A Nucleotide Analog Prevents Colitis-Associated Cancer via Beta-Catenin Independently of Inflammation and Autophagy

Abstract

Background & aims: Chronic bowel inflammation increases the risk of colon cancer; colitis-associated cancer (CAC). Thiopurine treatments are associated with a reduction in dysplasia and CAC in inflammatory bowel disease (IBD). Abnormal Wnt/β-catenin signalling is characteristic of >90% of colorectal cancers. Immunosuppression by thiopurines is via Rac1 GTPase, which also affects Wnt/β-catenin signalling. Autophagy is implicated in colonic tumors, and topical delivery of the thiopurine thioguanine (TG) is known to alleviate colitis and augment autophagy. This study investigated the effects of TG in a murine model of CAC and potential mechanisms.

Methods: Colonic dysplasia was induced by exposure to azoxymethane (AOM) and dextran sodium sulfate (DSS) in wild-type (WT) mice and mice harboring intestinal epithelial cell-specific deletion of autophagy related 7 gene (Atg7^ΔIEC). TG or vehicle was administered intrarectally, and the effect on tumor burden and β-catenin activity was assessed. The mechanisms of action of TG were investigated in vitro and in vivo.

Results: TG ameliorated DSS colitis in wild-type but not Atg7^ΔIEC mice, demonstrating that anti-inflammatory effects of locally delivered TG are autophagy-dependent. However, TG inhibited CAC in both wild-type and Atg7^ΔIEC mice. This was associated with decreased β-catenin activation/nuclear translocation demonstrating that TG's inhibition of tumorigenesis occurred independently of anti-inflammatory and pro-autophagic actions. These results were confirmed in cell lines, and the dependency on Rac1 GTPase was demonstrated by siRNA knockdown and overexpression of constitutively active Rac1.

Conclusions: Our findings provide evidence for a new mechanism that could be exploited to improve CAC chemoprophylactic approaches.

Keywords: Autophagy; Colon Cancer; Thioguanine; β-Catenin.

Graphical abstract

Figure 1

(A) Thiopurine pro-drug metabolism. TG and MP are purine analogue bases. Azathioprine (AZA) is non-enzymatically converted to MP. TGMP, TGDP, and TGTP are TGN. (B) TG, but not MP, inhibited β-catenin activity in vitro. β-catenin–driven transcriptional activity in HCT116 or Caco2 cells treated for 16 hours with either TG or MP in presence or absence of 1 μmol/L CHIR-99021 (highly selective inhibitor of glycogen synthase kinase 3, which activates β-catenin by promotion of Wnt signalling), IC50 TG ∼2 μmol/L. N = 4. (C) Immunofluorescence staining of HCT116 cells for β-catenin (green) and nuclei (DAPI, blue) ± 5 μmol/L TG treatment ± 1 μmol/L CHIR for 16 hours. Data are representative of N = 4 independent experiments. (D) Real-time imaging to 60 hours of HCT116 cells seeded into 96-well plates (7500 cells/well) by using a phase Cyte FLR live cell imager. (E) TG inhibited β-catenin in spheroid cells derived from an ulcerative colitis cancer, treated with vehicle control or TG (10 μmol/L) for 4 days. (F) TG conversion to TGN in Caco 2 and HCT116 cell lysates. MP was not converted to TGN in the acute in vitro experiments (N = 4 at each concentration).

Figure 2

TG inhibited colitis and colitis-associated colorectal tumor development and progression in mice. (A) Scheme showing induction procedure for AOM/DSS model of CAC. C57/BL6 WT were treated with DSS 1.5% in drinking water for 5 days, 3 cycles, ± daily intrarectal TG 1.5 mg/kg for 85 days (N ≥5 each group). (B) Daily assessments in TG or vehicle-treated mice (n ≥5) for diarrhea, body weight, rectal bleeding, and DAI score. (C) At death day 85: number of colon tumors (left panel); representative macroscopic images of colonic mucosa (right panel). (D) Average number of BrdU-staining cells per field of view in tumors and comparable areas of TG-treated mice, and representative immunohistochemical staining for BrdU in tumors (right panel). (E) Severity of dysplasia vs TG treatment in tumors 85 days after injection of AOM with representative H&E–stained sections of colonic tissue with tumors (scale bars are shown, right panel). (F) Tumor diameter and frequency in vehicle versus TG-treated mice. Statistics B–D and F: mean ± standard error of the mean, Mann-Whitney U test. ∗TG vs vehicle, ∗∗∗∗P < .0001 (B), ∗∗P < .01 (F) are P values shown (C and D).

Figure 3

TG inhibited colorectal tumor development in Atg7^ΔIEC conditional knockout mice. (A) Western blotting for ATG7 in colonic epithelial cells (IEC) to confirm deletion of ATG7 specifically in IECs in conditional knockout mice. (B–E) (N ≥10 each group) and figure legend as for Figure 2. (F) Combines data from 2E and 3C. Statistics B–D and F: mean ± standard error of the mean, Mann-Whitney U test. ∗TG vs vehicle, ∗∗∗P < .001, ∗∗P < .01, ∗P < .05. P values otherwise shown.

