MyD88-mediated signaling prevents development of adenocarcinomas of the colon: role of interleukin 18 - PubMed (original) (raw)
. 2010 Aug 2;207(8):1625-36.
doi: 10.1084/jem.20100199. Epub 2010 Jul 12.
Andrea Worschech, Marco Cardone, Yava Jones, Zsofia Gyulai, Ren-Ming Dai, Ena Wang, Winnie Ma, Diana Haines, Colm O'hUigin, Francesco M Marincola, Giorgio Trinchieri
Affiliations
- PMID: 20624890
- PMCID: PMC2916129
- DOI: 10.1084/jem.20100199
MyD88-mediated signaling prevents development of adenocarcinomas of the colon: role of interleukin 18
Rosalba Salcedo et al. J Exp Med. 2010.
Abstract
Signaling through the adaptor protein myeloid differentiation factor 88 (MyD88) promotes carcinogenesis in several cancer models. In contrast, MyD88 signaling has a protective role in the development of azoxymethane (AOM)/dextran sodium sulfate (DSS) colitis-associated cancer (CAC). The inability of Myd88(-/-) mice to heal ulcers generated upon injury creates an altered inflammatory environment that induces early alterations in expression of genes encoding proinflammatory factors, as well as pathways regulating cell proliferation, apoptosis, and DNA repair, resulting in a dramatic increase in adenoma formation and progression to infiltrating adenocarcinomas with frequent clonal mutations in the beta-catenin gene. Others have reported that toll-like receptor (Tlr) 4-deficient mice have a similar susceptibility to colitis to Myd88-deficient mice but, unlike the latter, are resistant to CAC. We have observed that mice deficient for Tlr2 or Il1r do not show a differential susceptibility to colitis or CAC. However, upon AOM/DSS treatment Il18(-/-) and Il18r1(-/-) mice were more susceptible to colitis and polyp formation than wild-type mice, suggesting that the phenotype of Myd88(-/-) mice is, in part, a result of their inability to signal through the IL-18 receptor. This study revealed a previously unknown level of complexity surrounding MyD88 activities downstream of different receptors that impact tissue homeostasis and carcinogenesis.
Figures
Figure 1.
Myd88−/− mice develop colonic polyps after AOM/DSS administration. Cohorts of 8–10 WT and _Myd88_−/− mice per group were injected i.v. with AOM on day 0 followed by two DSS cycles administered in drinking water. At the completion of the first DSS cycle, mice were monitored for bleeding (A) and diarrhea (B). 60 d after AOM administration, hematocrits were measured (C), mice were euthanized and colons were resected and measured (D), and macroscopic polyps were counted (E). Colon sections were fixed in formalin and stained with hematoxylin-eosin. Sections were analyzed for inflammation degree (F), extent of ulceration (G), and squamous metaplasia (H). The data shown correspond to a representative experiment out of four performed. Data represent means ± SE (and in some panels also individual mice results). *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 2.
AOM/DSS treatment of Myd88−/− mice results in decreased proliferation and enhanced apoptosis of colonic epithelial cells and increased expression of mitogenic, angiogenic, and pro-tumorigenic genes. Cohorts of 10 WT and _Myd88_−/− mice were injected i.v. with AOM on day 0 followed by 3 d of DSS administration in drinking water. Thereafter, colons were resected, fixed in formalin, and sections were stained with Ki67 or ApopTag antibodies. Photomicrographs of representative sections from the respective groups are shown at 400× magnification (A). The number of proliferating cells per crypt and the number of apoptotic cells was determined by counting 20 consecutive crypts/section (B and C). Gene expression profile in colon tissue after AOM/DSS treatment was analyzed by real-time RT-PCR. The gene expression was normalized to Gapdh levels, and the expression of each gene relative to untreated WT mice is depicted (D). The data shown in A–D correspond to a representative experiment out of two performed. Data represent means ± SE. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 3.
AOM/DSS treatment enhances mucosal expression of COX-2 and phospho-STAT3 in Myd88−/− mice. Cohorts of 5–10 mice were subjected to treatment as indicated in Fig. 2. At day 3 of DSS administration, colons were resected and fixed in formalin or snap frozen for protein extraction. Tissue sections were stained for COX-2 and pSTAT3. Photomicrographs of representative sections from the respective groups are shown at 200× (A). Protein extracts were analyzed by Western blotting for COX-2, STAT3, and pSTAT3 expression. Gapdh was used as a control (B). The data shown in A and B correspond to a representative experiment out of two performed.
Figure 4.
