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. 2015 Aug;21(8):906-13.
doi: 10.1038/nm.3908. Epub 2015 Jun 24.

Inflammasome-independent role of AIM2 in suppressing colon tumorigenesis via DNA-PK and Akt

Justin E Wilson  1 , Alex S Petrucelli  1 , Liang Chen  1 , A Alicia Koblansky  1 , Agnieszka D Truax  1 , Yoshitaka Oyama  1 , Arlin B Rogers  2 , W June Brickey  1 , Yuli Wang  3 , Monika Schneider  4 , Marcus Mühlbauer  5 , Wei-Chun Chou  1 , Brianne R Barker  6 , Christian Jobin  5 , Nancy L Allbritton  3 , Dale A Ramsden  7 , Beckley K Davis  8 , Jenny P Y Ting  1
Affiliations

Inflammasome-independent role of AIM2 in suppressing colon tumorigenesis via DNA-PK and Akt

Justin E Wilson et al. Nat Med. 2015 Aug.

Abstract

The inflammasome activates caspase-1 and the release of interleukin-1β (IL-1β) and IL-18, and several inflammasomes protect against intestinal inflammation and colitis-associated colon cancer (CAC) in animal models. The absent in melanoma 2 (AIM2) inflammasome is activated by double-stranded DNA, and AIM2 expression is reduced in several types of cancer, but the mechanism by which AIM2 restricts tumor growth remains unclear. We found that Aim2-deficient mice had greater tumor load than Asc-deficient mice in the azoxymethane/dextran sodium sulfate (AOM/DSS) model of colorectal cancer. Tumor burden was also higher in Aim2(-/-)/Apc(Min/+) than in APC(Min/+) mice. The effects of AIM2 on CAC were independent of inflammasome activation and IL-1β and were primarily mediated by a non-bone marrow source of AIM2. In resting cells, AIM2 physically interacted with and limited activation of DNA-dependent protein kinase (DNA-PK), a PI3K-related family member that promotes Akt phosphorylation, whereas loss of AIM2 promoted DNA-PK-mediated Akt activation. AIM2 reduced Akt activation and tumor burden in colorectal cancer models, while an Akt inhibitor reduced tumor load in Aim2(-/-) mice. These findings suggest that Akt inhibitors could be used to treat AIM2-deficient human cancers.

