We first addressed potential epigenetic changes in A20 expression in colon carcinogenesis by searching NCBI’s Genome Expression Omnibus 
. We found that A20 mRNA expression is significantly decreased in human adenomatous colonic polyps as compared to normal surrounding tissue (, Fig. S1
). These human colon tumors also express increased levels of the cell proliferation related genes c-myc and cyclinD1 (, Figure S1
). Several recent reports have also suggested genetic and epistatic changes in A20 expression in human colorectal cancer 
. To directly test A20’s potential cell-autonomous functions in IECs, we generated mice lacking A20 specifically in these cells by interbreeding A20FL
mice with villin-Cre transgenic mice 
. Compound A20FL/FL
villin-Cre mice effectively eliminated A20 protein expression from both small intestinal and colonic epithelial cells (). Previous reports have shown that mice harboring an intestinal epithelial cell-specific deletion of A20 were grossly normal and did not exhibit spontaneous inflammation or tumor development 
. However, these mice were more susceptible to induced models of colitis. Conversely, overexpression of A20 in intestinal epithelial cells protects mice from dextran sodium sulfate induced colitis, potentially through an enhancement of the epithelial barrier 
. To determine whether A20 expression might predispose to colon carcinogenesis, we interbred A20FL/FL
villin-Cre mice with APCmin
mice that harbor a mutation in the APC gene 
. Heterozygous APCmin/+
mice develop spontaneous intestinal tumors, while homozygous APCmin/min
mice are embryonically lethal 
. Importantly, this model does not involve gross inflammation such as that induced by repeated dextran sulfate sodium (DSS) and the mutagen azoxymethane (AOM). As previously reported, A20+/+
mice spontaneously developed multiple small intestinal tumors but infrequent colonic tumors (). Remarkably, A20FL/FL
mice possessed significantly increased numbers of colonic adenomas when compared to A20+/+
mice (). The aggregate size of colonic polyps was also increased in these mice (). The number of small bowel polyps was not affected by A20 deficiency in small intestinal IECs (). Thus, A20 appears to regulate colonic tumorigenesis.
Human colonic adenomas express less A20 than normal colonic mucosa.
A20 expression in IECs restricts colon tumorigenesis.
As A20 restricts NFκB signaling, A20 might restrict colon tumorigenesis by preventing spontaneous intestinal inflammation in A20FL/FL villin-Cre APCmin/+ mice. However, similar to A20FL/FL villin-Cre and A20+/+ villin-Cre mice, no signs of spontaneous inflammation were present in A20FL/FL villin-Cre APCmin/+ and A20+/+ villin-Cre APCmin/+ mice, and there were no differences in intestinal length or mouse weights (). Histologic examination of colonic neoplasms and surrounding normal intestinal tissues did not reveal significant differences between A20FL/FL villin-Cre APCmin/+ and A20+/+ villin-Cre APCmin/+ mice in inflammation, apoptosis, or proliferation rates (). Thus, A20 expression in IECs does not strongly restrict basal intestinal inflammation.
The APC gene is mutated in the majority of sporadic colorectal adenocarcinomas 
and in nearly all cases of familial adenomatous polyposis 
. The APC protein is part of a multi-protein complex termed the destruction complex. This complex, including the E3 ligase complex SCF/β-TRCP, causes phosphorylation, ubiquitination, and degradation of β-catenin 
. Stimulation of cells with wnt ligands, or mutations of proteins such as APC, interrupt this process, allowing β-catenin to migrate to the nucleus and induce transcription of β-catenin dependent genes 
. Recently, renewed interest has arisen concerning ubiquitination of the components of the classical wnt/β-catenin signaling cascade as a mechanism for its regulation 
Given A20’s pleiotropic functions in regulating ubiquitin dependent signals, we hypothesized that A20 might regulate wnt signaling. To investigate this possibility, we first used an siRNA approach to reduce A20 expression in RKO cells, a cell line with intact wnt induced β-catenin signaling 
. Acute knockdown of A20 expression caused a marked increase in TCF/LEF4 luciferase reporter activity after recombinant human wnt3a stimulation when compared to cells transfected with control siRNA (). These results suggest that A20 directly restricts wnt induced signaling.