Increased DNA damage is elicited by AOM/DSS treatment in Myd88−/− mice compared with WT mice. RNA was extracted from cohorts of 10 WT or _Myd88_−/− mice per group treated with AOM/DSS and from cohorts of six nontreated WT or _Myd88_−/− mice/group. Samples were processed using the Affymetrix microarray platform, and analyzed using the Ingenuity Pathway and Cluster and TreeView Programs. A significant decrease in DNA repair gene expression profile was observed in Myd88−/− mice after AOM/DSS treatment compared with treated WT mice (A and B). The data shown in A and B were obtained from the microarray experiment that was performed once. The pattern of DNA repair gene expression was subsequently confirmed using real-time RT-PCR. The gene expression was normalized to Gapdh levels, and the expression of each gene relative to untreated WT mice is depicted. One out of two representative experiments is shown (C). Data represent means ± SE. **, P < 0.01; ***, P < 0.001. For analysis of β-catenin mutations, cohorts of 15–20 mice/group were subjected to AOM/DSS treatment (two DSS/cycles). 60 d after AOM administration, individual polyps were resected, and DNA was extracted and analyzed for mutation of β-catenin using specific primers for exon 3. The numbers of polyps exhibiting point mutations at positions 33, 34, 37, and 41 in exon 3 are indicated in the scheme by the stars (D). The polyps analyzed for β-catenin mutations were obtained from multiple experiments and each polyp was sequenced for mutations once.
Figure 5.
AOM/DSS treatment induces increased frequency of colonic adenocarcinomas in Myd88−/− mice. Cohorts of 15 Myd88−/− mice and 20 WT mice were injected i.v. with AOM on day 0 followed by a single DSS cycle. Animals were monitored during 6 mo after treatment for survival (P = 0.0005, WT vs. _Myd88_−/−; A). All the colons were analyzed for polyps when mice reached any of the end points for euthanasia or at the completion of the experiment (***, P < 0.001; B). Colon sections were fixed and stained with hematoxylin-eosin. 17 polyps from the WT group and 52 polyps from Myd88−/− knockout mice were analyzed histologically. Photomicrographs of representative sections from the respective groups are shown at 20× (C). The percentage of mice developing adenocarcinomas is indicated (D). Photomicrographs of representative adenocarcinomas from multiple of those that developed in _Myd88_−/− mice are shown at 40× magnification (E–G). The data shown in this figure were obtained from a single experiment performed treating the mice with one DSS cycle but closely mimic the data obtained from two other independent experiments with mice treated with two DSS cycles that are shown in
Fig. S1
.
Figure 6.
Il18 deficiency mimics MyD88 deficiency susceptibility to polyp formation. Cohorts of 6–20 mice/group were subjected to AOM/DSS treatment as depicted by the scheme. At the end of the first DSS cycle, mice were analyzed for bleeding (A). 100 d after AOM administration, the _Il1r1_−/−, _Il18_−/−, and their corresponding controls were euthanized and colonic polyps were counted (B). For comparison, _Myd88_−/− mice were tested simultaneously. Because of their susceptibility to the treatment, the number of polyps was scored at the completion of the second DSS cycle (C). The data shown in A–C correspond to a representative experiment out of three performed. Il18−/− and Il1r−/− mice and their corresponding heterozygote and WT littermates were subjected to AOM/DSS treatment as indicated by the scheme. At the completion of the experiment, the number of colonic polyps was counted (D and E). The data shown in D and E correspond to a representative experiment out of two performed using littermate controls. Data represent means ± SE. **, P < 0.01; ***, P < 0.001.
Figure 7.
_Il18_-deficient mice exhibit enhanced expression of mitogenic/inflammatory cytokines and STAT-3 phosphorylation after AOM/DSS treatment. Cohorts of 8–10 WT and _Il18_−/− mice per group were injected i.v. with AOM on day 0, followed by 3 d of DSS administration in drinking water. Thereafter, colons were resected, RNA was extracted, and RT-PCR was performed using specific primers for the indicated genes. The gene expression was normalized to Gapdh levels, and the expression of each gene relative to untreated WT mice is depicted (A). Tissue sections were stained for pSTAT3. Photomicrographs of representative sections from the respective groups are shown at 200× magnification. Protein extracts were analyzed by Western blotting for COX-2, STAT3, and pSTAT3 expression. Gapdh was used as a control (C). Quantification of colonic epithelial cell proliferation and apoptosis was performed in tissue sections stained with Ki67 or ApopTag antibodies. The number of proliferating cells per crypt and the number of apoptotic cells/field are shown. The data shown in A–D correspond to a representative experiment out of two performed. Data represent means ± SE. *, P < 0.05; **, P < 0.01.
References
- Bartek J., Lukas J., Bartkova J. 2007. DNA damage response as an anti-cancer barrier: damage threshold and the concept of ‘conditional haploinsufficiency’. Cell Cycle. 6:2344–2347 - PubMed
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