighting epithelial IDO1 expression in β-catenin positive primary tumor cells from a WT mouse (top). IDO1−/− mouse tumor (bottom) shown for comparison.

Figure 2

Disrupted IDO1 activity affects tumor burden, but not colitis severity in AOM/DSS. A–C) Age-matched, co-caged, female C57Bl/6 WT and IDO1^−/− mice exposed to a single AOM (10 mg/kg) administration followed by 3 cycles of DSS (n=9/group, data from 1 of 3 replicate experiments with similar results). D–F) Female WT mice received either placebo or 20-day control-release 300 mg L-1mT tablet prior to second cycle of DSS (n=8–9/group, data from 1 of 2 replicate experiments with similar results). A, D) Relative change in weight B, E) Colon histology C, F) Average Tumor burden with representative image to right.

Figure 3

IDO1 expression supports epithelial proliferation in tumors and adjacent crypts. Colon tumors and adjacent upright crypts from WT, IDO−/− and L-1mT mice (from Figure2) were examined for BrdU uptake and β-catenin expression. A) Representative images showing greater tumor epithelial uptake of BrdU in WT than IDO−/− mice. B) Tumor proliferation index (BrdU+/total tumor crypt cells). C) Epithelial proliferation index (BrdU+/total crypt cells) in upright non-dysplastic crypts adjacent to tumors. D) β-catenin staining of WT and IDO1−/− tumors (representative) shown reduced nuclear β-catenin in IDO1−/− tumors. Examination was from 10– 20 crypts/tumor, 5–9 colons/group and 1–3 tumors/mouse for immunostaining and calculation of proliferation index. Tissue sections were formalin-fixed and paraffin embedded.

Figure 4

IDO1 activity is associated with increased tumor burden and proliferation independent of influence on adaptive immunity. Rag^−/− and Rag^−/−/IDO1^−/− (DbKO) mice received AOM (twice, one week apart) followed by 3 cycles of DSS. Similar trends were observed in clinical signs of colitis activity including A) Weight loss B) Disease activity index and C) Colon histology scores. However tumorigenesis and tumor proliferation was still lower in the mice lacking intact IDO1 activity. D) Representative colons with polyps E) Tumor quantification and F) Tumor proliferation index. (n=8/group, data shown for 1 of 3 replicate experiments.

Figure 5

IDO1 supports proliferation in constitutively expressing human colon cancer cells. A) IDO1 mRNA and B) protein is constitutively expressed by HCT 116 and HT-29 cells and is upregulated in response to IFNγ stimulation [0.1ng/ml ×48 hours]. C, D) Gene silencing with IDO1 specific siRNA (siIDO) effectively suppresses expression of IDO1 protein and mRNA in both cell lines [control siRNA or siIDO × 24 hours, followed by ± 24 hours of IFNγ 0.1 ng/ml]. E, F) Inhibition with L-1mT or gene silencing of IDO1 reduces cell proliferation in HCT 116 and HT-29 based on MTT incorporation. Cells grown in 48-well plates were exposed media alone, L-1mT (500mM), control siRNA or siIDO for 72 hours in the absence or presence of IFNγ 0.1ng/ml for 24 hours.

Figure 6

Metabolites of the IDO1 pathway stimulate β-catenin activation and proliferation in HCT 116 colon cancer cells. HCT 116 cells were incubated for 48 hours in control siRNA or siIDO + 500mM L-1mT ± IDO1 metabolites as indicated. A) Western blot [nuclear fraction] and immunofluorescence demonstrate that IDO1 inhibition reduces nuclear β-catenin, which is restored by addition of kynurenine (100 μM). B) Whole cell fraction Western blot for total and phosphorylated β-catenin [stabilization pathway = Ser 552; degradation pathway = Ser33/37/Thr41] as well as cell-cycle regulating genes Cyclin D1 and D3. C) Relative Axin2 mRNA a transcriptional target of Wnt/β-catenin/Tcf signaling. D) IDO1 pathway of tryptophan catabolism. E, F) Kynurenine and quinolinic acid but not picolinic acid rescue HCT 116 cell proliferation in setting of inhibited IDO1 activity based on MTT incorporation (E) and percent EdU incorporation (F) as marker of S-phase cells. Flow cytometry for EdU included >15,000 events/group, 4 replicates/group, data from 1 of 3 replicate experiments with similar results. P-values *,**,*** vs IDO1 inhibition state; ### (P<0.001) vs scramble siRNA control.

Figure 7

Kynurenine metabolites promote tumor growth and proliferation of malignant epithelium. Paired littermate female IDO1−/− mice received AOM and two cycles of 3% DSS. Following recovery, mice received kynurenine and QA IP (1mg each) four days/week for 4 weeks or vehicle control (PBS). A) Tumor burden. B) Tumor proliferation index (Brdu+/total crypt cells). C) Schematic depicting dual functions of IDO1 in colon cancer. IDO1 is expressed by the neoplastic epithelium and antigen presenting cells (APC) of the tumor draining lymph nodes or tumor microenvironment. A lowered tryptophan/kynurenine metabolite ratio suppresses tumor reactive effector T-cells and promotes T-reg activation to limit tumor immune-surveillance and thus facilitate tumor progression. Concurrently, elevated local levels of kynurenine metabolites directly stimulate proliferation in the neoplastic epithelium through activated nuclear β-catenin. Inhibition of IDO1 activity (1-mT) reduces tumor progression and proliferation.