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Figures

Figure 1
Figure 1
AIM2 is distinct from ASC during colitis-associated colon cancer. (a) Induction procedure for the AOM/DSS model of CAC. (b–d) Weight loss (b), % survival (d, days) (c) and average clinical scores (d) in Aim2−/− (n = 25), Asc−/− (n = 11) and wild-type (n = 30) mice following induction of CAC using AOM/DSS. Mock wild-type animals (n = 10) received a single i.p. injection of AOM and were given regular drinking water instead of DSS. Error bars, s.e.m. (e) H&E staining of colons from AOM/DSS-treated wild-type, Aim2−/− and Asc−/− mice and (f) semiquantitative scoring of inflammation in colons from AOM/DSS-treated wild-type and Aim2−/− mice (n = 6 for WT AOM/Mock and Aim2−/− AOM/DSS, and n = 5 for WT AOM/DSS and Aim2−/− AOM/Mock). Images were taken at 200× magnification; scale bars, 100 µm. (g) Inflammatory cytokine mRNA (AU, arbitrary units) (n = 3 for WT AOM/Mock and Aim2−/− AOM/Mock, and n = 6 for WT AOM/DSS and Aim2−/− AOM/DSS) and (h) protein expression in wild-type and Aim2−/− colons (n = 5 for WT AOM/Mock and Aim2−/− AOM/Mock, n = 10 for WT AOM/DSS and n = 8 for Aim2−/− AOM/DSS). (i) Caspase-1 activation in wild-type and Aim2−/− colons and (j) IL-1β and IL-18 production in wild-type, Aim2−/− and Asc−/− colons (n = 5 for WT AOM/Mock, Aim2−/− AOM/Mock and Asc−/− AOM/Mock, and n = 10 for WT AOM/DSS, n = 8 for Aim2−/− AOM/DSS and n = 5 for Asc−/− AOM/DSS). Error bars, s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001; b,d,j, one-way ANOVA (Tukey's multiple-comparisons test); c, log-rank test.
Figure 2
Figure 2
AIM2 protects against colon tumorigenesis. (a,b) Mini-endoscopy (a) and representative images (b) of colons from AOM/DSS-treated wild-type (n = 5), Asc−/− (n = 3) and Aim2−/− mice (n = 5). (c) Macroscopic polyp counts, average polyp size per mouse and tumor load (n = 21 for WT, n = 18 for Aim2−/− and n = 10 for Asc−/−). (d,e) Semiquantitative scoring of colon hyperplasia (d) and dysplasia (e) in AOM/DSS-treated wild-type and Aim2−/− mice (n = 6 for WT AOM/Mock and Aim2−/− AOM/DSS, and n = 5 for WT AOM/DSS and Aim2−/− AOM/Mock). (f) Colon polyp counts, average polyp size per mouse and tumor load in AOM/DSS-treated wild-type, Aim2−/−, Il1b−/− and Aim2−/−/Il1b−/− mice, with representative images of colons in the right panel (n = 5 for wild-type, Aim2−/− and Il1b−/−, and n = 4 for Aim2−/−/Il1b−/−). (g) Colon polyp counts, average polyp size per mouse and tumor load in AOM/DSS-treated wild-type and Aim2−/− radiation bone marrow chimeras, with representative images of colons in the right panel (n = 4 for WT>WT, n = 5 for WT>Aim2−/− and n = 7 for Aim2−/−>WT and Aim2−/−>Aim2−/−). (h) Colon polyp counts, average polyp size per mouse and tumor load in ApcMin/+ and Aim2−/−/ApcMin/+ mice representative images of colons in the right panel (n = 6 mice/group). Each symbol represents one animal. Error bars, s.e.m. ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001; c,g, one-way ANOVA (Tukey's multiple-comparisons test); d,e,f,h, unpaired t-test.
Figure 3
Figure 3
AIM2 negatively regulates Akt activity in vitro and in vivo. (a,b) Western blot analyses of PI3K pathway members in the colons of AOM/DSS-treated mice (representative of n = 6 mice/group). (c) Western blot analyses of Akt phosphorylation in colon homogenates from 12-week-old ApcMin/+ and Aim2−/−/ApcMin/+ mice (representative of n = 6 mice/group). (d–g) Western blot analyses of Akt phosphorylation in IGF-1–simulated MEFs from Aim2+/+ and Aim2−/− littermates (d), Asc+/+ and Asc−/− littermates (e), AIM2–3xFlag- or EV-transfected HCT-116 cells (f), and wild-type and Aim2−/− organoids (g). Actin (β-actin) and total Akt were used as loading controls. Results are representative of 1 of 3 experiments.
Figure 4
Figure 4
AIM2 restricts cell proliferation and promotes apoptosis. (a) Colon Ki-67 staining in AOM/DSS-treated wild-type and Aim2−/− mice (n = 6 for WT AOM/Mock and Aim2−/− AOM/DSS, and n = 5 for WT AOM/DSS and Aim2−/− AOM/Mock). 200× magnification; scale bars, 100 µm; error bars, s.e.m. (b) Cell numbers of Aim2+/+ and Aim2−/− MEFs (d, days). Error bars, s.e.m., from triplicate samples per group. (c) Western blot analyses of activated caspase-7 and procaspase-7 in colons from AOM/DSS-treated wild-type and Aim2−/− mice (n = 6 mice/group). (d) Western blot analyses of activated caspase-3 and procaspase-3 in staurosporine (Staur)-treated Aim2+/+ and Aim2−/− MEFs (h, hours). Results are representative of 1 of 3 experiments. (e) Representative images of colons from DMSO- or Akt inhibitor (API-2)-treated Aim2−/− mice. (f) Number of macroscopic colon polyps, average polyp size and tumor load in API-2-treated Aim2−/− mice. Each symbol represents one animal. Error bars denote standard error. ns, not significant. *P < 0.05, **P < 0.01, ***P < 0.001. a,b, unpaired t-test; f, Mann-Whitney U-test (due to the non-normal distribution of the data sets and small sample size).
Figure 5
Figure 5
AIM2 associates with DNA-PK and restricts its activity and Akt phosphorylation. (a) Immunoprecipitation of AIM2 and immunoblots of PI3K members and (b) reciprocal immunoprecipitation of DNA-PKcs and immunoblot of AIM2 in AIM2-Flag (Fg)- or empty vector (EV)-expressing HEK293T cells treated with IGF-1 (IGF) (c) Co-immunoprecipitation of DNA-PKcs and AIM2 in the presence of ethidium bromide (EtBr) in HEK293T cells. (d) Western blot analysis of phosphorylated DNA-PK (p-DNA-PK) in bleocin-treated AIM2-expressing HCT-116 cells. (e) Western blot analysis of p-Akt in 10 µM NU7026- or NU7441-treated Aim2+/+ and Aim2−/− MEFs. (f) Western blot analysis of activated caspase-3 in staurosporine (Stauro)-treated Aim2+/+ and Aim2−/− MEFs treated with NU7026 (5 µM; h, hours). (g) Cell number of Aim2+/+ and Aim2−/− MEFs cultured with 1 µM NU7026 (d, days). Error bars, s.e.m., from triplicate samples per group. Results are representative of 1 of 3 independent experiments (a,b,c,d,e,g) and representative of 1 of 2 independent experiments (f). **P < 0.01, ***P < 0.001. One-way ANOVA (Tukey's multiple-comparisons test).
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