Figure 4

Long-term daily intrarectal TG treatment was not associated with clinically significant systemic toxicity. (A) Sinusoidal obstructive syndrome was not present at day 85. Representative H&E liver histology for vehicle and TG-treated animals. (B) Hematologic parameters (white blood cell counts, hemoglobin, and platelets) in peripheral blood at day 85 from WT and Atg7^ΔIEC mice. (C) Fluorescence-activated cell sorter plots showing cell gating strategy and subsets of CD45+CD3e+ (T lymphocyte) and CD4+, CD8+populations. There was a small but statistically significant increase in CD8: CD4 mesenteric node lymphocytes with TG treatment in both WT and Atg7^ΔIEC mice.

Figure 5

TG inhibited β-catenin activity in CAC tumors in vivo. Measurements in TG or vehicle-treated tumors at day 85 (N ≥5 each group). (A) Immunohistochemical (IHC) analysis of β-catenin in WT mice and in Atg7^ΔIEC conditional knockout mice. Left panels: representative IHC staining for β-catenin. Right panels: β-catenin protein levels assessed using IHC and Visiopharm software (where higher intensity has a lower value) and expressed as percentage of β-catenin positive cells within the tumors (upper) and tumor β-catenin intensity (bottom) from each mouse (5 mice per group). (B) Immunoblot analysis of non-phosphorylated β-catenin and its downstream tumor promoting gene products, cyclin D1, C-Jun, CD44, MMP7, survivin in WT mice and (C) in Atg7^ΔIEC mice. Each lane represents distal colon or pooled tumors from distal colon in an individual mouse. Densitometry of proteins corrected for β-actin in bottom right panel. Statistics: box plots show median, quartiles, and range; Mann-Whitney U test. ∗TG vs vehicle, ∗∗P < .01. Data representative of 3 independent experiments.

Figure 6

TG inhibited β-catenin activity in Atg7^ΔIEC conditional knockout mice. Measurements in TG or vehicle-treated tumors at day 85 (N ≥5 each group). (A) Immunohistochemical (IHC) analysis of β-catenin in WT mice and in Atg7^ΔIEC conditional knockout mice. Left panels: representative IHC staining for β-catenin. Right panels: β-catenin protein levels assessed using IHC and Visiopharm software (where higher intensity has a lower value) and expressed as percentage of β-catenin positive cells within the tumors (upper) and tumor β-catenin intensity (bottom) from each mouse (5 mice per group). (B) Immunoblot analysis of non-phosphorylated β-catenin and its downstream tumor promoting gene products, cyclin D1, C-Jun, CD44, MMP7, survivin in WT mice and (C) in Atg7^ΔIEC mice. Each lane represents distal colon or pooled tumors from distal colon in an individual mouse. Densitometry of proteins corrected for β-actin in bottom right panel. Statistics: box plots show median, quartiles, and range; Mann-Whitney U test. ∗TG vs vehicle, ∗∗P < .01. Data representative of 3 independent experiments.

Figure 7

TG inhibits β-catenin activity in vitro independently of autophagy. (A) Knockdown of ATG7 in Caco2 and HCT116 cells 48 hours after transfection with ATG7 (#3) or ATG7 (#4) or control (-) siRNA was assessed by Western blotting. (B) Basal β-catenin–driven transcriptional activity in Caco2 and HCT116 cells transfected with ATG7 or control siRNA for 48 hours was increased with inhibition of the autophagy gene. (N = 4). (C) Treatment of the same cells in presence of 1 μmol/L CHIR-99021 and indicated dose of TG for 16 hours inhibited β-catenin–driven transcriptional activity. Data are representative of 4 independent experiments. RLU, relative light units.

Figure 8

TG inhibits β-catenin activity via TGN. (A) Knockdown of HPRT in Caco2 and HCT116 cells 48 hours after transfection with HPRT (#1) or HPRT (#2) or control (-) siRNA, assessed by Western blotting. (B) TG in presence of 1 μmol/L CHIR-99021versus β-catenin–driven transcriptional activity in cells silenced with HPRT siRNA. (C) WT and Hprt^-/- derived primary murine fibroblasts treated with 5 μmol/L CHIR ± 10 μmol/L TG or vehicle for 16 hours. Immunofluorescence staining for β-catenin (green) and nuclei (DAPI, blue). Data are representative of 4 independent experiments. (D) Western blotting of cytosol and nuclear compartments of WT and Hprt^-/- derived primary murine fibroblasts treated with 5 μmol/L CHIR ± 10 μmol/L TG or vehicle for 16 hours showing TG inhibiting β-catenin activity and nuclear translocation with CHIR only in WT cells.

Figure 9

TG at low concentrations suppressed β-catenin activation via inhibition of Rac1. (A) Knockdown of Rac1 in Caco2 and HCT116 cells 48 hours after transfection with Rac1 siRNA #6 or Rac1 siRNA #7 versus control (-) siRNA, assessed by Western blotting. (B) [TG] versus β-catenin–driven transcriptional activity in control cells versus Rac1-siRNA silenced cells. (C) Cartoon showing role of Rac1 in β-catenin–driven transcription of Wnt target gene: C1, normal physiology with GTP; C2, TG with WT Rac1; C3, TG with genetically inactivated GTPase Q61L Rac1. (D) Left panel: Western blotting for Flag in HCT116 and Caco2 cells confirms overexpression of Rac1 WT and Rac1 mutant in these cells. Right panels: [TG] versus β-catenin–driven transcriptional activity in WT and Q61L (ie, constitutively active Rac1). Caco2 and HCT116 cells. Data are representative of 3 independent experiments. (E) Modelling experiments showing TGTP docked with β-catenin protein crystal structure (left panel) and docked in the conserved Rac1 nucleotide binding site (right panel). RLU, relative light units.