A20 supports β-catenin ubiquitination and degradation through an interaction with the destruction box.
To better understand how A20 restricts β-catenin signaling, we investigated whether A20 binds to destruction complex proteins that regulate β-catenin ubiquitination. A20 co-precipitated with both heterologously expressed and endogenous Axin, the scaffolding protein that binds many destruction complex proteins (, Figure S2
). No change in this association was seen after wnt3a stimulation, suggesting a constitutive association between these two molecules. A20 utilizes a number of motifs to bind ubiquitin chains, other ubiquitin binding proteins, and other ubiquitin editing enzymes 
. We thus tested whether A20’s N-terminal or C-terminal domains mediate binding of A20 to Axin. Full length A20 co-precipitated with Axin, as did (to a lesser extent) the N-terminal OTU domain of A20 (). By contrast, the C-terminal portion of A20 containing its seven zinc fingers failed to co-precipitate with Axin, suggesting that A20 preferentially utilizes its N-terminal OTU domain to bind Axin (). These studies suggest that A20 interacts directly with the β-catenin destruction complex.
As β-catenin protein levels are tightly regulated by the SCF/β-TRCP E3 ligase complex, we tested the role of A20 in regulating β-catenin ubiquitination and stability. Stimulation of both A20 siRNA and control siRNA knockdown RKO cells with wnt3a led to increased levels of total β-catenin, however, A20 deficient RKO cells accumulated significantly greater levels of the active dephosphorylated form of β-catenin when compared to control RKO cells (). Notably, A20 protein levels did not increase after wnt3a stimulation, suggesting that wnt3a does not stimulate NFκB dependent induction of A20 expression (). In addition, increased β-catenin dependent TCF/LEF4 reporter activity in A20 deficient RKO cells did not result from increased β-catenin mRNA expression, suggesting that A20 regulates β-catenin via post-transcriptional or post-translational mechanisms (Fig. S3
). Cellular β-catenin protein levels are regulated at the post-translation level through K48-linked ubiquitination and subsequent proteasomal degradation. To determine whether A20 regulates β-catenin ubiquitination, we stimulated RKO cells with wnt3a ligand, immunoprecipitated β-catenin, and immunoblotted for ubiquitin. A20 deficient cells accumulated significantly decreased levels of ubiquitinated β-catenin than control cells, particularly at four hours after wnt3a stimulation (). These results correspond with increased β-catenin levels in A20 deficient cells (). Taken together, these studies indicate that A20 binds to the APC destruction complex and restricts wnt3a induced β-catenin signaling by promoting β-catenin ubiquitination and degradation.
Finally, to confirm that A20 expression in IECs regulates wnt/β-catenin signaling in vivo, we interbred A20FL/FL
mice with mice expressing a tamoxifen-sensitive Cre recombinase under the villin gene promoter elements (villin-ER/Cre) 
. Intra-peritoneal injection of tamoxifen into compound A20FL/FL
villin-ER/Cre and control A20+/+
villin-ER/Cre mice led to reduction of A20 mRNA expression and increased expression of the β-catenin dependent cyclinD1 and c-myc mRNAs in purified intestinal epithelial cells (). These genes are also increased in human colonic adenomas (Fig. S1
). Thus, A20 restricts β-catenin dependent signaling in vitro and in vivo.
Acute deletion of A20 from IECs leads to increased levels of Cyclin D1 and MYC mRNA in vivo.
In summary, we have discovered that the ubiquitin-modifying enzyme A20 is a tumor suppressor for colon carcinogenesis. This tumor suppressor function appears to be due to a novel function for A20 in restricting wnt induced β-catenin signaling. The relevance of our findings to human disease is high, as reduced A20 expression is a common epigenetic finding in human adenomas, and as the increased tumorigenesis of A20FL/FL APCmin/+ mice appears to be selective for colonic tissue, where most human intestinal cancers occur. Our findings extend A20’s tumor suppressor function from B cell lymphomas to colon cancers. Finally, these studies demonstrate that A20, a potent regulator of multiple human autoimmune and inflammatory diseases, is also broadly important for preventing malignant transformation.