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. 2015 Aug 14;6(23):19671-84.
doi: 10.18632/oncotarget.4284.

The MUTYH base excision repair gene protects against inflammation-associated colorectal carcinogenesis

Francesca Grasso  1   2 , Serena Di Meo  3   4 , Gabriele De Luca  5 , Luca Pasquini  5 , Stefania Rossi  5 , Monica Boirivant  6 , Mauro Biffoni  5 , Margherita Bignami  1 , Emma Di Carlo  3   4
Affiliations

The MUTYH base excision repair gene protects against inflammation-associated colorectal carcinogenesis

Francesca Grasso et al. Oncotarget. .

Abstract

MUTYH DNA glycosylase removes mismatched adenine opposite 7, 8-dihydro-8-oxoguanine (8-oxoG), which is the major mutagenic lesion induced by oxidative stress. Biallelic mutations in MUTYH are associated with MUTYH-Associated polyposis (MAP) and increased risk in colorectal cancer (CRC). We investigated cancer susceptibility associated with MUTYH inactivation in a mouse model of inflammation-dependent carcinogenesis induced by azoxymethane (AOM) and dextran sulphate (DSS). Mutyh-/- mice were more sensitive than wild-type (WT) animals to AOM/DSS toxicity and accumulated DNA 8-oxoG in their gastrointestinal tract. AOM/DSS-induced colonic adenomas were significantly more numerous in Mutyh-/- than in WT animals, and frequently showed a tubulo-villous feature along with high-grade dysplasia and larger size lesions. This condition resulted in a greater propensity to develop adenocarcinomas. The colon of untreated Mutyh-/- mice expressed higher basal levels of pro-inflammatory cytokines GM-CSF and IFNγ, and treatment with AOM/DSS induced an early decrease in circulating CD4+ and CD8+ T lymphocytes and an increase in myeloid-derived suppressor cells (MDSCs). Adenomas from Mutyh-/- mice had a greater infiltrate of Foxp3+ T regulatory cells, granulocytes, macrophages, MDSCs and strong expression of TGF-β-latency-associated peptide and IL6. Our findings indicate that MUTYH loss is associated with an increase in CRC risk, which involves immunosuppression and altered inflammatory response. We propose that the AOM/DSS initiation/promotion protocol in Mutyh-/- mice provides a good model for MAP.

Keywords: DSS; MUTYH; azoxymethane; colorectal cancer; inflammation.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare that there are no competing financial interests in relation to the work described.

Figures

Figure 1
Figure 1. Survival and DNA 8-oxoG levels following single and/or combined AOM and DSS exposures
A. Schematic representation of the treatment. B. Survival data of wild-type and Mutyh−/− mice treated with AOM, DSS or the combined treatment as indicated in A). C. Kaplan–Meier survival curves of AOM/DSS-treated wild-type (solid line) and Mutyh−/− (dotted line) mice. D. DNA 8-oxoG levels in the GI tract of wild-type (open bar) and Mutyh−/− (full bar) mice untreated or exposed to AOM, DSS or combined treatment. Data are mean ± SE of 5–10 animals/genotype. *P ≤ 0.05 (Student's t-test).
Figure 2
Figure 2. Analysis of leukocyte populations in the spleen or blood shortly after AOM/DSS exposures
A. Schematic representation of the treatment. B. A representative image of FACS analysis. C. Leukocyte populations as percentages of viable cells in the blood of untreated wild-type (WT) (open bar) and Mutyh−/− mice (full bar). D. Leukocyte populations as percentages of viable cells in the blood of untreated or AOM/DSS-treated mice. E. MDSCs populations (G: granulocytic, M: monocytic) as percentages of viable cells in the spleen of control or AOM/DSS treated mice. Untreated and treated wild-type mice (open and grey dashed bar, respectively); untreated and treated Mutyh−/− mice (full and black dashed bar). Data are mean ± SE of 5 animals/genotype/condition. *P ≤ 0.05 (Student's t-test).
Figure 3
Figure 3. Analysis of cytokines expression in the colon
Cytokines basal levels in the colon of wild-type (open bar) and Mutyh−/− (full bar) were measured with Bio-Plex Pro™ Mouse Cytokine 8-plex Assay. Proteins levels are expressed as concentration (pg/ml)/mg of total proteins. Data are mean ± SE of 4–5 animals/genotype. *P ≤ 0.05 (Student's t-test).
Figure 4
Figure 4. Morphologic features of colonic tumors following AOM/DSS treatment
A. Histologic alterations developed in the colon of wild-type and Mutyh−/− mice following AOM/DSS treatment. *P = 0.016 (Fisher's exact test). B. Average number of adenomas/mouse in wild-type (open bar) and Mutyh−/− (full bar). Data are mean ± SE of 7–14 animals/genotype. *P ≤ 0.05 (Student's t-test). Representative images of H&E stained sections of tubular adenomas with a mild/moderate dysplasia in wild-type (C) and Mutyh−/− (D) mice. Adenomas with severe dysplasia were more frequent in Mutyh−/− mice (E) which may also develop adenocarcinomas (F, G). Adenocarcinoma (ADK). Magnification: C, D, E, G, X400; F, X200. Scale bars: C, D, E, G, 30 μm; F, 50 μm.
Figure 5
Figure 5. Lymphocyte infiltrate, Foxp3 and TGFβ1-LAP expression in adenomas developed in AOM/DSS-treated wild-type and Mutyh−/− mice
A–F. Representative images of IHC performed on paraffin-embedded sections, showing CD3+ T lymphocyte infiltrate in the stromal component of adenomas developed in wild-type (A), and Mutyh−/− (B) mice. Foxp3+ lymphocytes were barely detected inside adenomas from wild-type mice (C), while they were more represented inside adenomas from Mutyh−/− mice (D). TGFβ1-LAP expression in wild-type (E), and Mutyh−/− (F) mice. Magnification: A–F, X400. Scale bars: A–F, 30 μm.
Figure 6
Figure 6. Myeloid cell infiltrate and IL6 expression in adenomas developed in AOM/DSS-treated wild-type and Mutyh−/− mice
A–H. Representative images of IHC performed on paraffin-embedded or frozen (C, D) sections of adenomatous lesions developed in wild-type (left column) or Mutyh−/− (right column) mice after AOM/DSS treatment. A–B) Granulocytes (GR-1+) staining in wild-type (A) and in Mutyh−/− mice (B) C–D) Macrophages (CD11b+/CD18+: Mac-1) staining in wild-type (C) and in Mutyh−/− mice (D) E–F) Double immunohistochemistry with anti-GR-1 (red), and anti-CD11b (brown) Abs, resulting in double stained MDSCs (brick red staining) inside adenomas from wild-type (E) and Mutyh−/− mice (F) G–H) Expression of IL6 in adenomas from wild-type (G) and Mutyh−/− mice (H), and within the nearby normal colonic crypts (H inset). Magnification: A and B, X200; C–H, X400. Scale bars: A and B, 50 μm; C–H, 30 μm.
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